The Production of High Affinity Monoclonal Antibodies to Human Chorionic Gonadotropin and Their Application to Immunoradiometric Assay

Abstract

Production of monoclonal antibodies against hCG has been studied using hCG as the antigen. This study reports the successful isolation of hybrid clones secreting monoclonal antibodies specific for hCG with an affinity constant higher than 10¹⁰M^?l. Of 23 fusions, only 17 fusions have produced positive clones which secrete antibodies giving high levels of binding with ^l25I-labelled hCG in the supernatant. Finally, 6 different monoclonal antibodies have been isolated; 4 of them, specific for the β-subunit, with a Ka approximately 1.1–4.0 ± 10¹¹M^?1 and 2 others, specific for the α-subunit, presenting an affinity of 2.5 ± 10¹⁰M^?l. When the antibodies specific for the β-subunit are used, specific and highly sensitive radioim-munoassays are obtained after only 3 hrs of incubation. Using iodinated monoclonal antibodies specific for the α-subunit and tubes coated with antibodies against the β-subunit, we have developped sensitive immunoradiometric assays.     

Monoclonal Antibodies Against Human Chorionic Gonadotropin (hCG): II. Affinity and Ability to Neutralize the Biological Activity of hCG

ABSTRACT: Monoclonal antibodies (MCA) against hCG have been characterized with regard to their affinity and their ability to neutralize the biological activity of hCG in vivo. The production and specificities of these reagents were described in the preceeding paper of this series. Equilibrium association constants (K_a) of the MCA, determined by radioimmunological saturation assays, ranged from less than 1 × 10⁸M⁻¹ up to 3.7 × 10⁹M⁻¹ whereas values for conventional polyclonal antisera against hCG ranged from 8.9 × 10⁹M to 1.8 × 10¹⁰M⁻¹. The ability of MCA to neutralize the biological activity of hCG was tested in a rat bioassay in vivo; 9 of 13 different MCA preparations tested could neutralize hCG. Surprisingly, this property did not correlate with affinity or specificity, and was not restricted to those MCA recognizing the hormone specific β-subunit. It could be demonstrated that determinants on each individual subunit as well as epitopes formed by both subunits are involved in the expression of the biological activity of hCG.     

Relative location of epitopes involved in synergistic antibody binding using human chorionic gonadotropin as a model

We systematically screened a large panel of well-characterized monoclonal antibodies (mAb) directed towards various epitopes on human chorionic gonadotropin (hCG) for synergistic binding of ¹²⁵I-hCG when they were adsorbed to a solid phase. The epitope locations involved in synergy were then related to the crystal structure of hCG and discussed in accordance with available data on the hCG epitopes. Enhanced binding of hCG was specific for certain pairs of mAb and was reflected in a 3–50-fold increased apparent functional affinity constant for hCG. Surface plasmon resonance revealed that when the mAb were captured by a polyclonal anti-IgG1 coupled to the Biacore chip, the off rates for hCG were significantly slower with synergistic mAb combinations than for the corresponding single mAb or nonsynergistic pairs of mAb, whereas the on rates did not differ appreciably. Each of the two antibodies involved in synergistic binding of hCG (more than 3-fold compared to additive binding of the two mAb) always belonged to a different epitope cluster in a separate antigenic domain on hCG. Synergistic epitope combinations on holo-hCG were located in similar structural planes. Combinations of mAb directed towards the epitope clusters α₂/β_3/5, α₂/hCGβ_CTP (C-terminal peptide) and β_3/5/hCGβ_CTP showed the strongest enhancement, with binding more than 10-fold greater than the sum of ¹²⁵I-hCG bound to the individual mAb, followed by pairs of mAb directed towards the epitope groups β₁/β_3/5, c_1/2/β_3/5, β₁/α₂, and α₂/α_3/5 (3–9-fold). The greater frequency of synergy obtained with the linear epitopes of the hCGβ_CTP can be ascribed to their greater molecular flexibility relative to the constrained discontinuous epitopes on hCGα and core-hCGβ (residues 1–112). In general, these studies provide a method for rapid screening of synergistic antibody pairs which also helps to identify non-overlapping epitopes that are accessible in similar structural planes. In turn, this facilitates the design of high-affinity bispecific antibodies targetted to a single antigen molecule.     

