Screening for fibrin specific monoclonal antibodies: the development of a new procedure.
The acquisition of monoclonal antibodies specific for human fibrin has been impaired by the similarity in chemical composition between fibrinogen and fibrin and the conformational difference between immobilised and soluble fibrinogen. Five monoclonal antibodies (mabs) with a known affinity for fibrin have been subjected to screening procedures which involved the presentation of different forms of both fibrinogen and fibrin to the test mabs. It was observed by scanning electron microscopy that dried fibrin (denoted fibrin D), immobilised on the wells of PVC plates was morphologically similar to the fibrin found in human clots whereas PVC-immobilised fibrin monolayers (fibrin M) and a homogenised form of fibrin (fibrin FF) presented two very different morphological appearances. It was shown that lack of cross reactivity of a mab with an antigen (e.g. fibrinogen) was validly demonstrated only when both mab and antigen were present in the soluble state. These findings have allowed the generation of a screening procedure which involves the use of fibrin D on PVC plates in conjunction with whole human plasma incubated with the test antibody. This screening procedure has been validated using two mabs, one of which has an exclusive fibrin affinity while the other has a broad spectrum crossreactivity with both fibrinogen and fibrin. This procedure would ensure the acquisition of all the five fibrin-specific mabs used in this study while other less reliable screening procedures might not.
Tymkewycz
P M
PM
National Institute for Biological Standards and Control (NIBSC), South Mimms, Potters Bar, Hertfordshire, UK.
Creighton-Kempsford
L J
LJ
Hockley
D
D
Gaffney
P J
PJ
D000911:Antibodies, Monoclonal / Q000032:analysis*; D000918:Antibody Specificity / Q000276:immunology*; D000937:Antigen-Antibody Reactions / Q000276:immunology; D000941:Antigens / Q000032:analysis / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005337:Fibrin / Q000032:analysis* / Q000276:immunology; D005340:Fibrinogen / Q000276:immunology; D005614:Freeze Fracturing; D006801:Humans; D006825:Hybridomas / Q000737:chemistry*; D007118:Immunoassay / Q000379:methods; D008855:Microscopy, Electron, Scanning; D011485:Protein Binding; D015203:Reproducibility of Results
D000911:Antibodies, Monoclonal; D000941:Antigens; D005337:Fibrin; D005340:Fibrinogen