Enzyme-linked immunosorbent assays for sperm antibody detection and antigenic analysis

Enzyme-linked immunosorbent assays for estimation of antibodies against human sperm and for determination of antigenic reactivity of spermatozoal proteins were established. Sperm immobilized on PHA-coated microtiter plates or solubilized spermatozoal antigens adsorbed on poly(L)-lysine coated microtiter plates were used as the solid phase. Assay of sperm antibodies was performed by incubation of the test samples with the solid phase followed by incubation with anti-Ig conjugated to peroxidase. Sigmoidal antibody dilution curves were obtained with rabbit and mouse anti-sperm sera. The ELISA was effectively used to screen production of anti-sperm antibodies by mouse myeloma x splenocyte hybridomas. The sensitivity of this ELISA for sperm antibodies was more than 1000-fold greater than the classical tray sperm immobilization test, and was comparable in sensitivity to a radioimmunoassay using 125I-labeled protein A as the tracer. Sperm immobilized on PHA-coated plates exhibited significantly greater antigenic reactivity in both the ELISA and RIA compared with methanol fixed sperm. In a competitive inhibition ELISA, linear Logit-log dose-response curves were obtained with detergent solubilized spermatozoal antigens. The assay was used to monitor the purification of the solubilized spermatozoal antigens by chromatofocussing; a more than 60-fold increase of antigenic potency of purified sperm antigen compared with unfractionated sperm extract was evident in the competitive ELISA.     

Use of monoclonal antibody in an ELISA test for the detection of antibodies to bovine leukaemia virus

A variant of the ELISA technique, involving a monoclonal anti-gp51 antibody yields a highly sensitive method for the detection of bovine leukaemia virus (BLV) antibodies. The gp51 antigen-coated microtitre plates are obtained by incubation of plastic-adsorbed monoclonal antibodies with a non-purified mixture of BLV antigens. Sera to be tested are incubated in the wells of the gp51-coated plates and bound antibodies are revealed by an enzyme-linked antibovine immunoglobulin reagent. This test is as sensitive as liquid phase radioimmunoassay using the same gp51 antigen and thus appears as a highly sensitive, practical, rapid and cheap method for the detection of BLV antibodies.     

A solid-phase immunoassay to screen anti-HLA monoclonal antibodies

A solid-phase assay to detect anti-HLA monoclonal antibodies was developed. In this assay microtiter plates are coated with antigens solubilized from cultured lymphoid cells by sonication and then incubated with anti-HLA monoclonal antibodies. The antigen-antibody interaction is indicated by the development of color following the addition of peroxidase-conjugated anti-mouse Ig xenoantibodies and its substrate. The assay is rapid since it does not require centrifugations during the washing steps. Furthermore the assay is simple, reproducible and suitable to screen large numbers of samples and to detect antibodies recognizing determinants not exposed on the membrane of viable cells. The sensitivity of the assay is influenced by the pH of the buffer used to coat plates with antigens, by the number of cells used to prepare soluble antigens, by the incubation time of antigen preparations with plates and by the incubation time of antibody preparations with antigen-coated plates. Titration of anti-HLA monoclonal antibodies with known specificity and screening of hybridomas generated with splenocytes from mice immunized with cultured human lymphoid cells indicate that the sensitivity of the solid-phase assay is similar to that of the ELISA with lymphoid cells.     

A biotin-labelled antigen radioimmunoassay (BILA) for antibodies to membrane antigens useful for monoclonal antibody screening

Viable cells or protein extracts were labelled with N-hydroxysuccinimidobiotin and used as target antigens in a biotin-labelled antigen radioimmunoassay (BILA). The binding of the biotinylated antigens to capture antibodies coated on the bottoms of 96-well plastic plates were measured using 125I-labelled streptavidin as the detection step. The assay circumvents some of the problems associated with solid-phase RIA and permits screening of antibodies to undefined protein antigens present in very small amounts in complex protein solutions.     

