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Dasgupta, Amitava, Amanda Peterson, Alice Wells, and Jeffrey K. Actor. "Effect of Indian Ayurvedic Medicine Ashwagandha on Measurement of Serum Digoxin and 11 Commonly Monitored Drugs Using Immunoassays: Study of Protein Binding and Interaction With Digibind." Archives of Pathology & Laboratory Medicine 131, no. 8 (August 1, 2007): 1298–303. http://dx.doi.org/10.5858/2007-131-1298-eoiama.
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Abstract Context.—Ashwagandha, a popular Ayurvedic medicine, is now available in the United States. Alkaloids found in this herb have structural similarity with digoxin. Objective.—To study potential interference of Ashwagandha with serum digoxin measurement by immunoassays. Potential interference was also investigated with immunoassays for 11 other commonly monitored drugs. In addition, interaction of components of Ashwagandha with the Fab fragment of antidigoxin antibody (Digibind) was investigated. Design.—Two different brands of liquid extract and 1 dry powdered form of Ashwagandha were used for this investigation. Aliquots of drug-free serum were supplemented with various concentrations of Ashwagandha and apparent digoxin concentrations were measured by 3 digoxin immunoassays. Mice were fed with Ashwagandha and apparent digoxin concentrations were measured 1 and 3 hours after feeding. Potential interference of Ashwagandha with immunoassays of 11 other drugs was also investigated. Interaction of components of Ashwagandha with Digibind was studied in vitro. Results.—Significant apparent digoxin concentrations were observed both in vitro and in vivo using the fluorescence polarization immunoassay of digoxin, whereas the Beckman and the microparticle enzyme immunoassay digoxin assay demonstrated minimal interference. Immunoassays of 11 other drugs tested were unaffected. When Ashwagandha extract was added to a serum pool containing digoxin, falsely elevated digoxin value was observed with fluorescence polarization immunoassay, but values were falsely lowered when measured by the microparticle enzyme immunoassay. Digibind neutralized digoxin-like immunoreactive components of Ashwagandha in vitro. Conclusions.—Components of Ashwagandha interfered with serum digoxin measurements using immunoassays. Digibind neutralized free digoxin-like immunoreactive components of Ashwagandha.
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Warkentin, Theodore E. "Challenges in Detecting Clinically Relevant Heparin-Induced Thrombocytopenia Antibodies." Hämostaseologie 40, no. 04 (October 22, 2020): 472–84. http://dx.doi.org/10.1055/a-1223-3329.
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AbstractHeparin-induced thrombocytopenia (HIT) is an antibody-mediated hypercoagulable state featuring high thrombosis risk and distinct pathogenesis involving immunoglobulin G-mediated platelet activation. The target of the immune response is a cationic “self” protein, platelet factor 4 (PF4), rendered antigenic by heparin. A key problem is that only a minority of anti-PF4/polyanion antibodies induced by heparin are pathogenic, i.e., capable of causing platelet activation and thereby clinical HIT. Since thrombocytopenia occurs frequently in hospitalized, heparin-treated patients, testing for “HIT antibodies” is common; thus, the problem of distinguishing between pathogenic and nonpathogenic antibodies is important. The central concept is that those antibodies that have platelet-activating properties demonstrable in vitro correlate well with pathogenicity, as shown by platelet activation tests such as the serotonin-release assay (SRA) and heparin-induced platelet activation assay. However, in most circumstances, immunoassays are used for first-line testing, and so it is important for clinicians to appreciate which immunoassay result profiles—in the appropriate clinical context—predict the presence of platelet-activating antibodies (Bayesian analysis). Clinicians with access to rapid, on-demand HIT immunoassays (e.g., particle gel immunoassay, latex immunoturbidimetric assay, chemiluminescent immunoassay) can look beyond simple dichotomous result interpretation (“negative”/“positive”) and incorporate semiquantitative interpretation, where, for example, a strong-positive immunoassay result (or even combination of two immunoassays) points to a greater probability of detecting platelet-activating antibodies, and hence supporting a diagnosis of HIT. Recent recognition of “SRA-negative HIT” has increased the importance of semiquantitative interpretation of immunoassays, given that strong immunoassay reactivity is a potential clue indicating possible HIT despite a (false) negative platelet activation assay.
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Cortez, Samuel, Kyle McNerney, and Ana Maria Arbelaez. "412 Cortisol cut off point to diagnose adrenal insufficiency (AI) using a monoclonal antibody immunoassay." Journal of Clinical and Translational Science 6, s1 (April 2022): 80. http://dx.doi.org/10.1017/cts.2022.239.
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OBJECTIVES/GOALS: AI is diagnosed when peak cortisol level after a cosyntropin stimulation test is <18 mg/dL using polyclonal antibody (pAb) immunoassay. However, the polyclonal assay is being replaced by a specific monoclonal antibody (mAb) immunoassay which yields lower cortisol levels, leading to the over diagnosis of AI and use of unnecessary steroid use. METHODS/STUDY POPULATION: We obtained 36 samples from patients undergoing 1 mcg cosyntropin stimulation tests for diagnosis of AI. Samples were analyzed using pAb immunoassay (Abbott Architect Cortisol), mAb immunoassay (Roche Elecsys Cortisol II), and mass spectrometry (MS). AI was diagnosed if serum cortisol level was <18 using the pAb immunoassay. Measurements by MS and mAb immunoassay were individually used in simple logistic regression models to predict AI. For each model, we calculated a cortisol level corresponding to a 50% probability (median) of AI and used the delta method to determine the standard error and 95% confidence interval of the median. We used receiver operator characteristic (ROC) curve, area under the curve, sensitivity, and specificity to evaluate the potential of the median values as thresholds for each predictor. RESULTS/ANTICIPATED RESULTS: Data showed a mean cortisol level of 17 mcg/dL using the pAb immunoassay, 12 mcg/dL using the mAb immunoassay, and 12.96 mcg/dL using MS. The mean difference in cortisol level between the mAb immunoassay and the pAb immunoassay was 5.12 mcg/dL (p-value <0.01). The ROC curve model indicated an area under the curve of 0.997 with a median value of 11.2 mcg/dL for the mAb immunoassay. This provides a sensitivity of 95%, specificity of 95%, positive predictive value of 95%, and negative predictive value of 94%. This new threshold has a Kappa coefficient of 0.89 when compared to the pAb immunoassay. DISCUSSION/SIGNIFICANCE: New and highly specific mAb immunoassays are being used more widely but yield lower cortisol results. This reflects the need for further studies to determine new cut off points for highly specific cortisol immunoassays. A cut off level of 11.2 mcg/dL would provide a sensitivity of 95% and specificity of 95%.
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Armbruster, D. A., R. H. Schwarzhoff, E. C. Hubster, and M. K. Liserio. "Enzyme immunoassay, kinetic microparticle immunoassay, radioimmunoassay, and fluorescence polarization immunoassay compared for drugs-of-abuse screening." Clinical Chemistry 39, no. 10 (October 1, 1993): 2137–46. http://dx.doi.org/10.1093/clinchem/39.10.2137.
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Abstract The newest formulation of the Syva EMIT assay for drugs of abuse, EMIT II, and a new immunoassay, OnLine (Roche), utilizing the kinetic interaction of microparticles in solution (KIMS) methodology, RIA tests, and TDx fluorescence polarization immunoassay (FPIA) procedures were compared for marijuana, cocaine, opiates, and barbiturates. Both EMIT II and OnLine immunoassays were performed with a Hitachi 717 analyzer. Calibration curves, the degree of separation between negative and cutoff calibrators, precision, likelihood of carryover from positive to negative samples, and overall ease and speed of analysis were evaluated. RIA and OnLine detected 99% of gas chromatography/mass spectrometry (GC/MS)-confirmed marijuana samples; TDx, 95%; and EMIT II, 88%. All four immunoassays detected approximately 99% of confirmed cocaine-positive urines. RIA, OnLine, and TDx all detected 100% of opiate-confirmed samples; EMIT II, 97%. Barbiturate assays exhibited the greatest disparity, with OnLine and TDx detecting 100% of confirmed positives; EMIT II, 88%; and RIA, 78%. For a variety of reasons, we prefer the fully automated EMIT II and OnLine assays for high-volume urine testing, in comparison with our laboratory's semiautomated RIA tests and the limited-throughput TDx system. The four immunoassays investigated delivered comparable performance in terms of detection rates for GC/MS-confirmed positives for some drugs but not for others.
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Kaufman, Bennett M., and Marion Clower. "Immunoassay of Pesticides: An Update." Journal of AOAC INTERNATIONAL 78, no. 4 (July 1, 1995): 1079–90. http://dx.doi.org/10.1093/jaoac/78.4.1079.
