A monoclonal mouse antiallotype antibody reacts with certain human and other vertebrate immunoglobulins: Genetic and phylogenetic findings

A new mouse monoclonal antibody, 18.1, recognizes an allotypic determinant on mouse IgG₁ of the a allotype. We found that this alloantibody reacts with immunoglobulins of evolutionary distant vertebrate species including man, and with only certain isotypes or allotypes in some of these species. Most mammalian sera are reactive, except those from lagomorphs, marsupials, and monotremes. Avian and reptilian sera are also positive, while the amphibian and fish sera tested were negative. In human sera, 18.1 detects an isotypic marker on IgG2 and an allotypic marker on IgG3. This reactivity parallels the distribution of the Gm non-g determinant. These findings are discussed in relation to the phylogeny of IgG and its subclasses.     

Localization of murine Igh-1a allotypic determinants by using a panel of mouse myeloma variant immunoglobulins

A number of monoclonal antibodies are available that are reactive with distinct mouse immunoglobulin allotypic determinants. By determining which ones are present on a panel of hybrid IgG2b-IgG2a immunoglobulins, we have localized some of the allotypic determinants present on the IgG2a heavy chain of the "a" allotype (Igh-1a proteins). In particular, one group of determinants--Ig(1a)9.8 (20.6B8), 17.2 (20.19.2), and 14.4 (21.74.4)--has been placed in the CH2 domain. A second group--Ig(1a)8.3 (20.8.3), 21.2 (20.11.2), and 15.3 (21.66.3)--is located in a segment spanning the C terminal 8 residues of the CH2 domain and the complete CH3 domain.     

The structural basis of rabbit VH allotypes: serologic studies on a1 H chains with defined amino acid sequence.

The amino acid sequences for the VH regions of three homogeneous antibodies elicited by type III pneumococcal vaccine were determined. All three antibodies had the group a allotype a1. Two of the antibody H chains (3372, 3381) had identical amino acid sequences in all framework positions that are considered correlates of the VH allotype, whereas the third H chain (3T72) differed from these at positions 15 and 16. The a1 allotypic specificities of the three homogeneous antibodies were compared by quantitative radiobinding and inhibition assays by using both insolubilized anti-a1 antisera and allotypic antiserum fractions rendered specific for the homogeneous antibody 3374. It was found that antibodies 3374 and 3381 are allotypically indistinguishable and have in common an a1 allotypic specificity that predominates in pooled a1 IgG. The allotypic specificity of the 3T72 antibody, on the other hand, was markedly deficient to those of 3374, 3381, and the a1 IgG pool. This correlation of allotypic difference with amino acid sequence variation at position 15 and 16 of the H chain indicates the involvement of these two residues in a major a1 allotypic determinant.     

Rabbit latent group a allotypes: characterization and relationship to nominal group a allotypic specificities.

Latent group a allotypes were detected with a sensitive radioimmune inhibition assay. Sera, IgG preparations, and antibody fractions containing these allotypes inhibited the binding of insolubilized allotypic antisera to various radiolabeled antigens including IgG pools, homogeneous antibodies, and, in the case of a3, a VH fragment from a3/b4 IgG. Several different group a antiallotypic sera were used in the assays and all gave similar results. Comparison of inhibition curves for nominal and latent allotypes indicated that the full spectrum of allotypic subspecificities may be expressed in latent allotypes. Hemagglutination studies carried out with five sera containing high levels of latent allotypes confirmed the results obtained with the radioimmunoassay and indicated that inhibition values did not, at least in four of the five samples studied, reflect the presence of antiallotype antibodies.     

