Studies of idiotypic antibodies. Production and characterization of autoantiidiotypic antisera.

Rabbits were immunized with a hapten-protein conjugate and sera were collected for 189 days. The antihapten antibodies were purified by affinity chromatography, then the same animal that synthesized the antibody was reinjected with polymerized F(ab')(2) fragments of antihapten antibodies. Sera were collected after autoimmunization and tested by an indirect radioimmunoassay technique for reaction with [(125)I]F(ab')(2) fragments of the original antihapten antibody. Results showed that each individual responded to its own F(ab')(2) and the antisera were specific for antihapten antibodies of that individual. Quantitative allotype assays established the immunoglobulin nature of the labeled test antigen. Inhibition assays showed that the reaction was specifically inhibitable with hapten. The relationship of this system with other idiotypic systems and the possible autoimmune implications of autoantiidiotypic antibodies are discussed.

Regulation of natural antiallotype antibody responses by idiotype network-induced auto-antiidiotypic antibodies.

This study was designed to determine whether natural immune responses could elicit immunoregulatory auto-antiidiotypic antibodies. Female rabbits heterozygous at the a and b Ig loci were bred to homozygous males. Offspring of one such breeding were studied for natural production of antibodies specific for the noninherited allotypes and for the production of immunoregulatory auto-antiidiotypic antibodies. All offspring mounted natural antiallotype responses. The anti-a1 responses cycled as a function of time whereas the anti-b5 responses were invariant. Anti-a1 responses from two offspring were shown to change specificity for different a1 subsets as they cycled. Anti-a1 was purified from the first cycle and was used to assay for auto-antiidiotypic responses. Auto-antiidiotypic antibodies were detected and were found to cycle in an inverse way with the anti-a1 cycles. The idiotopes detected using the natural auto-antiidiotypic antisera were strongly cross-reactive. Subsequent deliberate immunization showed that antibodies specific for all a1 subsets could be elicited after auto-antiidiotypic regulation had functioned. The data support the interpretation that idiotype network interactions indeed function in naturally occurring immunologic situations and are not merely laboratory curiosities or artifacts.

Naturally induced auto-anit-idiotypic antibodies. Induction by identical idiotopes in some members of an outbred rabbit family.

Naturally induced auto-anti-idiotypic (AAI) antibody responses specific for antimicrococcal antibody idiotypes were detected in 42% of the rabbits in a family immunized with Micrococcus lysodeikticus. The natural AAI response of each rabbit recognized only a portion (11-41%) of that individual's total antimicrococcal antibody population. Cross-reactions of idiotypes were observed within the group of rabbits exhibiting natural AAI responses. Examination of the basis for the cross-reactions showed that the natural AAI antisera recognized identical idiotopes on the antimicrococcal F(ab')2 fragments from each rabbit that made an AAI response. The cross-reactive idiotopes were shown to be of paternal origin and were found in the antimicrococcal antibodies of each offspring. The data strongly support the idiotypic network concept that naturally induced AAI responses may occur routinely in outbred normal individuals as a result of antigenic stimulation. Further, the data suggest that the induction of regulatory AAI antibody responses in outbred rabbits may depend on the expression of particular germ line idiotopes.

Autoregulation of an antibody response via network-induced auto-anti-idiotype.