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Abstract Measurement of levels of pesticide residues in foods and crops most often requires extensive cleanup and instrumental techniques such as gas chromatography. Immunoassay measurement techniques, on the other hand, may be used directly on the test portion or require only minimal cleanup. Further refinements of the common antibody–enzyme-based solid-phase assays, such as use of coated magnetic particles, antibody-coated crystals, and continuous-flow devices, have extended the measurement range and applicability of these assays. Likewise, new immunoassays for pesticides have been developed, and existing assays have been refined, optimized, and more completely characterized and validated. In addition to their ability to accurately and reliably measure amounts of residues present in food and crops, immunoassays can be readily used as rapid screening methods for contaminants in field samples. We have previously reviewed much of the work in the area of pesticide immunoassay; this report updates previous information and discusses some new immunoassay techniques.
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Ehm, M., and A. Kappel. "Immunoassays for diagnosis of coagulation disorders." Hämostaseologie 30, no. 04 (2010): 194–201. http://dx.doi.org/10.1055/s-0037-1619055.
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SummaryImmunoassays play a pivotal role in the clinical laboratory. In the coagulation section of the laboratory, they are used as an aid for diagnosis of deep vein thrombosis or pulmonary embolism, thrombophilia screening, or detection of coagulation factor deficiencies, respectively. Enzyme-linked immunosorbent assay (ELISA) and latex agglutination immunoassay technologies are currently most widely used, while Luminescent Oxygen Channeling Immuno - assay (LOCI®) and other chemiluminescencebased immunoassays are emerging technologies for the coagulation laboratory. However, not all immunoassay technologies employed are compatible with the workflow requirements of the coagulation laboratory, and, not all technologies are suitable for detection or quantification of every marker.This review focuses on technical and performance aspects of those immunoassay technologies that are most widely used in the coagulation laboratory, and provides a description of markers that are typically tested by immunoassays.
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Cortez, Samuel, Kyle McNerney, and Ana Maria Arbelaez. "PSAT085 Monoclonal Cortisol Immunoassay Cut Off Point to Diagnose Adrenal Insufficiency (AI)." Journal of the Endocrine Society 6, Supplement_1 (November 1, 2022): A115. http://dx.doi.org/10.1210/jendso/bvac150.234.
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Abstract Objective Adrenal Insufficiency (AI) is diagnosed when the peak cortisol level after a cosyntropin stimulation test is <18 mg/dL using a standard reference (e.g. polyclonal antibody (pAb) immunoassay). However, in most laboratories the polyclonal assay is being replaced by a specific monoclonal antibody (mAb) immunoassay which yields lower cortisol levels, leading to the over diagnosis of AI and unnecessary steroid use. Methods/Study Population We obtained blood samples from 36 patients undergoing 1 mcg cosyntropin stimulation tests for diagnosis of AI. Samples were analyzed using pAb immunoassay (Abbott Architect Cortisol), mAb immunoassay (Roche Elecsys Cortisol II), and mass spectrometry (MS). AI was diagnosed if serum cortisol level was <18 using the pAb immunoassay. Measurements by MS and mAb immunoassay were individually used in simple logistic regression models to predict AI. For each model, we calculated a cortisol level corresponding to a 50% probability (median) of AI and used the delta method to determine the standard error and 95% confidence interval of the median. We used receiver operator characteristic (ROC) curve, area under the curve, sensitivity, and specificity to evaluate the potential of the median values as thresholds for each predictor. Results The ROC curve model indicated an area under the curve of 0.997 with a median value of 11.2 mcg/dL for the mAb immunoassay. This provides a sensitivity of 95%, specificity of 95%, positive predictive value of 95%, and negative predictive value of 94%. This new value has a Kappa coefficient of 0.89 when compared to the pAb immunoassay. When using all the data points throughout the stimulation test, data showed a mean difference in cortisol level between the mAb immunoassay and the pAb immunoassay of 5.12 mcg/dL (p-value <0.01) at any point during the test. Discussion/Significance New and highly specific mAb immunoassays are being used more widely but yield lower cortisol results. This reflects the need for further studies to determine new cut off points for highly specific cortisol immunoassays. A cut off level of 11.2 mcg/dL for the mAb immunoassay would provide a sensitivity of 95% and specificity of 95%. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.
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Darwish, Ibrahim A. "Immunoassay Methods and their Applications in Pharmaceutical Analysis: Basic Methodology and Recent Advances." International Journal of Biomedical Science 2, no. 3 (September 15, 2006): 217–35. http://dx.doi.org/10.59566/ijbs.2006.2217.
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Immunoassays are bioanalytical methods in which the quantitation of the analyte depends on the reaction of an antigen (analyte) and an antibody. Immunoassays have been widely used in many important areas of pharmaceutical analysis such as diagnosis of diseases, therapeutic drug monitoring, clinical pharmacokinetic and bioequivalence studies in drug discovery and pharmaceutical industries. The importance and widespread of immunoassay methods in pharmaceutical analysis are attributed to their inherent specificity, high-throughput, and high sensitivity for the analysis of wide range of analytes in biological samples. Recently, marked improvements were achieved in the field of immunoassay development for the purposes of pharmaceutical analysis. These improvements involved the preparation of the unique immunoanalytical reagents, analysis of new categories of compounds, methodology, and instrumentation. The basic methodologies and recent advances in immunoassay methods applied in different fields of pharmaceutical analysis have been reviewed.
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Hashida, Seiichi, Setsuko Ishikawa, Kazuya Hashinaka, Ichiro Nishikata, Shinichi Oka, and Eiji Ishikawa. "Earlier Detection of Human Immunodeficiency Virus Type 1 p24 Antigen and Immunoglobulin G and M Antibodies to p17 Antigen in Seroconversion Serum Panels by Immune Complex Transfer Enzyme Immunoassays." Clinical Diagnostic Laboratory Immunology 7, no. 6 (November 1, 2000): 872–81. http://dx.doi.org/10.1128/cdli.7.6.872-881.2000.
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ABSTRACT For earlier diagnosis of human immunodeficiency virus type 1 (HIV-1) infection, the sensitivities of immune complex transfer enzyme immunoassays for HIV-1 p24 antigen and antibody immunoglobulin G (IgG) to HIV-1 p17 antigen were improved approximately 25- and 90-fold, respectively, over those of the previous immunoassays by performing solid-phase immunoreactions with shaking and increasing the serum sample volumes, and immune complex transfer enzyme immunoassay of antibody IgM to p17 antigen was also performed in the same way as the improved immunoassay of antibody IgG to p17 antigen. By the improved immunoassays, p24 antigen and antibody IgG to p17 antigen were detected earlier in 32 and 53%, respectively, of the HIV-1 seroconversion serum panels tested than before the improvements, and p24 antigen was detected as early as or earlier than HIV-1 RNA by reverse transcriptase-PCR (RT-PCR) in all of the panels tested. In 4 panels out of 19 tested, antibody IgG to p17 antigen or both antibodies IgG and IgM to p17 antigen were detected earlier than p24 antigen and RNA, although the antibody levels declined slightly before their steep increases usually observed after p24 antigen and RNA. Thus, the window period in diagnosis of HIV-1 infection can be shortened by detection of p24 antigen with the improved immunoassay as much as by detection of RNA with RT-PCR and, in some cases, more by detection of antibodies IgG and IgM to p17 antigen with the improved immunoassays than by detections of p24 antigen with the improved immunoassay and RNA with RT-PCR.
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Aslan, Kadir, and Tsehai AJ Grell. "Rapid and Sensitive Detection of Troponin I in Human Whole Blood Samples by Using Silver Nanoparticle Films and Microwave Heating." Clinical Chemistry 57, no. 5 (May 1, 2011): 746–52. http://dx.doi.org/10.1373/clinchem.2010.159889.
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BACKGROUND Cardiovascular diseases are among the leading causes of mortality in developed countries. It is widely recognized that troponin I (TnI) can be used for the assessment of a myocardial infarction. METHODS We investigated the use of the microwave-accelerated and metal-enhanced fluorescence (MA-MEF), a technique based on the combined use of low-power microwave heating, silver nanoparticle films (SNFs), and fluorescence spectroscopy for the detection of TnI from human whole blood samples. SNFs were deposited onto amine-modified glass microscope slides by use of Tollen's reaction scheme and characterized by optical absorption spectroscopy and scanning electron microscopy. The detection of TnI from buffer solutions and human whole blood samples on SNFs was carried out by using fluorescence-based immunoassays at room temperature (control immunoassay, 2 h total assay time) or microwave heating (MA-MEF–based immunoassay, 1 min total assay time). RESULTS We found that the lower limits of detection for TnI from buffer solutions in the control immunoassay and MA-MEF–based immunoassay were 0.1 μg/L and 0.005 μg/L, respectively. However, we were unable to detect TnI in whole blood samples in the control immunoassays owing to the coagulation of whole blood within 5 min of the incubation step. The use of the MA-MEF technique allowed detection of TnI from whole blood samples in 1 min with a lower detection limit of 0.05 μg/L. CONCLUSIONS The MA-MEF–based immunoassay is one of the fastest reported quantitative detection methodos for detection of TnI in human whole blood and has low detection limits similar to those obtained with commercially available immunoassays.
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Lukefahr, Ashley, and Barun De. "Laboratory Diagnosis of HIV in the Outpatient Setting: One Hospital’s Perspective." American Journal of Clinical Pathology 152, Supplement_1 (September 11, 2019): S20—S21. http://dx.doi.org/10.1093/ajcp/aqz112.040.