A rapid method for determining whether monoclonal antibodies react with the same or different antigens on the cell surface

A quick and simple method for determining whether monoclonal antibodies react with the same or different antigens is described which utilises an indirect radioactive binding assay against cells. Six antibodies were selected, BK19.45, BK20.20, GenOx4.17, GenOx4.21, W6-34 and W6-46, which detected antigens present either only on leukocytes (BK19.45 and BK20.20) or on a wider range of cell types including fibroblasts, liver cells and neuroblastoma cells. The saturation binding levels for each antibody, in terms of the amount of ¹²⁵I-anti-mouse immunoglobulin bound, were determined with respect to a fixed number of cells. The addition of two antibodies with different specities (BK19.45 or BK20.20 to either W6-34 or W6-46) resulted in a higher plateau value of ¹²⁵I-anti-mouse immunoglobulin bound per fixed number of cells than for either antibody singly. The increase in the amount of antibody bound is equivalent to the sum of the two individual saturation binding levels. In contrast, the addition of BK20.20 to BK19.45 or W6-45 produced no detectable rise in the saturation level. From these data it is concluded that BK20.20 and BK19.45, and W6-34 and W6-46 are bound to either identical epitopes which are in very close spatial relationship. On the other hand 19.45 and W6-34, as expected, detect unrelated antigens. Observations from autoradiograph binding studies on the semi-quantitative distribution on bone marrow cells of the antigens recognised by 19.45 and 20.20 supported the conclusion that the antibodies were recognising identical antigens.A study of the antibodies to HLA (2A1) and β₂ microglobulin (M3) showed an increase in the saturation level, when both antibodies were added together, which was less than the sum of the two individual saturation binding levels. The close association of β₂ microglobulin and HLA on the cell surface may to some extent prevent independent antibody binding. These data suggest that the above approach will differentiate between monoclonal antibodies which detect antigenic determinants that are located on closely associated surface molecules.     

Characterization of high-affinity antifluorescyl IgM antibodies

High-affinity IgM rabbit antibodies were elicited using the fluorescein hapten system. Purity and identification of IgM and IgG anti-fluorescyl antibodies was determined by immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis. Ultracentrifugation studies verified that liganded antibodies were of a high mol. wt (901 kd).Comparative analyses of IgM and IgG anti-fluorescyl antibody active sites substantiated the observation that purified IgM preparations, similar to their IgG counterparts, possessed high-affinity antibody active sites. Dissociation rate data confirmed that some IgM molecules within the purified antibody populations possessed association constants at 10¹¹M^?1, comparable with values of 10¹¹M^?1 or greater obtained for some anti-fluorescein IgG populations studied in this laboratory. A diffusion-controlled association rate of 4.6 × 10⁸M^?1 sec^?1 (Levison et al., 1971) was assumed in dissociation rate calculations. This is the first report of purified IgM antibodies possessing exceptionally high affinities.     

The Complement—Fixing Activity of Immune Complexes Containing IgG Antibodies of Different Functional Affinities: Effects on Superoxide Production by Rabbit Neutrophils

When neutrophil phagocytes are stimulated by IgG containing immune complexes (IgG‐IC), with or without the participation of the complement system, they show a sharp increase in oxygen uptake and begin to release large quantities of superoxide anions (O₂^?) and hydrogen peroxide (H₂O₂) into the surrounding medium. The aim of the present investigation was to provide insights into the production and release of O₂^? by rabbit neutrophils activated with immune complexes (IC) containing IgG antibodies of different functional affinity, opsonized and not opsonized by complement system components. For this purpose, two populations of polyclonal anti‐ovalbumin (OVA) IgG antibodies with different functional affinity, 5 × 10⁸ M^{? 1} and 2 × 10⁷ M^{? 1}, were prepared. The production of O₂^? was measured spectrophotometrically by a method using the superoxide dismutase‐inhibited reduction of ferricytochrome C to the ferrous form. The activation of complement by different IgG‐IC was determined by estimating the total residual haemolytic activity of the alternative and classical pathways in sera treated with different concentrations of anti‐OVA IgG/OVA immune complexes formed at equivalence. The results showed that: 1) antibody functional affinity influenced O₂^? production and the complement‐fixing activity induced by the IC. In general, the higher functional affinity antibodies were more efficient in stimulating the respiratory burst of neutrophils and in activating complement by the classical and alternative pathways than the lower functional affinity antibodies at all IC concentrations tested; 2) complement components incorporated into the immune complex lattice caused an increase in the stimulatory activity of both IgG antibodies to produce O₂^? (? 15% for the IC of IgG with K_a = 5 × 10⁸ M^{? 1} and ? 7% for the IC of IgG with K_a = 2 × 10⁷ M^{? 1}). This effect was dependent on antibody affinity and concentration; 3) there was a direct relationship between the overall level of complement activation, antibody affinity and superoxide production by neutrophils. Thus, we conclude that antibody affinity influences immune complex lattice formation, modulating its three‐dimensional structure and the disposition of Fc fragments interfering with the antibody's biological properties. These results can help understand the precise role of antibody functional affinity in antigen‐antibody complex diseases and define the immunochemical characteristics of pathogenic complexes.     