The antibody response of a single outbred rabbit was studied throughout three rounds of injections with Micrococcus lysodeikticus vaccine over a 31-mo period. The first-round response was characterized by a vigorous anti- micrococcus response and a strong anti-IgG rheumatoid factor response. The second-round response consisted of a triad of interacting molecules: anti- micrococcal antibodies, autoanti-idiotypic antibodies specific for distinct clonotypes of the first-round anti-micrococcal antibodies, and Fc-specific anti-IgG rheumatoid factor. The interacting triple complex was detected because of the formation of an immune complex that became insoluble upon dilution of the serum. Complex formation was inhibited in the presence of saccharide compounds known to be major immunodominant determinants of the micrococcal cell-wall carbohydrate polymer. The same saccharides did not affect the reaction of rheumatoid factor with IgG. Direct-binding radioimmunoassays ruled out mediation of the dilution-precipitation reaction by soluble micrococcal antigens. Specific absorption of rheumatoid factor inhibited the dilution-precipitation reaction. Auto-anti-idiotypic antibodies were specifically purified from second-round sera, directly confirming the presence of these antibodies. Suppressive effects of auto-anti-idiotypic antibodies on distinct antibody clonotypes were shown by gel isoelectric focusing of first-, second-, and third-round sera. Clonotypes expressed in the first round of immunizations were reduced in quantity or absent when auto-anti-idiotypic antibodies were detectable. Greatly enhanced levels or initial synthesis of new clonotypes of anti-micrococcal antibodies were detected during the period of auto-anti-idiotype synthesis. The third-round sera, devoid of detectable auto-anti-idiotype, contained clonotypes characteristic of both first- and second-round antisera. Thus, auto-anti- idiotypic-mediated suppression appeared to be reversible. The data are interpreted as lending strong support for concepts of autoregulation of immune processes in normal outbred animals via an idiotypic network.

Distinct functions of monoclonal IgG antibody depend on antigen-site specificities.

Intraveneous hyperimmunization of selectivity bred rabbits with streptococcal group A-variant vaccines elicits antibody responses of restricted heterogeneity at high antibody levels. All antisera contain two functionally distinct antibody populations, which can be isolated in single-band purity upon analytical isoelectric focusing. Typical examples of these two kinds of single-band antibodies were investigated in great detail for several parameters by a variety of methods. 85--99% of the streptococcal group A-variant polysaccharide (Av-CHO)-specific antibody in the antisera does not precipitate the isolated 5,000 daltons poly-L-rhamnose antigen, neither agglutinates nor lyses in the presence of complement Av-CHO-coated sheep erythrocytes (SRBC), binds the radio-labeled Av-CHO with an association constant in the ragne of 10(5)--10(6) M-1, and is of terminal specificity (nonreducing end) for the linear Av-CHO. In contrast, the minor fraction of Av-CHO-specific antibody (1--15%) does precipitate the linear Av-CHO, both agglutinates and lyses Av-CHO-coated SRBC in the presence of complement, has an affinity range of 10(8)--10(9) M-1, and is of internal specificity for the Av-CHO. The antigenic determinants of the Av-CHO for the antibodies are nonoverlapping, only one Fab of the low affinity antibody can be bound whereas four Fab of the high affinity antibody are accommodated. Hence, the determinant specificity explains the functional differences observed, for there is no indication of subclass differences. A mechanistic model of the A-variant carbohydrate presentation on the vaccine appears to account best for the unbalanced levels of low and high affinity antibody.

Quantitation of cytoplasmic tubulin by radioimmunoassay.

A radioimmunoassay has been developed for the quantitation of crytoplasmic tubulin. It measures tubulin between 20 and 1500 nanograms and does so independently of decay in colchicine-binding activity. In addition, the state of tubulin as subunit or polymer does not alter the measurement.

Induction and immunochemical properties of a novel anti-antibody.

This paper describes results which characterize an induced antibody in normal outbred rabbits which we have, for convenience, called parareactant (PR). PR resulting from autoimmunization of rabbits with either keyhole limpet hemocyanin-anti-tetanus toxoid F(ab')2 or with tetanus toxoid-anti-tetanus toxoid F(ab')2 complexes was studied. PR activity was directed solely to autologous, homologous or heterologous F(ab')2 fragments regardless of their specificity. PR failed to react with intact antibodies or with antigen-antibody complexes consisting of homologous antibody bound to specific antigen. Radioimmunoassay and ELISA inhibition assays showed that reactivity between PR and autologous anti-tetanus toxoid F(ab')2 or homologous anti-bovine serum albumin F(ab')2 fragments was specifically inhibited with antigen. Anti-allotypic antibodies specific for a2 and b6 markers strongly inhibited binding of 125I-anti-micrococcal carbohydrate F(ab')2 (a2, b6) with PR (a3, b4, b5). PR specificity thus appears to be directed against non-idiotypic determinants present in Fv regions. Affinity immunoblotting was used to analyze clonality of PR in the sera collected from individual rabbits during the course of an active immune response. PR-positive sera displayed clonally restricted spectrotype patterns. PR molecules were predominantly IgG with isoelectric points of 5.9-6.8. These results strongly suggest that these PR molecules are coded by a small number of V region genes.

