Tritrichomonas foetus extracellular cysteine proteinase cleavage of bovine IgG2 allotypes.

Bovine trichomoniasis is a sexually transmitted disease associated with reproductive failure. Systemic immunization results in protective IgG antibodies in uterine and vaginal secretions. Because bovine IgG2 is a better opsonin than IgG1, it is potentially important in defense. Yet, Tritrichomonas foetus extracellular cysteine proteinase (TFECP) cleaves bovine IgG2, evading protective IgG2 responses. Variations in resistance of the 2 IgG2 allotypes to digestion may explain inherited differences in protection. To address this hypothesis, TFECP was incubated with both IgG2 allotypes at different concentrations and times. The digestion products were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, stained, and quantitated by image analysis. IgG2a was digested faster by TFECP than IgG2b. Differences in the sizes and numbers of digestion products were observed, but the presence of bands the size of Fc and Fd fragments indicated that both allotypes were cleaved at the hinge. Cysteine in the digestion mixture reduced the antibody molecules and increased the rate of digestion, but IgG2a was still more susceptible to cleavage than IgG2b in the absence of cysteine. Thus, not only reduced H chains can be cleaved by cysteine proteinase secreted by T. foetus but also intact functional antibody molecules. Because parasites may evade protective antibody responses by cleaving IgG2, animals with the more resistant IgG2b allotype may be better protected by immunization than animals with the more readily digested IgG2a allotype.

Seroepidemiological study of hepatitis E virus in different population groups.

In order to determine the seroprevalence of hepatitis E virus, 1,993 sera (453 from healthy pregnant women, 491 from Moroccan subjects, 492 from blood donors, 321 from children, and 236 from intravenous drug users) were studied. IgG was measured by enzyme immunoassay (EIA), and positive results were confirmed by Western blot. The EIA detected antibodies in 3.96% of the subjects (5.6% of the Moroccans and drug users and 1.8% of the children). Fifty-four percent of these results were confirmed by Western blot, 11.4% were found to be negative, and 34.2% indeterminate. The overall prevalence after confirmation by Western blot decreased to 2.15%. When studying the Western blot pattern of the positive samples, 95% showed antibodies to SG-3, 65% to 8-5, and only 9.3% to CKS fusion protein. In the indeterminate Western blots, the results for these proteins were 96.3%, 62.9%, and 37%, respectively. When the epidemiological data were analysed, no statistically significant differences between women and men or between different age groups were found.

Heterogeneity of bovine IgG2. VI. Comparative specificity of monoclonal and polyclonal capture antibodies for IgG2a (A1) and IgG2a (A2).

The relative specificity of 26 randomly selected polyclonal and monoclonal anti-bovine IgG2 reagents for the A1 and A2 allotypic variants of IgG2a was evaluated in a direct RIA using the reagents as solid-phase capture antibodies (CAbs). More than 70% of these reagents were significantly allotype-biased and > 80% of those were positively biased to IgG2a (A1). Compared as the ratio of the ng of IgG2a (A1) bound versus ng IgG2a (A2) bound per 50 ng added (Krel), bias for IgG2a (A1) of six of these reagents was greater than two-fold. Compared in terms of their solid-phase equilibrium constants (Keq), differences as great as two-logs among these reagents were observed. Steward-Petty plots suggested that differences in Krel of a select panel of reagents was usually due to differences in Keq, but for two reagents with large differences in Krel, the existence of one population of CAbs recognizing an allotope and another recognizing common IgG2a determinants, was indicated. Eight of ten guinea pigs immunized with IgG2a (A1) responded with highly significant specificity bias for A1 whereas only two of 11 rabbits and two of ten guinea pigs immunized with IgG2a (A2) responded weakly with preference for IgG2a (A2). These results concur with the concept of the immunodominant nature of the A1 allotope, but also suggest that immunization with IgG2a (A2) might be a practical means of avoiding allotype bias in IgG2a reagents. The data indicate that the majority of randomly selected anti-bovine IgG2 reagents are allotype biased to the extent that when used as serological reagents to measure total IgG2 or bovine IgG2 antibody responses, the allotype of the animal tested rather than its total IgG2a concentration or IgG2 antibody titer, can determine the outcome of the serological test.

