L.R. Micklem

A monoclonal antibody enzyme linked immunosorbent assay (ELISA) directed towards a fibrin binding region of tissue-type plasminogen activator

Abstract A sensitive double monoclonal antibody ELISA has been developed for measurement of total t-PA antigen. The two mouse monoclonal antibodies employed recognise distinct epitopes at or near the kringle 2 fibrin binding region of t-PA and these clones have proved stable over several years. Complete recovery of t-PA in plasma when assayed against a standard t-PA diluted in buffer was achieved by dilution of the plasma sample in buffer containing ethylenediaminetetraacetic acid. The ELISA did not detect urokinase while t-PA complexed with endothelial plasminogen activator inhibitor was measureable in addition to free active t-PA. No discrimination between 1 and 2-chain forms of t-PA was evident. The concentration of t-PA antigen in basal human citrated plasma was found to be 3.8 ± 1.5 ng ml −1 (mean ± standard deviation, n =17) and good correlations were observed between t-PA antigen measured by ELISA and by radioimmunoassay or a functional assay. The ELISA provides a reliable and simple method for measuring t-PA antigen in plasma and other biological fluids and is based on stable reagents that recognise known epitopes on t-PA.

A mouse monoclonal antibody with anti-A,(B) specificity which agglutinates Ax cells

Abstract. A hybridoma (ES‐15) was obtained by fusing the NS‐1 cell line with spleen cells from a mouse immunised with soluble blood group A2 substance. The cloned hybridoma culture supernatant was shown to contain an IgM class antibody which strongly agglutinates group A cells and weakly agglutinates group B cells. The serological specificity of this antibody is described as anti‐A,(B) in this report. The abilities of unconcentrated monoclonal anti‐A,(B), a commercial human polyclonal anti‐A,B (group O serum) and a commercial monoclonal anti‐A reagent to detect 15 examples of Ax cells were compared by both slide and tube techniques. Using a slide technique monoclonal anti‐A,(B) agglutinated 14 examples of Ax cells, human anti‐A,B 2 examples, while monoclonal anti‐A failed to detect any of the Ax cells tested. Similar differences in the reactivity of the three antibodies were observed using a tube technique. Data are also presented which show that a 1:1 (v/v) mixture of monoclonal anti‐A,(B) with a monoclonal anti‐B reagent is an effective replacement for human anti‐A,B in ABO grouping procedures.

Monoclonal antibodies directed against human α-thrombin and the thrombin-antithrombin III complex

Human alpha-thrombin was poorly immunogenic in Balb/c mice. Nevertheless, following fusion of spleen cells from a responding mouse with NS-1 cells, 8 mouse monoclonal antibodies against alpha-thrombin were isolated, and 6 were characterised. Five of these were isotype IgG2a, and one was IgG1. One, EST 1, bound thrombin only minimally, and was directed against a neoantigen on the thrombin-ATIII (T-AT) complex. This antibody also recognised a site on prothrombin, though with much lower affinity. Its binding was markedly temperature-dependent, indicating a requirement for molecular mobility. A second antibody, EST 4, would not bind the T-AT complex. It inhibited both the clotting and amidase activities of thrombin, and modification of the active site histidine, but not the active site serine, reduced the affinity constant of binding to EST 4. This antibody appears to be directed against an epitope in the vicinity of the enzyme active site. The epitopes for EST 1 and EST 4 were both remote from those of the other monoclonal antibodies, EST 2, 6, 7 and 8. These four competed with each other for binding to thrombin, and all inhibited clotting but not amidase activity. Thrombin binding was not affected by modification of the active site, though formation of the T-AT complex reduced the affinity of binding to EST 6 and EST 8. These monoclonals recognise epitopes in the region of the fibrinogen binding site.

Human atrial natriuretic factor (ANF): characterisation of a monoclonal antibody panel and its use in radioimmunoassay

The production of nine monoclonal antibodies to human atrial natriuretic factor (ANF 1-28) is described. All possible combinations of two antibodies failed to reveal any which could simultaneously bind ANF. Studies with ANF analogues and the antibodies having the three highest affinity values (KD = 5, 25 and 21 pM) indicated that the antibodies are directed to the central portion of the antigen molecule. The highest affinity antibody was able to replace polyclonal antisera in the radioimmunoassay of ANF in extracts of plasma.