Destruction of antibody idiotopes with ultra-low concentrations of reducing agents.

The nature of the idiotopes present on F(ab')2 fragments prepared from rabbit anti-micrococcal carbohydrate antibodies and the loss of idiotypic reactivity of these F(ab')2 fragments upon iodination were examined. Rabbit anti-micrococcal idiotopes were shown to be exquisitely sensitive to treatment with very low concentrations of sodium metabisulfite or 2-mercaptoethanol. The treatment destroyed anti-micrococcal idiotopes, as shown by the loss of idiotopes on F(ab')2 fragments after reduction; the allotype epitopes and the antigen binding capacity of the F(ab')2 fragments were unaffected. The destruction of the idiotopes by very low concentrations of reducing agents indicated that an extremely labile disulfide bond is involved in the structure of the idiotope or in the maintenance of the conformation of the anti-micrococcal idiotopes. Identical reduction-sensitive anti-micrococcal idiotopes have been demonstrated in a number of related outbred rabbits, and in each case they induced a natural auto-anti-idiotype (AAI) antibody response. Recognition of the existence of these reduction-sensitive idiotopes and their properties could provide a basis for further study of these idiotopes and may lead to a better understanding of the idiotope network.

DNA hydrolysis by monoclonal anti-ssDNA autoantibody BV 04-01: origins of catalytic activity.

Monoclonal anti-DNA autoantibody BV 04-01 catalyzed hydrolysis of DNA in the presence of Mg2+ ions. DNA hydrolyzing activity was associated with BV 04-01 IgG, Fab, and SCA 04-01 proteins. Pronounced cleavage specificity for both ss and dsDNA was observed with efficient hydrolysis of the C-rich region of the oligonucleotide A7C7ATATAGCGCGT7 as well as preference for cleavage within CG-rich regions of double-stranded DNA. Data on specificity of ssDNA hydrolysis and kinetic data obtained from wild-type SCA 04-01 and two SCA 04-01 mutants (L32Phe and L27dHis) were used to model the catalytically active antibody site utilizing the previously resolved X-ray structure of (dT)3 liganded Fab 04-01. The resulting model suggested that BV 04-01 activates the target phosphodiester bond by induction of conformational strain. In addition, the antibody-DNA complex contained a potential Mg2+ ion coordination site composed of the L32Tyr and L27dHis amino acid side chains and a DNA 3'-phosphodiester group. Induction of strain and metal coordination could be constituents of a mechanism by which this antibody catalyzed DNA hydrolysis. Sequence data for BV 04-01 VH and VL genes suggested that the proposed catalytic antibody active site was germ-line encoded. This observation suggests the hypothesis that catalytic activity might represent an important but unspecified function of some antibody molecules.

Affinity immunoblotting. High resolution isoelectric focusing analysis of antibody clonotype distribution.

A sensitive and specific method has been developed for analyzing specific antibody clonotype changes during an immune response or comparing multiple sera for antibody clonotype similarities. Polyclonal serum antibodies were separated by flatbed acrylamide isoelectric focusing (IEF) and analyzed by an affinity immunoblotting method using antigen-coated nitrocellulose membranes. Antibodies present on the surface of the acrylamide gels after IEF bound the antigen on the nitrocellulose when the coated nitrocellulose was laid over the gels. Non-specific protein binding was inhibited with Tween 20. Bound IgG antibody clonotypes were detected using peroxidase-conjugated anti-IgG. This method has been used for the analysis of Ig clonotypes specific for five protein antigens and two carbohydrate antigens. Optimal antigen concentration for coating the nitrocellulose membranes ranged from 10 to 100 micrograms/ml. The reactions could be inhibited by saturating the nitrocellulose with soluble protein antigen or free hapten prior to immunoblotting. Antibodies of alternative allotypic forms were detected by probing the immunoblot with biotinylated anti-allotype antibodies instead of anti-IgG. The simultaneous analysis of alternative allelic forms of antibodies of defined specificity is not possible with methods which use labelled antigen for clonotype detection.

A method for preparing IgG F(ab')2 fragments using small amounts of serum.