A cell surface ELISA in the mouse using only poly-L-lysine as cell fixative

An ELISA using plates coated with mouse spleen cells has been developed for analysis of antibodies to cell surface antigens. Such assays have been used extensively with human cells or with tumor cells in various species, but application to normal mouse lymphocytes has been limited. Use of normal spleen cells allows access to the genetic resources offered by recombinant, congenic, and mutant mouse strains, in the preparation of cell-coated ELISA plates, the use of glutaraldehyde was found to be unnecessary and it was eliminated, thereby avoiding the destruction of some cell surface determinants. Poly-L-lysine, which was used to treat plates, was found to provide sufficient adherence and preservation of the cells. Binding of biotinylated monoclonal antibodies to cells could be detected at approximately 10 ng/well. In inhibition assays, unlabeled antibodies could be detected at approximately 10 ng/well. Cell-coated plates are stable once prepared, and can be stored for months before use. The assay described can be used to quantitate levels of antibody to a particular epitope, and can also be adapted for screening of fusions for monoclonal antibodies to cell surface antigens.     

Measurement of mouse anti-phospholipid antibodies to solid-phase microspheres by both flow cytofluorometry and Alcian blue-pretreated microtitre plates in an ELISA

Conventional solid-phase immunoassays measuring interactions between anti-phospholipid antibodies and phospholipids are generally characterized by problems of reproducibility and high levels of non-specific binding. Here we describe two immunoassays based on the use of phospholipids in the form of solid-phase microspheres to measure the presence of anti-phospholipid antibodies in sera. Following the production of antibodies in mice against liposomes containing lipid A, we show that flow cytofluorometric analysis provides a reproducible and sensitive way to detect anti-phospholipid antibodies. We also present a sensitive, rapid and reproducible enzyme-linked immunosorbent assay (ELISA) using Alcian blue pretreated microtitre plates and solid-phase microspheres as coating antigen. This ELISA permitted the detection of antibodies to 1/1000 dilution, while untreated plates gave negative results. Such modified ELISA procedures may be applicable to other types of molecule exhibiting solid-phase binding problems e.g. synthetic peptides (J. Immunol. Methods 175 (1994) 131–135).     

Comparison of two techniques for detecting antibody to HBsAg during a hepatitis B vaccine study

Two assays for the detection of antibody against hepatitis B surface antigen (anti-HBs) were compared. The first was a direct sandwich radioimmunoassay (RIA) which detects, in principle, antibody against any epitope of hepatitis B surface antigen (HBsAg). The second assay was an inhibition enzyme-linked immunosorbent assay (ELISA). In this assay a fixed amount of HBsAg which can be blocked by anti-HBs is measured in a direct sandwich test. Prevaccination screening sera (n = 191) and follow-up sera obtained from high risk groups (n₁ = 85; n₂ = 41) during two hepatitis B vaccine studies were compared in RIA and ELISA. In prevaccination sera either HBsAg or anti-HBs were detected by ELISA. Full agreement between the results of RIA and ELISA for anti-HBs was obtained in sera containing more than 10 IU/1 anti-HBs. Both tests showed variable results at low titres. Experiments with monoclonal anti-HBs indicated that ELISA is less sensitive for subtype specific antibodies (anti-d, anti-y), which may explain that there were consistent differences between RIA and ELISA in a minority of cases.     

Haptenated nylon-coated polystyrene plates as a solid phase for ELISA

An ELISA system, based on the novel use of a hapten-nylon conjugate as solid-phase coating antigen, has been applied in the screening of hybridoma cultures for anti-hapten monoclonal antibodies directed against the herbicide atrazine and its derivatives. Conjugation of a 2-aminocaproic acid derivative of atrazine with DCC to polyamide (Nylon 6) gave haptenated nylon which was soluble in aqueous cresol-ethanol mixtures and adsorbed efficiently on polystyrene microtitre plates. Reproducible ELISA results were obtained with culture supernatants of hybridomas derived from spleen cells of mice that had been immunized with atrazine-bovine serum albumin conjugates. Satisfactory results were also obtained with a water soluble peptide conjugated to nylon for use as a coating antigen in an ELISA. Plates coated with hapten-nylon as antigen have the added advantage that they can be stored at room temperature for at least 6 months without loss of activity. Nylon therefore appears to have general applicability as a carrier for both non-polar and polar haptens in the preparation and use of coating antigens.     

