Academic literature on the topic 'Immune serums – Purification'

There was described and efficient and economical approach for the removal of toxic substances from normal and immune sera from various species of animals with the use of human placenta tissue. Purification brings about to perceptible losses of neither serum-specific activity nor the original volume. Being simple the method does not require any special equipment and can be used in conditions of low-volume or in laboratory production of serum preparations. There are considered as well possible origins of serum toxicity as mechanism of antitoxic activity of placenta.

Lectins are involved in recognition phenomena and their ability to bind particular Carbohydrate structures are the key to their biological functions. Bacteria typically attaches to prospective host cell membranes in receptors with lectin like sugar specificity. This is of great importance as the adherence of bacteria to host tissue surfaces is the initial event in bacterial infection. Lectins are also known to play important roles in immune system by recognizing carbohydrates that are found exclusively on pathogens, or that are inaccessible on host cells. This ability of lectins to selectively bind or agglutinate specific sugars have made them useful tools for the characterization of certain cell types or fragments, to detect cells in different states of development, to distinguish normal from tumour cells and to separate different cell types by affinity chromatography. A total of 120 samples of local Achatina achatina snail specie were collected, authenticated at the Zoology Department of the University of Nigeria, Nsukka and 80mls of pooled crude Lectin extract was obtained. Purifications were performed on 20mls of the crude extract in three steps viz, Ammonium sulphate precipitation and Dialysis (Partial purifications), Con A Sepharose 4B affinity chromatography column (Complete purification). The affinity purified lectin was used in all the tests conducted in this research. The crude, partially and complete/affinity purified lectin extracts were subjected to Haemagglutination and Protein Assay tests. The Molecular weight was deduced by Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method. The microbial agglutination potentials of the lectin was assessed by testing typed bacterial organisms viz, Salmonella typhimurium, Escherichia coli, Lactobacilli acidophilus, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella aeruginosa and four typed fungal organisms: Aspergillus niger, Trichophyton mentagrophytes, Candida albicans and A. flavus. The lectin’s Lymphocyte blastogenesis activities was determined by its incubation with human lymphocytes for mitogenic stimulation assay. The results of the research showed as follows: On complete/affinity purification, 15mls of pure sample containing only the high molecular weight lectin was obtained. On standardization, the respective haemagglutination tests on the crude, partially and affinity purified lectin showed preferential agglutinations with Blood group A type. Only S. typhimurium (+++), E. coli (+) and L. acidophilus (+) reacted with the lectin but in different strengths. Incubation of the lectin with lymphocytes from human serum showed that it has the ability to stimulate lymphocytes to undergo mitosis. This research has therefore succeeded in assessing the Microbial agglutination and Lymphocyte blastogenesis potentials of the isolated and characterised A. achatina snail lectin.

High levels of granulocyte/macrophage–colony-stimulating factor (GM-CSF) autoantibodies are thought to cause pulmonary alveolar proteinosis (PAP), a rare syndrome characterized by myeloid dysfunction resulting in pulmonary surfactant accumulation and respiratory failure. Paradoxically, GM-CSF autoantibodies have been reported to occur rarely in healthy people and routinely in pharmaceutical intravenous immunoglobulin (IVIG) purified from serum pooled from healthy subjects. These findings suggest that either GM-CSF autoantibodies are normally present in healthy people at low levels that are difficult to detect or that serum pooled for IVIG purification may include asymptomatic persons with high levels of GM-CSF autoantibodies. Using several experimental approaches, GM-CSF autoantibodies were detected in all healthy subjects evaluated (n = 72) at low levels sufficient to rheostatically regulate multiple myeloid functions. Serum GM-CSF was more abundant than previously reported, but more than 99% was bound and neutralized by GM-CSF autoantibody. The critical threshold of GM-CSF autoantibodies associated with the development of PAP was determined. Results demonstrate that free serum GM-CSF is tightly maintained at low levels, identify a novel potential mechanism of innate immune regulation, help define the therapeutic window for potential clinical use of GM-CSF autoantibodies to treat inflammatory and autoimmune diseases, and have implications for the pathogenesis of PAP.