An alternative method devised to isolate functionally active antibody F(ab')2 fragments required fewer manipulations and used less serum than do methods generally used. The method involved pepsin digestion of the whole globulin fraction precipitated from as little as 3 ml of serum. Chromatographic separation of the digest on Sephadex G-150 yielded two distinct peaks: Peak I consisted of lipoprotein and showed no immunoglobulin determinants; Peak II, as established by immunodiffusion analysis, contained immunoglobulin F(ab')2 fragments. Radioimmunoassays performed on Peak II protein to determine the presence of allotypic markers revealed less than 10% contamination by non-immunoglobulin protein; coprecipitation tests, used to characterize Peak II further, also showed less than 10% contamination by non-immunoglobulin protein. Recovery of total serum IgG F(ab')2 was 90% or greater using this technique, compared with a potential 20-25% recovery using standard isolation procedures.

Comparison of the immune response to Ars-BGG in germfree or conventional piglets.

Neonatal germfree (GF) colostrum-deprived and conventional (CV) colostrum-fed piglets were immunized IP with p-azo-phenyl-arsonate-bovine gamma globulin (Ars-BGG) in Freund's adjuvant to study the development of the immune response in the absence or presence of maternal antibodies and environmental antigens. Overall, the immune response varied greatly within each group but did not differ in GF from CV piglets statistically. Affinity immunoblot analysis suggested that anti-Ars antibody was more restricted in GF than CV piglets and clonotype shifts occurred more in GF than CV piglets after each antigenic stimulation. In contrast, the clonotype pattern of the anti-BGG antibody was similarly heterogeneous in the two groups. Based on the affinity immunoblot data the antibodies generated to the Ars-haptenic group in CV piglets are more heterogeneous than GF piglets and suggest that clonotype generation is influenced by maternal antibodies and environmental antigens.

Adoptive immunity transferred by naive donor cells immunized in vitro.

Speedy restoration of immune responsiveness in bone marrow recipients has been the objective of studies in which the donor was immunized so that specific immunologic memory could be transferred adoptively and selectively. Using unrelated rabbits, matched for major histocompatibility antigens but mismatched for their immunoglobulin allotypes, it could be shown that recipients of lymphoid cells from naive donors became B cell chimeras but did not use donor-derived B cells for their antibody responses to test antigens. In contrast, cells from donors primed for such antigens dominated antibody production in recipients in response to specific challenge. Clonal restriction in such adoptive responses was demonstrated. We now show that the induction of effective memory in cells from naive donors can be achieved in vitro during the preparation of donor cells for transfer to the recipient. Early challenge of the recipient enhances expression of the transferred immune response quantitatively and also results in the establishment or preservation of a larger diversity of clones from the donor.

Microheterogeneity of S-glycoprotein of mouse hepatitis virus temperature-sensitive mutants.

Mouse hepatitis virus (MHV) strain JHM (MHV-JHM) is a neurotropic coronavirus that causes acute fatal encephalomyelitis in 75-99% of infected mice. The surviving animals may subsequently develop demyelinating disease. We compared the S peplomer protein of the wild type (wt) and five temperature-sensitive (ts) mutants of MHV-JHM. In contrast with the wt, none of these five cause fatal disease (mortality less than 10%). Three of these ts mutants did not induce any demyelinating disease, a fourth caused demyelinating disease in 5% of the animals and a fifth, designated ts8, exhibited strong demyelinating properties and caused demyelination in 99% of the animals. SDS-PAGE analysis revealed no differences in the molecular weight of S peplomer protein of wt or ts MHV-JHM mutants. However, isoelectric focusing of the S protein of these five ts mutants and the wt MHV-JHM, followed by transfer to nitrocellulose sheets and immunoblotting with anti-S specific antibody revealed significant differences in the microheterogeneity of the S protein.

Electrodissociation of high molecular weight complexes of interferon alpha during recycling isoelectric focusing.