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Abstract Traditional methods for the diagnosis of human immunodeficiency virus types 1 and 2 (HIV-1, HIV-2) involve initial screening by an immunoassay followed by a specific method Western blot. However, Western blot is not sensitive compared to third-generation immunoassay, which detects both IgG and IgM antibodies against viral envelope proteins causing false-positive results. In addition, neither initial screening nor confirmatory Western blot is capable of detecting acute infection earlier in the disease process, when the virus is more adaptable and highly infectious. To detect both acute and established infection, Abbott Architect platform introduced a new fourth-generation antigen/antibody initial screening assay in 2013. Also, to reduce false-negative results from Western blot, an alternate method Bio-Rad multisport immunoassay was recommended by the Centers for Disease Control and Prevention (CDC) that has higher sensitivity and can differentiate HIV-1 and HIV-2 infections. In 2014, the CDC released updated recommendations for the laboratory diagnosis of HIV. The CDC diagnostic algorithm recommends an initial combination immunoassay that detects HIV-1 and HIV-2 antibodies, followed by supplemental testing with an immunoassay that differentiates HIV-1 and HIV-2 antibodies. Specimens reactive on the initial antigen/antibody immunoassay but nonreactive or indeterminate on the differentiation immunoassay should be followed by nucleic acid testing (NAT) for resolution of this discrepancy. The objective of our study was to review the effect of this updated testing algorithm on HIV testing results in the outpatient setting at Banner University Medical Center–Tucson in Tucson, Arizona. Our study utilized laboratory information system queries to retroactively review all outpatient HIV laboratory testing results obtained from 2013 to 2017. A total of 17,397 HIV-1/2 antigen/antibody immunoassays were performed during this time period. Of the initial antigen/antibody immunoassays, 1.1% were reactive (n = 183). Of these reactive tests, 85% were collected from individuals with established HIV infections (n = 155). HIV-1/HIV-2 antibody differentiation assays were performed on 175 patients, with 86% reactive (n = 150), 2.3% indeterminate (n = 4), and 12% nonreactive (n = 21). Acute HIV-1 infections (antigen/antibody immunoassay reactive, antibody differentiation immunoassay nonreactive or indeterminate, and NAT positive) accounted for 2.7% of patients with reactive initial antigen/antibody immunoassays and 0.03% of all patients screened for HIV infection with the initial antigen/antibody immunoassay (n = 5). Viral loads in the patients with acute HIV-1 infection ranged from 173,000 to >10,000,000 copies/mL. No HIV-2 infections were identified in our patient population. Given that the previous CDC testing algorithm for HIV-1 failed to identify acute HIV-1 infections, our data, which confirm the ability of the current CDC algorithm to detect HIV-1 infections, represent important information for infectious disease practitioners, public health officials, and laboratory directors.
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Ningrum, Vitarani Dwi Ananda, Senya Puteri Amalia, and Ari Wibowo. "Vancomycin bioanalysis for TDM services by using immunoassay and HPLC: A scoping review." Pharmacy Education 24, no. 3 (May 1, 2024): 197–203. http://dx.doi.org/10.46542/pe.2024.243.197203.
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Background: Administration of vancomycin in treating infections caused by Methicillin-resistant Staphylococcus aureus (MRSA) requires therapeutic drug monitoring (TDM). The immunoassay method and high-performance liquid chromatography (HPLC) are the two methods of choice for examining vancomycin levels, with their respective advantages. Objective: This study aims to review the validity of immunoassay and HPLC methods, as well as consider which method is appropriate, effective, and efficient for TDM in the clinical setting. Method: Related articles were searched for using the keywords "immunoassay", "vancomycin", "HPLC", "bioanalysis", and "human" in the PubMed, Google Scholar, and Science Direct databases. Result: A total of 20 publications examined immunoassays, whereas 23 articles covered HPLC. Both the immunoassay and HPLC methods provided acceptable bioanalytical validation values. Conclusion: The immunoassay method is an option for routine sample analysis that requires fast results, but this method is not recommended for patients with high immunoglobulin levels. The HPLC method is a choice because it offers better selectivity and sensitivity.
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Eisenhofer, Graeme, Max Kurlbaum, Mirko Peitzsch, Georgiana Constantinescu, Hanna Remde, Manuel Schulze, Denise Kaden, et al. "The Saline Infusion Test for Primary Aldosteronism: Implications of Immunoassay Inaccuracy." Journal of Clinical Endocrinology & Metabolism 107, no. 5 (December 28, 2021): e2027-e2036. http://dx.doi.org/10.1210/clinem/dgab924.
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Abstract Context Diagnosis of primary aldosteronism (PA) for many patients depends on positive results for the saline infusion test (SIT). Plasma aldosterone is often measured by immunoassays, which can return inaccurate results. Objective This study aimed to establish whether differences in aldosterone measurements by immunoassay versus mass spectrometry (MS) might impact confirmatory testing for PA. Methods This study, involving 240 patients tested using the SIT at 5 tertiary care centers, assessed discordance between immunoassay and MS-based measurements of plasma aldosterone. Results Plasma aldosterone measured by Liaison and iSYS immunoassays were respectively 86% and 58% higher than determined by MS. With an immunoassay-based SIT cutoff for aldosterone of 170 pmol/L, 78 and 162 patients had, respectivel, negative and positive results. All former patients had MS-based measurements of aldosterone < 117 pmol/L, below MS-based cutoffs of 162 pmol/L. Among the 162 patients with pathogenic SIT results, MS returned nonpathologic results in 62, including 32 under 117 pmol/L. Repeat measurements by an independent MS method confirmed nonpathogenic results in 53 patients with discordant results. Patients with discordant results showed a higher (P < 0.0001) prevalence of nonlateralized than lateralized adrenal aldosterone production than patients with concordant results (83% vs 28%). Among patients with nonlateralized aldosterone production, 66% had discordant results. Discordance was more prevalent for the Liaison than iSYS immunoassay (32% vs 16%; P = 0.0065) and was eliminated by plasma purification to remove interferents. Conclusion These findings raise concerns about the validity of immunoassay-based diagnosis of PA in over 60% of patients with presumed bilateral disease. We provide a simple solution to minimize immunoassay inaccuracy-associated misdiagnosis of PA.
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McConeghy, Kevin W., Siyun Liao, Douglas Clark, Philip Worboys, Steven L. Barriere, and Keith A. Rodvold. "Variability in Telavancin Cross-Reactivity among Vancomycin Immunoassays." Antimicrobial Agents and Chemotherapy 58, no. 12 (September 15, 2014): 7093–97. http://dx.doi.org/10.1128/aac.03785-14.
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ABSTRACTTelavancin is a semisynthetic lipoglycopeptide with a dual mechanism of action against Gram-positive pathogens. Two brief reports have suggested potential cross-reactivity of telavancin with the vancomycin particle-enhanced turbidometric immunoassay (PETIA). The purpose of this study was to evaluate several commercially available vancomycin immunoassays (fluorescence polarization [FPIA], enzyme-multiplied immunoassays [EMIT], PETIA, and chemiluminescent immunoassay [CMIA]) for cross-reactivity with telavancin. Seven sites were selected to analyze serum samples for vancomycin. Each site received a set of samples (n= 18) which combined drug-free serum with telavancin, 7-OH telavancin metabolite, or vancomycin. Immunoassays demonstrating potential cross-reactivity were further evaluated by sending a duplicate sample set to multiple laboratories. Cross-reactivity was defined as the percent theoretical concentration (reported concentration/theoretical concentration × 100). No cross-reactivity was seen with FPIA or EMIT. Within the theoretical concentration range of 5 to 120 μg/ml of telavancin, the Synchron PETIA system reported vancomycin concentrations ranging from 4.7 to 54.2 μg/ml compared to vancomycin concentrations from 1.1 to 5.6 μg/ml for the Vista PETIA system. The Architect CMIA system reported vancomycin concentrations in the range of 0.27 to 0.97 μg/ml, whereas Advia Centaur XP CMIA reported vancomycin concentrations between 1.6 and 31.6 μg/ml. The Architect CMIA immunoassay had the lowest percent cross-reactivity (0.8 to 5.4%), while the Synchron PETIA immunoassay demonstrated the highest percent cross-reactivity (45.2 to 53.8%). Telavancin samples measured by liquid chromatography-mass spectroscopy were within 93.9 to 122% of theoretical concentrations. Vancomycin concentrations were not measured in any 7-OH telavancin-spiked sample. Vancomycin concentrations measured by liquid chromatography-mass spectroscopy were within 57.2 to 113% of theoretical concentrations. PETIA and CMIA measured vancomycin concentrations in telavancin-spiked samples. Significant variability in percent cross-reactivity was observed for each platform regardless of immunoassay method.
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Li, Li, Wenbo Lu, Chang Liu, Ying Wang, Jian Dong, and Weiping Qian. "Two Types of Immunoassay Based on Nile Blue Labeling Polydopamine Nanospheres." Nano 12, no. 08 (August 2017): 1750092. http://dx.doi.org/10.1142/s1793292017500928.