New experimental criteria for optimization of solid-phase antigen concentration and stability in ELISA

A peroxidase saturation technique for the determination of coating antigen concentration necessary to saturate polystyrene plates with a wide range of different antigens is described. The same technique has also been used to compare the stability of antigen-polystyrene bonds for native and denaturated antigens. Furthermore, the inappropriate selection of solid-phase antigen concentration and its influence on ELISA results is analyzed and an experimental criterion to select the optimum antigen concentration is proposed. Two different antigens, BSA-Ar and hydatid antigen, were used for ELISA determination of specific antibodies as a model system. Optimum solid-phase antigen concentration was determined by two different methods: peroxidase saturation, in which binding of peroxidase to non-antigen-occupied polystyrene surface sites was used to evaluate the degree of coating by antigen; chequer-board titration, using several immune sera of different affinity. Optimum antigen concentration selected by chequer-board titration using low affinity sera was similar to that selected with peroxidase saturation. On the other hand, lower antigen concentration would be selected by chequer-board titration using high affinity sera. For this reason, the concentrations of low affinity antibodies would be underestimated using the chequer-board titration. These results indicate that peroxidase saturation should be used to avoid avidity-dependent artifacts in ELISA.     

广州管圆线虫单抗结合抗原定位分布及在免疫诊断上的应用

目的 研制广州管圆线虫单克隆抗体诊断循环抗原提高诊断的特异性.方法 将广州管圆线虫分泌性抗原免疫小鼠,免疫鼠脾细胞与骨髓瘤细胞融合为杂交瘤细胞,用广州管圆线虫阳性患者血清筛选阳性杂交瘤细胞,培养阳性的杂交瘤细胞分离制备单克隆抗体命名为12D5和21B7,用免疫组织化学的方法分析12D5和21B7单抗结合抗原在广州管圆线虫体内的分布,并用筛选的双12D5和21B7单抗进行抗体夹心ELISA检测实验感染广州管圆线虫的大鼠、广州管圆线虫感染病人血清循环抗原(CAg),用其他寄生虫抗原鉴别单抗的特异性,并与抗体检测比较其敏感性和特异性.结果 经鉴定单抗12D5为IgG1,21 B7为IgM,两株单抗同时识别广州管圆线虫成虫相对分子质量为55 × 103的蛋白,两个单抗针对的抗原分布在虫体肠表面,12D5和21 B7双抗体夹心ELISA法对实验感染的广州管圆线虫的大鼠血清中CAg检出率为100%(48/48),广州管圆线虫感染病人血清CAg检出率为100%(32/32),与日本血吸虫、肝吸虫、肺吸虫、旋毛虫、蛔虫、包虫病人血清无交叉反应,与健康人血清无反应;而用抗原检测32个广州管圆线虫感染病人的抗体检出率为75%(24/32),同时抗体检测与其他寄生虫出现一定的交叉反应.结论 12D5和21 B7单抗结合的抗原为肠相关抗原,双抗体夹心ELISA法对感染广州管圆线虫人和动物血清中CAg检测的特异性强,敏感性高,优于抗体检测试剂,并能够确定现症感染.

