Assay principles of antiphospholipid antibodies and heterogeneity of the antibodies

The antiphospholipid syndrome(APS) is characterized by predominant clinical features of venous and arterial thrombosis and recurrent pregnancy loss accompanied by antiphospholipid antibodies(aPL) such as anticardiolipin antibodies(aCL) and lupus anticoagulant(LA). In 1990, three individual research groups, including us, first reported that a 50 kD plasma cofactor is required for the binding of aCL to cardiolipin(CL) and now, beta 2-glycoprotein I(beta 2-GPI), which binds to anionic phospholipids(PLs), is widely believed to be the major antigen for aCL. It was also reported that epitopes for such aCL are cryptic and that they appear only when beta 2-GPI interacts with lipid membranes containing anionic PLs, such as CL and phosphatidylserine, or with a polyoxygenated polystyrene surface. In contrast, prothrombin was recently identified as the "true" antigen for LA. In this review paper, we would like to describe on specificity of aPL and also on a possible mechanism on autoantibody-dependent development of atherosclerosis.     

Anti-actin antibodies. Chemical modification allows the selective production of antibodies to the N-terminal region

The specific properties of sera elicited by various native, unfolded or chemically modified actins were compared to provide a means of obtaining high titres of antibodies directed against the N-terminal (1-39) or the central regions of the actin sequence. The antigenic structure of the N-terminal region of actin was analyzed. It has at least 2 discrete epitopes, one of which appears to be species-specific and is composed of the hydrophilic N-terminal heptapeptide sequence.     

Avidities of hapten-specific antibodies when the responses are modulated by anti-carrier antibodies.

Either alone or in combination with antibodies having specificity for the carrier erythrocyte, TNP-ORBC were injected i.p. into CBA/J mice. Five days later, their spleens were removed and evaluated for TNP-specific PFC. The spleens from animals receiving 19S antibody (IgM) with carrier specificity displayed 3-4-fold more direct and indirect hapten-specific PFC than control animals receiving the TNP-erythrocyte conjugate only. Animals receiving 7S antibodies (IgG) with carrier specificity displayed very little change in their direct PFC response to the hapten. However, the indirect response to the hapten was suppressed as much as 16-fold by carrier specific IgG. Evaluation by haptenic inhibition of the relative avidity of the antibodies being secreted by these cells revealed the following: the relative avidity of antibodies secreted by indirect PFC in the spleens of animals receiving TNP-ORBC only was approximately 20-fold higher than antibodies secreted by the direct PFC. The 3-4-fold potentiation of the hapten-specific PFC response by carrier-specific IgM antibody did not result in a change in relative avidity of direct or indirect PFC. IgG with carrier specificity did not change the relative avidity of the antibodies secreted by direct PFC having specificity for the hapten. However, evaluation of the remaining PFC in spleens from animals whose indirect hapten-specific PFC response had been suppressed by carrier-specific IgG revealed that the remaining PFC had a lower avidity than indirect PFC from animals receiving TNP-ORBC only. In other words, carrier-specific IgG selectively induced suppression of high avidity hapten-specific IgG antibody secreting cells.     

Monoclonal antibodies against human erythrocyte membrane antigens and the antigens recognized by these antibodies

Six monoclonal antibodies (KOR-E1-E6) were raised against human erythrocyte membrane antigens. Aggregating reactions of normal human erythrocytes with or without enzyme treatment and specific antigen deficient (null type) erythrocytes were used for detection of the antigens. SDS-polyacrylamide gel electrophoresis with Western blotting and immunoperoxidase methods were also used to confirm the results. The antigen recognized by KOR-E1 and KOR-E5, which was sensitive to protease, trypsin, and neuraminidase, and only expressed on human erythrocytes, was identified as Pr1h. The antigen recognized by KOR-E2 and KOR-E6 was identified as the EnaTS portion of glycophorin A, because the antigen was sensitive to protease and trypsin, but resistant to neuraminidase, and was not present on En(a-) erythrocytes. The antigen recognized by KOR-E3 that was protease-, trypsin-, and neuraminidase resistant, and absent on En (a-) erythrocytes, was identified as Wrb antigen. As KOR-E4 reacted with all erythrocytes examined, the antigen it recognizes could not be determined.     

Further studies on the biological properties of guinea-pig IgG1 antibodies: anti-lymphocyte antibodies

Anti-rat lymphocyte serum was raised in guinea-pigs in order to study the ability of two 7S immunoglobulin classes to prolong the survival of rat skin homografts. Both classes, complement-fixing IgG2 and anaphylactic IgG1, are active, although the threshold for IgG2 activity is higher. This threshold can be lowered when a mixture of both classes is used. Thus, in this system, complement activation is not a requisite, and different mechanisms must come into play according to the class of anti-lymphocyte antibodies used. Moreover, these results have bearings on the postulated regulatory function of IgG1—antigen complexes.     

