Evaluation of monoclonal antibodies with specificity for human IgA, IgA subclasses and allotypes and secretory component. Results of an IUIS/WHO collaborative study.

51 monoclonal antibodies (McAb) with putative specificity for human IgA, the IgA subclasses, Am allotypes or secretory component (SC) were evaluated for immunoreactivity and specificity by nine laboratories employing immunodiffusion, agglutination, immunohistological assays, immunoblotting and direct binding and competitive inhibition enzyme immunoassays. McAbs specific for IgA PAN (n = 24), IgA1 (n = 7), IgA2 (n = 3), IgA2m(2) (n = 2), non-IgA2m(2) (n = 4) and SC or secretory IgA (n = 5) were identified that were immunoreactive and specific in the assays employed. The McAbs identified as IgA- or SC-reactive were shown to be non-reactive to human IgG, IgM, IgD, IgE, kappa and lambda by direct binding and competitive inhibition immunoassays. Interestingly, no McAbs with restricted specificity for IgA2m(1) were identified. Some McAbs displayed higher affinity and/or better performance in one or several of the assay groups. The IgA- and SC-specific McAbs identified in this international collaborative study have potential as immunochemical reference reagents to identify and quantitate monomeric and polymeric IgA in human serum and secretions.

The origin of IgG production and homogeneous IgG components after allogeneic bone marrow transplantation.

Pediatric recipients (n = 25) of an allogeneic bone marrow (BM) graft were selected on the basis of informative IgG allotype (Gm) differences between the BM donor and the recipient. To investigate the kinetics of the appearance of IgG of donor origin and the disappearance of IgG of recipient origin, G1m and G2m allotype levels were quantified in sera obtained at regular intervals between 3 months and 5 years after BM transplantation (BMT). For this quantification, a dot immunobinding assay (DIBA) has been developed. In 19 of 22 informative recipients, the Gm allotype distribution had reached the range of values expected on the basis of the Gm phenotype of the donor within 6 months after BMT. Remarkably, IgG of recipient origin persisted in 15 of 18 informative recipients until last follow up, ie, for several years after BMT. In addition to the origin of total IgG production, the origin of homogeneous IgG components (H-IgG) appearing after BMT was investigated. H-IgG of donor origin could be detected as early as 3 weeks after BMT, but also H-IgG of recipient origin were present in 8 of 13 informative recipients for a period of up to 1 year after BMT. We conclude that host-type IgG-producing cells were not eradicated by the (myeloablative) conditioning regimen and persisted in a high number of graft recipients. It is our hypothesis that lack of graft-versus-host disease (GVHD) in the majority of these recipients results in the persistence of IgG-producing cells of host origin. These observations may be relevant for the evaluation of patients who received allogeneic BMT for the treatment of multiple myeloma.

Molecular characterization of Gm(n+) and G2m(n-) allotypes.

Immunoglobulin (Ig) allotype typing is usually performed with serological methods based on hemagglutination inhibition. The recent development of molecular techniques has allowed the molecular typing of several Ig markers. The hinge, CH2, and CH3 domains of the G2 gene from six unrelated individuals (three G2m(n+) and three G2m(n-)) were amplified and cloned to establish the molecular basis of the G2mn+ and G2mn- . Comparison of the allele sequences revealed three changes: two (codons 308 and 437) are silent exonic substitutions, one is a G to A transition corresponding to an amino acid difference in position 282: Val (GTG) in G2mn- , Met (ATG) in G2mn+ . These substitutions were identified via two approaches: 282 polymorphism, after digestion of a specific polymerase chain reaction product with Nla III followed by acrylamide electrophoresis; 308 and 437, by a dot-blot technique using allele-specific oligonucleotides. These molecular typing results correspond exactly to those obtained serologically; moreover, the three substitutions defining the G2mn+ and G2mn- alleles are always associated in a strict linkage disequilibrium.

DNA sequences specific for Caucasian G3m(b) and (g) allotypes: allotyping at the genomic level.

