Serum or breast milk immunoglobulins mask the self‐reactivity of human natural IgG antibodies

B cells producing IgG antibodies specific to a variety of self‐ or foreign antigens are a normal constituent of the immune system of all healthy individuals. These naturally occurring IgG antibodies are found in the serum, external secretions, and pooled human immunoglobulin preparations. They bind with low affinity to antigens, which can also be targets for pathologic autoantibodies. An enhancement of naturally occurring IgG autoantibody activity was observed after treatment of human IgG molecules with protein‐destabilizing agents. We have investigated the interactions of human immunoglobulins that were obtained from serum or from breast milk of healthy individuals or IVIg with human liver antigens. Proteins from an individual serum or milk were isolated by two methods, one of which included exposure to low pH and the other did not. Purified serum, mucosal IgM, IgA, and the fraction containing immunoglobulin G F(ab’)₂ fragments each inhibited the binding of a single donor or pooled IgG to human liver antigens. Our study presents findings regarding the role of the breast milk or serum antibodies in blocking the self‐reactivity of IgG antibodies. It supports the suggestion that not IVIg only, but also the pooled human IgM and IgA might possess a potent beneficial immunomodulatory activity in autoimmune patients.     

Heme-Exposed Pooled Therapeutic IgG Improves Endotoxemia Survival

Antibody repertoires of healthy humans and animals contain a fraction of antibodies able to acquire additional polyspecificity following exposure to several biologically relevant redox molecules (free heme, reactive oxygen species, ferrous ions, HOCl, etc.). The physiological role of these “hidden” polyspecific antibodies is poorly understood. Similar to inherently polyspecific antibodies, those with induced polyspecificicty may also have immunoregulatory properties. We have previously shown that a pooled human IgG preparation, modified by the exposure to ferrous ions, acquires the ability to significantly improve survival of animals with polymicrobial sepsis or aseptic systemic inflammation induced by bacterial lipopolysaccharide or zymosan administration. In the present study, we have analyzed the effects of administration of heme-exposed pooled human IgG in the same models of sepsis and aseptic systemic inflammation. The administration of a single dose of heme-exposed pooled IgG has resulted in a significant increase in the survival of mice with endotoxinemia, but not in those with polymicrobial sepsis and zymosan-induced severe generalized inflammation. Finally, we have provided evidence that the anti-inflammatory effect of heme-exposed IgG can be explained by scavenging of pro-inflammatory mediators.     

Anti-inflammatory activity of human IgA antibodies and their Fab alpha fragments: inhibition of IgG-mediated complement activation

The interaction of human IgA antibodies with the classical pathway of complement activation was investigated in a homologous human system, by means of two IgA1 and three IgG1 myeloma proteins having antibody activity against a defined antigen, staphylococcal alpha-toxin. In a solid-phase antigen-dependent C3b-binding ELISA system, the monoclonal IgG antibodies were previously shown to activate the classical complement pathway synergistically, resembling polyclonal IgG antibodies, whereas IgA antibodies were unable to activate complement by either pathway. In the present study, IgA antibodies were found to inhibit significantly the activation of complement initiated by antigen-bound polyclonal or mixed monoclonal IgG antibodies, in relation to the amount of IgA antibodies applied and bound to antigen. IgA1 myeloma proteins devoid of antigen-binding activity were without effect. Inhibition was independent of the ability of the IgA antibodies to compete against the IgG antibodies in binding to antigen, and was demonstrable with physiological concentrations of antibodies. Similar results were obtained with polyclonal serum IgA having antigen-binding activity. However, the binding of C1q to antigen-complexed IgG was inhibited only by a monoclonal IgA antibody that could compete against one of the three monoclonal IgG antibodies that bound C1q synergistically. This observation implied that at least two mechanisms were involved in the inhibition of C3b fixation. Fab alpha fragments of monoclonal IgA antibodies, obtained by cleavage with IgA1 protease from Haemophilus influenzae type b, were found to have a similar inhibitory effect on C3b fixation to the intact IgA1 antibodies. This observation supports the hypothesis that IgA1 proteases contribute to the invasive pathogenicity of certain mucosal bacteria, by cleaving secretory IgA1 antibodies to antigen-binding Fab alpha fragments, which are not only defective in mucosal defense properties, but which also protect the organisms from other immune effector systems, such as complement activation.     