A novel approach to recover and identify immune complexes (ICs) was developed using size exclusion chromatography (SEC) and affinity chromatography on immunoglobulin binding columns (HiTrap Protein G). The purification process was monitored by 1D SDS-PAGE, protein staining, Western blotting and, finally, liquid chromatography tandem mass spectrometry (LC MS/MS) was used to identify the recovered antigens. This approach was applied to serum with artificially created immune complexes (ICs) comprising vaccine antigen (influenza) and antibody, which led to recovery and identification of influenza peptides within the recovered ICs. This approach was compared with the established method for IC detection and recovery, polyethylene glycol (PEG) precipitation, followed by LC MS/MS. Both approaches successfully enabled capture, recovery and characterization of immunoglobulins and influenza antigen(s) in complex with the immunoglobulins. However, PEG precipitation has the advantage of simplicity and is more suited for large scale studies.

A rapid method is described for the purification of human tissue inhibitor of metalloproteinases (TIMP) from plasma which involves immuno-affinity chromatography and gel filtration. The purified plasma inhibitor is immunologically identical with the TIMP previously purified from human amniotic fluid, human synovial fluid and human fibroblast culture medium. It is proposed that this inhibitor is identical with the plasma inhibitor previously named ‘B1 anticollagenase’, although the plasma inhibitor was shown to migrate as a gamma-serum component.

This study examined the effect of whole bee venom (BV) as a potential stimulant of the piglet immune system, on growth performance, blood parameters, plasma protein and immune globulin content of serum. Piglets (n = 97) received combinations of 0.5, 1.0, 1.5, 2.0 and 2.5 mg/kg of parenterally administered BV on 4 occasions between birth and Day 30. In the apipuncture group (n = 31), piglets were acupunctured with the worker honeybee. Two acupoints, GV-1 (Jiao-chao) and GV-20 (Bai-hui), were selected for apipuncture. All piglets (n = 128) in the treatment groups were treated 4 times throughout the study period of 60 days. The control piglets received no treatments. Blood was taken via jugular venipuncture on Day 30 after birth. Body weight and survivability were measured, and changes in hematological values were analyzed. Both the BV injection group and apipuncture group increased body weight and survivability by 26.6% and 21.8%, and 7.9% and 6.7% respectively compared to the controls. The numbers of leukocytes, erythrocytes, lymphocytes and monocytes were not influenced by treatments. However, a potential clinical benefit of high dose therapy was seen in increased populations of leukocytes, lymphocytes and monocytes compared with either the apipuncture or control groups. Other blood parameters such as total protein and albumin were not affected by treatment. However, IgG levels were generally higher in treated groups than in the controls. These findings indicate that BV might be useful to stimulate immuno-competence in pig production, possibly via the primary bioactive components of melittin, phospholipase A 2 and apamin. The administration of BV, either via injection or acupuncture, did not make any differences in growth performance of young pigs. These results would be useful for further purification and characterization of immune boosting agents from BV.

Phosphoenolpyruvate carboxylase (PEPC) from ripened banana (Musa cavendishii L.) fruits has been purified 127-fold to apparent homogeneity and a final specific activity of 32 mumol of oxaloacetate produced/min per mg of protein. Non-denaturing PAGE of the final preparation resolved a single protein-staining band that co-migrated with PEPC activity. Polypeptides of 103 (alpha-subunit) and 100 (beta-subunit) kDa, which stain for protein with equal intensity and cross-react strongly with anti-(maize leaf PEPC) immune serum, were observed following SDS/PAGE of the final preparation. CNBr cleavage patterns of the two subunits were similar, but not identical, suggesting that these polypeptides are related, but distinct, proteins. The enzyme's native molecular mass was estimated to be about 425 kDa. These data indicate that in contrast to the homotetrameric PEPC from most other sources, the banana fruit enzyme exists as an alpha 2 beta 2 heterotetramer. Monospecific rabbit anti-(banana PEPC) immune serum effectively immunoprecipitated the activity of the purified enzyme. Immunoblotting studies established that the 100 kDa subunit did not arise via proteolysis of the 103 kDa subunit after tissue extraction, and that the subunit composition of banana PEPC remains uniform throughout the ripening process. PEPC displayed a typical pH activity profile with an alkaline optimum and activity rapidly decreasing below pH 7.0. Enzymic activity was absolutely dependent on the presence of a bivalent metal cation, with Mg2+ or Mn2+ fulfilling this requirement. The response of the PEPC activity to PEP concentration and to various effectors was greatly influenced by pH and glycerol addition to the assay. The enzyme was activated by hexose-monophosphates and potently inhibited by malate, succinate, aspartate and glutamate at pH 7.0, whereas the effect of these metabolites was considerably diminished or completely abolished at pH 8.0. The significance of metabolite regulation of PEPC is discussed in relation to possible functions of this enzyme in banana fruit metabolism.