Assignment of the G3m(g) and (b) correlative amino acid residues was performed at the genomic level by direct sequencing of DNA from nine Caucasian individuals. Two oligonucleotide primers were used for subclass-specific enzymatic amplification of a DNA segment comprising a major portion of the second and third constant region domains (CH2 and CH3) of the human IgG3 heavy chain gene. Comparison of the sequences of amplified DNA from individuals serologically typed as homozygous for G3m(b) or G3m(g) or as heterozygous, G3m(b,g), revealed differences in the codons for the amino acid residues 291, 296, and 384. Proline, phenylalanine, and serine at these positions corresponded to G3m(b), and leucine, tyrosine, and asparagine to G3m(g). Heterozygotic individuals, typed G3m(b,g), displayed both the G3m(b) and G3m(g) codons at these three positions. The polymorphism at each of these three codons could be identified either as the appearance, or the loss, of recognition sites for the two restriction endonucleases, Nsp BII and Rsa I. This allowed the development of a polymerase chain reaction (PCR)-based assay permitting the distinction of G3mb and G3mg alleles by analyzing the electrophoretical mobility of the DNA fragments generated by digestion of the PCR-products with Nsp BII and Rsa I.

HTLV-II seroprevalence in pygmies across Africa since 1970.

HTLV-II-specific antibodies, with patterns similar to those in the Americas, were present in sera collected about 1970 from Bambuti pygmies in Zaire (14/102; 14%) and from pygmies in Cameroon (5/214; 2.3%), and were more prevalent than HTLV-I. In the Central African Republic, 504 pygmies were HTLV negative. After finding of 4 HTLV-II seropositives among 12 Bambuti pygmies sampled in 1991, this established that HTLV-II or a related retrovirus is present as an ancient endemic in some, but not all, insulated groups of African pygmies, similar to the HTLV-II distribution in Amerindian populations. The endemic among the oldest inhabitants of central Africa, and the occasional and scattered occurrence of apparent HTLV-II among predominant HTLV-I in other Africans, fit well with an ancient African virus and not with importation from the New World. Theories on the origin and evolution of the primate T-lymphotropic viruses (PTLVs) should take into account the longstanding presence of HTLV-II-type viruses in both the Old and New World. Present serology suggests identity of the African viruses with HTLV-II, but their assignment to a new HTLV type is open should genetic analysis show strong divergence from American HTLV-II. Clinical expression, if any, remains to be studied.

Transfer of specific immunity from donor to recipient of an allogeneic bone marrow graft: evidence for donor origin of the antibody producing cells.

A rapid recovery of specific humoral immunity in the recipient of an allogeneic bone marrow transplantation (BMT) can be observed after immunization of the donor before graft sampling. This has been attributed to transfer of specific immunity from donor to recipient. However, to maintain the concept of transfer the origin of the antibody producing cells in the recipient after BMT must be demonstrated. To this end, donor-recipient pairs with differences in Gm-allotypes were selected and immunized before BMT with the neo-antigen Helix pomatia haemocyanin (HPH) according to three immunization protocols. Additionally, the recipients were immunized at day 42 after BMT. Serum samples were weekly obtained from the recipients in the first 100 d after BMT. The origin of HPH-specific antibody producing cells was assessed by two approaches: (1) determination of the Gm-allotypes of anti-HPH antibodies within a distinct IgG subclass, (2) analysis of anti-HPH antibody spectrotypes by isoelectric focusing combined with immunoblotting. The results obtained with these two approaches show concordance in most instances and led to the conclusion that the antibody producing cells are of donor origin.

Evaluation of monoclonal antibodies having specificity for human IgG subclasses: results of the 2nd IUIS/WHO collaborative study.

Following the 1st IUIS/WHO Collaborative Study of monoclonal anti-IgG subclass antibodies, a panel of WHO Specificity Reference Reagents (SRR) was established [Jefferis, R., et al. (1985) Immunol. Lett., 10, 223]. At the time, the hope was expressed that further reagents particularly for IgG2, and other allotypic specificities would become available which could be applied in a wide range of assay protocols. The 2nd study reports the evaluation of nineteen anti-subclass and seven anti-allotype monoclonal antibodies. The anti-IgG1 antibody HP6187 was equivalent in performance to the SRR. Others, that were not of the mouse IgG1 isotype, may be useful for particular applications. The anti-IgG2 antibody HP6200 could be a valuable addition to the WHO SRR; it is specific for an epitope in the Fab region but does not have the light chain bias of HP6014. Antibodies of putative allotype specificity exhibited the claimed specificity when used within protocols similar to those employed by the originating laboratory. It appears to be inherent in the nature of the epitopes (allotopes) recognized that it will take several years before reagents applicable to a wide range of techniques will become available.

The first monoclonal anti-G1m(x) antibody: production and usefulness in haemagglutination-inhibition assay.