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The sandwich-type immunoassays have been developed by using electrochemical and surface-enhanced Raman scattering (SERS) techniques for the detection of carcinoembryonic antigen (CEA). Nile blue as a kind of Raman dye has been decorated on nanospheres with polydopamine resin (PDR) via [Formula: see text]-stacking interaction. The Nile blue displays the strong SERS signals as well as a characteristic electrochemical reduction peak at [Formula: see text]0.33[Formula: see text]V (versus Ag/AgCl). The implementation of the PDR nanospheres mixing with Au nanoparticles (AuNPs/PDR) exhibits a better electrical conductivity and large SERS enhancement. The immunoassays based on Nile blue-labeled AuNPs/PDR nanospheres have been fabricated by using electrochemical and SERS techniques for the detection of CEA. The proposed immunoassay shows higher sensitivity, high selectivity, lower detection limit and long-term stability. The performances of the electrochemical immunoassay are better than that of SERS immunoassay. For the electrochemical immunoassay, the linear range occurs from 1[Formula: see text]pg/mL to 100[Formula: see text]ng/mL ([Formula: see text]) with a detection limit of 0.68[Formula: see text]pg/mL and signal-to-noise ratio of 3 in the detection of CEA. The data for the analysis of human serum samples by using the electrochemical method are acceptably consistent with those obtained from the enzyme-linked immunosorbent assay (ELISA). The proposed immunoassay exhibits a promising potential for application in clinical diagnosis.
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Parker, K. M., V. Rawlings, and K. E. Blick. "Cyclosporine monoclonal immunoassays: fluorescence polarization immunoassay vs RIA." Clinical Chemistry 37, no. 11 (November 1, 1991): 2016. http://dx.doi.org/10.1093/clinchem/37.11.2016.
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Kubotsu, K., S. Goto, M. Fujita, H. Tuchiya, M. Kida, S. Takano, S. Matsuura, and I. Sakurabayashi. "Automated Homogeneous Liposome Immunoassay Systems for Anticonvulsant Drugs." Clinical Chemistry 38, no. 6 (June 1, 1992): 808–12. http://dx.doi.org/10.1093/clinchem/38.6.808.
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Abstract We developed automated homogeneous immunoassays, based on immunolysis of liposomes, for measuring phenytoin, phenobarbital, and carbamazepine from serum. Liposome lysis was detected spectrophotometrically from entrapped glucose-6-phosphate dehydrogenase activity. The procedure was fully automated on a routine automated clinical analyzer. Within-run, between-run, dilution, and recovery tests showed good accuracies and reproducibilities. Bilirubin, hemoglobin, triglycerides, and Intrafat did not affect assay results. The results obtained by liposome immunoassays for phenytoin, phenobarbital, and carbamazepine correlated well with those obtained by enzyme-multiplied immunoassay (Syva EMIT) kits (r = 0.995, 0.986, and 0.988, respectively) and fluorescence polarization immunoassay (Abbott TDx) kits (r = 0.990, 0.991, and 0.975, respectively). The proposed method should be useful for monitoring anticonvulsant drug concentrations in blood.
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Ellington, Allison A., Iftikhar J. Kullo, Kent R. Bailey, and George G. Klee. "Antibody-Based Protein Multiplex Platforms: Technical and Operational Challenges." Clinical Chemistry 56, no. 2 (February 1, 2010): 186–93. http://dx.doi.org/10.1373/clinchem.2009.127514.
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AbstractBackground: The measurement of multiple protein biomarkers may refine risk stratification in clinical settings. This concept has stimulated development of multiplexed immunoassay platforms that provide multiple, parallel protein measurements on the same specimen.Content: We provide an overview of antibody-based multiplexed immunoassay platforms and discuss technical and operational challenges. Multiplexed immunoassays use traditional immunoassay principles in which high-affinity capture ligands are immobilized in parallel arrays in either planar format or on microspheres in suspension. Development of multiplexed immunoassays requires rigorous validation of assay configuration and analytical performance to minimize assay imprecision and inaccuracy. Challenges associated with multiplex configuration include selection and immobilization of capture ligands, calibration, interference between antibodies and proteins and assay diluents, and compatibility of assay limits of quantification. We discuss potential solutions to these challenges. Criteria for assessing analytical multiplex assay performance include the range of linearity, analytical specificity, recovery, and comparison to a quality reference method. Quality control materials are not well developed for multiplexed protein immunoassays, and algorithms for interpreting multiplex quality control data are needed.Summary: Technical and operational challenges have hindered implementation of multiplexed assays in clinical settings. Formal procedures that guide multiplex assay configuration, analytical validation, and quality control are needed before broad application of multiplexed arrays can occur in the in vitro diagnostic market.
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Jockenhövel, F., S. A. Khan, and E. Nieschlag. "Varying dose–response characteristics of different immunoassays and an in-vitro bioassay for FSH are responsible for changing ratios of biologically active to immunologically active FSH." Journal of Endocrinology 127, no. 3 (December 1990): 523–32. http://dx.doi.org/10.1677/joe.0.1270523.
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ABSTRACT Serum FSH levels in fertile and infertile men were determined by applying the Sertoli cell in-vitro bioassay and six different immunoassays. Bioassay and immunoassay estimates were significantly correlated (r ranging from 0·78 to 0·86; P<0·01). On average, all immunoassays measured lower FSH concentrations in samples with low FSH levels and higher FSH concentrations in those with high FSH levels compared with the bioassay. Ratios of bioactivity to immunoreactivity (B/I) were highest in fertile men and lowest in men with severe disturbances of testicular function. Depending on which immunoassay was used these differences were either significant or only marginal. Dose–response characteristics for WHO FSH standard preparation 78/549, used in the bioassay as well as in the immunoassays, were different between immunoassays and the bioassay, suggesting that decreasing B/I ratios with increasing FSH serum levels were method-related and reflected different slopes of the dose–response characteristics of the assays, rather than being true changes in the molecular composition of FSH. The present investigation underlines the necessity of choosing the immunoassay used for comparison with the bioassay carefully and of validating the system in regard to parallelism between dose–response characteristics. B/I ratios must be interpreted with great caution and previous studies which report changing B/I ratios in various endocrine situations may have to be reevaluated. Journal of Endocrinology (1990) 127, 523–532
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Lin, Yen-Heng, Chih-Ching Wu, Wan-Ling Chen, and Kai-Ping Chang. "Anti-p53 Autoantibody Detection in Automatic Glass Capillary Immunoassay Platform for Screening of Oral Cavity Squamous Cell Carcinoma." Sensors 20, no. 4 (February 11, 2020): 971. http://dx.doi.org/10.3390/s20040971.
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The incidence of oral squamous cell carcinoma (OSCC), which is one of the most common cancers worldwide, has been increasing. Serum anti-p53 autoantibody is one of the most sensitive biomarkers for OSCC. Currently, the most commonly used method on clinical screening platforms is the enzyme-linked immunosorbent assay, owing to its high specificity and repeatability. However, conducting immunoassays on 96-well plates is typically time consuming, thereby limiting its clinical applications for fast diagnosis and immediate prognosis of rapidly progressive diseases. The present study performed immunoassays in glass capillaries of 1-mm internal diameter, which increases the surface to volume ratio of the reaction, to shorten the time needed for immunoassay. The immunoassay was automated while using linear motorized stages and a syringe pump. The results indicated that, when compared with the 96-well plate immunoassay, the glass capillary immunoassay decreased the reaction time from typical 120 min to 45 min, reduced the amount of reagent from typical 50 µL to 15 µL, and required only simple equipment setup. Moreover, the limit of detection for glass capillary anti-p53 autoantibody immunoassay was 0.46 ng mL−1, which is close to the 0.19 ng mL−1 value of the conventional 96-well plate assay, and the glass capillary method had a broader detection range. The apparatus was used to detect the serum anti-p53 autoantibody concentration in clinical patients and compare its results with the conventional 96-well plate method results, which suggested that both of the methods detect the same trend in the relative concentration of serum anti-p53 autoantibody in healthy individuals or patients with OSCC.
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Jeong, Hee-Jin. "Quenchbodies That Enable One-Pot Detection of Antigens: A Structural Perspective." Bioengineering 10, no. 11 (October 30, 2023): 1262. http://dx.doi.org/10.3390/bioengineering10111262.
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Quenchbody (Q-body) is a unique, reagentless, fluorescent antibody whose fluorescent intensity increases in an antigen-concentration-dependent manner. Q-body-based homogeneous immunoassay is superior to conventional immunoassays as it does not require multiple immobilization, reaction, and washing steps. In fact, simply mixing the Q-body and the sample containing the antigen enables the detection of the target antigen. To date, various Q-bodies have been developed to detect biomarkers of interest, including haptens, peptides, proteins, and cells. This review sought to describe the principle of Q-body-based immunoassay and the use of Q-body for various immunoassays. In particular, the Q-bodies were classified from a structural perspective to provide useful information for designing Q-bodies with an appropriate objective.
