Studies of the 'hook' effect in the one-step sandwich immunoassay.

The one-step sandwich immunoassay is increasingly replacing the traditional two-step immunoassay due to obvious advantages such as assay speed. However, the one-step sandwich immunoassay suffers from the 'hook' effect irrespective of the analyte characteristics. The 'hook' effect is dependent primarily on the analyte concentration. Three different model analytes, human growth hormone (hGH), the dimeric form of hGH (D-hGH, having a discrete number of repeating epitopes) and ferritin (multiple epitopes) having different immunological properties have been employed in studies of the one-step sandwich immunoassay. The characteristics of each of the model analytes offer new insights into general guidelines for assay procedures. These guidelines permit rapid optimization of assay conditions for an immunoassay without a priori knowledge of the immunological characteristics of the antibody or antigen. Both experimental and theoretical data show several instances where high capacity solid-phase antibodies can effectively shift the 'hook' to relatively higher analyte concentrations. The effect of the concentration of labeled antibody on assay response was examined theoretically.     

Studies of the low dose 'hook' effect in a competitive homogeneous immunoassay.

The interactions of two monoclonal antibodies with human growth hormone (hGH) have been investigated. The individual antibodies showed normal behavior in a competitive binding assay, but mixtures of the antibodies demonstrated a 'hook' attributable to cooperative interactions. Cooperativity was observed in titrations which preceded the competitive binding assay. Size exclusion chromatographic data suggest that the cooperativity is explained by the formation of higher molecular weight complexes (up to 700 kDa). The major complex is probably linear, consisting of three antibody molecules. Circular and linear complexes with four antibody molecules (octameric complexes) are also possible. Theoretical models also support the formation of cyclic complexes in a competitive binding assay.     

Discrimination of epitopes identified by monoclonal antibodies by competitive binding to nitrocellulose bound antigens

A new method is described for determining the distribution of epitopes identified by monoclonal antibodies. The method utilizes nitrocellulose membranes as a solid support for antigens which are rapidly adsorbed to nitrocellulose by vacuum-blotting and then used in competitive antibody binding assays. The distribution of epitopes is established by the reciprocal cross-blocking of radiolabeled antibody by increasing concentrations of unlabeled antibody. When unlabeled antibody does not block the binding of labeled antibody to antigen, the 2 antibodies recognize distinct epitopes. When unlabeled antibody blocks the binding of labeled antibody to antigen, the 2 antibodies recognize the same epitope. The method is rapid, sensitive and should be applicable to screening monoclonal antibodies to any epitope.     

Topographical analysis of bovine herpesvirus type-1 glycoproteins: use of monoclonal antibodies to identify and characterize functional epitopes

Monoclonal antibodies, specific for two of the major bovine herpesvirus type-1 (BHV-1) glycoproteins, GVP 3/9 and GVP 6/11a/16, have been previously produced and characterized. The reactivity of each monoclonal antibody was determined by enzyme-linked immunosorbent assay (ELISA), the ability to neutralize viral infectivity, and the capacity to mediate complement-dependent (AbC') lysis of virus-infected cells. The topography of epitopes on GVP 3/9 and GVP 6/11a/16 was analyzed, using selected monoclonal antibodies in a competitive antibody binding assay (CBA). Nine epitopes were identified on GVP 3/9. Comparison of the biological activities and epitope specificities of the monoclonal antibodies against GVP 3/9 showed that one epitope was involved in virus neutralization, whereas six epitopes mediated AbC' lysis of the virus-infected cell. Of the seven epitopes identified on GVP 6/11a/16, six were involved in neutralization and three participated in AbC' lysis. In some cases, partial competition between monoclonal antibodies was observed, indicating that they were directed against overlapping or closely adjacent epitopes. In other instances, asymmetrical competition between monoclonal antibodies suggested conformational changes in the glycoprotein molecule induced by antibody binding. Mixtures of two monoclonal antibodies with different epitope specificities resulted in higher neutralizing activity than either antibody alone, suggesting that multiple monoclonal antibodies can exert a synergistic effect on virus neutralization.     

