Clonal diversity and homology of latent and nominal group a immunoglobulin allotypes in the rabbit.

Latent VH immunoglobulin allotypes are expressed unexpectedly and transiently at low concn in the serum of rabbits. Latent group a1 molecules in sera from rabbit colonies in Philadelphia (U.S.A.) and Birmingham (U.K.) were examined for a1 specificity and clonal diversity using reference nominal allotypic reagents and isoelectric focusing (IEF) autoradiography. Latent a1 molecules from rabbits of both colonies had diverse spectrotypic patterns in the pI range 5.5-8.3, as identified with 125I-labelled affinity-purified specificity-tested, anti-nominal a1 antibody. Comparisons of spectrotypes between nominal a1 antigen and latent a1 focused molecules revealed a marked correspondence in banding over the pI range. Reference anti-nominal a1 antibodies could be absorbed out substantially by the IgG fraction of serum from two rabbits containing latent a1 molecules; in a reciprocal fashion absorption with nominal a1 molecules reduced the binding of focused latent a1. The latent a1 molecules from both U.S.- and U.K.-bred rabbits displayed strikingly similar IEF spectra and their antigenic similarities were confirmed by similar absorption capacities of the reference anti-a1 serum. When sequential serum samples from one (U.S.) latent a1 rabbit were compared by IEF, some bands, e.g. those between pH 7.75 and 8.3, appeared to fluctuate in their presence, whereas others, e.g. between pH 5.3 and 7.4, were expressed continuously. We can conclude that latent a1 molecules are clonally complex and some are consistently produced in small amounts. As they also show antigenic similarity, if not identity with nominal a1, we believe that they are probably the product of the same gene (or genes) with an equivalent capacity to be associated with specificity-determining genes even though the level of synthetic activity is lower and possibly governed differently. Anti-a1 antibody was raised in a rabbit in which latent a1 allotype had been previously detected. This antibody was of low avidity and, while inhibitable on RIA by nominal a1 it was not inhibitable by the donor's latent a1 or by a second latent a1 of the same (partially inbred) U.K. colony, but was inhibitable by a latent a1 serum from the Philadelphia, U.S.A. colony. This result suggests that a1 molecules are the products of more than one gene.

Presence of kappa 2 light chain in normal rabbits and as induced auto anti-allotype antibody in kappa 1 light chain suppressed subjects.

Using an antiserum raised in b(bas)/b(bas)-suppressed rabbits to kappa 2 light chain we have shown that the kappa 2 light chain is an isotype of kappa 1, present in the majority of, if not all, rabbits. It probably exists in at least 2 allelic forms. It is capable of producing a functional antibody molecule (auto anti-b6) and compensates for the absence of kappa 1 light chain in homozygous b6-suppressed rabbits.

The idiotypy of auto anti-allotype antibody induced in immunoglobulin allotype suppressed rabbits.

Rabbit anti-rabbit idiotype antibody was raised to both clonally heterogeneous and restricted auto anti-b6 antibodies induced in b6 allotype-suppressed (b6)/(b6) homozygous and b4/(b6) heterozygous rabbits. In every case the anti-idiotypic antibodies were specific only for the inducing antibody as shown by direct binding solid-phase RIA. Anti-idiotypes directed to the same antibody preparation had a similar but not identical specificity. It was demonstrated by IEF that the same idiotype specificity (spectrotype) was present throughout the anti-b6 response in individual rabbits.

Escape from b locus allotype suppression in the rabbit is mediated by IgM molecules bearing an allotope-restricted variant of kappa light chain.

Rabbits homozygous for b6 at the kappa light chain b locus were suppressed for the expression of the b6 allotype and then induced to produce auto anti-b6 antibody. Rabbits which subsequently escaped suppression produced auto antibody with restricted allotype specificity. Escape from allotype suppression was mediated by IgM bearing a kappa chain variant with a restricted number of b6 allotopes and having a diminished interaction with auto anti-b6 antibodies from the same and other rabbits escaping b6 suppression. This suggested that there were allelic variants or subpopulations of the b6 light chain which were under independent regulation of expression, clearly influenced by the specificity of auto anti-allotype antibody. Since escape from suppression was mediated by IgM it is proposed that a normal pathway of B cell differentiation occurs during recovery from suppression.

The use of cellulose carbonate-based immunoadsorbents in the isolation of minor allotypic components of rabbit immunoglobulin populations.

