Number Of Antigenic Sites Research Articles

In vitro studies of the effect of MAb NDA 4 linked to toxin on the proliferation of a human EBV-transformed lymphoblastoid B cell line and of gibbon MLA leukemia cell line

The rejection of allografts is mediated by cytolytic T cells and antibody-secreting B cells. Selective ablation of these activated cells from peripheral blood lymphocytes may offer a method of controlling allograft rejection, An immunotoxin was prepared from the monoclonal antibody (mAb) NDA 4, which recognizes a differentation antigen (NDA 4) common to activated B and T cells. MAb NDA 4 was conjugated to the ribosome-inhibiting protein gelonin via a cleavable disulfide bond provided by a crosslinking reagent. The purified immunotoxin was evaluated for in vitro cytotoxicity on NDA 4 positive T and B cell lines, Conjugation of mAb NDA 4 to gelonin increased the in vitro cytotoxicity by a concentration factor of 1000, compared to gelonin alone. The specificity and saturability of mAb NDA 4 binding, as well as the number of antigenic sites per cell on resting versus activated T lymphocytes, were also evaluated. Resting T cells expressed 400–800 sites per cell. PHA-activated T cells and the MLA T cell leukemia expressed 10,000 to 80,000 sites per cell, Peripheral blood mononuclear cells obtained from allografted baboons in quiescence or undergoing rejection were compared for NDA 4 expression by flow cytometry. Lymphocytes obtained from baboons rejecting a heart allograft expressed NDA 4, whereas transplant recipients in quiescence showed no detectable NDA 4, These results suggest that mAb NDA 4-derived immunotoxins may be valuable for the selective depletion of activated lymphocytes while sparing the resting population.

Measurement of antibody affinity for cell surface antigens using an enzyme-linked immunosorbent assay

We present a fast, simple, and accurate method to determine the affinity constants of antibodies that bind to cell surface antigens. This procedure utilizes intact cells and native, unmodified antibody in a conventional enzyme-linked immunosorbent assay. Target cells are incubated with serial dilutions of antibody and allowed to reach equilibrium. Cells are then pelleted by centrifugation, and aliquots of unbound antibody in the supernatant are added to a microtiter plate precoated with capture antibody and measured in a conventional enzyme-linked immunosorbent assay (ELISA). We measured the affinity constant of murine monoclonal antibody CLB-1H-gran2, which binds to K562 cells (a human erythroleukemia line), and compared the ELISA-based results to those obtained by flow cytometric determination of antibody affinity. The affinity constants obtained by the two methods are in good aggreement. The affinity constant is calculated utilizing only the concentrations of bound and free antibody, so that the actual antigen concentration (or number of antigenic sites per cell) need not be known. However, the number of antibody molecules bound per cell can be estimated from the results.

Immunohistochemical detection of 1,25-dihydroxyvitamin D3 receptors and estrogen receptors by monoclonal antibodies: comparison of four immunoperoxidase methods.

We developed an immunohistochemical method for visualization of vitamin D (VDR) and estrogen receptors (ER) in cryostat sections, using monoclonal antibodies (MAb) to the vitamin D receptor and estrogen receptor, respectively. This method is based on an avidin-biotin labeling technique (LAB). To establish a reliable and sensitive method which can be used easily as a routine diagnostic procedure, we systematically compared four different immunoenzymatic methods with respect to their efficiency in detecting vitamin D and estrogen receptors. Compared to the indirect bridged avidin-biotin (IBRAB), the peroxidase- anti-peroxidase (PAP), and the avidin-biotin complex (ABC) methods, the LAB method produced stronger staining intensities and had higher detection efficiency for both vitamin D and estrogen receptors. In addition, the LAB method had a higher spatial resolution compared to the ABC technique in detection of VDR in normal human skin biopsies. In the case of steroid receptors, i.e., nuclear antigens, immunohistochemistry must deal with a relatively low number of antigenic sites per cell, restricted accessibility of the antigens, and slight differences in antigen concentrations among cells. Under these particular conditions, the chemical properties of the conjugates used in the LAB method may explain why it is superior to the other methods. Consequently, the LAB method is recommended for visualization of ER and VDR.