三株抗rhIL－6单克隆抗体识别表位的鉴定及IL－6的双单克隆抗体夹心RIA检测方法的建立

采用放射免疫标记抗体竞争结合法，对３株抗ｒｈＩＬ－６单克隆抗体识别的表位进行了鉴定。按识别表位的不同，将３株单克隆抗体分为两组：一组ＳＩ８，ＳＩ９识别相同或相近的表位，另一组ＳＩ７。在表位鉴定的基础上，选择识别不同表位的单克隆抗体，由此获得最佳组合，灵敏度为８０ｐｇ／ｍｌ。通过对肾脏病人血清ＩＬ－６水平检测，所得结果与以ＩＬ－６依赖株Ｂ９生物学检测结果一致，且重复性好，特异性强。     

Monoclonal antibodies to human chorionic gonadotropin and their application to two-site sandwich radioimmunoassay

Monoclonal antibodies were prepared against human chorionic gonadotropin (HCG). One monoclonal antibody recognized a conformational determinant expressed only on native HCG molecule and another monoclonal antibody had the specificity for the epitopes located on the beta-subunit of HCG. Monoclonal antibodies reacting with different antigenic determinants on the HCG molecule were used to develop a simplified 2-site sandwich radioimmunoassay in which one monoclonal antibody was immobilized and another labeled with 125iodine. This assay was highly specific for HCG and there was no cross-reactivity with alpha, beta-subunit of HCG, luteinizing hormone and follicle stimulating hormone.     

Use of monoclonal antibodies to identify the distribution of A and M epitopes on smooth Brucella species.

The smooth types of Brucella species express two lipopolysaccharides (LPSs) (A and M) which are antigenically distinct. Their existence as cross-reactive antigenic complexes makes definitive serology difficult. Murine hybridomas were produced and selected for their ability to produce monoclonal antibodies to the specific A- or M-LPS epitopes. The specificity of the monoclonal antibodies was determined by microplate enzyme-linked immunoassay, binding inhibition assay, microplate agglutination, and dot blot assay. Monoclonal antibody 12AE6 was specific for an epitope on the A LPS of Brucella spp., which was also expressed on Yersinia enterocolitica O:9. A unique epitope of M LPS was detected by monoclonal antibody 33.1.5. The two monoclonal antibodies did not exhibit cross-reactions when assayed with whole Brucella cells or purified M- and A-LPS preparations. Cross-reactive serology with polyvalent sera can be attributed to the presence of common antigenic determinants on both molecules. The use of A- and M-LPS-specific monoclonal antibodies has the potential to replace the more laborious methods involved in the production of monospecific sera.     

A convenient method for epitope competition analysis of two monoclonal antibodies for their antigen binding

Choosing optimum pair of capturing antibody and detecting antibody when developing monoclonal antibody (MAb)-based, sandwich enzyme-linked immunosorbent assays is a time-consuming process requiring the coupling of individual antibodies to an enzyme like horseradish peroxidase or alkaline phosphatase. The MAbs required for the two-site sandwich ELISA should bind to distinct epitopes of the antigen, and their binding should not be mutually exclusive. To determine if two monoclonal antibodies would occupy distinct sites of their antigen in binding, and enzyme-linked immunosorbent assay was devised, which is easy-to-use and does not require any coupling of monoclonal antibodies to enzymes. Microplate wells are coated with rabbit polyclonal antibodies raised against the same antigen of MAbs. After blocking, a limited amount of the antigen is added for incubation with the rabbit antibodies. Mouse monoclonal antibody 1 (Mab 1) is added to saturation. A serial dilution of MAb 2 (for analysis) or MAb 1 (for control) is added subsequently. An enzyme-labeled, goat anti-mouse secondary antibody and its substrates are added for color development. Thus, the epitope competition of two MAbs for their antigen binding is easily determined by the measurement and comparison of color development between the two MAb additions.     

Production and characterization of monoclonal antibodies against human Cu/Zn superoxide dismutase and the establishment of a super-rapid enzyme-linked immunosorbent assay (SURALISA).

Murine monoclonal IgG1 antibodies directed against four different epitopes of human Cu/Zn superoxide dismutase (SOD) were produced by immunization with recombinant Cu/Zn SOD. The antibodies reacted well with the recombinant protein and Cu/Zn SOD purified from human erythrocytes, with binding constants ranging from 8.8 X 10(9) to 2.2 X 10(10) l/mol. When mixed, these antibodies completely prevented the binding of rabbit and sheep polyclonal antibodies raised against erythrocyte Cu/Zn SOD. Whereas one antibody was directed against a common homology region of bovine and human Cu/Zn SOD, all the other antibodies reacted exclusively or preferentially with human Cu/Zn SOD. Only one epitope on the human Cu/Zn SOD molecule was accessible at two different sites as demonstrated in a homologous two-site assay with one and the same antibody used as both capture and indicator antibody. In the indirect two-site assay with unlabelled monoclonal antibodies, and additive effect with a steeper dose-response curve was obtained by mixing antibodies against different epitopes. A super-rapid one-step two-site enzyme immunoassay (overall duration 20 min) was established with antibodies against two different epitopes. Its detection limit was 0.5 micrograms SOD/l.     