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Rossi, Brian, Francesca Freni, Claudia Vignali, Cristiana Stramesi, Giancarlo Collo, Claudia Carelli, Matteo Moretti, Dario Galatone, and Luca Morini. "Comparison of Two Immunoassay Screening Methods and a LC-MS/MS in Detecting Traditional and Designer Benzodiazepines in Urine." Molecules 27, no. 1 (December 24, 2021): 112. http://dx.doi.org/10.3390/molecules27010112.
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Sensitive and specific immunoassay screening methods for the detection of benzodiazepines in urine represent an important prerequisite for routine analysis in clinical and forensic toxicology. Moreover, emerging designer benzodiazepines force labs to keep their methodologies updated, in order to evaluate the reliability of the immunochemical techniques. This study aimed at evaluating the sensitivity and specificity of two different immunoassay methods for the detection of benzodiazepines in urine, through a comparison with the results obtained by a newly developed liquid chromatographic tandem mass spectrometric (LC-MS/MS) procedure. A cohort of authentic urine samples (N = 501) were processed, before and after a hydrolysis procedure, through two immunoassays and an LC-MS/MS method. The LC-MS/MS target procedure was optimized for monitoring 25 different molecules, among traditional and designer benzodiazepines, including some metabolites. At least one of the monitored substances was detected in 100 out of the 501 samples. A good specificity was observed for the two immunoassays (>0.99), independently of the cut-offs and the sample hydrolysis. The new kit demonstrated a fairly higher sensitivity, always higher than 0.90; in particular, a high cross-reactivity of the new immunoassay was observed for samples that tested positive for lorazepam and 7-aminoclonazepam. The two immunoassays appeared adequate to monitor not only traditional benzodiazepines but also new designer ones.
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Kachab, Edward. "Homogeneous assays: a paradigm shift in immunodiagnostics." Microbiology Australia 27, no. 2 (2006): 66. http://dx.doi.org/10.1071/ma06066.
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Immunoassay methods are either heterogeneous or homogeneous and rely on the interaction between an antibody and antigen. Historically, diagnostic immunoassays assays have relied on heterogeneous assay formats for analyte detection.
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Mutayoba, B. M., H. H. D. Meyer, D. Schams, and E. Schallenberger. "Development of a sensitive enzyme immunoassay for LH determination in bovine plasma using the streptavidin-biotin technique." Acta Endocrinologica 122, no. 2 (February 1990): 227–32. http://dx.doi.org/10.1530/acta.0.1220227.
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Abstract Using the biotin-streptavidin amplification technique, highly sensitive specific second-antibody enzyme immunoassays for determining LH in bovine plasma with long (48 h) and short (4 h) incubation periods were developed. Biotin was linked to bLH by the N-hydroxysuccimidine method and the product (biotinyl-bLH) used to bridge between streptavidin-peroxidase and the immobilised bLH antibody in competitive tests. The assays were validated and their performance compaired with a radioimmunoassay currently in use. The sensitivities of the long and short incubation enzyme immunoassays (8 pg and 15 pg/well, respectively) were superior to that of 5-day incubation radioimmunoassay (100 pg/tube). Plasma interference in both assays were acceptable and volumes of 5 to 40 μl gave parallel standard curves and comparable LH levels, 10–20 μl plasma was sufficient to measure LH baseline levels by the long incubation enzyme immunoassay. The mean recovery of added standard bLH to plasma samples containing different endogenous LH was >90% (range 91.7–112) in both assays. The intra- and inter-assay variations of both assays were less than 10 and 17%, respectively. When both enzyme immunoassay and radioimmunoassay were used to measure LH in cyclic cows, the basal levels measured by enzyme immunoassay were lower than that measured by radioimmunoassay. Enzyme immunoassay offers an attractive alternative to the lengthy radioimmunoassay in current usage, with an added advantage of employing non-isotopic label.
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Chang, Yaw-Jen, Hong-Wei Yang, Len-Hao Yao, and Wen-Tung Yang. "Droplet-Based Immunosensor for Simultaneous Immunoassays of Multiplex Histidine-Tagged Proteins." SLAS TECHNOLOGY: Translating Life Sciences Innovation 25, no. 2 (October 4, 2019): 132–39. http://dx.doi.org/10.1177/2472630319879647.
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This paper presents a droplet-based immunoassay chip allowing each droplet to be positioned in a passive droplet-positioning cavern under continuous flow. In addition, the chip surface can immobilize any kind of histidine-tagged capture agents for performing simultaneous multiplex immunoassays. Distinct families of monodispersed droplets were generated since a diaphragm, which is a thin elastomeric flap film suspended from the top of the main channel, forms a double T junction for shearing the aqueous liquids by the carrier flow. These two types of monodispersed droplets traverse the main channel to the downstream detection area and enter the passive positioning caverns for further immunoassay. A layer of Ni–Co film was coated on the substrate by electrodeposition in order to immobilize the multiplex histidine-tagged capture molecules. In this study, the tumor suppressor protein p53 and the extracellular signal-related kinase 1 (ERK1) were used as the capture agents. Then, both histidine-tagged proteins p53 and ERK1 were immobilized by the Ni–Co layer in a microarray format for subsequent immunoassay and fluorescence detection. The experimental results show that the detected fluorescence intensity is proportioned to the concentration of the encapsulated content in a small droplet. This proposed droplet-based immunoassay chip can immobilize multiplex histidine-tagged proteins, irrelevant to the species of proteins, to carry out simultaneous immunoassays and allow the operation sequence to be conducted automatically through the manipulation of droplets.
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Bielohuby, Maximilian, Martin Bidlingmaier, and Uwe Schwahn. "Control of (pre)-analytical aspects in immunoassay measurements of metabolic hormones in rodents." Endocrine Connections 7, no. 4 (April 2018): R147—R159. http://dx.doi.org/10.1530/ec-18-0035.
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The measurement of circulating hormones by immunoassay remains a cornerstone in preclinical endocrine research. For scientists conducting and interpreting immunoassay measurements of rodent samples, the paramount aim usually is to obtain reliable and meaningful measurement data in order to draw conclusions on biological processes. However, the biological variability between samples is not the only variable affecting the readout of an immunoassay measurement and a considerable amount of unwanted or unintended variability can be quickly introduced during the pre-analytical and analytical phase. This review aims to increase the awareness for the factors ‘pre-analytical’ and ‘analytical’ variability particularly in the context of immunoassay measurement of circulating metabolic hormones in rodent samples. In addition, guidance is provided how to gain control over these variables and how to avoid common pitfalls associated with sample collection, processing, storage and measurement. Furthermore, recommendations are given on how to perform a basic validation of novel single and multiplex immunoassays for the measurement of metabolic hormones in rodents. Finally, practical examples from immunoassay measurements of plasma insulin in mice address the factors ‘sampling site and inhalation anesthesia’ as frequent sources of introducing an unwanted variability during the pre-analytical phase. The knowledge about the influence of both types of variability on the immunoassay measurement of circulating hormones as well as strategies to control these variables are crucial, on the one hand, for planning and realization of metabolic rodent studies and, on the other hand, for the generation and interpretation of meaningful immunoassay data from rodent samples.
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Kugler, Alan R., Thomas M. Annesley, Gerald D. Nordblom, Jeffrey R. Koup, and Stephen C. Olson. "Cross-reactivity of fosphenytoin in two human plasma phenytoin immunoassays." Clinical Chemistry 44, no. 7 (July 1, 1998): 1474–80. http://dx.doi.org/10.1093/clinchem/44.7.1474.
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Abstract The cross-reactivity of fosphenytoin, a phosphate ester prodrug of phenytoin, was investigated in the Abbott phenytoin TDx®/TDxFLxTM fluorescence polarization immunoassay (TDx) and the Behring Diagnostics phenytoin Emit® 2000 enzyme-multiplied immunoassay (Emit). The first part of our study investigating cross-reactivity utilized in vitro correlation of the two immunoassays with a validated and specific phenytoin HPLC method used to assay plasma samples prepared in several phenytoin and fosphenytoin concentration combinations. Fosphenytoin cross-reacted with both immunoassays, but to a greater extent with TDx. In the second part of the study, empirically-derived models that best explained the in vitro data were used to predict “immunoassay-derived” phenytoin concentrations in plasma samples collected from actual patients after intravenous (IV) or intramuscular (IM) fosphenytoin dosing. The greatest degree of phenytoin concentration overestimation occurred at times when fosphenytoin concentrations were highest: within 1 to 2 h after IV infusion or during the first 2 to 4 h after IM injection. It is recommended that phenytoin concentrations not be monitored using these or other potentially nonspecific immunoanalytical methods for at least 2 h after IV fosphenytoin infusion or 4 h after IM fosphenytoin injection.
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Schooley, Elizabeth, Graham Bilbrough, and Tiffany Glavan. "Art of measuring progesterone: understanding immunoassays." Clinical Theriogenology 11, no. 4 (December 1, 2019): 627–31. http://dx.doi.org/10.58292/ct.v11.9464.