Natural human interferon alpha has been separated by selective ultrafiltration into low molecular weight components and the molecules exceeding 100K daltons. Interferon associated with a higher molecular weight fraction showed partial pH sensitivity and resisted dissociation after treatment with urea, mercaptoethanol, sodium chloride or significant changes in pH. However, interferon activity was released from high molecular weight components during recycling isoelectric focusing. Electrodissociation was carried out in 1% ampholytes for 574 watt-hours. The interferon activity was concentrated in a pH range of 6-6.5, whereas, the majority of proteins were generally found in a more acidic position. The dissociated interferon was neutralized by polyclonal antibody to human interferon alpha (IFN alpha) and showed no presence of pH labile form. A pH sensitivity of high molecular weight interferon (HMW-IFN) may reflect an aggregation phenomenon rather than intrinsic structural differences.

Antibody synthesis induced by endogenous internal images.

In this study, immunization with a vaccine consisting of multiple F(ab')2 fragments of affinity-purified antitetanus toxoid antibodies covalently bound to a carrier protein successfully induced antitetanus toxoid antibodies. Further studies showed that this vaccine preparation contained no biologically detectable tetanus antigen. The induced antitetanus antibody (Ab1') titer was higher than the titer of antibodies binding control antigens. The immunizing F(ab')2 preparation did not elicit a secondary antitetanus response from mice primed with tetanus toxoid and, hence, appeared free of tetanus epitopes. The specificity of Ab1' was established by absorption and inhibition with antigen. Immunization with antitetanus F(ab')2 (Ab1') fragments appears to have elicited naturally occurring autologous antitetanus toxoid antibody (Ab1') through an idiotypic pathway. As predicted by network theory, anti-idiotype (Ab2) and antitetanus (Ab1') cycled reciprocally. Clonotypic characterization of Ab1' using isoelectric focusing and affinity immunoblotting showed increases in Ab1' titer to be the result of increased synthesis by limited subsets of antitetanus toxoid B-cell clones and not increased synthesis by multiple clones, as is characteristic of antigen-driven Ab1 responses. Many Ab1 and Ab1' clonotypes had identical pIs, suggesting that they either share V region genes or are the product of the same B-cell clones. These findings indicate that immunization with polyclonal multivalent Ab1 preparations can trigger active synthesis of antibodies with the same specificity. The results provide further evidence for naturally occurring idiotypic cascades that could be exploited for studies of catalytic antibodies.

DNA hydrolysis by monoclonal autoantibody BV 04-01.

Monoclonal anti-DNA autoantibody BV 04-01 catalyzed hydrolysis of DNA in the presence of Mg2+. Catalysis was associated with BV 04-01 IgG, Fab, and single-chain-antibody (SCA) proteins. Cleavage of both ss and dsDNA was observed with efficient hydrolysis of the C-rich region of A7C7ATATAGCGCGT2, as well as a preference for cleaving within CG-rich regions of dsDNA. Data on specificity of ssDNA hydrolysis and kinetic data obtained from wild-type SCA, and two SCA mutants were used to model the catalytically active antibody site using the previously resolved X-ray structure of BV 04-01. The resulting model suggested that the target phosphodiester bond is activated by induction of conformational strain. In addition, the antibody-DNA complex contained a Mg2+ coordination site composed of the L32Tyr and L27dHis side chains and a DNA 3'-phosphodiester group. Induction of strain along with the metal coordination could be part of the mechanism by which this antibody catalyzes DNA hydrolysis. Sequence data for BV 04-01 V(H) and V(L) genes suggested that the proposed catalytic-antibody active site was germline-encoded. This observation suggests that catalytic activity might represent an important-rarely examined-function for some antibody molecules.

Quality control of murine monoclonal antibodies using isoelectric focusing affinity immunoblot analysis.