Immunoelectron microscopic localization of idiotypes and allotypes on immunoglobulin molecules

We have developed a novel approach to the analysis of antigenic (allotypic and idiotypic) determinants on intact immunoglobulin molecules. Immune complexes composed of IgG in combination with anti-idiotype or anti-allotype antibody were "visualized" by transmission electron microscopy. Individual Fab fragments of anti-idiotype or anti-allotype antibody, when bound to the IgG, altered the "Y" configuration in a reproducible and interpretable manner. Anti-idiotype antibody (either as Fab or IgG) bound to the terminus of the presumed V region of the IgG molecule, thus extending the apparent length of the Fab arms. Analysis of a rabbit VH framework allotype (a1) revealed that the determinant(s) is (are) located on the lateral portion of the V region of IgG. Binding of the anti-a1 Fab fragments was always at approximately right angles to the axis of the Fab arms of IgG. Fab antibody to the rabbit kappa light chain (b4) allotype bound to the lateral portion of the terminal half of the IgG Fab arms. This technique should be of value in localizing less well defined immunoglobulin determinants.     

Comparative analysis of the specificity of poly- and monoclonal antiallotypic antibodies and allotype-recognizing T-lymphocytes in rats

Using concurrent solid-phase radioimmunoassay, it has been shown that rat immunoglobulin k chain, Igk-lb allotype, is represented by a single serologically defined determinant. Anti-Igk-lb T cells do not recognize this determinant and poly- and monoclonal anti-Igk-lb antibodies show no influence on Igk-lb-specific proliferative T-cell response in vitro to IgG (Igk-lb)-pulsed splenic antigen-presenting cells. It is suggested that T cells recognize allotypic determinant(s) of processed Ig (Igk-lb) molecule.     

An allotypic specificity presumably of the a series in rabbit immunoglobulins, different from a1, a2, and a3

From the serum of a wild rabbit lacking all the known allotypic specificities of the a series, IgG showing an allotypic specificity named. A100 has been isolated and antisera against it prepared in domestic rabbits. The determinants responsible for the A100 allotypic specificity are present both on IgG and IgM. They are located on the heavy chain and the Fab fragment of IgG.Evidence for the genetic determinism of A100 suggests that it is the product of a new allele at the $\text{a}$ locus.     

A reverse hemolytic plaque assay for the detection and the enumeration of immunoglobulin allotype-secreting cells

We describe a reverse hemolytic plaque assay to enumerate rabbit immunoglobulin allotype-secreting cells. This technique makes use of sheep red blood cells (SRBC) sensitized with goat anti-rabbit IgG and rabbit anti-allotypic sera as revealing antisera. We have used the assay to compare at the IgG molecule level and at the immunoglobulin allotype-secreting cell level, the preferential expression (pecking order), in heterozygous rabbits, of one of the two alleles either at the a or the b locus, respectively, governing the a series allotypic specificities carried by the variable region of the heavy chains and the b series allotypic specificities essentially carried by the constant region of the K light chains.     

Surface immunoglobulin on rabbit lymphoid cells. III. Double expression and separate endocytosis of surface immunoglobulin allotypes on heterozygous lymphocytes demonstrated by immunoelectron microscopic labeling.

The expression of immunoglobulin b locus (k chain) allotypes on the surface of rabbit peripheral blood lymphocytes (PBL's) is examined using an indirect double immunoelectron microscopic labeling technique. Ferritin and whelk hemocyanin individually conjugated to allotypically specific IgG are used as ultrastructurally identifiable molecular markers. These indicators are coupled to lymphocyte surface immunoglobulin (Ig) allotypic determinants by an antiallotype antibody linkage. Human red blood cells, conjugated with IgG of a specific allotype and used as test cells, demonstrate the absolute specificity and high efficiency of the ultrastructural labeling technique. Specific labeling on rabbit PBL's shows that 65–75% of the cells are positive for surface Ig. Lymphocytes from homozygous donors (b4b4 or b6b6) are labeled specifically with only the appropriate allotypic labeling system. Thirty-three percent of the PBL's from heterozygous donors (b4b6) express both allotypes (allelic inclusion) on the cell surface; the remaining proportion of Ig-bearing cells have only one detectable allotype present (allelic exclusion). We conclude that approximately 50% of the Ig-bearing PBL's demonstrate allelic inclusion for the b locus allotypes. On allelically included heterozygous lymphocytes, both allotypic determinants can undergo specific endocytosis. Endocytosis of one allotype on heterozygous cells can be induced by stimulation with antiallotypic serum without affecting the surface appearance of the other allelic marker (separate endocytosis).     