The heterogeneity of bovine IgG2--V. Differences in the primary structure of bovine IgG2 allotypes.

The partial amino acid sequences of the gamma chains of the bovine IgG2a(A1) and IgG2a(A2) allotypes were determined. Sequence differences were found in the CH1 domain, the hinge region, and the CH3 domain. The hinge regions displayed only 71.4% similarity and all of the differences were of a radical nature. The A2 hinge has isoleucine instead of serine at 229, histidine for asparagine at 235, proline for histidine at 238, and cysteine instead of proline in position 234; the latter has the potential for forming an additional interheavy chain disulphide bridge. The occurrence of such a bridge could explain the presence of a pepsin fragment consisting of the hinge region and the Fc. A corresponding fragment is not obtained with the A1 allotype. Both allotypes have a shortened hinge region and a truncated CH2 domain. This feature is characteristic of all reported sequences of IgG2 proteins but not IgG1 in cattle and the goat. This structural feature may be important in subclass-specific recognition by Fc gamma receptors in ruminants. A surprising discovery was the occurrence of five substitutions in the CH3 domain of the IgG2a(A2) in comparison with the A1, which are shared with the CH3 of IgG1. These permit the occurrence of isoallotypic determinants and can explain the difficulty encountered in preparing A2-specific antisera during which adsorption with IgG1 is a routine procedure. The primary sequence data we report confirm the presence of major structural differences between the A allotypes of cattle that was suggested by previous work. The sequence of the A1 allotype most closely agrees with the two IgG2 sequences deduced from their nucleotide sequences whereas the sequence differences in the hinge and C-terminal CH3 make IgG2a(A2) unique. The structural differences between allotypes could have major consequences for such biological activities as phagocytosis, transepithelial transport, lymphocyte and complement activation.

Abbott HCV EIA 2nd generation: a new screening assay.

An enzyme immunoassay for the detection of antibodies against structural and nonstructural domains of the hepatitis C virus (EIA-2) was compared to a first generation test (EIA-1) that can only detect antibodies against one nonstructural antigen (c100). EIA-2 revealed elevated detection rates as assessed in high risk and patient populations as well as lower repeat reactive rates in volunteer blood donors. Research test systems proved an increase in apparent specificity.

Human rheumatoid factor cross-idiotypes. III. Bla monoclonal rheumatoid factor, prototype of the BLA cross-idiotype group, has distinct kappa chains related to the V kappa III subgroup and VH4 heavy chains.

The BLA cross-idiotype (XId) is present on a unique subset of rheumatoid factors (RF) that cross-react with DNA-histone. In this study, prototype Bla monoclonal RF was shown from serologic investigations and N-terminal amino acid sequence analysis to have distinct kappa chains related to the V kappa III subgroup and VH4 heavy chains. The amino terminus of the heavy chain was cyclized, rendering the protein resistant to Edman degradation and providing a possible investigator bias to the published Ig sequence data to date. This appears to be the first definitive report of a serum IgM that expresses the VH4 gene. RF with DNA cross-reactivity have been reported to be produced by human and mouse cloned cells that have the VH4 or homologous mouse Vh36-60 gene.

Multiple mutations in the variable region of the kappa light chains of three monoclonal human IgM with anti-myelin-associated glycoprotein activity.

Human monoclonal IgM having an antibody activity directed to myelin-associated glycoprotein have distinctive features. Amino-terminal sequence of light and heavy chains from 6 IgM kappa that we have previously studied indicated that heavy chains belong to the VHIII subgroup, whereas light chains belong to 3 different subgroups of variability (V kappa I 2, V kappa II 1, and V kappa IV 3). We report here the complete sequence of the variable domain of 3 L chains: 2 V kappa IV and 1 V kappa II subgroups. Strikingly an unusually high degree of mutations clustered in the complementarity-determining regions (CDR) 1 and CDR 3 was found and the variable regions were joined to three different JK segments. Amino acid substitutions did not yield similar sequence in the CDRs suggesting that the kappa chains had no predominant role in the unique binding activity of these IgM or alternatively they are directed against different epitopes. Data are consistent with the previously reported lack of easily demonstrated public idiotopes common to anti-myelin-associated glycoprotein IgM. The pathogenesis of these IgM autoantibodies is most likely different from that of previously studied monoclonal rheumatoid factors or cold agglutinins where a genetic restriction of L or H chains or both has been observed.