Murine monoclonal antibodies against active site epitopes on human endothelial plasminogen activator inhibitor (PAI-1)

Summary A panel of 10 murine monoclonal antibodies was produced against human endothelial PAI-1 that had been purified to homogeneity in the absence of denaturants. All the antibodies bound to iodinated PAI-1 but no binding was observed to iodinated α1-antitrypsin (α1-AT), antithrombin III (ATIII) heparin cofactor II or α2-antiplasmin (α2-AP). PAI-1 was presented in the form of a complex with t-PA immobilised to microplate wells. Three of the 10 monoclonal antibodies (ESPI-1, ESPI-2 and ESPI-12) bound to the complexed PAI-1 while the others did not. Effects of the antibodies upon the interaction between PAI-1 and t-PA were measured by determining the distribution of 125I-labelled t-PA between free and complexed forms after SDS-PAGE and autoradiography. Two clones (ESPI-4 and ESPI-6) reduced complex formation markedly when incubated with PAI-1 prior to its reaction with 125I t-PA. ESPI-4 and ESPI-6 were also effective at quenching the activity of PAI-1 measured as reduction in t-PA activity in a chromogenic peptide substrate assay. Partial quenching of PAI-1 activity was achieved with ESPI-1 and ESPI-12. Thus 4 types of antibody reactivity towards PAI-1 could be distinguished. ESPI-3, 8, 10, 11 and 14 bound free PAI-1 (by ELISA or tracer binding assay) but did not bind complexed PAI-1 nor influence PAI-1 biological activity. ESPI-4 and 6 did not bind complexed PAI-1 but inhibited PAI-1 activity and prevented complex formation, ESPI-1 and 12 reacted with complexed PAI-1 and exhibited some inhibition of PAI-1 activity. Finally, ESPI-2 reacted with complexed PAI-1 but had no effect upon PAI-1 activity. The high affinity binding of PAI-1 by 3 of the antibodies immobilised on Sepharose 4B suggested their suitability for preparing PAI-1 depleted plasma and reagents. The other antibodies were effective as immunoaffinity purification reagents. They retained their capacity to bind PAI-1 over several cycles and the PAI-1 could be eluted with 0.1 M glycine-HC1 pH 3.0. The panel of antibodies will have a wide range of applications as assay, immunoaffinity purification and immunocytochemical reagents.

Stability of murine monoclonal anti-A, anti-B and anti-A, B. ABO grouping reagents and a multi-centre evaluation of their performance in routine use

Abstract. We have previously reported the production of 3 murine monoclonal reagents for ABO typing (designated ES‐9, ES‐4 and ES‐15). This study presents results of tests of stability of these 3 reagents, together with a fourth murine monoclonal antibody (LM103/107). In addition, data are also presented from a multi‐centre evaluation of the performance of the murine monoclonal reagents in routine ABO typing of both donors and patients using a wide variety of techniques, both manual and automated. The potency and stability of the 4 monoclonal antibody based reagents is compared with a broad selection of monoclonal and polyclonal ABO typing reagents. The reagents used for comparison were produced by European and United Stated manufacturers in both the public and private sector and are widely used in routine ABO typing. The Scottish monoclonal reagents have been used successfully to ABO type over 500,000 blood samples in 7 centres within the UK, with no discrepant results.

Characterisation of mouse monoclonal antibodies produced by immunisation with a single serotype component of a polyvalent Pseudomonas aeruginosa vaccine

Mouse monoclonal antibodies raised by immunisation with a protective antigen extract from Pseudomonas aeruginosa serotype 1 varied in immunoglobulin isotype, in passive protective properties against infection by homologous P. aeruginosa serotype 1, and in cross-reactions in ELISA against antigen preparations from 15 other P. aeruginosa serotypes. All monoclonal antibodies with specificity in ELISA for the immunising antigen gave some degree of protection to mice against lethal infection by the homologous P. aeruginosa serotype. The IgG antibodies were more protective than the IgM antibodies.