Prevalence of high affinity naturally occurring IgG2 antibodies against human chorionic gonadotropin and its subunits in patients with ovarian cyst

Naturally occurring antibodies to tumour antigens are gaining interest as clinically important cancer biomarkers for early diagnosis, prognosis and for the development of anti-cancer therapeutics. The glycoprotein αβ heterodimer hormone human chorionic gonadotropin (hCG) and its β subunit (hCGβ) are produced by various cancers, and their increased serum levels correlate with poor prognosis. We have previously reported that patients with benign ovarian cysts, but not the malignant tumours, were characterized by augmented serum levels of naturally-occurring IgG antibodies to hCG and hCGβ. Here we further characterise these antibodies in patients with ovarian cysts.IgG and IgM antibody binding to whole hCG, hCGβ, hCGα, hCGβ C-terminal peptide (hCGβCTP), and the hCGβ core fragment (hCGβCF) were measured in the sera from 36 patients with ovarian cysts and 12 healthy non-pregnant women using a standard ELISA. IgG subclass usage and affinity was also determined together with cross-binding to whole hCG and its subunits of four selected commercial monoclonal antibodies generated against ovarian cyst mucins.Our results showed that 91.7% of the sera tested contained elevated IgG, but not IgM antibodies to one or several antigens, with an overwhelming prevalence of high affinity IgG2 indicating their binding to carbohydrate epitopes and possibly ovarian cyst mucins. Anti-mucin commercial antibody ab212418 (Abcam) produced against Gal1-3GalNAc, exhibited strong cross-binding to hCGαβ, hCGβ, hCGα and hCGβCTP. The protective anti-cancer potential of these antibodies will be further investigated and could lead to the development of novel treatment strategies for ovarian cancer.     

The molecular relationship between antigenic domains and epitopes on hCG

Antigenic domains are defined to contain a limited number of neighboring epitopes recognized by antibodies (Abs) but their molecular relationship remains rather elusive. We thoroughly analyzed the antigenic surface of the important pregnancy and tumor marker human chorionic gonadotropin (hCG), a cystine knot (ck) growth factor, and set antigenic domains and epitopes in molecular relationships to each other. Antigenic domains on hCG, its free hCGα and hCGβ subunits are dependent on appropriate inherent molecular features such as molecular accessibility and protrusion indices that determine bulging structures accessible to Abs. The banana-shaped intact hCG comprises ∼7500 Å² of antigenic surface with minimally five antigenic domains that encompass a continuum of overlapping non-linear composite epitopes, not taking into account the C-terminal peptide extension of hCGβ (hCGβCTP). Epitopes within an antigenic domain are defined by specific Abs, that bury nearly 1000 Å² of surface accessible area on the antigen and recognize a few up to 15 amino acid (aa) residues, whereby between 2 and 5 of these provide the essential binding energy. Variability in Ab binding modes to the contact aa residues are responsible for the variation in affinity and intra- and inter-species specificity, e.g. cross-reactions with luteinizing hormone (LH). Each genetically distinct fragment antigen binding (Fab) defines its own epitope. Consequently, recognition of the same epitope by different Abs is only possible in cases of genetically identical sequences of its binding sites. Due to combinatorial V(D)J gene segment variability of heavy and light chains, Abs defining numerous epitopes within an antigenic domain can be generated by different individuals and species. Far more than hundred Abs against the immuno-dominant antigenic domains of either subunit at both ends of the hCG-molecule, the tips of peptide loops one and three (Ł1 + 3) protruding from the central ck, encompassing hCGβŁ1 + 3 (aa 20–25 + 64 + 68–81) and hCGαŁ1 (aa 13–22; Pro16, Phe17, Phe18) plus hCGαŁ3 (Met71, Phe74), respectively, have been identified in the two “ISOBM Tissue Differentiation-7 Workshops on hCG and Related Molecules” and in other studies. These Abs recognize distinct but overlapping epitopes with slightly different specificity profiles and affinities. Heterodimeric-specific epitopes involve neighboring αŁ1 plus βŁ2 (hCGβ44/45 and 47/48). Diagnostically important Abs recognize the middle of the molecule, the ck (aa Arg10, Arg60 and possibly Gln89) and the linear hCGβCTP “tail” (aa 135–145; Asp139, Pro144, Gln145), respectively. Identification of antigenic domains and of specific epitopes is essential for harmonization of Abs in methods that are used for reliable and robust hCG measurements for the management of pregnancy, pregnancy-related disease and tumors.     