Abstract：

Objective To detect infection of Angiostrongylus cantonensis and examine effection of treatment to prepare monoclonal antibodies(McAbs). Methods Six-week-old BALB/c mice were imrnunized by the intraperitoneal injection of e/s antigens of Angiostrongylus cantonensis. Fusion of splecn cells from immunized mice with prepared SP2/0-Ag14 myeloma cells was performed in RPMI 1640. Fused cells were suspended in RPMI 1640 containing 1% HAT and 20% fetal calf serum and dispensed into 96-well cell culture plates. The supernatants of clones were screened by ELISA with sera of patients of angiostrongyliasis.Distribution of cohere antigen of 12D5 and 21B7 monoclonal antibodies was analyzed with immunohistochemistry. Two McAbs ( 12D5 and 21B7) were applied to detect the circulating antigen (CAg) in the sera of rats infected with A. cantonensis and angiostrongyliasis patients respectively by double antibody sandwich ELISA.Results 12D5 McAb was identified as IgG1 and 21 B7 McAb was IgM. Western blot result showed two McAbs could used to identified 55 × 103 protein of adult worms of A. cantonensis. Cohere antigen of 12D5 and 21B7 monoclonal antibodies were distributed on intestine surface of A. cantonensis. The detection rates of CAg in the sera of infected rats 100% (48/48), the detection rates of CAg in the sera of angiostrongyliasis patients was 100% (32/32). No cross-reaction to sera of patients with other infection of parasites, such as clonochiasis, fasiolopsiasis, ancylostomiasis, trichinosis, anisakiasis as well as schsitosomiasis, and health srea did not reacted with 12D5 and 21B7 McAbs,and detaction rate of antibody of angiostrongyliasis patients only reached 75% (24/32) with antigen of A. cantonensis. Conclusion Cohere antigen of 12D5 and 21B7monoclonal antibodies were antigens of enteric epithelium. Sandwich ELISA with 12D5 and 21B7 McAbs showed high specificity act as detecting CAg of A. cantonensis in sera of infection animal and patients. It is apparent that Sandwich ELISA with 12D5 and 21 B7 is not only rapid and simple without requirement of special instrument, but also rather sensitive and specific for the detection of current infection with A. cantonensis.     

Pea lectin as a universal cell binding agent for ELISA (screening) tests

A cell binding assay (CBA) was developed in which plant lectins are used as binding agents between microtiter plates and human cells. After binding, the cells were fixed by mild glutaraldehyde treatment. Their antigenic activity was investigated by enzyme-linked immunosorbent assay (ELISA) with several monoclonal antibodies. The binding method resulted in cell layers that remained firmly attached to the plates during the washing and incubation procedures of the ELISA. A comparative phenotype analysis, performed by indirect membrane fluorescence, showed that cells bound by this method, do not lose their antigenic activity. This binding assay can be used as a rapid, large scale screening test for monoclonal antibodies to membrane antigens of malignant and normal cells.     

多种单抗联合检测HIV抗原

目的 建立多种单抗联合早期检测HIV抗原的夹心ELISA方法.方法 以SAS盐析沉淀法和亲和层析法纯化抗HIV-1 p24、gp41、gp120及抗HIV-2 gp36的腹水型单克隆抗体(McAb),用高碘酸钠法将纯化的McAb以HRP进行标记.建立针对单个抗原的双抗体夹心ELISA法,对其灵敏度及特异性进行检测.将筛选得到的4株捕获McAb按比例混合作为捕获抗体,4株酶标McAb按比例混合作为检测抗体,建立多种单抗联合检测HIV抗原的夹心ELISA方法,检测混合HIV抗原.结果 按确定的最优反应条件建立的多种McAb联合夹心ELISA方法,检测到的最高稀释度的HIV混合抗原中各抗原的终浓度分别为:重组HIV-1 p24:0.625 pg/ml,gp41:6.25 ng/ml,gp120:6.25 ng/ml;HIV-2 gp36:9.25 ng/ml.结论 建立了具有高度敏感性的鸡尾酒式多种单抗联合检测HIV抗原的夹心ELISA法,为早期榆测HIV抗原提供了新的思路,为后续的研究奠定了一定基础.     

Lymphokines,vol. 5, monoclonal T cells and their products: edited by M. Feldmann and M.H. Schreier. Academic Press,New York, 1982 (xviii + 496 pp., illus.) $55.00

HLA-A and -B antigens were detected on fresh and dried peripheral blood lymphocytes by an enzyme-linked immunosorbent assay. Intact cells fixed to plates with glutaraldehyde were used as antigen and anti-HLA alloantisera as a source of antibodies. Determination of HLA antigens by the ELISA technique was comparable with the complement-dependent cytotoxicity test. The relative stability of HLA antigens as shown in this report and the extensive polymorphism of the HLA system make the ELISA technique a promising tool for the analysis of HLA antigens on non-living cells including, for example, medicolegal investigation of blood stains.     