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Measurement of progesterone has become an essential component of canine reproductive medicine. Reliable measurement of steroid hormones is challenging. In veterinary medicine, we depend on immunoassays to determine concentrations of circulating progesterone. Immunoassay is the general term for a diagnostic technique that relies on antibodies to detect a compound within a biologic sample. There are several types of immunoassays available, which have innate differences. Therefore, to correctly apply the results to a clinical scenario, it is essential for the practicing veterinarian to understand the method they are utilizing. Data from development of a new in-house progesterone test are reviewed and a variety of immunoassay methods, assay validation, and future trends within diagnostic industry are discussed. Understanding these principles will help the clinician choose an assay which will best suit their diagnostic needs.
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Apple, F., L. Preese, R. Bennett, and A. Fredrickson. "Clinical and analytical evaluation of two immunoassays for direct measurement of creatine kinase MB with monoclonal anti-CK-MB antibodies." Clinical Chemistry 34, no. 11 (November 1, 1988): 2364–67. http://dx.doi.org/10.1093/clinchem/34.11.2364.
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Abstract We examined the clinical and analytical performance of two immunoassays (Becton Dickinson CK-MB; Ciba-Corning Magic Lite CK-MB) in which monoclonal anti-CK-MB antibodies are used for directly measuring creatine kinase (EC 2.7.3.2) isoenzyme MB (CK-MB) in serum, and also one electrophoretic method (Ciba-Corning). Within- and between-assay precision for both immunoassays was good at the upper reference limits (less than 10% CV). Analytical recoveries ranged from 102 to 114%. Both immunoassays were free from interference by CK-BB, mitochondrial-CK, macro-CK, adenylate kinase, and CK-MM. Retrospectively, we evaluated four categories of patients, using both immunoassays and electrophoresis: normal controls, acute myocardial infarction (AMI) patients, severe skeletal muscle trauma patients, and acutely ill patients known not to have AMI. In general, there were excellent correlations among all three methods. CK-MB activity (U/L) measured by the Becton Dickinson immunoassay was approximately 50% of the mass concentration (microgram/L) of the Magic Lite immunoassay and 50% of the activity concentration (U/L) determined by electrophoresis. Both immunoassays were easy to perform and sensitive to the low CK-MB concentrations often found with low total-CK activities.
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Borisova, O. V., E. B. Fayzuloev, A. A. Marova, V. I. Kukushkin, and V. V. Zverev. "PROSPECTS AND PROBLEMS OF USING THE EFFECT OF SURFACE-ENHANCED RAMAN SCATTERING IN THE DIAGNOSIS OF VIRAL INFECTIONS." Journal of microbiology epidemiology immunobiology, no. 3 (June 28, 2017): 106–14. http://dx.doi.org/10.36233/0372-9311-2017-3-106-114.
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This review presents the latest advances in the use of surface-enhanced Raman scattering (SERS) immunoassay, which can be used to detect viral markers. As in the case of conventional immunoassays, these methods are often based on «sandwich-type» solid phase immunoassay. In recent years the necessary components of the immunochemical methods with SERS detection is SERS-active substrates to create a variety of approaches have been developed. Despite the difficulty of achieving high sensitivity and specificity in the analysis of clinical samples, a number of successful examples with promising results have been demonstrated.
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RONALD, A., and W. H. STIMSON. "The evolution of immunoassay technology." Parasitology 117, no. 7 (November 1999): 13–27. http://dx.doi.org/10.1017/s0031182099004989.
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Since they were first utilized, immunoassays have witnessed phenomenal growth in the range and scope of their applications. A vast array of different labels and assay strategies has been developed to meet the requirements of sensitivity, accuracy and convenience. The development of increasingly sensitive labels and detection equipment has seen a drastic improvement in the sensitivity of immunoassay systems, allowing an ever-increasing range of analytes to be measured accurately. At the same time, simple to use, inexpensive assay systems have been developed with the necessary reliability, accuracy and sensitivity to bring immunoassay technology to much more diverse areas such as home testing, near-patient monitoring, and large screening programmes in developing countries. Recent developments in molecular biology techniques have made possible the production of fusion antibody conjugates, which can lead to further improvements in sensitivity and cost of reagents, as well as possibly revolutionizing the production of monoclonal antibodies. However, dissatisfaction with various aspects of existing immunoassay technology will necessarily lead to the continued development of this already widely diverse subject.
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Wang, Yuan Kai, Qi Zou, Jian He Sun, Heng An Wang, and Ya Xian Yan. "Detection of Zearalenone by Chemiluminescence Immunoassay in Corn Samples." Advanced Materials Research 550-553 (July 2012): 1911–14. http://dx.doi.org/10.4028/www.scientific.net/amr.550-553.1911.
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A chemiluminescence immunoassay with polyclonal antibody for detecting zearalenone in corn samples was developed. The optimized reaction conditions include the concentration of coating antigen (0.253 μg/ml), the polyclonal antibody (1:7500) and HRP labeled goat anti-mouse antibody (1:40000), the competition time (60 min), pH of dilution buffer (7.4), the concentration of methanol (10%) and the ratio of luminol to ρ-iodophenol (2:5) were all investigated. Based on optimum conditions, the chemoluminscence ELISA detect range was f 0.05 - 54.34 ng/ml, with IC50 was 2.93 ng/ml, and the limit of detection was 0.02 ng/ml. The chemiluminescence immunoassay shows a good agreement with commercial ELISA kit for detection zearalenone in the spiked samples. The chemiluminescence immunoassay is easier in pretreating, and more sensitive when compared with other detection methods include chromatography methods and immunoassays.
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Banno, Yuto, Takuma Nomiyama, Shoma Okuno, Sachiko Ide, and Noritada Kaji. "Quantitative Evaluation of Interleukin-4 by Immunowall Devices Made of Gelatin Methacryloyl Hydrogel." Molecules 28, no. 12 (June 8, 2023): 4635. http://dx.doi.org/10.3390/molecules28124635.
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Immunoassays, which use antigen–antibody reactions, are the primary techniques used to selectively quantify specific disease markers in blood. Conventional immunoassays, such as the microplate-based enzyme-linked immunosorbent assay (ELISA) and paper-based immunochromatography, are widely used, but they have advantages and disadvantages in terms of sensitivity and operating time. Therefore, in recent years, microfluidic-chip-based immunoassay devices with high sensitivity, rapidity and simplicity, which are compatible with whole blood assays and multiplex assays, have been actively investigated. In this study, we developed a microfluidic device using gelatin methacryloyl (GelMA) hydrogel to form a wall-like structure in a microfluidic channel and perform immunoassays inside the wall-like structure, which can be used for rapid and highly sensitive multiplex assays with extremely small sample amounts of ~1 μL. The characteristics of GelMA hydrogel, such as swelling rate, optical absorption and fluorescence spectra, and morphology, were carefully studied to adapt the iImmunowall device and immunoassay. Using this device, a quantitative analysis of interleukin-4 (IL-4), a biomarker of chronic inflammatory diseases, was performed and a limit of detection (LOD) of 0.98 ng/mL was achieved with only 1 μL sample and 25 min incubation time. The superior optical transparency over a wide range of wavelengths and lack of autofluorescence will help to expand the application field of the iImmunowall device, such as to a simultaneous multiple assay in a single microfluidic channel, and provide a fast and cost-effective immunoassay method.
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Kroner, Grace M., Kamisha L. Johnson-Davis, Kelly Doyle, and Gwendolyn A. McMillin. "Cannabinol (CBN) Cross-Reacts with Two Urine Immunoassays Designed to Detect Tetrahydrocannabinol (THC) Metabolite." Journal of Applied Laboratory Medicine 5, no. 3 (March 24, 2020): 569–74. http://dx.doi.org/10.1093/jalm/jfaa020.
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Abstract Background The psychoactive component of cannabis, tetrahydrocannabinol (THC), is one of many cannabinoids present in the plant. Since cannabinoids have extensive structural similarity, it is important to be aware of potential cross-reactivity with immunoassays designed to detect THC metabolite. This is especially important as cannabinoid products are increasingly marketed as legal supplements. The objective of this study was to assess the cross-reactivity of 2 commercial immunoassays designed to detect THC metabolite with 4 cannabinoids: cannabidiol, cannabinol, cannabichromene, and cannabigerol. Methods Deidentified residual patient urine samples that tested negative for THC metabolite on initial testing were pooled and fortified with the above compounds to detect cross-reactivity. We next tested a range of CBN concentrations to determine what concentration of CBN was required to trigger a positive immunoassay result. Finally, we tested whether CBN has an additive effect with THC in the immunoassay by adding CBN to 21 samples weakly positive for THC by a mass spectrometry method but negative by the EMIT II Plus immunoassay. Results Both the EMIT II Plus assay and the Microgenics MultiGent assay demonstrated cross-reactivity with CBN. For the EMIT II Plus assay, about 5-fold more CBN than THC metabolite was required to produce an assay signal equivalent to the cutoff concentration, and CBN displayed an additive effect with THC metabolite. For the Microgenics assay, 20-fold more CBN than THC metabolite was required to cross the cutoff concentration. Conclusions These data may help guide the need for confirmatory testing when results of THC metabolite testing by immunoassay are inconsistent with expectations.