Functional and neutralization profile of seven overlapping antigenic sites on the HN glycoprotein of Newcastle disease virus: monoclonal antibodies to some sites prevent viral attachment

We have previously identified five antigenic sites on the hemagglutinin-neuraminidase (HN) glycoprotein of the Australia-Victoria isolate of Newcastle disease virus (Iorio and Bratt J. Virol. 48, 440–450; Iorio et al. J. Gen. Virol. 67, 1393–1403). Two additional sites (designated 12 and 23) are now described, bringing to a total of seven the number of antigenic sites defined by our panel of neutralizing anti-HN antibodies. Competition antibody binding and additive neutralization assays reveal that each of these newly-identified sites overlaps two previously-defined ones. The seven HN antigenic sites thus form a continuum in the three-dimensional conformation of the molecule. Studies on the inhibition of hemagglutination (HA), neuraminidase (NA) and the attachment of virus to chick cell monolayers have been used to construct a functional profile of each antigenic site. Monoclonal antibodies (mAbs) to three overlapping sites (12, 2 and 23) inhibit HA and NA and prevent viral attachment to chick cell monolayers. These findings are consistent with the domains recognized by these mAbs being close to the NA and receptor-binding sites. MAbs to two other overlapping sites, 14 and 1 (which in turn, overlap site 12), inhibit HA quite effectively, and attachment to a lesser extent. Sites 14 and 1 probably identify a second domain involved in receptor recognition. MAbs to the two remaining sites (3

Structural features of T-cell recognition: applications to vaccine design

T lymphocytes, in contrast to antibodies, appear to recognize primarily a limited number of antigenic sites on any given antigenic protein. We find that a single site can so dominate the T-cell repertoire that the presence or absence of a response to one immunodominant site can make the difference between a high responder and a low responder, even though low responders respond to other sites almost as well as high responders. Besides interaction with major histocompatibility complex (MHC) molecules, the mode by which the antigen is processed into fragments for T-cell recognition also determines which sites are seen. The products of natural processing of the protein appear to be larger than the synthetic peptides and contain structures which hinder binding to certain MHC molecules or to the T-cell receptor. A third factor in immunodominance is the intrinsic structure of the antigenic site. We have shown that amphipathic helices have a higher than random chance of being immunodominant, and have developed a computer program to locate such structures in protein amino acid sequences. We prospectively predicted sites in the malaria circumsporozoite protein and found that the four most widely recognized sites in an endemic area of West Africa were all predicted. Similarly, we identified two helper T-cell sites from the HIV (AIDS virus) envelope, and have now shown that immunization with these elicits enhanced antibody responses to the whole envelope when injected into monkeys. These sites are also recognized by human T cells from volunteers who had been immunized with a recombinant vaccinia virus expressing the HIV envelope. Also, because cytotoxic T lymphocytes (CTLS) may play a critical role in defence against AIDS, we have used a recombinant vaccinia virus and transfectants expressing the HIV envelope gene to induce specific CTLS against the HIV envelope. Using synthetic peptides, we were able to identify the first CTL recognition site in the AIDS virus. These results may contribute to the rational design of vaccines.

Binding parameters of monoclonal antibodies reacting with ovarian carcinoma ascites cells.

Binding parameters were determined for four mouse monoclonal antibodies reacting with three antigens on the surface of fresh human ovarian carcinoma ascites cells, under nearly physiological conditions. The object of these experiments was to aid in the selection of the optimal monoclonal antibodies for intraperitoneal immunotherapy. The number of antigenic sites per cell, the effective equilibrium association constant (affinity) and the half-life for dissociation were: for Ab MH99, 1.2 x 10(6) sites/cell, (1.9-4.1) x 10(8) M-1, and 4 h; for Ab MX35, (3.2-4.1) x 10(5) sites/cell, (3.4-4.8) x 10(8) M-1, and greater than 10 h; and for Ab MW207, 1.3 x 10(5) sites/cell, (3.6-4.1) x 10(9) M-1, and 3.1 h, respectively. One of the antigens, MH99, is recognized by five different monoclonal antibodies, and competitive inhibition experiments demonstrated that two distinct determinants are present; this antigen is also recognized by the previously described Ab 17-1A. These binding data will aid the rational design of immunotherapy strategies.