Screening for epitope specificity directly on culture supernatants in the early phase of monoclonal antibody production by an ELISA with biotin-labeled antigen

This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious advantages using this assay, are that it can be performed directly on culture supernatants in the early phase of monoclonal antibody production, and also works for antigens with repetitive epitopes. Moreover, the bonus effect, i.e., a signal in excess of the reference signal when sets of monoclonal antibodies with different epitope specificity are compared, gives a relative measure of affinity.     

Induction of high levels of antibodies recognizing the neutralizing epitope ELDKWA and the D- or K-position-mutated epitopes by candidate epitope vaccines against HIV-1

Monoclonal antibody 2F5 recognizing the ELDKWA epitope on HIV-1 gp41 has a significant neutralization potency against 90% of the investigated viruses of African, Asian, American, and European strains, but the antibody responses to the epitope 2F5 in HIV-1-infected individuals were very low. We attempted to induce high levels of epitope-specific antibodies to ELDKWA and its three mutated epitopes by candidate epitope vaccines. The four candidate epitope vaccines all induced strong antibody responses at dilutions from about 1:6,400 to 1:25,600. We tested the cross-reactions between these antisera and four epitope peptides. The ELDKWA-specific antisera showed strong cross-reactivity with three neutralizing-resistant mutated epitopes which contain changes in the D or K positions of the epitope sequence. Virus variants containing these changes could escape neutralization by monoclonal antibody 2F5. In immunoblotting analysis, the ELDKWA, ELDEWA, and ELEKWA epitope specific antibodies all recognized rsgp41 which confirms that the antibodies against both mutated epitopes, ELDEWA and ELEKWA, could cross-react with the native epitope on rsgp41. Although it is not clear whether the polyclonal antibodies induced by the ELDKWA epitope vaccine could neutralize the mutated viruses containing these mutated epitopes, it is conceivable that epitope vaccines based on mutated epitopes could induce strong antibody responses with predefined epitope specificity to neutralize mutated viruse containing the mutated epitope. An epitope vaccine, using different epitopes including mutated epitopes, could provide a new concept for developing a new vaccine against HIV-1.     

Monoclonal antibodies generated against an affinity-labeled immune complex of an anti-bile acid metabolite antibody: an approach to noncompetitive hapten immunoassays based on anti-idiotype or anti-metatype antibodies

Conventional immunoassays for haptens such as steroids and synthetic drugs are dependent on the competitive reaction between an unlabeled antigen (analyte) and a labeled antigen against a limited amount of anti-hapten antibody. Although noncompetitive immunoassay procedures such as two-site immunometric assays offer a much higher sensitivity, direct application of this principle to haptens has been difficult due to their small molecular mass precluding simultaneous binding by two antibody molecules. Here, we have attempted to develop a noncompetitive immunoassay system based on anti-idiotype or anti-metatype antibodies. Ursodeoxycholic acid 7-N-acetylglucosaminide (UDCA 7-NAG), which is a bile acid metabolite (molecular weight, 595.8), was selected as the model hapten. A/J mice were immunized with a monoclonal antibody against UDCA 7-NAG, which had been affinity-labeled with a relevant hapten derivative. The fusion between the immune spleen cells and P3/NS1/1-Ag4-1 myeloma cells yielded four kinds of alpha-type and two kinds of beta-type monoclonal anti-idiotype antibodies, each recognizing the framework region and paratope of the anti-hapten antibody. The use of a selected combination between alpha-type and beta-type antibodies together with the anti-hapten antibody provided a noncompetitive assay system with a subfemtomole order sensitivity (detection limit, 118 amol) and a practical specificity.     

Recognition of a high molecular weight glycoprotein (HMW-GP) associated with rat colon cancer cells by rat monoclonal antibodies

In this report we describe an auto-immunogenic tumor associated high molecular weight glycoprotein (HMW-GP) present on most rat colon carcinomas as detected by syngeneic monoclonal IgM antibodies. The HMW-GP has an apparent molecular weight of more than 10(6) D and carries several epitopes for various lectins, carbohydrate specific mouse monoclonal antibodies and four rat syngeneic monoclonal antibodies. The epitope defined by the 10B 12 rat monoclonal antibody is present in colon carcinoma tissue, but only in very low amounts in normal gastro-intestinal-tissue. The epitope is present at multiple sites on the large molecule and is shown to be sterically related, but not identical to determinants for Dolichos biflorus lectin and a mouse mAB binding to blood group A. The 10B 12 mAB binding is insensitive to boiling, reduction with mercaptoethanol and treatment with trypsin, but abolished by pretreatment of the antigen with sodium periodate or neuraminidase. The epitopes defined by 1 G6 and 1F6 rat monoclonal antibodies are present both in tumor tissue and in normal colon mucosa and the antibody binding is increased after treatment with both neuraminidase and sodium periodate.     