Monoclonal antibodies to human alpha-foetoprotein: analysis of the behaviour of three different antibodies

Three mouse monoclonal antibodies directed to different epitopes on human alpha-foetoprotein have been produced. Two are of IgG1 subclass and the third is IgA. The polyethylene glycol-dependent immunoprecipitation system, designed for conventional antisera, had to be adapted before reproducible results could be obtained with the reagents. In this adapted system, as well as in a radioimmunoassay using solid-phase second antibody, a mixture of the 3 monoclonal antibodies exhibits cooperativity. However, the sensitivity of the radioimmunoassay with pooled monoclonals is not good as that of conventional antiserum. Low affinity monoclonal antibodies have been used for immunopurification of the antigen, whilst the high affinity one is useful for antigen quantitation in a labelled antibody-dependent system which requires absolute antibody specificity.     

Antinuclear antibodies

The presence of abnormal levels of autoantibodies to intracellular antigens is a hallmark of systemic connective tissue disease. The indirect immunofluorescence assay is the most commonly used routine test for the detection of antinuclear antibodies. In this text, several representative patterns of fluorescence are reviewed and some pitfalls for application of the results to the clinical field are mentioned.     

Human monoclonal anti-DNA antibodies react as lymphocytotoxic antibodies

Two out of 25 monoclonal anti-DNA autoantibodies that were produced by human-human hybridoma were found to have lymphocytotoxic activity. The antibodies reacted with normal B and T lymphocytes at cold (4 degrees C) as well as at warm (37 degrees C) temperatures. The lymphocytotoxic activity of the monoclonal anti-DNA antibodies could be inhibited by prior incubation of the antibodies with either polynucleotides, e.g. poly(I), poly(dT) or anti-idiotypic antibodies, that had been raised against a dominant anti-DNA antibody. The cross-reactivity between nuclear material and lymphocyte membrane raises the question whether these apparently diverse materials have a shared epitope. The cross-reactivity between anti-DNA antibodies and lymphocyte membrane may account in part for the lymphopenia observed in systemic lupus erythematosus patients.     

Anti-idiotypic antibodies against antibodies to the herpes simplex virus and its early antigens]

Polyclonal anti-idiotypic antibodies (anti-Id-AT or AT2) were produced to AT idiotopes of virion antigens--structural proteins of herpes simplex virus (HSV) and of early virus-induced antigens (VIA). The method of SPRIA established that anti-Id-AT seemed to be associated with AT active centers. Besides, immunization with anti-Id-AT produced anti-anti-Id-AT (AT3) similar in their specificity with antiviral AT or anti-VIA. It was shown that AT2 could be used as a diagnostic preparation instead of specific antigen for the detection of antibody to HSV or VIA in human sera by indirect RIA.     

Antiphospholipid antibodies

OBJECTIVE: To review the role of lupus anticoagulants in the pathogenesis of both venous and arterial thromboembolic events, as well as in recurrent spontaneous abortions. The pathophysiology of lupus anticoagulants and associated antiphospholipid antibodies (eg, anticardiolipin antibodies) is also discussed. DATA SOURCES: Review of the recent medical literature. DATA EXTRACTION AND SYNTHESIS: Key articles in the recent medical literature dealing with lupus anticoagulants and their role in pathogenesis of thromboembolic events were reviewed. Plasma proteins that have an affinity for binding to "perturbed cellular membranes" have been identified as the antigenic targets for antiphospholipid antibodies. Thus, the concept of antiphospholipid antibodies needs to be reevaluated. Perhaps a better term is antiprotein-phospholipid antibodies. The principal antigenic protein targets are beta(2)-glycoprotein I, prothrombin, and a wide range of additional proteins that interact with activated cellular membranes, including protein C, protein S, annexin V, etc. Most research reported in the literature has focused on beta(2)-glycoprotein I and human prothrombin.     

Antiphospholipid antibodies

The concept of antiphospholipid syndrome(APS) has been widely accepted. Antiphospholipid antibodies originally included anticardiolipin antibodies and lupus anticoagulants as serological marker of APS. However, recent advances have shown that most pathogenic antiphospholipid antibodies are directed to phospholipid binding proteins such as beta 2-glycoprotein I and prothrombin as well as phospholipids. The preliminary classification criteria for definite APS have been advocated as the "Sapporo criteria". Further prospective investigations are required to re-evaluate the clinical significance of so-called antiphospholipid antibodies.