SummaryHemophilia A is the inherited bleeding disorder that results from mutation of blood coagulation factor VIII (fVIII). Described here is the generation of a regulated expression system producing recombinant murine fVIII. Murine B-domainless fVIII was expressed at a peak level of 4 units/106 cells/24 h in serum-free media. Subsequently, a two-step purification procedure resulted in 5,300-fold enrichment and a 70% yield. Highly purified recombinant murine fVIII had a specific coagulant activity of 660 units per nanomole. It underwent proteolytic processing by thrombin to yield an activated heterotrimer that demonstrated significantly greater stability than activated human fVIII. Recombinant murine fVIII was utilized to generate an anti-fVIII polyclonal antibody. Intravenous injection of recombinant murine fVIII into hemophilia A mice failed to induce a significant anti-fVIII immune response using a schedule that yielded high titer inhibitory antibodies to human fVIII. This may provide an important model for the study of immune tolerance to fVIII.

The purification to homogeneity of hexokinases B and C from the cytosol of rat Novikoff hepatoma was achieved by a protocol using an initial chromatography on Blue 2-agarose to separate the isoenzymes from each other. After that step each hexokinase was subjected to chromatography on DEAE-cellulose, hydroxyapatite and Sephacryl S-300, followed by re-chromatography on hydroxyapatite. The final preparations of hexokinases B and C had specific activities of 86 and 23.5 units/mg of protein respectively, and gave single bands on electrophoresis under non-denaturing conditions or in SDS/polyacrylamide gels. Mr values of about 100,000 were found for both isoenzymes either by Sephacryl S-300 chromatography or by SDS/polyacrylamide-gel electrophoresis. Values of apparent Km for glucose and ATP of pure hexokinase B were similar to those reported for the enzyme from other sources. The apparent Km value for glucose of hexokinase C was 0.025 mM. Marked inhibition of hexokinase C by glucose concentrations above 0.2 mM was found. The effect was partially relieved by ATP concentrations above 1 mM and was independent of pH. Glucose 6-phosphate was inhibitory, but the Ki value (0.18 mM) is higher than those reported for other animal hexokinases. The amino acid composition of hexokinase C was found to be similar to those reported for hexokinases B and D. Also, an immune serum directed against hexokinase A was able, at low dilutions, to bind hexokinases B and C. An immune serum directed against hexokinase C was able, at low dilutions, to bind hexokinase B and also, but weakly, hexokinase A.