IgA-associated renal diseases: Antibodies to environmental antigens in sera and deposition of immunoglobulins and antigens in glomeruli

Levels of IgA1, IgA2, IgM, and IgG antibodies specific for 10 ubiquitous food and bacterial antigens were examined by radioimmunoassay in the sera of 29 patients with IgA-associated renal diseases and 22 normal individuals. No significant differences were observed between patient and normal groups in the levels of IgA1 antibodies, and IgA2 antibodies were detected in only a few individuals in either group. Minor differences in IgM or IgG antibodies were seen against some antigens. Significant positive correlations between IgA1 and IgG and between IgA1 and IgM antibodies to casein were found in the patient group. Analysis of the molecular form of serum IgA1 antibodies revealed that although the pattern of polymeric and monomeric forms varied between individuals and between antibody specificities, there was no preponderance of one form in either patient or normal groups. Examination of kidney biopsies from 50 patients with IgA-associated renal diseases revealed that IgA1 represented the predominant subclass deposited in the glomerular mesangium; glomeruli from three patients contained both IgA1 and IgA2. Seventy-eight percent of the patients also had deposits of IgM, although IgA and IgM deposits did not always coincide. When IgG was present in glomeruli (45% of patients), the IgG1 subclass predominated. J chain was detectable in glomeruli of only four patients. C3 was detected in glomeruli of 95% of the patients, although the distribution of C3 did not always coincide with that of IgA. Indirect immunofluorescence staining with rabbit antisera to various environmental antigens showed that milk protein antigens could be deposited in association with IgA in the glomerular mesangium.     

Monoclonal IgA class-switch variants against bacterial surface antigens: molecular forms and transport into murine respiratory secretions

The present study describes a new model for passive immunization of the respiratory tract with IgA in comparison to other isotypes. Monoclonal IgA-isotype-switch variants were isolated from different IgG-producing hybridoma clones specific for surface epitopes of bacterial respiratory tract pathogens. Analysis of the molecular form of the IgA variants revealed the simultaneous production of monomeric, dimeric and higher polymeric IgA by a single-cell line with predominance of the polymeric forms. The specificities of the IgA variants were identical to the parent IgG antibodies as demonstrated by inhibition experiments. The IgA variant antibodies were separated into monomers and polymers by gel filtration. Intravenous injection of the different molecular forms of IgA and of IgG into mice were used to investigate the transport characteristics of IgA into murine upper and lower respiratory tract secretions by the physiological route in comparison to IgG. Polymeric IgA variant, monomeric IgA variant and IgG were detected in immunologically active form in both nasal secretion and bronchoalveolar fluid as evidenced by binding to their antigens in an enzyme-linked immunosorbent assay. The relative contribution of the specific exogenous monoclonal IgA and monoclonal IgG to total IgA and IgG, respectively, was determined in secretions. Comparison of the secretion to serum transport ratios clearly indicates selective transport of polymeric IgA variant into nasal secretions relative to IgG parent antibody. Molecular and functional characteristics of the IgA variants make them ideal for passive mucosal immunization experiments and identification of protective epitopes in mucosal immunity.     

Both VH and VL chains of polyreactive IgM antibody are required for polyreactivity: expression of Fab in Escherichia coli.

Monoclonal polyreactive antibodies can bind to many structurally dissimilar self and non-self antigens. Neither the precise antigen-binding site on the polyreactive antibody molecule nor the molecular basis of polyreactivity has been elucidated. The present study was initiated to see whether antibody genes encoding the Fab fragment of a human monoclonal polyreactive IgM antibody (MoAb 67) could be efficiently expressed in Escherichia coli, and whether the bacterially expressed Fab fragments possessed biological activity. cDNA encoding the variable domains of the heavy and light chains of MoAb 67 were cloned, amplified by polymerase chain reaction (PCR) and expressed in E. coli. Neither the recombinant heavy nor light chain showed antigen-binding activity. In contrast, the recombinant Fab 67 fragment showed the same antigen-binding reactivity profile as the native IgM antibody. It is concluded that the antigen-binding activity of polyreactive antibodies resides in the Fab fragment, and that both the heavy and light chains are required for activity.     