Quality control of murine hybridoma secretory products was performed using two variations of the isoelectric focusing affinity immunoblot analysis. The first approach employed antigen-coated nitrocellulose placed on top of an acrylamide gel containing isoelectrically focused ascites to bind antigen specific monoclonal antibody (MoAb). Murine antibody bound to the insolubilized antigen was then detected with enzyme-conjugated anti-mouse IgG. In a second variation, focused ascites proteins were passively blotted onto nitro-cellulose and specific monoclonal antibody was detected with enzyme-conjugated antigen. Several batches of ascites containing anti-human IgG antibodies that were produced by 6 hybridomas over a 1-3 year period were assessed by IEF-affinity immunoblot analysis. Both immunoblot approaches permitted effective monitoring of immunoreactive antibody for pI microheterogeneity. IEF-affinity immunoblot patterns of unprocessed ascites displayed specific MoAb banding patterns with narrow pI ranges (less than or equal to 0.6 pH units), in contrast to the reported 5.5-8.0 pI range of polyclonal mouse IgG. Banding patterns obtained in the IEF affinity immunoblot typically displayed 3-5 major dense bands flanked by 2-4 minor fainter bands. Batches of ascites obtained years apart produced similar immunoblot patterns, indicating constant antibody production and confirming the stability of these hybridoma clones. Minor bands appeared in 2 earlier lots of ascites, suggesting possible modification of antibody during storage. IEF affinity immunoblot analysis is a useful tool for monitoring MoAb pI microheterogeneity as an indicator of antibody quality without the need for isolation of monoclonal antibody from culture medium or ascites.

Molecular mimicry between Fc receptor and S peplomer protein of mouse hepatitis virus, bovine corona virus, and transmissible gastroenteritis virus.

We have previously demonstrated molecular mimicry between the S peplomer protein of mouse hepatitis virus (MHV) and Fc gamma R (Fc gamma R). A monoclonal antibody (MAb) to mouse Fc gamma R (2.4G2 anti-Fc gamma R MAb), purified rabbit immunoglobulin, but not their F(ab')2 fragments, as well as mouse and rat IgG, immunoprecipitated (1) recombinant S peplomer protein expressed by a vaccinia virus recombinant in human, rabbit, and mouse cells, and (2) natural S peplomer protein from cells infected with several strains of MHV and MHV escaped mutants. We report here results of studies documenting molecular mimicry between Fc gamma R and S peplomer protein of viruses representing three distinct antigenic subgroups of the Coronaviridae. We have shown a molecular mimicry between the S peplomer protein of bovine corona virus (BCV) and Fc gamma R. The 2.4G2 anti-Fc gamma R MAb, rabbit IgG, but not its F(ab')2 fragments, as well as homologous bovine serum, free of anti-BCV antibodies, immunoprecipitated S peplomer protein of BCV (Mebus strain). In contrast, we did not find molecular mimicry between S peplomer protein of human corona virus (HCV-OC43) and Fc gamma R. Although the OC43 virus belongs to the same antigenic group as MHV and BCV, MAb specific for human Fc gamma RI or Fc gamma RII and purified human IgG1, IgG2, and IgG3 myeloma proteins did not immunoprecipitate the S peplomer protein from HCV-OC43-infected RD cells. In addition, we did demonstrate molecular mimicry between the S peplomer protein of porcine transmissible gastroenteritis virus (TGEV) and Fc gamma R. TGEV belongs to the second antigenic subgroup of coronaviridae.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphologic evaluation of IgM cells of the canine small intestine by fluorescence microscopy.

Biopsies of small intestine from 7 dogs were examined by fluorescence microscopy to determine the number of IgM-containing cells in the lamina propria. Biopsies were taken from duodenum, jejunum, and ileium. (Cell counts were made by 2 persons to demonstrate reproducibility.) There were 452.24 +/- 60.09 cells per mm2 in the duodenum 572.68 +/- 62.13 cells per mm2 in the jejunum, and 107.47 +/- 59.57 cells per mm2 in the ileum. All sections were cut at 6 micrometer. The ileum had fewer cells than either duodenum or jejunum (P = 0.000038 and 0.00001, respectively), whereas duodenum and jejunum did not differ significantly in numbers of cells (P = 0.17528). Quantifying autofluorescent cells in the same sites showed no significant differences among the 3 tissues (P = 0.24697). The autofluorescent cells differed in intensity and morphology from the IgM cells. These two observations tend to support the contention that the autofluorescent cells did not bias the IgM cell counts at the 3 sites. Total autofluorescence (cells, collagen, and vessels) was higher in the ileum than in either the jejunum or the duodenum (P = 0.04967 and 0.03050, respectively). However, all 3 categories counted (IgM cells, autofluorescent cells, and autofluorescent structures) had significant dog-tissue interactions. This will necessitate determining normals for each age-sex-breed category of dog studied.