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Dissertacao (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Yau Wing Yiu, Winifred.
On t.p. "alpha"s appear as the Greek letter.
Thesis submitted in: October 2006.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2007.
Includes bibliographical references (leaves 91-98).
Abstracts in English and Chinese.
Abstract --- p.i
Abstract (Chinese version) --- p.iv
Acknowledgements --- p.vi
Table of Contents --- p.vii
List of Abbreviations --- p.xii
List of Figures --- p.xiv
List of Tables --- p.xvi
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- Peroxisome proliferator-activated receptors (PPARs) --- p.1
Chapter 1.1.1 --- What are PPARs? --- p.1
Chapter 1.1.2 --- PPAR ligands - peroxisome proliferators --- p.1
Chapter 1.1.3 --- PPAR isoforms --- p.2
Chapter 1.2 --- Biological roles of PPARα --- p.3
Chapter 1.2.1 --- Lipid metabolism --- p.3
Chapter 1.2.2 --- Glucose metabolism --- p.4
Chapter 1.2.3 --- Inflammation --- p.5
Chapter 1.2.4 --- Oxidative stress --- p.5
Chapter 1.2.5 --- Cell proliferation and apoptosis --- p.6
Chapter 1.3 --- PPARα in health and diseases --- p.6
Chapter 1.3.1 --- Wound-healing --- p.6
Chapter 1.3.2 --- Anti-atherogenesis --- p.7
Chapter 1.3.3 --- Neuroprotection --- p.7
Chapter 1.3.4 --- Carcineogenesis --- p.7
Chapter 1.4 --- PPARα-regulated and starvation inducible gene (PPSIG) --- p.8
Chapter 1.4.1 --- PPSIG is a PPARα target gene --- p.8
Chapter 1.4.2 --- Computer-assisted predictions on PPSIG --- p.9
Chapter 1.4.3 --- Current characterization of PPSIG --- p.10
Chapter 1.5 --- Objectives of the present study --- p.11
Chapter Chapter 2 --- Materials and Methods --- p.12
Chapter 2.1 --- Materials --- p.12
Chapter 2.2 --- Animals and treatment --- p.13
Chapter 2.3 --- Cloning of PPSIG into pThioHis and pTYB expression vectors --- p.13
Chapter 2.3.1 --- PCR amplification of PPSIG cDNA insert --- p.13
Chapter 2.3.1.1 --- PPSIG cDNA insert for pThioHis vector --- p.13
Chapter 2.3.1.2 --- PPSIG cDNA insert for pTYB vector --- p.15
Chapter 2.3.2 --- Restriction enzyme digestion of PPSIG cDNA insert and pThioHis vector --- p.18
Chapter 2.3.3 --- Restriction enzyme digestion of PPSIG cDNA insert and pTYB vector --- p.20
Chapter 2.3.4 --- Ligation and transformation --- p.20
Chapter 2.3.5 --- Screening for recombinants by phenol/chloroform method --- p.21
Chapter 2.3.6 --- Confirmation of recombinant plasmid by restriction enzyme digestion --- p.22
Chapter 2.3.6.1 --- Digestion of pThioHis-PPSIG plasmid with Xba I and Sac II --- p.22
Chapter 2.3.6.2 --- Digestion of pTYB-PPSIG plasmid with EcoR V --- p.22
Chapter 2.3.7 --- Transformation into expression E. coli strains --- p.23
Chapter 2.4 --- Over expression of PPSIG proteins in E. coli --- p.23
Chapter 2.5 --- Semi-purification of PPSIG fusion proteins by preparative SDS-PAGE --- p.24
Chapter 2.6 --- Rabbit immunization --- p.25
Chapter 2.7 --- Northern blotting analysis --- p.26
Chapter 2.7.1 --- Probe preparation --- p.26
Chapter 2.7.2 --- "Formaldehyde-agarose gel electrophoresis, blotting of RNA and hybridization" --- p.26
Chapter 2.8 --- Subcellular fractionation --- p.29
Chapter 2.9 --- Western blotting of liver microsomes --- p.31
Chapter 2.10 --- Immunoprecipitation --- p.32
Chapter 2.11 --- Mass spectrometry --- p.33
Chapter 2.11.1 --- Trypsin digestion and peptide extraction --- p.33
Chapter 2.11.2 --- Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry --- p.34
Chapter Chapter 3 --- Results --- p.36
Chapter 3.1 --- Cloning of PPSIG into pThioHis and pTYB vectors --- p.36
Chapter 3.1.1 --- Cloning of PPSIG into pThioHis vector --- p.36
Chapter 3.1.2 --- Cloning of PPSIG into pTYB vector --- p.36
Chapter 3.2 --- Protein expression of Thio-PPSIG and Intein-PPSIG --- p.41
Chapter 3.3 --- Identification of recombinant Thio-PPSIG and Intein-PPSIG by mass spectrometry --- p.49
Chapter 3.4 --- Preparation and characterization of Thio-PPSIG and Intein-PPSIG antisera --- p.61
Chapter 3.5 --- Identification of native PPSIG and its induction pattern --- p.65
Chapter 3.5.1 --- PPSIG was highly inducible upon 72-h starvation in a PPARα dependent manner --- p.65
Chapter 3.5.2 --- "PPSIG showed slight induction upon 2-wk Wy-14,643 treatment" --- p.71
Chapter 3.6 --- Confirmation of the specificity of PPSIG antiserum --- p.74
Chapter Chapter 4 --- Discussion --- p.81
References --- p.91
Appendix A Deduced amino acid sequences of PPSIG fusion proteins --- p.99
Chapter A1 --- Deduced amino acid sequence of Thio-PPSIG from pThioHis-PPSIG plasmid --- p.99
Chapter A2 --- Deduced amino acid sequence of Intein-PPSIG from pTYB-PPSIG plasmid --- p.101
Appendix B Mass spectra of trypsin digested native PPSIG --- p.104
Chapter B1 --- Mass spectrum of trypsin digested native PPSIG immunoprecipitated from liver microsomes from PPARα wild-type mice fed with normal diet (starvation experiment) --- p.104
Chapter B2 --- Mass spectrum of trypsin digested native PPSIG immunoprecipitated from liver microsomes from PPARα wild-type mice starved for 72 hours (starvation experiment) --- p.105
Chapter B3 --- "Mass spectrum of trypsin digested native PPSIG immunoprecipitated from liver microsomes from PPARα wild-type mice fed with control diet (Wy-14,643 feeding experiment)" --- p.106
Chapter B4 --- "Mass spectrum of trypsin digested native PPSIG immunoprecipitated from liver microsomes from PPARα wild-type mice fed with 0.1% (w/w) Wy-14,643 for 2 weeks (Wy-14,643 feeding experiment)" --- p.107