Systemic immune response after mucosal immunization in patients with IgA nephropathy

Increased IgA production has been proposed as a portion of the etiology of IgA nephropathy. Indirect human data suggest that IgG and complement may be equally important. We have immunized 17 patients with IgA nephropathy and 27 controls with tetanus toxoid. They were nasally immunized and, 2 weeks later, received an im booster immunization. This protocol has been shown to result in an increased serum IgA1 antibody response to tetanus toxin (TT). Patients had higher serum IgG antibodies to TT before and after the im immunization than did controls (pre, 42 vs 13 U; post, 155 vs 71 U;P=0.004). Patients also had a greater increase in serum IgG antibodies (118 vs 58;P=0.02). After the im TT, patients had lower levels of serum IgA1 antibody to TT (115 vs 180;P=0.005) but the change in IgA1 antibodies was not significant. These data suggest that patients with IgA nephropathy may produce inappropriately large amounts of serum IgG antibodies to antigens encountered in the upper respiratory tree. Such antigens also induce a serum IgA1 response. Such a response could result in the formation of potentially nephritogenic immune complexes containing IgG, IgA1, and C3.     

Molecular determinants of polyreactive antibody binding: HCDR3 and cyclic peptides

Human monoclonal antibody 63 (mAb63) is an IgM/lambda polyreactive antibody that binds to multiple self and non-self antigens. The molecular basis of polyreactivity is still unclear. The present study was initiated to prepare a recombinant Fab of mAb63 and use it to study the determinants involved in polyreactivity. The baculovirus system was employed to express large amounts of mAb63 Fab in Sf9 cells. Our experiments showed that infected Sf9 cells secreted a soluble 50-kD Fab heterodimer that bound to multiple self and non-self antigens. The antigen-binding activity of mAb63 Fab was inhibited by both homologous and heterologous antigens. To study in more detail the molecular determinants involved in polyreactivity, the heavy chain complementarity-determining region 3 (HCDR3), which is known to play a key role in the binding of monoreactive antibodies to antigens, was subjected to site-directed mutagenesis. A single substitution, alanine for arginine, at position 100A resulted in complete loss of antigen-binding activity. The 19 amino acids comprising the HCDR3 of mAb63 were then synthesized and a cyclic peptide prepared. The cyclic peptide showed the same antigen-binding pattern as the parental mAb63 and the recombinant mAb63 Fab. A five amino acid motif (RFLEW), present in the HCDR3 of mAb63, was found by searching the GenBank in three of 50 other human polyreactive antibodies, but in none of nearly 2500 human antibodies thought to be monoreactive. It is concluded that HCDR3 plays a major role in polyreactivity and that in some cases cyclic peptides comprising the HCDR3, by themselves, may be polyreactive.     

Anti-IgA antibodies in IgA-deficient children

IgG and IgM isotype antibodies to polyclonal human IgA, myeloma IgA₁, and myeloma IgA₂ were estimated in 38 IgA-deficient children aged between 0.9 and 15 years. All children had IgM anti-IgA antibodies. IgG antibodies against either polyclonal IgA, IgA₁, or IgA₂ were present in 63% of the IgA-deficient children. IgG anti-IgA antibodies were detected against all three antigens in 8 of 11 severely IgA-deficient children and in 7 of 27 partially IgA-deficient children, but in only 1 of 23 healthy adult controls. The proportion of children with IgG anti-IgA antibodies was significantly greater in the severely IgA-deficient group in comparison with the partially IgA-deficient group and the adult controls (chi-square test,P<0.01 andP<0.005, respectively). There was a strong correlation within each IgG subclass between antibody responses toward each of the three IgA antigens. Twenty-four children were followed over a period ranging from 0.9 to 11 years (mean, 2.3 years). Three children who were initially IgG anti-IgA antibody negative became antibody positive and three who were antibody positive became antibody negative. Five children with severe IgA deficiency remained severely IgA deficient and IgG antibodies to IgA persisted in all five at follow-up. The presence of IgG anti-IgA antibodies did not influence the normalization of serum IgA at follow-up in 14 of 19 children who were initially partially IgA deficient.     