Characterization of the D, c, E and G antigens of the Rh blood group system with human monoclonal antibodies

The human MAbs, anti-D, -c, -E and -G of the Rh blood group system, produced by Epstein-Barr virus transformed B-cell lines, were purified by protein A-Sepharose chromatography and used to characterize the Rh antigens of human red cells. Scatchard plot analyses performed with the radiolabelled MABs indicated that each R 2R 2 red cell carries 0.43, 0.32 and 0.38 × 10 5 D, c and E binding sites, respectively. About half this number of antigen sites are present on erythrocytes from heterozygote individuals using the appropriate antibody. We found, however, that only 0.18 × 10 5 G antigenic sites were present on each R 1R 1 red cell. The affinity constants of the anti-D, -E and -G were similar varying from 0.6 to 1.5 × 10 8 M −1 whereas that of the anti-c was much lower (0.035 × 10 8 M −1). The blood group specificity and binding properties indicate that the MAbs behave like the polyclonal anti-Rh reagents. Immunoprecipitation experiments carried out with membranes from R 2R 2 red cells show that a 30–32 kDa component can be identified whatever the antibody used. The immune complexes involving anti-c, -E or -G antibodies could be formed with the detergent lysates from red cell membranes. In contrast, membrane integrity was a prerequisite for the binding of the anti-D antibodies. Finally, from extraction studies of immunocomplexes with non-ionic detergents it was concluded that all the Rh-active components are bound to the membrane skeleton, suggesting that these molecules may have important function for maintaining red cell shape and viability.

Transmissible gastroenteritis.

Transmissible gastroenteritis (TGE) virus, a coronavirus, causes an acute infection of the small intestine of the pig. The disease is rarely fatal in adult animals but can cause extensive mortality in the neonate. Since its first description in Britain in the 1950s, the disease has been epizootic but recently it has become established on some farms and an antigenically related respiratory virus has become endemic in the national herd over the past two years. Piglet immunity to TGE, which relies on passive protection from milk, has been impossible to achieve with vaccines and research has aimed at understanding the nature of the interaction between virus and the pig. Following infection of the pregnant sow, antibody-producing cells migrate to the mammary gland where they secrete virus neutralising antibodies into the milk; prospective vaccines will need to stimulate a similar response. The location and number of antigenic sites on the virus particle associated with neutralisation have been established with monoclonal antibodies and the role of the other viral genes in pathogenesis and immunity is being studied with genetic engineering techniques.

Human Monoclonal Antibody Against Rh(D) Antigen: Partial Characterization of the Rh(D) Polypeptide From Human Erythrocytes

A human monoclonal anti-Rh(D) antibody produced by an Epstein-Barr virus (EBV)-transformed B-cell line (IgG1(lambda), clone H2D5D2) has been purified on protein A-Sepharose column and used for binding studies and immune precipitation of the blood group rhesus (Rh) antigens. Scatchard plot analyses show that the 125I-labeled antibody (iodo-gen procedure), binds to 1.09 X 10(5), 0.43 X 10(5), and 0.32 X 10(5) antigen sites on each D--/D--, R2R2 and R1R1 RBC, respectively, with an association constant of approximately 0.6 X 10(8) mol/L-1. Immune precipitation studies indicate also that the Rh(D) antigen of the Rh(D)-positive RBCs is carried by a 29 kd polypeptide as deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). No material could be precipitated from Rh(D)-negative or Rhnull RBCs. These results indicate that the monoclonal and the polyclonal human anti-Rh(D) behave similarly. A sample (Blo., presumed genotype R2r or R0R2) showing an increased number of antigen sites (0.76 X 10(5)/cell) and a high binding constant (5.7 X 10(8) mol/L-1) was used, as well as D--/D-- RBCs, for further purification of the 29-kd component. Extraction by Triton X-100 (0.1% to 5%) of the immune complexes formed between the membrane-bound Rh(D) antigens and the monoclonal antibody as well as a direct quantitative estimate of the 29-kd component, suggest that the Rh(D) polypeptide is loosely bound to the skeleton, since less than or equal to 80% can be solubilized from the membrane. In similar conditions, glycophorin A showed a slight association with the Triton-insoluble residue, whereas glycophorin B was easily and completely extracted. In contrast, both the minor RBC sialoglycoproteins, glycophorin C and glycoprotein gamma, remained predominantly bound to the membrane skeleton. The purified Rh(D) polypeptide obtained from Blo. and D--/D-- RBCs by immunoprecipitation and preparative gel electrophoresis was homogenous as judged by SDS-PAGE. Amino acid composition indicated that the Rh(D) protein contained sulfhydryl groups which are essential for biological activity.