Monoclonal antibodies against the human acetylcholine receptor

Monoclonal cell lines synthesizing antibodies against partially purified acetylcholine receptor from human muscle (H.AChR) were produced. Eleven clones secreted antibodies against H.AChR. Four were obtained in ascitic form. Two of them have been exhaustively studied. Specificity and affinity for H.AChR were demonstrated. Cross-reactivity with mouse AChR was shown but not with torpedo or porcine AChR at the same concentration. Purified IgG injected intravenously provoked an obvious muscular weakness. Inhibition experiments on myasthenia gravis sera binding have demonstrated that monoclonal antibody specificity is directed against an antigenic determinant shared by human and mouse AChR.     

Human immunoglobulin allotypes: Possible implications for immunogenicity

More than twenty recombinant monoclonal antibodies are approved as therapeutics. Almost all of these are based on the whole IgG isotype format, but vary in the origin of the variable regions between mouse (chimeric), humanized mouse and fully human sequences; all of those with whole IgG format employ human constant region sequences. Currently, the opposing merits of the four IgG subclasses are considered with respect to the in vivo biological activities considered to be appropriate to the disease indication being treated. Human heavy chain genes also exhibit extensive structural polymorphism(s) and, being closely linked, are inherited as a haplotype. Polymorphisms (allotypes) within the IgG isotype were originally discovered and described using serological reagents derived from humans; demonstrating that allotypic variants can be immunogenic and provoke antibody responses as a result of allo-immunization. The serologically defined allotypes differ widely within and between population groups; therefore, a mAb of a given allotype will, inevitably, be delivered to a cohort of patients homozygous for the alternative allotype. This publication reviews the serologically defined human IgG allotypes and considers the potential for allotype differences to contribute to or potentiate immunogenicity.Key words: human IgG, polymorphisms, IgG allotypes, antibody therapeutics, immunogenicity, anti-therapeutic antibody, IgG glycosylation     

Identification of goat allotypic determinants and generation of goat-mouse hybridomas secreting goat IgG2

Five allotypic determinants controlled by independent genes have been identified in goat. Of these determinants, four have been detected with alloimmune antisera and one with monoclonal antibodies. The specificities A1, C1 and D1 are lipoproteins; B1 is possibly an alpha 2 macroglobulin and E1 and IgG2. The specificity B1 is not expressed until the age of 3-4 months. The gene controlling the specificity E1 is present at about the same frequency (0.38-0.41) in goat, sheep, cattle and water buffalo. Stable hybridomas secreting goat IgG2 have been obtained by the fusion of goat peripheral lymphocytes with mouse myeloma cells.     

Evidence for two B-cell alloantigen loci in theHLA-D Region

Antigenic determinants recognizable by human antisera (Hon 7 and 2075abs sera) were found in a partially purified antigen preparation obtained from an HLA-D and -DR homozygous cell line (EBV-Wa). Sequential coprecipition tests showed that two determinants detectable with Hon 7 and 2075abs sera (Hon 7 and 2075abs determinants) were present on different molecules. These two antigenic determinants were shown to be allotypic and were expressed predominantly in the B-cell-rich fraction. Family studies showed that both antigenic determinants segregated concordantly with respectiveHLA haplotypes. In the population study, the 2075abs and Hon 7 determinants were shown to be in strong linkage disequilibrium and the 2075abs determinant perfectly correlated with the HLA-DRw4 specificity. The results indicate that the Hon 7 determinant is coded for by a gene distinct from alleles at theHLA-DR locus. Furthermore, the locus (Hon 7) coding for the Hon 7 determinant is suggested to be very closely linked with theHLA-DR locus.     