Subclass restriction pattern of antigen-specific antibodies in donors with defective expression of IgG or IgA subclass heavy chain constant region genes

We have developed a method for the measurement of the IgG and IgA subclass distribution of antigen-specific human antibodies. The controls for the specificity of the assay include the use of a number of monoclonal human antibodies and sera from individuals with deletions of particular immunoglobulin heavy chain constant region genes. The system was used to determine the shift in immunoglobulin subclass patterns of specific antibodies against a variety of protein and polysaccharide antigens in individuals with a regulatory deficiency of a given IgG or IgA subclass. Normally, the pattern is quite distinct and antibodies against protein antigens are mainly of the IgG1 subclass, whereas antibodies against polysaccharide antigens are mainly of the IgG2 subclass. The results on serum from an IgG1 deficient donor suggested that IgG3 and IgG4 appear to compensate for a lack of IgG1, whereas isolated deficiencies of IgG3, IgG4, or IgA2 do not markedly influence the expected distribution of specific antibodies. In IgG2-deficient individuals a more complex pattern was observed where antibodies against protein antigens were retained, whereas levels of antibodies against polysaccharide antigens could vary markedly between donors, which appeared to be dependent on whether the IgG2 deficiency was an isolated defect or combined with IgG4/IgA deficiency. However, all the IgG2-deficient donors had a skewed pattern of anti-polysaccharide antibodies with a shift to IgG1 to IgG3.     

Binding Specificities of a Polyreactive and a Monoreactive Human Monoclonal IgG Rheumatoid Factor: Role of Oligosaccharides

The immunological specificites of two human rheumatoid factor-reactive IgG monoclonal antibodies derived from unstimulated rheumatoid synovial lymphocytes have been analysed. A malaria antigen-reactive IgG monoclonal antibody from an immune donor served as a control. Purified IgG monoclonal antibody from one IgG-RF hybridoma (L1), but not from the other IgG-RF hybridoma (D1) or the anti-malaria monoclonal antibody, exhibited dose-dependent binding to multiple self and non-self antigens such as ds-DNA, cytochrome-c, bovine thyroglobulin, transferrin, cellulose and lipopolysaccharide and therefore was considered polyreactive. The immunological specificity was confirmed by inhibition experiments using the same soluble antigens as inhibitors. The polyreactivity of the IgG-RF MoAb was markedly inhibited by absorption with glycoproteins such as thyroglobulin, a commonly used target for xenoreactive natural antibodies, and cytochrome-c, indicating that the monoclonal antibody is reactive with epitopes expressed on these ligands. Since some naturally occurring antibodies are carbohydrate specific, the authors tested the IgG-RF MoAb for possible carbohydrate specificity. Absorption with certain polysaccharides containing only one or two different sugar moieties did not inhibit the binding reactivities to any of the tested antigens. Polyreactivity of the monoclonal antibody, unlike most xenoreactive natural antibodies, was not caused by reactivity with (galα1-3gal) as indicated by the remaining binding reactivity after α-galactosidase treatment of the antigen. Removal of the N-linked glycosylation sites within the Fc portion of target IgG markedly reduced the antibody binding. The findings suggest that the carbohydrate content of the antigen is necessary for binding of the polyreactive IgG-RF MoAb. Reactivity to carbohydrate antigens may readily explain the so-called multispecificity of certain antibodies.     

IgA hybridomas: A method for generation in high numbers

An immunization regimen has been developed which yields a high frequency of hybridomas producing IgA isotype, antigen-specific antibody when spleen cells from immunized mice are fused with non-immunoglobulin secreting murine myeloma cells. Germfree BALB/c mice were carrier-primed with sheep erythrocytes (SRBC) by gastric intubation (GI) for 2 consecutive days followed 1 week later by GI with trinitrophenyl (TNP)-haptenated SRBC. After 7 days, spleen cells were fused with non-immunoglobulin secreting myeloma cells (X63-Ag8.653), and 2–3 weeks later, culture wells were scored for hybrid clones. Of 240 culture wells plated, 157 wells (65.4%) exhibited clones producing anti-TNP antibodies as determined by enzyme-linked immunosorbent assay. A total of 50 specific cell lines were established, of which 27 clones (54%) produced IgA isotype anti-TNP antibodies, while the anti-TNP antibodies produced by the remaining 23 clones were approximately equally distributed between the IgM and IgG isotypes. The IgA and IgM monoclonal antibodies were more effective in hemagglutinating TNP-SRBC than were IgG isotype antibodies. This study describes a method for production of a high number of antigen-specific IgA hybridomas which will allow production of IgA monoclonal antibodies to important antigens on mucosally-associated pathogens, and thus allow elucidation of functions of IgA antibody at mucosal surfaces.     