Human monoclonal antibody against Rh(D) antigen: partial characterization of the Rh(D) polypeptide from human erythrocytes

A human monoclonal anti-Rh(D) antibody produced by an Epstein-Barr virus (EBV)-transformed B-cell line (IgG1(lambda), clone H2D5D2) has been purified on protein A-Sepharose column and used for binding studies and immune precipitation of the blood group rhesus (Rh) antigens. Scatchard plot analyses show that the 125I-labeled antibody (iodo-gen procedure), binds to 1.09 X 10(5), 0.43 X 10(5), and 0.32 X 10(5) antigen sites on each D--/D--, R2R2 and R1R1 RBC, respectively, with an association constant of approximately 0.6 X 10(8) mol/L-1. Immune precipitation studies indicate also that the Rh(D) antigen of the Rh(D)-positive RBCs is carried by a 29 kd polypeptide as deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE). No material could be precipitated from Rh(D)-negative or Rhnull RBCs. These results indicate that the monoclonal and the polyclonal human anti-Rh(D) behave similarly. A sample (Blo., presumed genotype R2r or R0R2) showing an increased number of antigen sites (0.76 X 10(5)/cell) and a high binding constant (5.7 X 10(8) mol/L-1) was used, as well as D--/D-- RBCs, for further purification of the 29-kd component. Extraction by Triton X-100 (0.1% to 5%) of the immune complexes formed between the membrane-bound Rh(D) antigens and the monoclonal antibody as well as a direct quantitative estimate of the 29- kd component, suggest that the Rh(D) polypeptide is loosely bound to the skeleton, since less than or equal to 80% can be solubilized from the membrane. In similar conditions, glycophorin A showed a slight association with the Triton-insoluble residue, whereas glycophorin B was easily and completely extracted. In contrast, both the minor RBC sialoglycoproteins, glycophorin C and glycoprotein gamma, remained predominantly bound to the membrane skeleton. The purified Rh(D) polypeptide obtained from Blo. and D--/D-- RBCs by immunoprecipitation and preparative gel electrophoresis was homogenous as judged by SDS- PAGE. Amino acid composition indicated that the Rh(D) protein contained sulfhydryl groups which are essential for biological activity.

Using a Monoclonal Antibody to Identify Patients with Type I and Type II von Willebrand’s Disease

Three monoclonal antibodies produced against vWF:Ag by conventional hybridoma technique did not inhibit factor VIII coagulant activity (F. VIII:C) but did inhibit VIII ristocetin cofactor activity. The antibodies were used in an indirect competitive ELISA for quantifying von Willebrand's antigen (vWF:Ag) and compared with values obtained by the Laurell technique using commercial antibody by means of a ratio: ELISA/Laurell. For one monoclonal BD2-CC9, vWF:Ag values obtained in the two assays were in good agreement for normal and hemophilia A plasmas (normal, n = 19, ratio = 1.13 +/- .17, hemophilia A, n = 10, ratio = 0.91 +/- .15). However, type II vWD patients had a disproportionately low value of vWF:Ag with the ELISA. Use of the ratio normalized the difference among individual plasma values and allowed a significant separation of type II vWD plasma (n = 9, ratio = 0.46 +/- .19) from normal plasma (p = .0001) and type I vWD plasma (n = 8, ratio = 1.52 +/- .34) from type II vWD plasma (p = .0003) using BD2-CC9. Although the sample size was small, the greater degree of discrimination among the vWD plasmas tested with BD2-CC9 (compared with the other two antibodies [CA3-AE4, CC6-BG10]) suggests that this antibody may recognize conformational epitopes that reflect the degree of multimeric polymerization of the vWF molecule rather than simply recognize a decreased number of antigenic sites in a basic subunit. BD2-CC9 may be valuable in investigating the various types of vWD and/or the process of polymerization of this complex protein.

Change in binding reactivity of an anti-tumor monoclonal antibody after the introduction of 2-pyridyl disulphide groups.

The use of monoclonal antibodies for in vivo therapeutic approaches depends largely on their specificity. During the characterization of ricin A-chain-murine monoclonal antibody conjugates we found that the binding specificity of a monoclonal antibody raised against human ovarian carcinoma (MOv2) seemed altered. Therefore, the binding reactivity of the unmodified antibody (MOv2), the conjugation intermediate (MOv2-PDP) and the conjugate (MOv2-A chain) was titrated by solid-phase radioimmunoassay on 11 human tumor cell lines belonging to seven different histotypes. The three reagents bound with the two reference cell lines (SW626:ovary carcinoma and HT-29:colon carcinoma). The MOv2-PDP and the Mov2-A chain also reacted with seven other cell lines which were unreactive with the unmodified MOv2. In addition MOv2-PDP exhibited reactivity on all normal cells tested (peripheral blood lymphocytes and skin fibroblasts). To elucidate the significance of these findings the following experiments were performed: cross inhibitions between the unmodified and modified monoclonal antibodies; comparative absorption tests with different cell lines; and immunoblotting analysis of the target antigens. The results suggest that after chemical modification with SPDP the monoclonal antibody MOv2 increases its binding activity, so that even a low number of antigenic sites can be detected. This study underlines the need to redefine the specificity of a conjugate before considering therapeutic applications.