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Tao, Xiaoqi, Song Zhou, Xiameng Yuan, and Hongjun Li. "Determination of chloramphenicol in milk by ten chemiluminescent immunoassays: influence of assay format applied." Analytical Methods 8, no. 22 (2016): 4445–51. http://dx.doi.org/10.1039/c6ay00792a.
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Development of 10 chemiluminescent immunoassays for the detection of CAP for a comparison study. First systematic assessment in sensitivity and robustness of different immunoassay formats based on diverse combinations of coating, reverse reaction and antibody source.
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Drame, Papa M., Sasisekhar Bennuru, and Thomas B. Nutman. "Discovery of Specific Antigens That Can Predict Microfilarial Intensity inLoa loaInfection." Journal of Clinical Microbiology 55, no. 9 (June 21, 2017): 2671–78. http://dx.doi.org/10.1128/jcm.00513-17.
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ABSTRACTAntigen-based immunoassays are currently needed for point-of-care quantification ofLoa loamicrofilariae (mf). Coupling transcriptomic approaches with bioinformatic analysis, we have identified 11 specific putative proteins (coding mRNAs) with potential utility as biomarkers of patent (mf+)L. loainfection. We successfully developed antigen capture immunoassays to quantify 2 (LOAG_14221 and LOAG_15846) of these proteins in individual plasma/serum samples. Of the 2 quantifiable circulating biomarkers, LOAG_14221 showed the highest degree of specificity, particularly with a monoclonal antibody-based immunoassay. Moreover, the levels of LOAG_14221 inL. loamf+patients were positively correlated to the mf densities in the corresponding blood samples (r= 0.53 andP= 0.008 for polyclonal assay;r= 0.54 andP= 0.004 for monoclonal assay). Thus, LOAG_14221 is a very promising biomarker that will be exploited in a quantitative point-of-care immunoassay for determination ofL. loamf densities.
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Immunoassay Research Society of Jap. "Immunoassay." RADIOISOTOPES 44, no. 10 (1995): i—xiv. http://dx.doi.org/10.3769/radioisotopes.44.10_i.
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Immunoassay Research Society of Jap. "Immunoassay." RADIOISOTOPES 45, no. 10 (1996): i—xiv. http://dx.doi.org/10.3769/radioisotopes.45.i.
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Immunoassay Research Society of Jap. "Immunoassay." RADIOISOTOPES 46, no. 9 (1997): i—xxii. http://dx.doi.org/10.3769/radioisotopes.46.i.
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Ngo, That T. "Immunoassay." Current Opinion in Biotechnology 2, no. 1 (February 1991): 102–9. http://dx.doi.org/10.1016/0958-1669(91)90067-f.
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Haugen, Linda M., and Frank H. Tainter. "Immunoassay." Journal of Forestry 85, no. 4 (April 1, 1987): 15–20. http://dx.doi.org/10.1093/jof/85.4.15.
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Smith, J., and G. Osikowicz. "Abbott AxSYM random and continuous access immunoassay system for improved workflow in the clinical laboratory." Clinical Chemistry 39, no. 10 (October 1, 1993): 2063–69. http://dx.doi.org/10.1093/clinchem/39.10.2063.
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Abstract We describe a new clinical laboratory instrument, the Abbott AxSYM, which provides random- and continuous-access testing for immunoassays, 20 onboard reagents, primary tube sampling, and a throughput of 80 to 120 tests per hour. The AxSYM incorporates three separate analytical technologies for processing immunoassays: microparticle enzyme immunoassay, fluorescence polarization immunoassay, and a novel technology known as ion-capture immunoassay. The system incorporates both common and technology-specific subsystems controlled by a real-time software scheduling processor. Tests can be processed in one- or two-step sandwich or competitive formats, with variable pipetting steps, incubation periods, optical read formats, and wash sequences. Menu capabilities include tests for hepatitis, retrovirus, tumor markers, fertility markers, thyroid functions, and therapeutic drugs. The time to first result is approximately 15-25 min for most routine assays and < or = 15 min for stat assays (i.e., creatine kinase MB isoenzyme, human chorionic gonadotropin beta subunit, and therapeutic drugs). AxSYM assay performance for 23 assays was comparable with that of the Abbott IMx and TDx analyzers; specimen correlation data had correlation coefficients ranging from 0.97 to 0.99 and slopes ranging from 0.99 to 1.10. Within-run imprecision (CV) was 1.5% to 11.4%, with most assays (19 of 23) demonstrating CVs < or = 8.0%.
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Diamandis, E. P. "Multiple labeling and time-resolvable fluorophores." Clinical Chemistry 37, no. 9 (September 1, 1991): 1486–91. http://dx.doi.org/10.1093/clinchem/37.9.1486.
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Abstract A new time-resolved fluorescence immunoassay system involving use of the europium chelate of 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid as label is reviewed. This stable chelate by itself is not very fluorescent but, used in multiple labeling strategies, improves the achievable detection limits. By using multiple labeling, streptavidin tailing, and Eu3+ activation, one can create a very stable, easy-to-use reagent that is suitable for devising highly sensitive immunoassays and other biotechnological assays. This reagent, a streptavidin-based macromolecular complex, is able to detect approximately 300,000 molecules (approximately 0.5 amol) of alpha-fetoprotein in a model noncompetitive immunoassay.
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Gold, David V., David E. Modrak, Zhiliang Ying, Thomas M. Cardillo, Robert M. Sharkey, and David M. Goldenberg. "New MUC1 Serum Immunoassay Differentiates Pancreatic Cancer From Pancreatitis." Journal of Clinical Oncology 24, no. 2 (January 10, 2006): 252–58. http://dx.doi.org/10.1200/jco.2005.02.8282.
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Purpose To evaluate a new immunoassay for identification and quantitation of MUC1 in the sera of patients with pancreatic cancer or pancreatitis. The sensitivity and specificity of the assay are examined and compared to results from a CA19-9 immunoassay. Methods An in vitro enzyme immunoassay was established with monoclonal antibody PAM4 as the capture reagent, and a polyclonal anti-MUC1 antibody as the probe. Patient sera were obtained from healthy, adult patients with acute and chronic pancreatitis, and those with pancreatic and other forms of cancer, and were measured for PAM4-reactive MUC1. Results At a cutoff of 10.2 units/mL, 41 (77%) of 53 pancreatic cancer patients, none of the healthy individuals (n = 43), and only four (5%) of 87 patients with pancreatitis were positive above this value. Among nonpancreatic cancers investigated, colorectal cancers gave the highest percentage of positives (14%; five of 36). Overall, the sensitivity and specificity of the immunoassay for pancreatic cancer were 77% and 95%, respectively. Receiver operator characteristic analyses for discrimination of pancreatic cancer from pancreatitis provided an area under the curve of 0.89 (95% CI, 0.82 to 0.93), with a specificity of 95.4% and a positive likelihood ratio of 16.8. A direct pair-wise comparison of PAM4 and CA19-9 immunoassays for discrimination of pancreatic cancer and pancreatitis resulted in a significant difference (P < .003), with the PAM4 immunoassay demonstrating superior sensitivity and specificity. Conclusion The high sensitivity and specificity observed suggest that the PAM4-based immunoassay of circulating MUC1 may be useful in the diagnosis of pancreatic cancer.
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Armbruster, D. A., E. C. Hubster, M. S. Kaufman, and M. K. Ramon. "Cloned enzyme donor immunoassay (CEDIA) for drugs-of-abuse screening." Clinical Chemistry 41, no. 1 (January 1, 1995): 92–98. http://dx.doi.org/10.1093/clinchem/41.1.92.
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Abstract Large numbers of specimens (5000-18,000) were screened for amphetamines, barbiturates, cocaine, marijuana, opiates, and phencyclidine by RIA (Roche), Emit II (Syva), and a new immunoassay, CEDIA (cloned enzyme donor immunoassay, Microgenics). All immunoassays performed equivalently for cocaine, opiates, and phencyclidine. All immunoassays detected the same amphetamine/methamphetamine-positive specimens, but all also detected numerous specimens containing cross-reacting sympathomimetic amines. CEDIA detected 100%, Emit II 93%, and RIA 82% of the barbiturate-positive specimens. Emit II and CEDIA detected 86-88% of the specimens found by RIA to be marijuana positive. A subset of specimens was additionally screened by OnLine (Roche) and TDx (Abbott) for amphetamines, cocaine, and marijuana. OnLine and TDx also detected all of the amphetamine-positive specimens and numerous specimens containing cross-reacting sympathomimetic amines. All immunoassays performed equivalently for cocaine, and the four nonisotopic tests detected 86-89% of the marijuana positives found by RIA. Interfering sympathomimetic amine drug compounds can be eliminated by using an oxidizing agent, thus decreasing the number of unconfirmable amphetamine presumptive positives. The CEDIAs for all of the major drugs of abuse are reliable and effective for large-volume urine screening programs.