Complement-coated antibody-transfer (CCAT); serum IgA1 antibodies intercept and transport C4 and C3 fragments and preserve IgG1 deployment (PGD)

In periodontal disease, IgG1 and IgA1 antibodies produced in situ deposit on antigens in the affected tissues. Thus, there is an interest in the effect of co-deposited IgA1 antibodies on complement activation by IgG1-immune complexes. In the present study, we first analyzed the effect of IgA1-immune complexes on complement using human IgA1 antibodies to dansyl (with dansylated human serum albumin serving as the immobilized antigen). It was observed that these IgA1-immune complexes when incubated for prolonged times with 33% human serum as a source of complement received C4b and C3b deposition. As C4b and C3b deposited on the IgA1 antibodies and on the antigenic surface, the complement-coated IgA1 antibodies departed. These fluid-phase complement-coated IgA1 antibodies were transferred to antigen-coated microtiter-ELISA plates, where they became bound to the antigens. Thus, the complement-coated IgA1 antibodies retained their antigen-binding function, especially as a proportion of their covalently bound C3b progressively degraded to iC3b and C3d. Genetically engineered carbohydrate-deficient mutant human IgA1 antibodies were used to assess the role of carbohydrate in accepting the C4b and C3b depositions, and these studies indicated that the carbohydrate on the Fc-region of IgA1 played a positive role. Another interesting finding generated by this study was that when IgA1 was co-deposited with IgG1 antibodies, and serum complement was added, the IgG1 antibodies tended to remain on the antigenic surface. The co-deposited IgA1 antibodies not only controlled (reduced) the rate of the consumption of the first component of complement (C1) and of classical complement pathway activation by IgG1-immune complexes (and therein reduced the rate of complement-mediated dissolution of the IgG1-immune complexes), but also the co-deposited IgA1 antibodies simultaneously intercepted/accepted C4b and C3b, then departed, as complement began to cover the antigenic surfaces. The process in which complement-coated IgA1 antibodies transferred to non-complement-coated antigens is termed complement-coated antibody-transfer/transport (CCAT). In this way, IgA1 antibodies extended the efficiency of the complement system by insuring the specific IgA1 antibody-mediated transport of the captured biologically active complement fragments to those antigens stimulating the IgA1 antibody response but not yet neutralized (completely coated) with complement. Simultaneously by impeding the rate of C1 consumption and by intercepting C4b and C3b, IgA1 antibodies slowed C4b and C3b deposition on the antigenic surface and on the co-deposited IgG1 antibodies. Thus, in the presence of ongoing complement activation, the deposition of serum IgA1 antibodies enabled the co-deposited IgG1 antibodies to better maintain their ability to interact with antigens. We termed this latter phenomenon, preservation of IgG antibody deployment (PGD). In summary, co-deposited IgA1 antibodies maximized the efficiency of the complement system, transported their covalently bound complement fragments to specific antigens and sustained the effective deployment of IgG1 antibodies directed to those same antigens.     

Immunoglobulin A-mediated hepatobiliary transport constitutes a natural pathway for disposing of bacterial antigens.

The possibility that hepatobiliary transport of immunoglobulin A (IgA) immune complexes might eliminate bacterial antigens was investigated in mice with pneumococcal type III capsular polysaccharide and C carbohydrate and corresponding monoclonal antibodies. Although all isotypes of antibody caused uptake by the liver, only IgA, but not IgG or IgM, antibodies transported these substances into bile where they were detected in the form of immune complexes and as free antigens. Small doses (10 micrograms or less) of passively administered IgA antibody were sufficient to induce measurable transport of capsular polysaccharide into bile. Transport of C carbohydrate was significantly correlated with the level of naturally occurring IgA antibodies specific for the phosphocholine determinant, but not with IgM or IgG antibodies. These results suggest that the continual process of hepatic uptake of circulating polymeric IgA may function to eliminate bacterial macromolecular products that are not readily susceptible to other mechanisms of disposal.     

Complement-Fixing Properties of Human IgA Antibodies Alternative Pathway Complement Activation by Plastic-Bound, But Not Specific Antigen-Bound, IgA

The complement-fixing properties of human IgA antibodies bound to specific antigen, or coated directly on plastic surfaces, were examined in comparison with those of IgG antibodies. Use was made of antigen-binding (anti-staphylococcal alpha-toxin) IgA and IgG monoclonal antibodies and normal polyclonal IgA and IgG, purified greater than 99.9% by avoidance of denaturing processes. Complement-fixation ELISA was used, with a high density of biotin-conjugated staphylococcal alpha-toxin bound to avidin-coated plates for the efficient capture of antibodies, and conditions were adjusted for the assessment of classical and alternative pathways of complement activation. Although IgA coated directly on plastic surfaces activated the alternative complement pathway in a dose-dependent manner, IgA antibodies bound to antigen failed to fix complement by either classical or alternative pathways. In contrast, IgG antibodies, either bound to antigen or coated directly on plastic, activated complement mainly by the classical pathway. It was concluded that the complexation of IgA antibodies with antigen is insufficient to elicit complement activation: rather a degree of denaturation seems to play a part in the expression of alternative complement pathway-activating properties by IgA.     