Complement C1q binding affects spin-labeled heterosaccharides of rabbit antibodies in immune but not artificial immunoglobulin G aggregates

IgG anti-hapten antibodies were purified from the sera of rabbits homozygous for allotypic determinants d11 and d12 in the constant region of the heavy chain. Correlative with this determinant is the absence (d11) or presence (d12) of an oligosaccharide chain just below the hinge region of the IgG molecule. Both d11 and d12 molecules contain a complex heterosaccharide chain located near the carboxyl terminus of the second constant region domain. The two populations of IgG antibodies were thus selectively labeled with the spin probe Tempamine in their second constant region domains by reductive amination primarily of terminal N-acetylneuraminic acid residues. Chemical and enzymatic cleavages showed about 80% of the attached spin labels were N-acetylneuraminic acid-associated. Analysis of probe adducts by ESR spectrometry showed the presence of slower and faster moving subcomponents. Formation of immune complexes by antigen induces slight but significant restrictions of spin label mobility for both d11 and d12 IgG molecules. This restriction is qualitatively different from that seen in glutaraldehyde-, carbodiimide-, or ethanol-induced aggregates of the same IgG antibodies. The addition of purified complement C1 subcomponent C1q to immune aggregates resulted in marked immobilization of spin labels, the rotational correlation time of which was 30-40 mu s for both d11 and d12 molecules (evaluated by saturation transfer spectroscopy). A similar spin probe immobilizing effect is not seen when C1q binds to chemically aggregated IgG antibodies (which also do not activate C1). A novel model is proposed in which C1q is hypothesized to juxtapose Fc moieties in a discrete fashion required for subsequent C1 activation processes mediated by immune complexes.     

Allotype-related sequence variation of the heavy chain of rabbit immunoglobulin G

The heavy chain of rabbit immunoglobulin G exists in three major allotypic patterns, Aa1–Aa3. A comparison of the amino acid compositions of the heavy chains isolated from immunoglobulin IgG homozygous for each allotypic determinant revealed the presence of an additional methionine residue per chain in the Aa3 allotype relative to the Aa1 and Aa2 allotypes. The position of the additional methionine residue was determined by cyanogen bromide cleavage and by tryptic digestion of the γ-chains; it coincided with the inter-Fd–Fc area of the chain. Isolation and characterization of the corresponding tryptic peptides of 31 amino acid residues from each of the allotypes showed the presence of a methionine-for-threonine replacement in the Aa3 allotype, but only in about 70–80% of the molecules. No other allotypic variations were seen in this tryptic peptide. Allotypically related variations in composition were also detected in the N-terminal cyanogen bromide-cleavage peptide.     

Serum Antigens of Cattle. IV. Immunogenetics of a Soluble Antigenic Determinant Derived from Lysed Erythrocytes

This paper describes an allotypic system in cattle called Ec (Erythrocyte cattle). The antigenic determinant is derived from red cells and is detected in lysates by gel precipitation with alloimmune serum. The Ecl specificity is controlled by an autosomal dominant gene and occurs with variable frequencies in different cattle breeds. The Ec system is a very useful genetic marker because the homozygous and heterozygous genotypes show a dosage effect and can be distinguished by the size of the precipitin rings in double diffusion gel precipitation tests.     

Heavy chain variable region allotypic sub-specificities of rabbit immunoglobulins. I. Identification of three subpopulations of a1 IgG molecules.

Four anti-al Ab subpopulations were isolated from an anti-al antiserum by sequential immunoadsorption chromatography. These four anti-al Ab subpopulations were differentially bound by two "limited heterogeneity" Abs having different components of the al allotypic specificity. Each of the four anti-al Ab subpopulations reacted with al IgG molecules obtained from a2 and a3 rabbits. A subpopulation designated anti-al Ab reacted with 100% of al IgG molecules. Thus, the anti-al-A Ab recognizes al determinants common to all al IgG molecules. Each of the other three subpopulations, designated anti-al-B Ab, anti-al-C Ab, and anti-al-D Ab, reacted with only a fraction of the al IgG molecules but the sum of the percentages of al IgG molecules which reacted with each of these three anti-al Ab subpopulations approximated 100% of the al IgG molecules. Thus each of the anti-al-B Ab, anti-al-C Ab, and anti-al-D Ab recognizes non-common determinants distinct for each of three subpopulations of al IgG molecules. Although 65 to 90% of IgG molecules in al homozygous rabbits have the al allotypic specificity, these IgG molecules are heterogeneous with respect to their antigenic determinants comprising the al allotype; at least three kinds of al IgG molecules are identified. This heterogeneity probably reflects variation in the amino acid sequence of the Vh region of al IgG molecules and, therefore, poses a similar argument which had led to the hypothesis of two genes for one polypeptide chain and to the theory of episomal insertions for the genetic control of immunoglobulin synthesis.     