The interaction of antibody with the major surface glycoprotein of vesicular stornatitis virus II. Monoclonal antibodies to nonneutralizing and cross-reactive epitopes of Indiana and New Jersey serotypes

Eleven monoclonal antibodies reactive with the major surface glycoprotein (G-protein) of vesicular stomatitis virus serotypes Indiana and New Jersey (VSV-Ind, VSV-NJ) were used to map antigenic sites found on one or both serotypes. The antibodies used were unable to neutralize infectivity of the virus in vitro although they were able to bind to the G-protein. Six of the antibodies bound to the G-proteins of both serotypes and delineated three nonoverlapping epitopes as determined by a competitive binding assay. In addition, one monoclonal antibody bound to both serotypes and could neutralize infectivity in vitro of only VSV-Ind. This antibody could compete with several cross-reactive nonneutralizing antibodies which could not neutralize either VSV-Ind or VSV-NJ. Three monoclonal antibodies were serotype specific for VSV-NJ and exhibited no overlap among themselves or with the cross-reactive antibodies. One VSV-Ind serotype-specific antibody was isolated which could compete with a cross-reactive antibody. Enhancement of antibody binding by the binding of a second antibody was observed in some cases. This phenomenon appeared to be due to an increase in availability of antigenic sites caused by allosteric modifications.     

Topological mapping of murine leukemia virus proteins by competition-binding assays with monoclonal antibodies

Monoclonal antibodies produced by hybrid cells in culture are chemically homogeneous reagents that react with constant avidity to single antigenic determinants (epitopes). As such, these antibodies are remarkably specific probes for small regions of complex antigenic proteins. We have used a panel of monoclonal antibodies in competition binding assays to investigate the arrangement of six epitopes on the envelope proteins of AKR leukemia virus. It was reasoned that if two epitopes were adjacent to each other on a single protein, the binding of antibody to one epitope would sterically hinder the binding of antibody to the other epitope. On the other hand, if the two epitopes were at distant sites on the protein, the binding of antibody to one epitope would not influence the binding of antibody to the other epitope. The results of these competition binding assays demonstrated the presence of two distinct antigen sites on both the gp70 and p15(E) envelope proteins. With the gp70 protein, one antigen site contained the gp70^b and gp70^c epitopes; the other antigen site on this protein contained the gp70^a epitope. With p15(E), one of the antigen sites contained the p15(E)^b and p15(E)^c epitopes, while the other site contained the p15(E)^a epitope. These findings demonstrate the utility of this type of serological analysis for the study of the tertiary structure of individual viral proteins.     

The complete amino acid sequence of the light chain variable region of two monoclonal anti-p-azobenzene-arsonate antibodies bearing the cross-reactive idiotype

Two monoclonal anti-p-azobenzene-arsonate antibodies produced by cell fusion of A/J spleen lymphocytes were selected. It had previously been shown that they both expressed the cross-reactive idiotype (CRI) and that the amino terminal sequence of their heavy chain variable region differed by only one amino acid substitution within the first 55 residues, at residue 41 in the second framework region. A novel, sensitive peptide-mapping method had indicated that the light chain of these two antibodies differed by at least three amino acid substitutions. Here, the complete amino acid sequence of the light chain variable region is presented. There are only 3 amino acid substitutions between the two antibodies, located in the first and second complementarity determining regions and the third framework region respectively. Although the variable region of the light chains of these two monoclonal antibodies show such a high degree of homology they differ by 26 and 27 substitutions from the reference sequence of the light chain of CRI⁺ anti-ABA serum antibodies. In addition, they are homologous to 4 other such CRI⁺ monoclonal antibody light chain sequences published so far, in which only 2 of the above 3 substitutions are not represented. The contribution of the light chain to the CRI is discussed.     