The separation of human serum IgG into subclass fractions by immunoaffinity chromatography and assessment of specific antibody activity

Murine monoclonal antibodies ( McAbs ) with specificity for subclass-specific or subclass-restricted determinants on human IgG have been coupled to Sepharose to generate affinity columns. The judicial use of positive and negative chromatography and the exploitation of the special properties of individual McAb affinity columns has allowed the preparation of individual IgG subclasses from polyclonal IgG containing less than 1% contamination by any other IgG subclass. The specificity of the antibodies present in each polyclonal IgG subclass preparation has been assayed against a bacterial toxoid (tetanus), 2 bacterial cell wall antigens (E. coli and pneumococcal) and coat antigen(s) of a DNA virus (CMV). Antibodies were predominantly IgG1 to tetanus toxoid, IgG2 to pneumovax and E. coli cell walls, and IgG1, 2 and 3 to CMV coat antigens.     

Preparation of patient-related allergens for hyposensitization

An affinity chromatography method for preparation of patient-related antigens from commercially available allergen extracts has been investigated. IgG1,2,4 from a patient previously hyposensitized with dog hair and dandruff allergen was bound to protein A-sepharose. Secondly, commercial allergen extract was applied to the immunosorbent, and patient-related antigens were selectively absorbed by the specific antibodies from the patient serum. Finally, immune complexes containing antigen and IgG were eluted. Alternatively, the antigens alone were purified by retaining the IgG on the column by means of a covalent reinforcement of the protein A-IgG-binding. Purified, patient-related antigens were investigated in crossed immunoelectrophoresis and identity to IgG- and IgE-binding antigens (as determined by the CRIE-technique) was suggested. The affinity purified IgG was unaltered with regard to protein A-binding, binding of antigen and affinity for monoclonal antibody to human IgG-subclasses. Further, it was demonstrated that the antigen-binding capacity of IgG in the immune complexes was intact as evidenced by strong affinity to antigens even at low pH. The antigens eluted together with IgG were predominantly found in immune complexes with a molecular weight of greater than 300 kdalton equivalent to 2 or more molecules of IgG. The possibility of employing a similar method with IgE instead of IgG for preparation of patient-related allergens instead of antigens, is discussed.     

IgA polyspecific autoantibodies in IgA nephropathy.

The specificity of circulating and kidney-bound IgA during IgA nephropathy is still a matter of discussion. In the present study, high levels of IgA antibodies directed against a panel of self and non-self antigens were found in the serum from patients with IgA nephropathy and were eluted from four out of the seven kidney biopsies studied. After immunoadsorption of pooled selected serum samples on TNP and actin-coated columns, polyspecific IgA antibodies were eluted. This supports the hypothesis that IgA-bearing B cells clones most probably producing polyspecific antibodies are a major feature of human IgA nephropathy. These findings also suggest that it may be hazardous to draw conclusions from the finding of apparently monospecific IgA antibodies in this condition.     

Half-life of polyreactive antibodies

Monoclonal polyreactive antibodies bind to a variety of self and foreign antigens. In contrast, monoclonal monoreactive antibodies bind to a single or restricted number of known antigens. The rate at which polyreactive antibodies are removed from the circulation compared to monoreactive antibodies has not been determined. In the present experiments, human monoclonal polyreactive and monoreactive antibodies of different isotypes were injected intravenously into mice and the clearance from the circulation was determined. The halflife of polyreactive IgM, IgA, and IgG antibodies was 8.0, 8.2, and 9.8 hr, respectively, compared to 35.4, 26.6, and 280 hr for monoreactive IgM, IgA, and IgG antibodies, respectively. Examination of tissue sections from animals given intravenous antibody showed substantial deposition of polyreactive, but not monoreactive, antibodies in several organs, the liver being the principal site of deposition. It is concluded that polyreactive antibodies are cleared from the circulation substantially faster than monoreactive antibodies.     

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.