Species and epitope specificity of two 65 kDa glutamate decarboxylase time-resolved fluorometric immunoassays

The 65 kDa isoform of human glutamate decarboxylase (GAD65) is a major autoantigen in type 1 diabetes (T1D). In the present study, we have developed a sensitive sandwich time-resolved fluorescence immunoassay (TRFIA) for the quantification of GAD65 in cell extracts, cell media and serum. The monoclonal antibody GAD-6 is used to selectively capture GAD65 but not the slightly larger isoform GAD67, and the utilization of different detecting antibodies with distinct GAD65 epitope specificity allows modulating the specificity of the assay. To this effect we have biotinylated a recombinant antigen-binding fragment (rFab) with epitope specificity for the N-terminal region of rat and human GAD65 (rFab N-GAD65) and another rFab that selectively binds to the middle part of human GAD65 (rFab b96.11). In the assay the biotinylated rFabs are recognized by Europium labeled streptavidin. The obtained time-resolved fluorescence (TRF) is directly proportional to the concentration of GAD65 over a large measuring range (0.1 to >100 ng/mL). Based on total error estimation including both bias and imprecision, the lower limit of quantitation (LLOQ) of GAD65 in cell extracts is 0.33 ng/mL with the N-GAD65 TRFIA, and 0.10 ng/mL with the b96.11 TRFIA, but the latter is suitable for human GAD65 only, whereas the N-GAD65 TRFIA has equal sensitivity with rat and human GAD65. Specificity was further checked with GAD65/67 fusion proteins, confirming that the presence of intact capture as well as detection epitope on the analyte is a prerequisite for recognition in both assays. We show that the beta cell-specific marker GAD65 can be quantified in pancreatic cell extracts and in serum, allowing studies on discharge during cell death in vitro as well as in vivo.     

抗人甲胎蛋白单克隆抗体的研制及其应用

本文报告将自制的人甲胎蛋白(AFP)抗原免疫小鼠,采用常规杂交瘤技木融合小鼠的脾细胞和SP2/0细胞。经有限稀释法克隆出九株分泌抗人AFP单克隆抗体(McAb)的杂交瘤细胞株。分别用血凝抑制试验、琼脂双扩散、ELISA和放射免疫分析等方法鉴定了McAb对抗原的表位特异性、亲和力及其稳定性和小鼠Ig亚类属性。实验结果表明几种McAb分别作用于AFP抗原上相同或不同的抗原表位。其中大多数McAb具有较高的亲和力。抗原表位特异性不同的McAb在免疫沉淀反应中有协同作用。本文报告的AFP McAb双位点夹心ELISA,对一些临床标本的检测结果与AFP放射免疫试剂盒的结果有着良好的相关性。     

A comparison of ELISA screening methods for the production of monoclonal antibodies against soluble protein antigens

A library of monoclonal antibodies against pig heart citrate synthase has been raised. Twelve solid-phase immunoassay systems, employing different methods of antigen immobilization, have been compared for their ability to detect the various members of this library. It was found that a sandwich immunoassay, in which the citrate synthase antigen was immobilized on the solid support via a polyclonal antibody preparation, was the only system capable of detecting all the monoclonal antibodies tested. It is suggested that such a sandwich assay should be used in preference to direct assays for the initial screening of monoclonal antibodies.     

Use of the average antibody-antigen bond concept and probability theory to simplify modeling of linear and circular antibody-antigen complex formation

We illustrate use of a simple approach to describe the equilibrium interactions of mixtures of monoclonal antibodies and antigens. This procedure is based on elementary concepts in probability theory and is readily suited to describing interactions of antibodies and antigens which form circular as well as linear complexes. The method is also suited to describing the inhibitory effects of antibodies which compete for overlapping epitopes and an example is provided to show how the procedure can be used to describe the interactions of antibodies which inhibit circular complex formation. We also outline simple strategies for preparing computer programs to simulate binding of antigens to defined antibody mixtures. The methods described should facilitate design of immunoassay procedures based on the use of defined mixtures of monoclonal antibodies.     