Cellulose trans-2,3-carbonate has been used as a new insoluble matrix for the simple coupling of a1- and b4-positive rabbit immunoglobulin to make immunoadsorbents capable of purifying from serum, with great efficiency, alloantibodies to these allotypic determinants. The antibodies have themselves been conjugated to prepare specific antibody immunoadsorbents of high binding activity for their allotypic target molecules. With these anti-allotypic solid-phase reagents it has been possible to affinity purify a1- and b4-positive immunoglobulin molecules and to deplete serum immunoglobulin of these molecules to leave in the eluates only the allotypically uncontaminated minor immunoglobulin components which are a-negative or b-negative (lambda chain-bearing) molecules. lambda chain molecules were also purified in very small quantities by affinity chromatography on a sheep anti-rabbit lambda chain column. This method of purifying minor populations of rabbit immunoglobulin from normal serum by special immunoadsorbent applications offers new opportunities to study the products of rarely expressed immunoglobulin genes in normal rabbits.

A quantitative study of the distribution of IgG sub-classes in a group of normal human sera.

The radial immunodiffusion method of Mancini has been applied to quantitative study of IgG subclasses in a normal population. The method was assessed in terms of both reproducibility and antiserum consumption. The distribution of IgG subclasses in a group of normal individuals was studied, and compared with their incidence in a series of monoclonal proteins investigated by previous workers and particularly with other quantitative studies on groups of normal individual's sera.

Demonstration of the clonality and specificity of an auto-antibody response to a suppressed immunoglobulin allotype of the rabbit kappa chain b locus.

A regulatory idiotypic network is proposed to control allotype expression in normal rabbits. We have used suppression of the kappa chain b6 allotype in an attempt to restrict the number of regulatory idiotypes involved in an induced auto anti-allotype response. These auto anti-b6 antibodies were examined by an agarose imprint immuno-fixation IEF technique using iodinated allotype-bearing IgG. All totally b6-suppressed rabbits produced a clonally complex response which was generally spectrotypically unique - thus contradicting previous claims of a dominant common idiotypic pattern. The compensating light chains thus have available as many regulatory V genes as does the kappa 1 light chain. However, the b6/b6 homozygotes breaking b6 suppression produce an auto anti-b6 antibody which does not interact with their "escaping" molecules and which is clonally restricted. We propose a regulatory mechanism limiting the V genes utilised to produce the autoantibody, the latter then allowing only molecules bearing non-interactive allotopes to "escape" suppression.

Improved methodology for the production of monoclonal antibodies against parasites.

A modification of the standard fusion methodology is described which results in greatly increased yields of monoclonal antibodies against certain organ-specific parasites. Several fusions were carried out using mice infected with Schistosoma mansoni or Nematospiroides dubius, using B lymphocytes harvested from either the spleen or the mesenteric lymph nodes. Results indicated a greatly improved yield of positive clones using the lymph nodes as a source of B cells for fusion. A 7-fold increase in the number of positive clones was seen with N. dubius injections, while S. mansoni fusions showed a 2-fold increase.

Transmission of immunoglobulin to foetal and neonatal mice.

Transmission of immunoglobulin (Ig) classes and subclasses from mother to foetus and to neonate, and the survival of maternal Ig in the circulation of the young mouse up to 40 days after birth, has been quantitated in Balb/c homozygous and (Balb/c X SJL/J)F1 matings using isotype-specific heteroantisera in radial immunodiffusion in gel assays. The transfer of anti-allotype (anti-Ig-1b (gamma 2a] antibodies from immunised Balb/c mothers (Ig-1a) to F1 heterozygote (Ig-1ab) offspring was measured by passive haemagglutination of Ig-1b target allotype-coated sheep red blood cells. A small but significant level of transmission of Ig to the foetus occurs by the 15th day of gestation (5 days before birth) but the bulk of passively acquired Ig is derived from the milk after birth. All Ig acquired in utero and later across the intestinal barrier is exclusively of IgG isotypes (gamma 1, gamma 2a, gamma 2b) even though the milk has a large predominance of IgA. An appreciable level of maternally derived antibody is maintained in the circulation of the young mouse 24 days or more after gut 'closure' on the 16th day post-partum.

Spectrotypic analysis of passively acquired and newly synthesised IgG antibodies in the neonatal and young mouse.