Antigen valence and Fc-localized secondary forces in antibody precipitation

A tetravalent dinitrophenyl hapten, 1,6,11,16-tetra-(?-N-Dnp)-l-lysine₁₆3 was synthesized. Upon interaction with specific antibody of moderate affinity (K_t = 6 × 10⁶ M^?1), a maximum of 80% precipitation occurred. Low pH or 2.5 M guanidine hydrochloride completely inhibited precipitation without significantly affecting hapten binding. Anti-Dnp (Fab')₂ fragments also formed precipitates when mixed with the tetravalent hapten, but these were not soluble in 2.5 M guanidine hydrochloride, and the precipitation itself occurred over a much narrower range of hapten/antibody ratios than observed with intact molecules. These results suggest that a site located in the Fc region assists aggregation and preciptation in intact antibody molecules.     

Investigation of interaction between two neutralizing monoclonal antibodies and SARS virus using biosensor based on imaging ellipsometry

Two neutralizing human scFv, b1 and h12 were identified initially using ELISA,employing highly purified virus as the coating antigen. The biosensor technique based on imaging ellipsometry was employed directly to detect two neutralizing monoclonal antibodies and serial serum samples from 10 SARS patients and 12 volunteers who had not SARS. Further, the kinetic process of interaction between the antibodies and SARS-CoV was studied using the real-time function of the biosensor. The biosensor is consistent with ELISA that the antibody h12 showed a higher affinity in encountering the virus than antibody b1. The affinity of antibody b1 and antibody h12 was 9.5 × 10⁶ M⁻¹ and 1.36 × 10⁷ M^{− 1}, respectively. As a label free method, the biosensor based on imaging ellipsometry proved to be a more competent mechanism for measuring serum samples from SARS patients and the affinity between these antibodies and the SARS coronavirus.     

Serological analysis of antigenic determinants on the env gene products of AKR dualtropic (MCF) murine leukemia viruses

A panel of eight monoclonal antibodies recognizing individual antigenic determinan (epitopes) on the envelope proteins (gp70 and p15(E)) of murine leukemia virus (MuLV) has been used to probe the serological properties of recombinant dualtropic (MCF) viruses. Since six of these monoclonal antibodies showed specificity for epitopes that were exclusive to endogenous N-ecotropic MuLV, it was reasoned that they might provide a means to determine the relative genetic contribution of ecotropic information in the env gene of dualtropic isolates. It was anticipated that the loss of ecotropic-specific sequences in these viruses would be reflected by a loss of reactivity with the ecotropic-specific monoclonal antibodies. This expectation proved to be correct, as several of the monoclonal antibodies that reacted with endogenous ecotropic MuLV of AKR mice failed to react with isolates of recombinant dualtropic MuLV from the same strain. In addition, it was noted that the “nonreactivity” of the monoclonal antibodies with different dualtropic viruses occurred in characteristic patterns. A common feature of the patterns was coordinate loss of expression of the gp70^a, gp70^d, and gp70^e epitopes from all AKR dualtropic MuLV. In contrast to the common deletion of ecotropic-specific epitopes, the gp70^b, gp70^c, and p15(E)^a epitopes were variably deleted in different recombinant viruses.     