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Hinds, J. A., C. F. Pincombe, H. Morris, and P. Duffy. "Competitive solid-phase enzyme immunoassay for measuring digoxin in serum." Clinical Chemistry 32, no. 1 (January 1, 1986): 16–21. http://dx.doi.org/10.1093/clinchem/32.1.16.
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Abstract In this clinically useful enzyme immunoassay of digoxin in serum, we mix sample, beta-galactosidase-labeled digoxin, and anti-digoxin Fab fragments for 30 min at room temperature, then use Sepharose-bound second antibody for phase separation, and measure the unbound enzyme activity directly in the supernate of the equilibrium reaction mixture. The immunoassay buffer--phosphate-buffered isotonic saline with added rabbit globulin (4 g/L), hydrolyzed gelatin (2 g/L), Brij 96 detergent (5 g/L), glycerol (0.25 mol/L), and N-acetyl-8-anilinonaphthalene-1-sulfonic acid (2 mmol/L)--minimizes serum matrix effects for convenient measuring of unbound enzyme--digoxin conjugate. The immunoassay developed with Fab fragments has better displacement characteristics than that with intact antibody. Performance of the assay compares favorably with that of other manual digoxin immunoassays; in comparison studies with EMIT involving 110 clinical specimens, the coefficient of correlation was 0.97.
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Uenoyama, Yuta, Atsushi Matsuda, Kazune Ohashi, Koji Ueda, Misaki Yokoyama, Takuya Kyoutou, Kouji Kishi, et al. "Development and Evaluation of a Robust Sandwich Immunoassay System Detecting Serum WFA-Reactive IgA1 for Diagnosis of IgA Nephropathy." International Journal of Molecular Sciences 23, no. 9 (May 5, 2022): 5165. http://dx.doi.org/10.3390/ijms23095165.
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Aberrant glycosylation of IgA1 is involved in the development of IgA nephropathy (IgAN). There are many reports of IgAN markers focusing on the glycoform of IgA1. None have been clinically applied as a routine test. In this study, we established an automated sandwich immunoassay system for detecting aberrant glycosylated IgA1, using Wisteria floribunda agglutinin (WFA) and anti-IgA1 monoclonal antibody. The diagnostic performance as an IgAN marker was evaluated. The usefulness of WFA for immunoassays was investigated by lectin microarray. A reliable standard for quantitative immunoassay measurements was designed by modifying a purified IgA1 substrate. A validation study using multiple serum specimens was performed using the established WFA-antibody sandwich automated immunoassay. Lectin microarray results showed that WFA specifically recognized N-glycans of agglutinated IgA1 in IgAN patients. The constructed IgA1 standard exhibited a wide dynamic range and high reactivity. In the validation study, serum WFA-reactive IgA1 (WFA+-IgA1) differed significantly between healthy control subjects and IgAN patients. The findings indicate that WFA is a suitable lectin that specifically targets abnormal agglutinated IgA1 in serum. We also describe an automated immunoassay system for detecting WFA+-IgA1, focusing on N-glycans.
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Debeljak, Željko, Ivana Marković, Vatroslav Šerić, Vesna Horvat, Sanja Mandić, Dario Mandić, Iva Lukić, and Jasna Pavela. "Analytical bias of automated immunoassays for six serum steroid hormones assessed by LC-MS/MS." Biochemia medica 30, no. 3 (October 12, 2020): 422–31. http://dx.doi.org/10.11613/bm.2020.030701.
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Introduction: There is a growing amount of evidence showing the significant analytical bias of steroid hormone immunoassays, but large number of available immunoassays makes conduction of a single comprehensive study of this issue hardly feasible. Aim of this study was to assess the analytical bias of six heterogeneous immunoassays for serum aldosterone, cortisol, dehydroepiandrosterone sulphate (DHEAS), testosterone, 17-hydroxyprogesterone (OHP) and progesterone using the liquid chromatography coupled to the tandem mass spectrometry (LC-MS/MS). Materials and methods: This method comparison study included 49 serum samples. Testosterone, DHEAS, progesterone and cortisol immunoassays were performed on the Abbott Architect i2000SR or Alinity i analysers (Abbott Diagnostics, Chicago, USA). DiaSorin’s Liaison (DiaSorin, Saluggia, Italy) and DIAsource’s ETI-Max 3000 analysers (DIAsource ImmunoAssays, Louvain-La-Neuve, Belgium) were chosen for aldosterone and OHP immunoassay testing, respectively. All immunoassays were evaluated against the LC-MS/MS assay relying on the commercial kit (Chromsystems, Gräfelfing, Germany) and LCMS-8050 analyser (Shimadzu, Kyoto, Japan). Analytical biases were calculated and method comparison was conducted using weighted Deming regression analysis. Results: Depending on the analyte and specific immunoassay, mean relative biases ranged from -31 to + 137%. Except for the cortisol, immunoassays were positively biased. For none of the selected steroids slope and intercept 95% confidence intervals simultaneously contained 0 and 1, respectively. Conclusions: Evaluated immunoassays failed to satisfy requirements for methods’ comparability and produced significant analytical biases in respect to the LC-MS/MS assay, especially at low concentrations.
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Cabrera, Carlos, Lei Chang, Mars Stone, Michael Busch, and David H. Wilson. "Rapid, Fully Automated Digital Immunoassay for p24 Protein with the Sensitivity of Nucleic Acid Amplification for Detecting Acute HIV Infection." Clinical Chemistry 61, no. 11 (November 1, 2015): 1372–80. http://dx.doi.org/10.1373/clinchem.2015.243287.
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Abstract BACKGROUND Nucleic acid testing (NAT) has become the standard for high sensitivity in detecting low levels of virus. However, adoption of NAT can be cost prohibitive in low-resource settings where access to extreme sensitivity could be clinically advantageous for early detection of infection. We report development and preliminary validation of a simple, low-cost, fully automated digital p24 antigen immunoassay with the sensitivity of quantitative NAT viral load (NAT-VL) methods for detection of acute HIV infection. METHODS We developed an investigational 69-min immunoassay for p24 capsid protein for use on a novel digital analyzer on the basis of single-molecule-array technology. We evaluated the assay for sensitivity by dilution of standardized preparations of p24, cultured HIV, and preseroconversion samples. We characterized analytical performance and concordance with 2 NAT-VL methods and 2 contemporary p24 Ag/Ab combination immunoassays with dilutions of viral isolates and samples from the earliest stages of HIV infection. RESULTS Analytical sensitivity was 0.0025 ng/L p24, equivalent to 60 HIV RNA copies/mL. The limit of quantification was 0.0076 ng/L, and imprecision across 10 runs was <10% for samples as low as 0.09 ng/L. Clinical specificity was 95.1%. Sensitivity concordance vs NAT-VL on dilutions of preseroconversion samples and Group M viral isolates was 100%. CONCLUSIONS The digital immunoassay exhibited >4000-fold greater sensitivity than contemporary immunoassays for p24 and sensitivity equivalent to that of NAT methods for early detection of HIV. The data indicate that NAT-level sensitivity for acute HIV infection is possible with a simple, low-cost digital immunoassay.
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Hesberg, Jakob, Sentot Santoso, Gregor Bein, Tamam Bakchoul, and Ulrich Sachs. "Evaluation of a new nanoparticle-based lateral-flow immunoassay for the exclusion of heparin-induced thrombocytopenia (HIT)." Thrombosis and Haemostasis 106, no. 12 (2011): 1197–202. http://dx.doi.org/10.1160/th11-06-0390.
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SummaryHeparin-induced thrombocytopenia (HIT) is an adverse complication of heparin caused by HIT antibodies (abs) that recognise platelet factor 4-heparin (PF4/hep) complexes. Several laboratory tests are available for the confirmation and/or refutation of HIT. A reliable and rapid singlesample test is still pending. It was the objective of this study to evaluate a new lateral-flow immunoassay based on nanoparticle technology. A cohort of 452 surgical and medical patients suspected of having HIT was evaluated. All samples were tested in two IgG-specific ELISAs, in a particle gel immunoassay (PaGIA) and in a newly developed lateral-flow immunoassay (LFI-HIT) as well as in a functional test (HIPA). Clinical pre-test probability was determined using 4T's score. Platelet-activating antibodies were present in 34/452 patients, all of whom had intermediate to high clinical probability. PF4/hep abs were detected in 79, 87, 86, and 63 sera using the four different immunoassays. The negative predictive values (NPV) were 100% for both ELISA tests and LFI-HIT but only 99.2% for PaGIA. There were less false positives (n=29) in the LFI-HIT compared to any other test. Additionally, significantly less time was required to perform LFI-HIT than to perform the other immunoassays. In conclusion, a newly developed lateral-flow assay, LFI-HIT, was capable of identifying all HIT patients in a cohort in a short period of time. Beside an NPV of 100%, the rate of false-positive signals is significantly lower with LFI-HIT than with other immunoassay(s). These performance characteristics suggest a high potency in reducing the risk and costs in patients suspected of having HIT.