Isoelectric focussing with autoradiography has been used to analyse the selective nature of passive transmission of specific anti-Ig allotypic antibodies from the mother to the young mouse, and to study the generation of spectrotypic (clonal) diversity of autologous IgG2a (carrying the paternal inherited Igh-1b allotype) in BALB/c X SJL/J F1 (Igh- 1ab heterozygote) mice. Transmission of anti-allotypic antibody to neonatal mice was found to be pI restricted, with selection favouring electrophoretically fast IgG. Comparison of the antibody spectrotype in maternal serum, milk and neonatal serum revealed that the pI restriction in transmission operates at the level of the neonatal gut. Analysis of the paternally inherited Igh-1b IgG2a molecules as they are first synthesised and secreted into the neonatal serum revealed an extensive polyclonality on first detection by the very sensitive focussing assay. It can be deduced that IgV region diversity is generated by IgG2a-synthesising cells prior to, or at the time of, the first secretion of this class of antibody into the serum.

Two enzyme linked immunosorbent assays for detecting antibodies against meningococcal capsular polysaccharides A and C.

AIMS: To evaluate two of the recent methods of coating microtitre plates in the enzyme linked immunosorbent assay (ELISA) for detecting human antibodies against meningococcal capsular polysaccharides A and C with a view to validating a specific meningococcal antibody assay for routine clinical use. METHODS: Two four-layer ELISA protocols were standardised: one method utilised meningococcal polysaccharides conjugated to poly-L-lysine polypeptide for coating the microtitre plates; another used polysaccharides mixed with methylated human serum albumin (mHSA). Titration curves were plotted for the ELISAs and the squared Pearson correlation coefficient (R2) was used to determine the degree of accuracy of fit of the curves. Specificity tests were performed by inhibition and adsorption studies. RESULTS: Both methods gave good titration curves with a high R2 of > 0.98, indicating a high degree of accuracy in forming the curves. The titration end point after vaccination, obtained by the mHSA method, was 20 times higher, however, than that obtained by the poly-L-lysine method. Specificity tests showed that in the ELISA using polysaccharide/poly-L-lysine, antibody activity of a pre-vaccination serum sample was inhibited by 37%, and of post-vaccination serum by 50% with 1000-fold excess antigen. Antibody activity (post-vaccination) was reduced by 51% and 59%, respectively, by adsorption with antigen-coated Sepharose beads or adsorption with suspensions of killed meningococci. In contrast, antibody activity of a pre-vaccination serum was inhibited by 60% and a post-vaccination serum by 90% in ELISA employing polysaccharides mixed with mHSA. Reproducibility was better with the use of methylated human serum albumin than with poly-L-lysine; the former showed intrabatch and interbatch coefficients of variation of 4% and 2%, respectively, compared with 43% (intrabatch) and 16% (interbatch) obtained with the poly-L-lysine. CONCLUSION: It is concluded that the antibody assay using meningococcal polysaccharides groups A and C mixed with mHSA is much better than that using polysaccharides coupled with poly-L-lysine.

Anti-epithelial (anti-A549) antibodies: their nature, specificity and relevance to transplantation.

Several studies have addressed the possible importance of anti-epithelial cell antibodies in kidney transplantation using the A549 cell line as an in vitro model. In this paper we report our results using for the first time an enzyme-linked immunosorbent assay (ELISA) to detect the anti-A549 cell antibodies. Sera from 129 kidney transplant patients were tested for IgM anti-epithelial cell antibodies directed against the A549 cell line prior to transplantation; only three sera were positive (2.3%). 101 of these patients were then followed-up post-transplantation; sera were collected routinely at 2, 6 and 12 weeks and at the time of rejection episodes. All samples were also tested for cytomegalovirus (CMV) IgM antibodies. Sixteen patients developed anti-A549 IgM antibodies, and there was no correlation with acute graft rejection. Anti-epithelial antibodies showed no binding to sections of normal kidney or biopsies of rejected kidneys. Eleven patients were positive for anti-CMV IgM antibodies. In nine cases both IgM anti-A549 and IgM anti-CMV antibodies were found, which was a highly significant association (p < 0.001). Analysis of A549 cellular proteins by immunoblotting gave evidence for the presence of CMV polypeptides in the cell lysate. Electron-microscopic examination of A549 cell preparations revealed intracellular particles which were compatible in size with CMV. Polymerase chain reaction analysis confirmed the presence of a specific CMV DNA sequence in A549 cells of several batches from different sources. Our data strongly suggest that the A549 cell line used in several published reports is infected with CMV and that in the majority of cases the anti-A549 'anti-epithelial' antibodies found in renal transplant patients are anti-CMV antibodies.

Species-specific detection of venom antigens from snakes of the Bothrops and Lachesis genera.