Binding Characteristics of Anti-Rh0(D) Antibodies to Rh0(D)-Positive and Du Red Cells

The relation of human erythrocyte Rh0(D) to Du sites is an important unresolved question in the field of immunohematology. To compare the immunological reactivity of Rh0(D)-positive and Du erythrocytes, the binding characteristics of two anti-Rh0(D) antisera to human Rh0(D)-positive and Du ("low-grade") erythrocytes were studied. 14C-Protein A and direct antibody-labeled techniques were used to generate binding curves and to derive double-reciprocal plots. The results show that the number of antigen sites differ by a factor of 10 to 15 between the Rh0(D)-positive and Du red cells, but that the dissociation constants between anti-Rh0(D) and the Rh0(D) and Du antigens are indistinguishable when studied by the two labeling methods and two different anti-Rh0(D) antibodies. The extent of binding to 112 different Du samples showed a normal distribution and was independent of apparent phenotype. These data suggest immunologic identity of Rh0(D) and Du ("low-grade") sites and that the difference between the antigens of Rh0(D) and Du cells is quantitative only. The data are incompatible with the "missing mosaic" and gene interaction theories of mechanism.

Binding characteristics of anti-Rh0(D) antibodies to Rh0(D)-positive and Du red cells

The relation of human erythrocyte Rh0(D) to Du sites is an important unresolved question in the field of immunohematology. To compare the immunological reactivity of Rh0(D)-positive and Du erythrocytes, the binding characteristics of two anti-Rh0(D) antisera to human Rh0(D)- positive and Du (“low-grade”) erythrocytes were studied. 14C-Protein A and direct antibody-labeled techniques were used to generate binding curves and to derive double-reciprocal plots. The results show that the number of antigen sites differ by a factor of 10 to 15 between the Rh0(D)-positive and Du red cells, but that the dissociation constants between anti-Rh0(D) and the Rh0(D) and Du antigens are indistinguishable when studied by the two labeling methods and two different anti-Rh0(D) antibodies. The extent of binding to 112 different Du samples showed a normal distribution and was independent of apparent phenotype. These data suggest immunologic identity of Rh0(D) and Du (“low-grade”) sites and that the difference between the antigens of Rh0(D) and Du cells is quantitative only. The data are incompatible with the “missing mosaic” and gene interaction theories of mechanism.

Citrate synthase: an immunochemical investigation of interspecies diversity

Rabbit antibodies have been raised to pig heart citrate synthase. Using purified IgG, competitive enzymelinked immunoassays and assays of citrate synthase activity indicate the presence of antibodies to a number of antigenic sites on the enzyme, only some of which are essential for catalytic activity. From a comparison of citrate synthases from prokaryotic and eukaryotic organisms, the degree of interaction between antibody and enzyme was in the order: pig heart > pigeon breast > Bacillus megaterium > Escherichia coli. These findings are discussed in terms of the known interspecies diversity of the enzyme. Citrate synthase Immunochemistry Enzyme diversity

Immunochemistry of scorpion α-neurotoxins. Determination of the antigenic site number and isolation of a highly enriched antibody specific to a single antigenic site of toxin II of Androctonus australis Hector

Scorpion neurotoxins active on mammals, i.e. α- and β-toxins, have been divided into several groups according to structural and also immunological criteria. A study of the α-toxins has been performed in order to determine the number of antigenic sites; a minimum of four Fab fragments were found to bind simultaneously to toxins I and II of Androctonus australis Hector, and toxin I of Buthus occitanus tunetanus. Taking advantage of the loss of one common antigenic site between toxin II of Androctonus australis Hector and toxin III of Buthus occitanus tunetanus, a highly purified antibody population towards a single site of toxin II of Androctonus australis Hector was isolated and characterized. This result is discussed in terms of sequence homologies between the two proteins as the amino acid sequence of toxin III of Buthus occitanus tunetanus has been determined using standard procedures: the two proteins differ at three positions, two of which (positions 10 and 64) are in the vicinity of the disulfide bridge Cys 12-Cys 63, the third (position 51) is the only one to be conservative.

Elution of group-specific substance A from RBC of various subgroups of A and its effect on the agglutination of AX RBC.