Increased specificity in human cardiac-myosin radioimmunoassay utilizing two monoclonal antibodies in a double sandwich assay

In some instances, even the increased resolution that may be afforded in immunoassays by the use of monoclonal antibodies fails to effect resolution among molecules that share many epitopes. An immunoradiometric assay that simultaneously measured two different epitopes on the same molecule was devised to overcome this difficulty in the differentiation between cardiac- and skeletal-myosin light chains. Three monoclonal antibodies were examined that were 100% (1C5), 25% (2B9) and 17% (4F10) cross reactive, respectively, between the two antigens. One antibody of the pair to be studied was immobilized to cyanogen bromide-activated Sepharose 4B while the other was iodinated with 125I using the lactoperoxidase method. The antigen was mixed with the immobilized antibody, the labeled antibody was added and the precipitate then washed and counted in a gamma counter. When both antibodies of the pair to be studied (immobilized and labeled) were the same (2B9), no radioactivity above background was bound to the precipitate, indicating that the second antibody could not bind to an already occupied epitope. When two different antibodies were employed, the specificity of the assay increased over that of a single antibody. The cross reactivity of a pair approximated the product of the cross reactivities of the individual antibodies. Thus, 1C5 and 2B9 were 25% cross reactive together, 1C5 and 4F10 17% cross reactive, and 2B9 and 4F10 4.3% cross reactive.     

Complement activation by individual and combinations of monoclonal antibodies to Actinomyces viscosus T14V fimbriae: a probe for epitope distribution on these polymeric proteins.

The spatial requirements for IgG activation of the classical complement pathway has provided a basis for utilizing complement consumption by individual and pairs of monoclonal antibodies (mAbs) to compare the repeating epitope patterns of the type 1 and type 2 fimbriae of Actinomyces viscosus T14V and to examine the co-operative effects of mAbs against these polymeric proteins. Three of five mAbs specific for the type 1 fimbriae consumed complement when assayed individually. Four patterns of complement consumption were detected with pairs of these mAbs: inhibition, addition, enhancement or synergy. Inhibition occurred when both members of a pair reacted with the same epitope but only one consumed complement. A strictly additive effect was observed if both mAbs consumed complement and, in addition, recognized the same epitope. Complement consumption by mAbs against certain epitopes was enhanced by non-complement consuming mAbs that reacted with different epitopes. Synergy was observed with extremely low concentrations of two mAbs each of which reacted with a different epitope and consumed complement. In contrast to the anti-type 1 mAbs, only one of seven mAbs against the type 2 fimbriae consumed more than 20% of the available complement. Pairs of anti-type 2 mAbs exhibited only inhibition or synergy. The latter effect was particularly striking as pairs containing mAbs that reacted with different epitopes and failed to consume complement or were minimally active when assayed individually were extremely efficient. These data indicated that the spatial arrangements of individual mAbs bound to repeating epitopes in the type 1, but not the type 2, fimbriae were appropriate for activation of complement. Thus, the repeating epitope patterns of the two types of fimbriae apparently differ.     

Monoclonal antibodies to human growth hormone induce an allosteric conformational change in the antigen.

We re-investigated the properties of a monoclonal antibody (mAb), 4D11, to human growth hormone (hGH) that showed a very weak affinity, recognizing hGH only when the hormone was solubilized on a solid surface. MAb4D11 did not significantly bind 125I-hGH. It was found that three mAb directed to different hGH epitopes (mAb 3C11, 10C1 and NA71) were able to induce the binding of the soluble antigen to mAb 4D11. The co-operative effect could be demonstrated by the formation of binary complexes (Ag:Ab, 1:2) detected by high-performance liquid chromatography (HPLC) and by the increase of radioactivity found when the synergistic mAb were added to 125I-hGH incubated with mAb 4D11 immobilized on polyvinyl microplates. Other possible explanations, such as the formation of cyclic complexes or the generation of a new epitope in the Fc fragment of the first antibody (Ab), were dismissed because the Fab fragment of one of the enhancing mAb (3C11) gave the same effect as the intact Ab. The data suggest that the hGH molecule undergoes a localized conformational change after binding to mAb 3C11, NA71 or 10C1 and that mAb 4D11 binds with high affinity to the modified region of the hormone. The formation or not of ternary complexes (Ag:Ab, 1:3) was used to localize the 4D11 epitope on the surface of the Ag. It is suggested that mAb 4D11 recognizes a conformational change produced in the region defined by the AE5/AC8 epitopes, which is close to the hGH antigenic domain only expressed when the protein is immobilized on plastic surfaces.