Murine monoclonal anti-human Ig allotype antibodies have been described for a limited number of specificities. Now the first monoclonal anti-G1m(x) antibody has been produced by fusion of lymph gland cells of a mouse injected with G1m(zax)-positive myeloma proteins with cells of the myeloma cell line SP2/0. Several limiting dilutions resulted in one antibody-producing hybridoma, clone 3A12. The specificity of monoclonal antibody (mAb) 3A12 in the direct haemagglutination assay, as well as in the haemagglutination-inhibition (HAI) assay, is described. Neither cross-reactivity nor nonspecific inhibition with G1m(x)-negative Ig was seen. Typing for the G1m(x) allotype with mAb 3A12 gave results which are identical to those obtained with the conventional human antisera. With the production of this mAb, it now is possible to discriminate between the four main G1m alleles not only by HAI assay but also by other assay systems, of which the preliminary results are promising.

Immunoglobulin allotypes in systemic lupus erythematosus--results of a central European multicenter study. SLE Study Group.

Immunoglobulin heavy chains (G1m, G2m, G3m, A2m) and kappa light chain (Km) allotype and phenotype frequencies were examined in 323 central European Caucasian patients with systemic lupus erythematosus (SLE). No significant differences were found between the different allotype or phenotype frequencies of the SLE patients and a control group of healthy individuals. Our results indicate that Gm, A2m and Km allotypes do not represent susceptibility factors for SLE in Caucasians.

Immunoglobulin allotypes are not associated with HLA-antigens, autoantibodies and clinical symptoms in systemic lupus erythematosus. Members of the SLE Study Group.

Immunoglobulin heavy chain (G1m, G2m, G3m, A2m) and kappa light chain (Km) allotype and phenotype frequencies of 323 central European Caucasian patients with systemic lupus erythematosus (SLE) were examined and correlated with various genetic, serologic and clinical markers of SLE. No significant associations were found between immunoglobulin allotypes or phenotypes and all 20 parameters tested (nephritis, vasculitis, arthralgias, photosensitivity, discoid lesions, central nervous system disease, Raynaud's phenomenon, sex, anti-Ro, anti-La, anti-nRNP, HLA-DR1-DR7, HLA phenotypes B8-DR3, B7-DR2). It could therefore be assumed that Gm, A2m and Km allotypes were not associated with HLA-antigens and had no influence on the serologic and clinical expression of SLE.

Size calculation of restriction enzyme HaeIII-generated fragments detected by probe YNH24 by comparison of data from two laboratories: the generation of fragment-size frequencies.

Restriction fragment-length polymorphism of locus D2S44 detected by the highly polymorphic probe YNH24 and restriction endonuclease HaeIII can be used to improve parentage testing when representative fragment-size frequencies can be obtained. By joining the results of different laboratories, it is possible to set up a meaningful databank. Therefore, the same randomly chosen samples were tested for the HaeIII RFLP detected by probe YNH24 in Düsseldorf (DUS) and Amsterdam (AMS). The results of the different fragment-size calculations obtained by using internal markers and a computerized system (DUS-cad and AMS-cad), and by using external markers and manual calculations (DUS-man), were analyzed. Comparing these results, no statistically significant differences were seen. The results obtained with probe YNH24 and enzyme HaeIII in Düsseldorf and Amsterdam can be used to attain a sufficient number of samples to generate relevant fragment-size frequencies.

Multiple levels of analysis of an IGHG4 gene deletion.

Human immunoglobulin heavy chain constant region (IGHC) genes constitute a typical multigene family, usually comprising eleven genes on the telomere of chromosome 14 (14q32). In this region, deleted and duplicated haplotypes have been reported to exist with considerable frequency. Their origin is the result of either unequal crossing-over or looping out excision. In this paper, we report the characterization of a new type of deletion, involving the IGHG4 gene, in a subject who also carries a larger deletion of a previously described type on the second chromosome. Employment of several methods (polymerase chain reaction, standard Southern blot, pulsed field gel electrophoresis, serological techniques) to analyze these deleted haplotypes has resulted in a level of accuracy in their characterization that has not been achieved in previous cases. The site of recombination responsible for the IGHG4 deletion was restricted to a 2.5-kb region 3' of the G4 gene; this rules out any possible involvement of the S regions in the recombination process. The usefulness of the various techniques in the characterization of the deletions is also discussed, together with possible future applications in the field.