HUMAN ‘Ia’ ANTIGEN POPULATIONS DEFINED BY MONOCLONAL ANTIBODIES

The relationships between the antigens recognized by four monoclonal anti-human ‘Ia’-like antibodies were investigated using sequential immunoprecipitation and capping techniques. Two of the antibodies were ‘monomorphic’ and have previously been shown to recognize epitopes in which carbohydrate residues are involved, whereas the two ‘polymorphic’ antibodies recognized protein-defined epitopes—one of these epitopes being present on MB⁺DR^- molecules. In the absence of an indisputable anti-DR monoclonal antibody, it was not possible to conclusively verify which ‘Ia’-encoded antigens were detected by the anti-‘Ia’-like monoclonal antibodies. Nevertheless, several firm conclusions could be drawn: (a) so-called ‘monomorphic’ antibodies do not necessarily react with all ‘Ia’ molecules encoded by a single locus—from the results using the two monomorphic antibodies, B5.1 and 3F1.1, described herein, two populations of antigens being B5.1⁺3F1.1⁺ and B5.1⁺3F1.1^- were identified; (b) cross-reactivity of a polymorphic determinant expressed on antigenically-separable ‘Ia’ molecules was noted—using the two polymorphic antibodies, 26.1 and F5C9, molecules which were 26.1⁺F5C9⁺ and 26.1^-F5C9⁺ were identified; and (c) the data clearly point to the existence of at least two loci coding for ‘Ia’-like antigens (one of which may or may not be the HLA-DR locus). Given that polymorphisms can now include protein- and carbohydrate-defined epitopes, that cross-reactions occur and that the definition of DR itself by monoclonal antibodies is not clear, the complexity of the human ‘Ia’ antgens is apparent.     

Neutralization of tetanus toxin by distinct monoclonal antibodies binding to multiple epitopes on the toxin molecule.

Fifty-seven hybridomas producing antibodies to tetanus toxoid or to the Ibc or B-IIb fragment of the toxin were isolated independently. Competitive inhibition studies demonstrated that monoclonal antibodies from mice immunized with the toxoid bound to at least 20 different epitopes on the toxoid molecule. Similar competitive binding studies revealed eight distinct epitopes on the B-IIb fragment and three to five epitopes on the Ibc fragment of the toxin. Neutralization of toxicity was effected by nine distinct monoclonal antibodies from hybridomas of toxoid-immunized mice and by one monoclonal antibody from B-IIb-immunized mice. Mixtures of two, three, and four different monoclonal antibodies in a variety of combinations exerted a synergistic effect of ca. 200-fold over that observed with individual monoclonal antibodies, indicating that efficient neutralization may involve the simultaneous binding of at least two antibody molecules to different specific regions of the toxin molecule. Only one toxoid-induced monoclonal antibody failed to bind to tetanus toxin. All neutralizing antibodies bound to epitopes on the heavy chain of tetanus toxin. Six of these were directed toward epitopes on the NH2-terminal half, whereas four bound to epitopes on the carboxy-terminal half of the heavy chain. Only one monoclonal antibody bound preferentially to the light chain, but two other monoclonal antibodies appeared to bind to both chains, indicating some homology between these two chains.     

Mapping of the binding sites of human histamine <Emphasis Type="Italic">N</Emphasis>-methyltransferase (HNMT) monoclonal antibodies

Objective

Recently, we characterized mouse monoclonal antibodies that allow the specific and sensitive detection of human histamine N-methyltransferase (HNMT). To understand differences in binding characteristics and recognition of enzyme variants we mapped the antibody binding sites.

Methods

Fragments of human HNMT were expressed as glutathione S-transferase fusion proteins that were used for testing antibody binding on immunoblots. Combined information from species cross-reactivity, sequence comparison, protein structure, and binding site prediction software were used to localize the epitope recognized by each antibody.

Results

All eight monoclonal HNMT antibodies bound to linear epitopes in the C-terminal domain of the 292 amino acid protein. Of the five antibodies cross-reacting with HNMT from other species, one bound region L¹⁸²–T²²³, three region M²²⁴–E²⁶¹, and one region L²⁶²–A²⁹². All three antibodies recognising only human HNMT bound the C-terminal region L²⁶²–A²⁹² that contains residues present only in the human protein.

Conclusions

Our HNMT monoclonal antibodies bind in three different regions of the protein and those binding the same putative epitope exhibit similar binding characteristics and species cross-reactivity. Antibodies binding non-overlapping epitopes will facilitate analyses of all clinically relevant variants described for HNMT.

     

Epitopes of Human Chorionic Gonadotropin and Their Relationship to Immunogenicity and Cross-Reactivity of β-Chain Mutants