A novel enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to HIV-1 envelope glycoproteins based on immobilization of viral glycoproteins in microtiter wells coated with concanavalin A

We have developed a novel method that greatly simplifies the preparation of solid-phase HIV-1 envelope glycoproteins for use in an ELISA that detects serum antibodies to HIV envelope antigens. This method utilizes concanavalin A absorbed to wells of microtiter plates to affinity immobilize detergent-solubilized viral glycoproteins released in culture fluids of HIV-1 infected cell lines grown in serum free medium. Antibodies binding to ConA-immobilized viral antigens are detected by peroxidase-conjugated antibodies and appropriate enzyme substrates. Unlike most commercial HIV ELISAs, which utilize gp120 depleted-purified virus as the source of antigens and thus favor detection of antibodies to core antigens, the ConA envELISA is highly sensitive for detecting antibodies to native gp120, as evidenced by the strong reactivity of gp120-specific human monoclonal antibodies. Our results also suggest that representation of gp41 in the assay varies and depends on which virus infected cell lines are used for antigen production. Since this assay accurately identified 14 HIV-1 antibody positive patient sera and no false positives were detected among 16 HIV-1 negative sera, the ConA envELISA shows promise as an inexpensive assay for the serologic diagnosis of HIV infections.     

Binding of sperm-reactive antibodies in human sera to surface-associated antigens on human sperm compared by indirect immunobead, immunofluorescence, and immunogold assays

Human sera were identified as positive or negative for sperm-reactive antibodies in a solid-phase enzyme-linked immunosorbent assay (ELISA). Of these, 28 positive and 22 negative sera were blind-coded and used as first antibody to compare three immunoassays, a modified liquid-phase indirect immunobead assay (IBA); a liquid-phase indirect immunofluorescence assay (IFA); and a solid-phase indirect immunogold assay (IGA). These three immunoassays perform both as sperm-reactive antibody detection assays and as sperm-associated antigen localization assays. As antibody detection assays, the IBA, IFA, and IGA gave 37, 27, and 28 positives and 13, 23, and 22 negatives, respectively. The usefulness of the IBA as an antigen localization assay was limited by the size of the marker, while the smaller IFA and IGA markers enabled increased resolution of binding patterns of sperm-reactive antibodies to surface-associated sperm antigens. Although the antigen-antibody binding patterns were almost identical for IFA and IGA, suggesting the same sperm-associated antigens were detected by both assays, the IGA reaction product was stable, higher in resolution, and visible by light microscopy.     

Use of solid-phase C1Q to remove soluble antigen/antibody complexes in an inhibition ELISA for streptococcal cell membrane antigens

Solid-phase C1q was used to remove antigen/antibody complexes in an inhibition ELISA for low molecular weight streptococcal cell membrane (SCM) polypeptide antigens. To selectively fix IgM monoclonal antibody bound to antigen, binding was carried out in C1q-coated ELISA plates; transfer of supernatants to SCM-coated plates for ELISA permitted measurement of residual antibody. When inhibition occurred in the presence of C1q, the maximal binding was 72-98%. In the absence of C1q the maximum apparent binding was only 45-50%, which we attribute to displacement of the initially bound SCM antigen by solid phase SCM antigen. Removal of antigen/antibody complexes by solid-phase Clq during inhibition assays may facilitate analysis of low affinity antigen/antibody interactions.     

Sensitive detection of hydrophobic antigens using a novel lipid-aggregate based ELISA