Isolation and purification of antithrombin III (AT III) by affinity chromatography on immobilized heparin is a standard method for the large scale preparation of this protein from human or animal plasma. Hence, after AT III became available by gentechnological methods, we tried to adapt this procedure for the isolation of AT III from supernatants of mammalian- and yeast-cells. Indeed, it was possible to use this method also for the isolation of the recombinant gene products. Since, however, the cell growth media contain heterologous protein or peptide mixtures like fetal calf serum, the method had to be improved to avoid the adsorption of non human proteins or peptides. We are now able to purify AT III from CHO-cell-superna-tants to more than 95 % purity. The characterization of this AT III-product by double immuno diffusion revealed that it is immunologically totally identical with the authentic material from plasma. AT III antigen content, progressive inhibitor activity and heparin cofactor activity compare very well in the final product; hence, it is totally active compared to AT III from plasma.In polyacrylamidegel electrophoresis most of the material migrated differently to the authentic material showing 9 bands in equal distance to each other, instead four in the At III from plasma. After degradation with sialinidase from both AT III preparations identical cleavage products were obtained migrating predominantly as a single band. Hence, the electrophoretic heterogeneity seems to be due to a different degree of sialinyla-tion of the products.

Splenocytes from a Balb/c mouse immunized with purified human protein S (PS) were fused with murine hybridoma cell line SP2/0-Agl4 and cultured in Iscove's medium without addition of fetal bovine serum. Hybrid supernatants were screened for the presence of specific antibodies by solid-phase ELISA and cloned by the limiting dilution technique. Pour clones, named S2, S3, S8, and S10, were selected, recloned several times, and injected intraperitoneally into Balb/c mice for the production of antibody-rich ascitic fluid. The monoclonal antibodies (Mabs), all of IgGl subclass with k light chain, were purified from ascitic fluid by Protein-A chromatography. The specificity of Mabs was controlled by the immunoblotting technique: the Mabs appeared to react only with two plasma proteins, one with a MW of about 70.000 dal tons comigrating with purified PS, and the other with a MW >300.000 da that is likely to be the C4b-binding protein-PS complex. No interaction has been observed with PS-depleted plasma. As tested by a fluid phase radio immunoassay, the binding of Mabs to PS was significantly higher in the presence of EDTA while was totally inhibited in the presence of Ca2+. Scatchard plot analysis of the binding between 125I-PS and Mabs showed that the binding affinity of the antibodies ranged from 108 to 109 1/mol. Each EDTA-dependent Mab was then immobilized on Sepharose 4B-CNBr and used to purify PS from barium precipitation of citrated plasma. The fraction eluted with 2 mmol of CaCl2 from the immunoadsorbent appeared to contain only two proteins when stained with Cocmassie Blue. By immuno blotting, all Mabs reacted with both proteins, one comigrating with purified PS and the other with a MW >300.000. Essentially homogeneous PS, free from the high MW component, was obtained when the barium citrate adsorbate was applied to a DEAE-Sephadex column and the protein peack containing the balk of PS was sussequently applied to the immunoadsorbent and eluted with 2 mmol CaCl2.In summary, we have described an unusual set of EDTA-dependent monoclonal antibodies to PS and their use for the purification of homogeneous protein S from human plasma.