Venom-insoluble adsorbents were employed to absorb out the cross-reacting antibodies from monovalent polyclonal antivenoms. The absorbed antivenoms were tested by enzyme-linked immunosorbent assay against homologous and heterologous venoms and showed species-specificity throughout a range of venom concentrations. The same absorbed antisera were used in immunoblots under non-reducing conditions as probes to reveal species-specific antigens. In all cases studied this was achieved. The range of mol. wts of specific antigens was between 20,000 and 120,000, approximately. Venoms added to human serum experimentally were specifically detected by their homologous absorbed antivenom antibodies. The work here described could be important in the development of diagnostic assays for envenomings involving snakes from the Bothrops and Lachesis genera.

IgA antibodies in human milk: epidemiological markers of previous infections?

The concept of an enteromammary link in secretory IgA (SIgA) antibody production was tested by hypothesising that specific SIgA antibody profiles in human milk might be an epidemiological marker for enteropathogens in a community. Milk from three subject groups was studied: 64 Sri Lankan women living in poor suburbs of Colombo, 20 Asian immigrant women domiciled in Birmingham, for a median period of five years (range 14 days-16 years), and 75 white women living in Birmingham. An enzyme linked immunosorbent assay (ELISA) was developed for the detection and measurement of SIgA antibodies to a panel of 14 crude O and 10 pure lipopolysaccharide antigens of diarrhoeagenic Escherichia coli strains well known to be endemic in the Indian subcontinent. The number of Sri Lankan and Asian immigrant women with SIgA antibodies to all 14 diarrhoeagenic E coli antigens (except O127 in Asian women) was significantly higher than in the white controls. The amount of E coli O antigen specific SIgA antibody activity as a percentage of total SIgA also gave significantly higher median values in Sri Lankan (6%) and in Asian immigrant (4%) women than in white controls (0.7%). SIgA antibodies were highly O serogroup specific and showed excellent concordance between crude O and the corresponding purified lipopolysaccharide antigens. These results suggest that milk antibody profiles represent an epidemiological marker of exposure to enteral pathogens. The continuing specific milk antibody response in Asian women who have been domiciled in the United Kingdom for many years may indicate 'memory' in the human secretory immune system.

Presence of secretory IgA antibodies to an enteric bacterial pathogen in human milk and saliva.

The concept of a common mucosal immune system in man was tested by examining the concurrent presence of specific-secretory IgA (SIgA) antibodies in human milk and saliva from three groups of subjects: 64 Sri Lankan women living in Sri Lanka; 20 immigrant Asian women living in Birmingham (median duration of residence in the United Kingdom five years); and 75 Caucasian women living in Birmingham (controls). Enzyme linked immunosorbent assays (ELISA) were developed to detect enterotoxigenic Escherichia coli (ETEC) colonisation factor/1 (CFA/1) specific SIgA antibodies in milk and saliva. ETEC CFA/1 specific SIgA antibody activity was detectable in milk (37.5% and 25%) and saliva (42.1% and 35%) of Sri Lankan and immigrant Asian women, respectively, but not in any of the Caucasian controls. Eighty five point two per cent of subjects who were positive had specific antibodies detectable in both milk and saliva; 5% of all Sri Lankan women and 10% of all immigrant Asian women had detectable antibody only in saliva. These observations lend further strong support to the idea that a common mucosal immune system exists in man. The continuing presence of specific SIgA antibodies in Asian immigrants to previously encountered antigens suggests that there may be an 'immunological memory' in the human secretory immune system.

Development of a species-specific ELISA for Brazilian pit-viper venoms.

Antigenic cross-reactivity between venoms of the genus Bothrops has been shown to be an extensive problem. However, some venom components are species-specific. In this study we have produced species-specific antivenoms against some members of the genus Bothrops. Monospecific rabbit antivenoms (IgG) were absorbed on venom affinity adsorbents. The species-specificity was tested by ELISA assays and immunoblots. The results of both assays showed complete species-specificity in some cases and highly increased species-specificity in others. These reagents can be used to determine the envenomating species in snake bite patients as an aid to improved serotherapy.

Infection and reinfection of chickens with Salmonella typhimurium: bacteriology and immune responses.