Antibodies against hydrophobic antigens are common in several autoimmune diseases. However, detection of such antibodies by standard immune-assays, such as ELISA, is problematic, in part because of the problems with coating hydrophobic molecules onto polystyrene multi-well plates. We describe a novel method of stably associating hydrophobic antigens to ELISA plates. By mixing the antigen with a hydrophobic molecule containing a hydrophilic anchor, we generate mixed lipid aggregates that can attach to ELISA plates, and are resistant to detergent wash. Using the ganglioside GM-1 and phosphatidylethanolamine conjugated to the hapten DNP (dinitrophenyl) as model antigens, we show that hydrophobic antigens incorporated into mixed lipid aggregates expose their antigenic determinants in a correct configuration. The detection limit of both GM-1 and DNP-PE was considerably improved compared to when these antigens were coated on ELISA plates using organic solvents. Furthermore GM-1 incorporated into mixed lipid aggregates can be detected by specific antibodies in patient serum. The method of incorporating hydrophobic antigens into mixed lipid aggregates for stable association to ELISA plates can presumably be applied to a vast array of hydrophobic antigens, and may well be developed into a large scale screening system for serum reactivity towards different hydrophobic antigens.     

Caveats for the use of surface-adsorbed protein antigen to test the specificity of antibodies.

Rabbit antisera against apo-cytochrome c, which was prepared by removal of the covalently bound heme prosthetic group from yeast iso-1 cytochrome c, were tested for reactivity against native yeast iso-1-cytochrome c. When the antigen was adsorbed to a microtiter plate in a conventional enzyme-linked immunosorbent assay (ELISA), the antisera were unable to distinguish between their cognate antigen apo-cytochrome c, a random coil protein, and native cytochrome c, a small globular protein of remarkable conformational stability in solution. However, when the assay was conducted under conditions where antigen and antibody were free to associate in solution, that is in a solution-phase radioimmunoassay (RIA), the antisera were highly specific for apo-cytochrome c. Similarly, antibodies induced by native cytochrome c and discriminating strongly between native and apo-cytochrome c in a solution-phase RIA, did not distinguish between native and apo-cytochrome c in a solid-phase ELISA. This discrepancy of results obtained by different immuno assay procedures clearly indicates that adsorption to plastic alters the antigenic structure of even a conformationally stable protein such as cytochrome c. A conventional solid-phase ELISA strongly selects for those antibodies that recognize the unfolded antigen. The results presented warrant serious thoughts about previous reports on anti-peptide antibodies reacting with native whole protein molecules, as tested by those ELISA procedures that have the protein antigen adsorbed to plastic.     

A small scale indirect 125iodine-labelled protein A binding assay for detection of monoclonal antibodies against avian oncoviral proteins

A small scale solid-phase radioimmunoassay employing 125I-labelled protein A is described which is suitable for screening large numbers of monoclonal antibodies directed against antigens which can be prepared in small amounts only, for example oncoviral proteins. The use of polystyrene Terasaki microtest plates instead of 96-well microtitre plates reduces the amount of antigen required for screening hybridoma supernatants to less than 30 ng/well. This method facilitates washing procedures and reduces the quantity of radioactive waste. The sensitivity and specificity of the method is demonstrated by the isolation and initial characterization of monoclonal antibodies specific for avian oncoviral transforming, structural and polymerase proteins.     

Variants of ELISA in plant virus diagnosis

Variations of enzyme-linked immunosorbent assay (ELISA) were compared with respect to their ability to detect and to differentiate serologically related plant viruses. The broadest range of serologically related viruses was detected by an indirect ELISA on unprecoated plates. Coating the plates with F(ab')2 fragments led to narrowing of the specificity in heterologous reactions of tymo-, tombus- and tobamoviruses in indirect ELISA. With Andean potato latent virus (APLV) heterologous reactions were weaker on plates precoated with F(ab')2 fragments than on those precoated with intact antibodies. Even on plates precoated with F(ab')2 fragments the indirect ELISA detected a broader range of serologically related viruses than the direct double antibody sandwich method. Heterologous reactions in indirect ELISA procedures on plates precoated with either intact antibodies or F(ab')2 fragments were always weaker than homologous reactions independent of the concentration of coating reactants and detecting antibodies. Attempts to differentiate closely related strains of APLV or radish mosaic virus by direct ELISA using F(ab')2 fragments either for coating the plates or after labelling with alkaline phosphatase for detecting the trapped antigens failed. Under suitable conditions, the additional working step usually necessary for indirect ELISA could be avoided by using a short procedure which at low concentrations of detecting antibodies was more sensitive than the conventional procedure.