The isotypic, allotypic and idiotypic heterogeneity of bovine IgG2.

The antigenic heterogeneity of bovine IgG2 observed by single radial diffusion, when different cattle sera are tested with a panel of IgG2-specific reagents, was examined by a combination of biochemical and serological assays. Using an autologous anti-A1 allotypic reagent, the major antigenic heterogeneity detected by swine, rabbit and goat anti-IgG2 reagents was due to their propensity to recognize the AI allotope. All heterologous reagents and most monoclonals so far test are biased in their specificity toward this determinant. A second type of serological heterogeneity, recognized by only certain heterologous reagents, was their specificity for what we have called the IgG2b isotype of IgG2. This isotype is found in all cattle, has a restricted ion-exchange elution behaviour and does not bear a-locus allotypic determinants; molecules bearing the latter are now designated IgG2a. IgG2a elutes from anion-exchange columns in subpopulations over a wide range of ionic strength including fractions which contain IgG2b and IgG1 as well. Using IgG2a from an AI homozygous steer, these subpopulations were shown to result from idiotypic variation which appears to primarily reside in their VH-regions.

The heterogeneity of bovine IgG2--III. The ion-exchange heterogeneity of IgG2a is the result of VH-region variation.

Bovine IgG2a from an animal homozygous at the A-locus could be fractionated on DEAE columns into subpopulations; studies using analytical size columns showed that the number of subpopulations was dependent on the ionic strength of equilibration buffer. Subpopulations of IgG2a could be purified by this method which did not differ in their carbohydrate content but had mean Ips consistent with their elution behavior. The spectrotype of their H-chains but not their L-chains, paralleled the spectrotype of the corresponding intact IgG2a subpopulations. When papain digests of each subpopulation were fractionated by chromatofocusing, their Fc fragments eluted homogeneously with a pI ca 6.0 while their Fabs eluted heterogeneously with pIs ranging from 8 to 5. The distribution of carbohydrate among such fractions corresponded to the distribution of the Fcs. The behavior of the IgG2a Fabs on chromatofocusing paralleled the elution behavior of the parent IgG2a subpopulations from DEAE-Sephadex and the isoelectric behavior of their H-chains. The antibody activity of Fab fragments, separated by chromatofocusing, are consistent with the concept of idiotypic variation. The differential antibody activity of each intact IgG2a subpopulation for seven different antigens suggests that the ion-exchange behavior of IgG2a, in which the influence of allotypes and sub-isotypes has been excluded and for which charge-related L-chain heterogeneity is minimal, must reside in the VH-regions of the different IgG2a subpopulations. Investigators are cautioned against assigning ruminant subclass designations on the basis of subpopulations isolated by ion-exchange chromatography.

The heterogeneity of bovine IgG2--IV. Structural differences between IgG2a molecules of the A1 and A2 allotypes.

The major product of digestion of bovine IgG2a(A1) with immobilized pepsin is F(ab')2 while similar treatment of IgG2a(A2) yields Fab, Fc and other products. It is postulated that structural differences in the hinge region, including the absence of the most N-terminal disulfide bridge in IgG2a(A2) and a displacement of the primary pepsin cleavage site toward the Fd, can explain this effect. The immunodominant A1 allotope(s) appears to be located in the CH3 domain of IgG2a(A1) while the A2 allotopes are located elsewhere and are apparently affected by digestion. The allotypic bias of rabbit anti-IgG2a is also present in anti-IgG2a raised in goats. However, the A2 allotypic determinants of bovine IgG2a are recognized by goat precipitins although precipitins of this specificity are not detectable in rabbits immunized with IgG2a(A2). Rabbit anti-A2 antibodies are detectable using the ELISA in rabbits immunized with IgG2a(A2).

The heterogeneity of bovine IgG2. II. The identification of IgG2b.