PROBLEM: Human chorionic gonadotropin (hCG) is a placental glycoprotein hormone, a heterodimeric molecule, consisting of α and β chains. It induces the synthesis of progesterone, which is essential for the maintenance of the fertilized egg. Antibodies directed against hCG can, therefore, prevent pregnancy and serve as a vaccine. hCG belongs to the glycoprotein hormone family and shares the α chain with the other members. The β chain is a hormone-specific subunit that is unique to hCG, but still possesses 85% amino acid homology with the β chain of luteinizing hormone (LH), which means that prolonged immunization with hCG produces antibodies that cross-react with LH. METHOD OF STUDY: We have taken an approach involving the mutation of βhCG to eliminate cross-reactive epitopes without affecting the natural folding of the polypeptide chain and thus the unique βhCG-specific epitopes. RESULTS: Several mutants have been constructed that have maintained the binding to hCG-specific monoclonal antibodies (mAbs) but have lost the ability to bind to a panel of LH cross-reactive mAbs. To investigate the immunogenicity of selected mutants, mice were immunized with expression plasmid DNA, containing the gene for wild-type βhCG and two mutants: mutant 3, with four amino acid substitutions (68 Arg→Glu; 74 Arg→Ser; 75 Gly→His; 79 Val→His), and mutant 7, with a single amino acid substitution (68 Arg→Glu). CONCLUSIONS: Athough both mutants were able to elicit antibody responses in at least some animals, the levels were less than those seen with the wild-type βhCG DNA, and there seems still to be a residual cross-reacitivity with LH. Attempts to improve the immunogenicity of the mutants and to further modify the sequence to remove the cross-reacitivty are currently underway.     

Characterization of a Monoclonal Antibody that Binds to Apolipoprotein E and to Lipoprotein of Human Plasma Containing APO E. Applications to ELISA Quantification of Plasma APO E

Abstract

This study describes the development of an enzyme-linked immunosorbent assay for human apolipoprotein E (apo E).

A mouse monoclonal IgG₁ antibody named E01 against apolipoprotein E was selected from five antibodies secreted by hybridomas. This antibody had a high affinity for apo E ((K a 1.2 × 10⁷ L.M^?1 for purified apo E and K = 1.05 × 10⁷ L.M^?1 for native apo E in very low density lipoproteins) in liquid phase and recognized every isoform of apo E but not other proteins in VLDL. Competition experiments with ¹²⁵I apo E showed that its binding affinity for the E in every density class (VLDL, HDL, LDL) and in serum was the same.

This antibody was used for the quantification of human apo E in serum by enzyme linked immunoassay. E01 was coated on microtiter plates and a polyclonal peroxidase-conjugate was used as second antibody. A good correlation was observed between the values obtained for apo E using both monoclonal and polyclonal antibodies.     

Isolation and Characterization of Human Monoclonal Autoantibodies to Glutamic Acid Decarboxylase

Production of human monoclonal autoantibodies to glutamic acid decarboxylase M_r 65,000 (GAD₆₅), characterization of their isotype, binding affinity, V region sequences and competition with autoantibodies in patients' sera is described. Lymphocytes from a patient with Addison's disease who had GAD₆₅ autoantibodies without diabetes were immortalised and fused to a mouse/human hybridoma. In addition, mouse monoclonal antibodies to GAD₆₅ were produced using standard techniques. F(ab')₂s from our monoclonals and the GAD6 mouse monoclonal were used in competition with intact monoclonals and sera from diabetic patients for binding to ¹²⁵I-labelled GAD₆₅ (amino acids 46-586). Reactivities of the human monoclonals with GAD 65,000/67,000 M_r chimeras were also studied. Variable region genes of human monoclonals were sequenced and analysed. The human monoclonals (n = 3) had affinity constants for GAD₆₅ of 2.2 &#50 10⁹, 5.8 &#50 10⁹, 1.3 &#50 10¹⁰ mol/l^{&#109 1}; affinities of the mouse monoclonals (n = 5) ranged from 1.1 &#50 10⁸ to 5.4 &#50 10¹⁰ mol/l^{&#109 1}. The binding of each of the human monoclonals was inhibited by GAD6 F(ab')₂ and the binding of GAD6 antibody was inhibited by the human monoclonal F(ab')₂s suggesting that the epitopes for these antibodies were overlapping. Studies with GAD₆₅/GAD₆₇ chimeras indicated that the human monoclonals reacted with C-terminal epitopes. The human monoclonals, GAD6 and 3/5 mouse monoclonals inhibited serum autoantibody binding to ¹²⁵I-labelled GAD₆₅. Overall, the human monoclonals were of high affinity, reacted with C-terminal epitopes and showed evidence of antigen driven maturation; they represented only a proportion of the repertoire of autoantibodies to GAD₆₅ in the donor's serum and in the sera of patients with type-1 diabetes.