Four-day-old chickens infected orally with a spectinomycin-resistant (Spcr) mutant of a highly invasive avian Salmonella typhimurium strain excreted salmonellae in the feces for at least 10 weeks. When these chickens were reinfected at this time with a nalidixic acid-resistant (Nalr) mutant of the same strain, they excreted this mutant in significantly smaller numbers (P less than 0.01) than did a previously uninfected control group. The Nalr mutant had a shorter survival rate in the tissues of the immunized chickens than in tissues of the control birds. The Spcr mutant stimulated strong IgG, IgA, and IgM responses in serum, small-intestinal contents, and bile. These were detected by enzyme-linked immunosorbent assay (ELISA) against antigens of crude whole bacterial cell protein sonicate, lipopolysaccharide, flagella, and outer-membrane proteins. There was some evidence of an anamnestic response with IgA in bile following reinfection with the Salmonella. The peak response of antibody-producing cells from the spleens of infected chickens, assayed by solid-phase ELISA, occurred at 3 weeks postinoculation. A strong delayed hypersensitivity reaction, detected by foot-pad swelling after inoculation with either whole-cell or outer-membrane proteins, was observed between 2 and 5 weeks after infection with the Spcr mutant. The data indicate that outer-membrane proteins are major immunogens for both humoral and cell-mediated arms of the immune system.

Multiple Ig classes on rabbit B lymphocytes.

Rabbit PBL were studied regarding the presence of different classes of s-Ig, under experimental conditions, ensuring the endogeneous origin of these proteins. About 40% of the lymphocytes are B cells (Fab positive and a1 positive in a1 homozygous rabbits). IgG positive lymphocytes could be found, but only using and anti dll conjugate (allotype located on the Fd gamma region of IgG). Anti Fc gamma conjugates were negative. Most of the B cells are IgM positive, most of these IgM cells however were also positive for either IgG or IgA. Np lymphocytes were found bearing both IgG and IgA. Careful analysis of the percentages of various isotypes found on B cells suggests that some "IgD" positive lymphocytes could be present. Results are discussed in relation to B cell differentiation.

Study the presence of fibronectin binding protein (FnBp) in tuberculosis (TB) patients by elisa.

Fibronectin-binding protein (FnBp) antigens are a prominent secretory protein of short term culture supernatants of M. tuberculosis and M. bovis (BCG) and is conserved within the genus Mycobacterium. The 30/31 kDa antigen of M. tuberculosis is one of the major secretory molecules and is probably routinely recognised by the host immune system in the early stage of tuberculosis infection. Serum immune complexes, prepared from TB patients and normals, were analysed for the presence of FnBp by ELISA using an anti-30/31 kDa (FnBp) monoclonal antibody (CF8) and by western blotting using Fibronectin-HRP. A significant difference was seen between normals and TB patients (p < 0.05). This test was found to have a specificity of 80% and a positive predictive value of 73%. This is a preliminary finding and the test needs to be evaluated further for its performance on a larger number of confirmed TB patients and controls.

Study of the mechanisms of killing of Mycobacterium bovis BCG by apoptosis in J774 murine macrophages.

OBJECTIVE: To determine the relationship between the mechanism of apoptosis and intracellular killing of Mycobacterium bovis BCG. Apoptosis, "programmed cell death"--a physiologically beneficial and distinct form of cell death. SETTING: In vitro study was carried out in murine macrophage cell line J774 that was infected with Mycobacterium bovis BCG in different set of conditions. Percentage of surviving BCG and apoptotic cells was determined. METHODS: IFN-g and/or LPS-activated and non-activated J774 mouse macrophage cells were infected with BCG in a ratio of 1:5. The morphology of the host cells was studied after 4 hours, 24 hours and 48 hours of infection in cytospins stained with Jenner-Giemsa. Surviving bacteria were counted by incorporation of radiolabelled-uridine after cell lysis. RESULTS: Both in the activated and non-activted J774 cultures some cells undergo apoptosis. In cells activated with IFN-g or LPS without BCG, less than 10% of cells were found to be apoptotic. More apoptosis was seen when LPS-activated cells were infected with BCG. In the cells activated with IFN-g or LPS-activated cells the percentage of apoptotic cells was much higher than in non-activated cells or cells activated with either INF-g or LPS alone. After 24 hours culture, without BCG, about 15% of the cells were found to be apoptotic and with BCG infection this increased to 23% (p < 0.01). The level further increased after 48 hours of infection. BCG growth inhibition was observed in both non-activated J774 cells and cells activated with LPS, INF-g or both and was sustained to 48 hours of co-culture. CONCLUSION: It is evident that BCG-infected J774 cells undergo apoptosis in the presence of a high concentration of RNI and/or ROI. During this process the cells shrink considerably in volume with the removal of water that may concentrate toxic products in the cell. The increased concentration of toxic species and the disorganisation of the phagocytic vacuoles may account for the enhanced stasis and/or death of the intracellular micro-organisms. We conclude that host cell apoptosis may arrest the growth and account for the death of the intracellular mycobacterial pathogen.