Mouse immunoglobulin allotypes: multiple differences between the nucleic acid sequences of the IgE a and IgE b alleles

To clarify the allotypic difference of the IgE antibody molecule, we determined the complete nucleotide sequence of the genes encoding the constant portion of mouse IgE of a (BALB/c) as well as b (B10.A) allotypes. A comparison of the sequences revealed that there were 12 single-base changes: 2 single-base changes in CH1 and CH2, 3 in CH3, and 7 in CH4. Five of them were silent changes, but seven resulted in amino acid substitutions. Although the silent changes are scattered through CH1 to CH4, the nonsilent substitutions were found only in CH3 (two substitutions) and CH4 (five). The allotypic determinant(s) that conventional antisera detect most likely reflects an amino acid difference(s) in CH3 and/or CH4.     

Quantitative measurement of mouse IgG subclasses with the use of heteroantisera: the importance of allotype considerations.

Double antibody radioimmunoassays have been used to determine the quantities of IgG1, IgG2a and IgG2b in samples of normal serum IgG from BALB/cJ, AKR/J and C57BL/6J inbred mice. The assays employed subclass-specific goat antisera which had been prepared with BALB/c myeloma proteins as immunogens and as immunoabsorbents. 125I-labeled BALB/c myeloma proteins were used as probes. Results indicate that partial resolution of mouse IgG subclasses was achieved by ion exchange chromatography on DEAE-Sephadex. Nearly all of the protein in BALB/cJ and AKR/J IgG fractions could be accounted for as IgG1, IgG2a and IgG2b, and IgG2a was the predominant species observed. However, considerably less protein in C57BL/6J IgG fractions of purity similar to the BALB/cJ fractions could be accounted for as these three subclasses, and virtually no IgG2a was detected. Furthermore, an IgG2a myeloma protein bearing the C57BL/6 allotype failed to inhibit the IgG2a-specific assay significantly. Thus the IgG2a-specific antibody in the goat heteroantiserum employed appeared to consist nearly exclusively of antibody to BALB/c Ig-1a allotypic determinants. These findings point to the importance of allotype considerations in the use of heteroantisera to quantitate IgG subclasses.     

Mouse Ly-31.1 is an alloantigenic determinant of alkaline phosphatase predominantly expressed in the kidney and bone

The mouse lymphocyte surface alloantigen, Ly-31, defined by monoclonal antibody N1.10 (IgG2b, k) and controlled by a gene locus closely linked to theAkp-2 locus on chromosome 4, was biochemically investigated. By employing a quantitative immunoassay system, it was found that the Ly-31.1-specific antibody detected an allotypic determinant of mouse alkaline phosphatase. Ly-31.1, i. e., mouse alkaline phosphatase, was expressed predominantly in kidney and bone and was also detected in placenta, lung, and testis. Concerning tumor cell lines, they varied in the amount of antigen present, with both T and B lymphoid lineages selectively possessing the antigen. In normal lymphoid tissues, lesser amounts of antigen were detected. The binding of mouse alkaline phosphatase to Ly-31.1-specific monoclonal antibodies was specific in nature. The Ly-31.1 antigen was immunoprecipitated from the lysates of surface-radiolabeled YAC-1 moloney leukemia cells, and appeared as a single band of about 78 000 under both reduced and nonreduced conditions on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, treatment of tumor cell lines with phosphatidylinositol-specific-phospholipase C resulted in the removal of Ly-31 antigen from the cell surface. These results suggest that a gene cluster containing theLy-31 andAkp-2 loci which control the alkaline phosphatase is formed on mouse chromosome 4. The Ly-31 antigen is the first enzyme demonstrated to be a lymphocyte surface alloantigen.