Goat and rabbit polyclonal reagents can be raised which recognize a new isotype of bovine antibodies. The polyclonal goat reagent was raised against a preparation enriched in the major IgG2 isotype (IgG2a) which contained the new isotype as a contaminant. The polyclonal rabbit reagent was prepared against a trypsin-derived Fc fraction of bovine IgG1 which contained the Fc of the new isotype as a contaminant. This new isotype is present in the sera of the cattle of all breeds tested regardless of their IgG2a allotype and is antigenically distinct from IgG2a, IgG1, IgA, IgM and IgE. The new isotype coelutes from DEAE anion exchangers with IgG1 and the more acidic populations of IgG2a. The isotype is tentatively designated IgG2b. The distribution of IgG2b antibody activity to E. coli K99 and phosphorylcholine among 15 cattle of different A allotypes is not correlated with the IgG2a or IgG1 antibody activity in these animals.

The immunochemistry of sandwich ELISAs--I. The binding characteristics of immunoglobulins to monoclonal and polyclonal capture antibodies adsorbed on plastic and their detection by symmetrical and asymmetrical antibody-enzyme conjugates.

Radiolabelled bovine IgG1, IgG2, SIgA and IgM and heavy-chain specific polyclonal and monoclonal antibodies to these isotypes were employed as models to investigate immunochemical aspects of sandwich enzyme immunoassays (ELISAs). The titration plots obtained by measuring enzyme activity paralleled those obtained when the binding of radiolabelled immunoglobulins to solid-phase capture antibodies was quantitated. As predicted from the Mass Law, the percentage of labelled immunoglobulin which was bound remained constant over the range in which the sandwich ELISA titration was linear on a log-log plot. Also as predicted from the Mass Law, increasing the solid-phase concn of polyclonal antibodies by affinity purification increased the linear region of the log-log ELISA plot and the corresponding region over which a constant percentage of immunoglobulin binding was observed. When used as capture antibodies adsorbed on plastic at equal concns, the best monoclonal antibodies were 1/8- less than 1/16 as effective as their polyclonal counterparts in binding iodinated bovine immunoglobulins; these differences can be directly interpreted to result from an 8 and greater than 16-fold higher functional, relative affinity of the polyclonal reagents. Steric hindrance was shown to occur when symmetrical sandwich ELISAs, i.e. capture and detection antibody are both heavy-chain specific, are used to measure monomeric but not IgM immunoglobulins. The use of an asymmetrical configuration, i.e. anti-Fab antibody-enzyme conjugates, avoids this problem. Symmetrical conjugates based on the avidin-biotin system, horseradish peroxidase or alkaline phosphatase, were less effective than their asymmetrical (anti-Fab) counterparts. Evidence that the lower activity of symmetrical conjugates was due to steric hindrance was illustrated using horseradish peroxidase-antibody conjugates of different sizes. Sandwich assays using affinity-purified, polyclonal solid-phase antibodies and an asymmetrical conjugate were judged to be immunochemically and economically optimal. Using an asymmetrical configuration, the non-linear nature of sandwich ELISA titration plots is the predictable result of changing antibody to antigen ratios in an antibody-limiting system, and not the result of steric hindrance of the detection system.

Heterogeneity of bovine IgG2--I. The A1 allotypic determinant is the major antigenic determinant recognized on bovine IgG2a by polyclonal rabbit anti-IgG2a.

Rabbit antisera specific for the IgG2a subclass of bovine immunoglobulins also contain antibodies which recognize the A1 allotype of this immunoglobulin subclass. In many sera anti-Al constitutes the major specificity of these rabbit antisera and all of the 10 rabbits immunized with Al(+) IgG2a produced some antibodies which recognized the allotype. The determinant is the same as recognized by a bovine alloantiserum to Al. The Al allotype is shared by the intact and the Fc portion of IgG2a, but rabbits immunized with the Fc fragment also recognize an Al-related determinant which is not exposed on intact Al(+) IgG2a. None of the four rabbits immunized with Al(-) IgG2a produced precipitating antibodies which recognized the gene product of the Al(-), i.e. A2 allele, suggesting that only Al is an immunodominant antigenic determinant for rabbits. Data reported here help to explain the antigenic heterogeneity seen among the IgG2 populations from different cattle when they are tested by immunoprecipitation using rabbit anti-IgG2 reagents.