Attempt to calculate the allele frequency distribution of a TaqI RFLP detected by the highly polymorphic probe YNH24.

To improve the analysis of parentage testing with the additional technique of DNA polymorphisms, the usefulness of probe YNH24 was studied. The allele frequency distribution of restriction fragments detected by probe YNH24 on TaqI-digested genomic DNA from 100 unrelated individuals was determined. For this purpose, the size of the fragments was calculated by making use of HindIII-digested lambda DNA as an internal marker and of a digitizing tablet coupled to a computer. The size of the fragments ranged from 2.53 kb to 5.89 kb. The mean standard deviation was 0.05 kb. The differences between the fragment sizes appeared to be smaller than the standard deviation. For this reason, it was not possible to calculate the allele frequency distribution of this highly polymorphic genetic system.

A sandwich ELISA with monoclonal anti-IgG allotype antibodies permitting the deduction of the G1m genotype.

With murine monoclonal antibodies specific for the human IgG1 allotypes G1m(z), G1m(a), G1m(f) and the IgG1 subclass, sandwich enzyme-linked immunosorbent assays (ELISAs) were developed to determine whether different allotypic determinants are located on the same molecule. With the conventional haemagglutination-inhibition test, it is only possible to determine the phenotype. With this ELISA system, it is now possible to deduce the G1m genotype.

Evaluation of monoclonal antibodies with putative specificity for human IgG allotypes.

Fourteen monoclonal antibodies (MAbs) of putative specificity for human IgG allotypes and isoallotypes were evaluated for reactivity and specificity in 8 different assay systems. The study showed that the MAbs tested could be classified into 1 of 4 groups: those exhibiting allotypic specificity regardless of the assay system, allotypic specificity dependent on the assay system, isoallotypic specificity, and those showing neither allotypic nor isoallotypic specificity. These observations were presumably dependent on antigen presentation, epitope integrity and/or antibody multispecificity. For the G1m(a), G1m(f), G1m(z), G3m(g) and G3m(u) specificities, MAbs have been produced which can be used for routine typing purposes in defined haemagglutination and enzyme-linked immunosorbent assay systems. MAbs are also available that show 'non-g' isoallotypic specificity.

Gm haplotypes in inflammatory demyelinating polyneuropathies.

The possible association of Gm haplotypes with inflammatory neuropathies has been studied in 59 patients with Guillain-Barré syndrome and 55 patients with chronic inflammatory demyelinating polyradiculoneuropathy. The frequency of Gm haplotype 1,2,17;21 (or z,a,x;g) was significantly raised in the patients with Guillain-Barré syndrome. These findings provide further evidence for an association of chromosome 14 genes with inflammatory neuropathies.

Molecular and serologic analysis of IgG1 deficiency caused by new forms of the constant region of the Ig H chain gene deletions.

Selective IgG1 deficiency is a rare disease. We report a familial form of IgG1 deficiency, in which IgG1 was undetectable in a 5-yr-old girl with a history of asthma and respiratory tract infections. Her father had an IgG1 level that was one-third of the mean amount found in normal healthy controls. The defect in the proband was caused by a homozygous deletion of the structural gene for C gamma 1. A Southern blot analysis demonstrated that the maternal haplotype contained a deletion encompassing C gamma 1, C psi epsilon 1, C alpha 1, C psi gamma, and C gamma 2, whereas the deletion on the paternal haplotype was confined to the C gamma 1 gene. Neither of these deletions has previously been reported. IgG1 normally constitutes the dominant isotype for antibodies directed against protein Ag, including viral proteins. We have analyzed the immune response to a number of different protein and polysaccharide Ag in the patient and her parents. In the proband, antiviral antibodies were restricted to the IgG3 and IgG4 subclasses. However, the total amount of IgG directed against several viruses was below the concentration found in normal seropositive individuals. The father and the paternal grandfather, both with low serum IgG1 levels, also had asthma, thus indicating a possible causal relationship.

Gm and Km frequencies in West Germans: usefulness of typing Gm(1,2,3,10,21) and Km(1,3) in paternity cases.

The immunoglobulin allotypes G1m(1,2,3) and G3m(10,21) were typed in 2855 unrelated West German adult individuals. 1455 individuals were typed for the factors Km(1,3). Phenotype and haplotype frequencies are reported. The usefulness of this routine typing programme in paternity tests is demonstrated in three case reports.