A high titer, boost responding inhibitor was present in a patient and his nephew, both with severe hemophilia B. On digests of their DNA, Southern blots hybridized to a cDNA were normal. They had no detectable IX antigen, including immuno-radiometric assays with a calcium-requiring polyclonal antibody fraction, in either serum or urine (less than 0.03 U/dl).Plasmas from the patient and his nephew had 12 and 25 NIH U/ml inhibitor titers, respectively. They were fractionated over IX-agarose with calcium. Unlike fractionation of rabbit polyclonal antibodies, EDTA eluates did not bind IX. Purified patient inhibitors were eluted at low pH and contained detectable IgG1, IgG2 and IgG4 but not IgG3, IgA or IgM by radial immunodiffusion. On immunoradiometric assay, each patient's 125I-inhibitor bound to IX on the same solid phase inhibitor, indicating recognition of more than one epitope. Factor IX Ag was readily detected in these assays. The patients' inhibitors competed with binding of 125I-IX binding to each of 3 monoclonal antibodies. Each monoclonal antibody blocked 125I-IX binding to its own, insolubilized species. Similar results were obtained on an inhibitor plasma provided by H. Reisner (Chapel Hill, NC) which, unlike the present cases, contained a calcium-requiring antibody fraction (Briet E, et al Prog Clin Biol Res 150:123-139, 1984). Our patient's inhibitor was tested with two rabbit polyclonal fractions; the calcium-dependent fraction (epitopes in the light chain of IXa) and a specific heavy chain-binding fraction. The latter was from immunoaffinity purification of the non-calcium binding rabbit polyclonal fraction over an insolubilized synthetic peptide containing residues 256 through 269 of the IX sequence. Both fractions, in fluid phase, inhibited binding of 125I-IX to their own but not the other solid phase fraction. The patient's inhibitor did not block 125I-IX binding of the calcium-requiring fraction but did compete with the fraction prepared from the peptide column. These data suggest that the defect in our patient is a mutation between the Gla domain's 2nd or 3rd exons and the second growth factor-like region in the 5th exon.

In order to develop specific immunologic reagents for mapping functionally important sites on FVIII, we have prepared rabbit polyclonal antibodies against synthetic peptides of FVIII derived from regions along the entire FVIII amino acid sequence. To date, a total of 70 peptides have been synthesized and characterized by amino acid and HPLC analysis. The peptides were coupled to keyhole limpet hemocyanin with glutaraldehyde as a linkage reagent and used to immunize rabbits. Antisera were tested by ELISA assay on polystyrene microtiter plates coated with either the peptide immunogen, or purified FVIII. The antisera were also tested for their ability to inhibit FVIII clotting activity and to react with separated FVIII polypeptides on immunoblots.Of the 70 peptides, all reacted with the peptide immunogen, 45 reacted with purified FVIII and 33 reacted with FVIII on immunoblots. Because we had obtained evidence that cleavage of the amino terminal region of the 80 kDa polypeptide may play a role in FVIII activation by thrombin, a series of partially overlapping peptides, 15 residues in length, were synthesized in this area. After affinity purifying these antibodies on columns of FVIII immobilized on agarose, adjusting the antibodies to equal antigen binding titers by dot immunoblotting and testing for inhibition of FVIII activity, only one antibody could strongly inhibit FVIII clotting activity. This inhibition could be blocked by the peptide itself at nanomolar concentrations and no significant inhibition could be shown by antibodies to partially overlapping peptides individually, or in combination. These data suggest that a site important to FVIII function can be localized to a 15 amino acid residue region of the 80 kDa polypeptide of FVIII. In addition, a second inhibitoryantibody was identified which was produced against a peptide in the carboxy terminal region of the 54 kDa thrombin fragment of FVIII and this area is currently being studied in a similar manner. In addition, two monoclonal anti-FVIII synthetic peptide antibodies have been produced which react with purified FVIII on immunoblots. One of these antibodies also functions as an immunoadsorbent when linked to agarose and FVII can be purified in this manner, using the synthetic peptide as eluant. It is evident that antibodies to synthetic peptides of FVIII can be useful probes of FVIII structure, function and interactions as well as being of use in FVIII purification.