Experiments are described which demonstrate that when the Landsteiner heat elution technique is used to prepare eluates from group A cells, some A substance as well as anti-A is released into the eluates. The concentration found is greatest from A1 RBC and least from Ax, suggesting a relationship with the number of antigen sites on RBC of the various subgroups. Further experiments have shown that the presence of this A-specific substance in eluates accounts for the well-know phenomenon that anti-A from selected group O sera, when eluted from A1 RBC, will not agglutinate Ax even though the original serum showed an appreciable titre against Ax. The presence of the eluted antigen also suggests an explanation for the behaviour of antibody recovered from the red cells of various subgroups of A towards Ax.

Determination of the number of antigenic sites of scorpion neurotoxins. The purification of Fab specific of one determinant of [formula omitted] toxin II (AaH II) : Mohamed EL AYEB, Pierre DELORI and Hervé ROCHAT. Laboratoire de Biochimie Faculté de Médecine Secteur Nord Boulevard Pierre Dramard 13326 Marseille Cedex 3

Determination of the number of antigenic sites of scorpion neurotoxins. The purification of Fab specific of one determinant of [formula omitted] toxin II (AaH II) : Mohamed EL AYEB, Pierre DELORI and Hervé ROCHAT. Laboratoire de Biochimie Faculté de Médecine Secteur Nord Boulevard Pierre Dramard 13326 Marseille Cedex 3

A shared alloantigenic determinant on Ia antigens encoded by the I-A and I-E subregions: evidence for I region gene duplication.

Two monoclonal antibodies to mouse Ia antigens were produced by fusion of xenoimmune rat spleen cells with the NSI myeloma. These monoclonal antibodies detect polymorphic determinants present on B cells and activated T lymphocytes from mice carrying the H-2b, H-2d, H-2k, H-2r, and H-2q haplotypes but not from mice carrying the H-2s or H-2r haplotypes. Antigenic site number determinations showed the positive haplotypes can be divided into 2 groups. Mice bearing the H-2b, H-2d, and H-2q haplotypes express a high number--40,000 to 80,000--of antigenic sites per B lymphocyte, and monoclonal antibody plus complement can lyse B cells from these mice. In contrast, mice bearing the H-2k and H-2r haplotypes express a low number of antigenic sites--about 5000 per cell. Spleen cells from mice carrying the latter haplotypes are not lysed with monoclonal antibody and complement. Genetic mapping demonstrated that high and low expression map to the I-A and I-E subregions, respectively. The monoclonal antibodies detect an Ia specificity on I-Ab, I-Ad, I-Ed, and I-Ek molecules. These observations were confirmed using several different experimental approaches, i.e., cytotoxicity, fluorescent staining, competitive inhibition of monoclonal antibody binding, and 2-dimensional gel electrophoresis of immunoprecipitates. The avidity for A alpha b A beta b and E alpha k E beta k is 5 to 7 x 10(-9) M-1. The antigenic determinant is heat labile, which suggests that it is not carbohydrate. The results imply that Ia antigens encoded by distinct subregions share sequence homology, which may be a consequence of ancestral gene duplication.

Immunoenzymometric assays for simultaneous quantitation and distribution of erythrocyte antigen.

We describe an adaptation and modification of the enzyme-labeled immunosorbent assay (ELISA), which yields information on the quantity and distribution of surface antigens on erythrocytes. The distribution assay, named "ELADA" (for Enzyme-Linked Antigen Distribution Assay), involves use of an alkaline phosphatase-immunoglobulin conjugate with alpha-naphthol phosphate and a diazonium salt as substrate. The insoluble reaction product forms a complex on the cell surface at the location of the antigen. Antigens in aliquots from the same enzyme-immunoglobulin conjugate are quantified with the soluble substrate p-nitrophenyl phosphate. One can thus establish whether the distribution of erythrocyte antigens is random by examining the ELADA-stained cells with a scanning electron microscope. Combining the quantitative ELISA with measurements of cell numbers makes it possible to estimate the number of antibody molecules-and thus the number of antigenic sites per cell. By using various modified antisera, substituted naphthol phosphates with higher avidities, and reaction conditions designed to optimize the kinetic reaction, we have been able to resolve the enzyme product deposited on the antigenic site to within 20 nm. It is not always possible to decide whether each deposit represents a cluster of antigenic determinants or a single determinant; however, if the number of antibody sites is known from ELISA quantitation, information about the number of antibody sites per deposit can be ascertained.