Dot-based ELISA and RIA: Two rapid assays that screen hybridoma supernatants against whole live cells

Two assays are described that are suitable for the screening of large numbers of hybridoma supernatants as well as for the screening of a wide range of tissues for the distribution of a given cell surface antigen. These assays use whole live cells as targets, avoiding fixation or extraction, which could alter the antigenic structural profile. The assays are rapid, simple and inexpensive. Their advantages and applications are discussed.     

Immunoperoxidase slide assay (IPSA)--a new screening method for hybridoma supernatants directed against cell surface antigens compared to other binding assays

A modified immunoperoxidase assay on microscope slides (IPSA) was adopted as a screening procedure for hybridoma supernatants with specificity for human lymphocyte subpopulations. The method proved to be suitable for testing a large number of hybridomas with a minimum of cells and reagents. In addition, the slide-technique yields considerable information about the type and morphology of target cells. This allows a first differentiation between cell types in the assayed preparation, e.g. between lymphocytes and monocytes. Compared to indirect immunofluorescence, solid-phase radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) with intact cells as antigen, IPSA turned-out to be as sensitive as RIA and superior to indirect immunofluorescence and ELISA. Nevertheless, none of the assays used detected all supernatants binding to cells in the right manner and could, thus, be considered ideal. In our hands, the combination of RIA and IPSA proved to be an excellent system for screening procedures on lymphocytes.     

Use of a monoclonal antibody in a double-sandwich ELISA for detection of IgM antibodies to Toxoplasma gondii major surface protein (P30)

A double-sandwich ELISA, developed for detection of IgM antibodies to the major surface protein of Toxoplasma gondii (P30), is proposed for the diagnosis of acute acquired toxoplasmosis. The method is based on the capture of serum IgM antibodies, which are revealed indirectly by the sequential addition of a Toxoplasma extract and a beta-galactosidase-conjugated anti-P30 monoclonal antibody. All 57 patients tested with serological characteristics of recently acquired toxoplasmosis showed high levels of IgM anti-P30 antibodies. In addition, 5 out of the 24 patients with chronic toxoplasmosis and all 7 patients with a clinical acute infection in which the classical IgM serology was negative, also presented significant anti-P30 IgM antibodies. Patients with either rheumatoid factor or antinuclear antibodies were all negative. In view of its simplicity, specificity and sensitivity, this method is recommended for the current diagnosis of T. gondii infection.     

Specificity of monoclonal antibodies directed against human and murine class II histocompatibility antigens as analyzed by binding to HLA-deletion mutant cell lines

The specificity of 70 monoclonal anti-Ia monoclonal antibodies (MoAbs) (18 mouse allo-induced and 52 rodent anti-human) was studied with a panel of 17 HLA-deletion mutants that were derived from a single parent line and vary in expression of Ia antigens due to deletion of different subregions of HLA. MoAb binding was analyzed both by ELISA and flow microfluorometry. Characterization of the MoAbs with respect to specificity for products of subregions of a DR1-DC1-SB2 haplotype revealed great complexity. Many antibodies were quite specific for DR-linked determinants (26 MoAbs), DC-linked determinants (5 MoAbs), or determinants indistinguishable from SB (4 MoAbs). However, many MoAbs bound to products of more than one subregion: DR + SB (± weak DC) (22 MoAbs); Dr + DC (3 MoAbs); or DR + DC + SB (1 MoAb). Furthermore, a number of the MoAbs bound unequally to products of the two HLA haplotypes analyzed, particularly among those recognizing DC1-linked determinants and the murine alloinduced MoAbs. Finally, despite strong structural homologies of murine I-A to human DC and murine I-E to human DR, the intraspecies cross-reactions of MoAbs do not closely follow that pattern. These data: (1) illustrate the usefulness of HLA-deletion mutant cell lines for analysis of the specificity of MoAbs and for delineation of HLA subregions; (2) demonstrate the great diversity of MoAbs specific for class II molecules and the high frequency of MoAbs that bind to products of more than one Ia subregion, particularly DR and SB. In view of such complexity, many (perhaps most) MoAbs cannot be relied on to unambiguously identify products of a particular Ia subregion, without extensive characterization.     

Development of a sensitive solid-phase radioimmunoassay technique for quantification of class-specific Escherichia coli antibodies

A specific and sensitive, quantitative solid-phase radioimmunoassay (RIA) has been developed for the detection of Escherichia coli antibodies in serum and secretions. Preparation of affinity-purified anti-E. coli standards allows accurate quantification of G, M and A classes of antibody down to 10 ng/ml using only 20 microliters of sample. This technique has considerable advantages over indirect haemagglutination in sensitivity and accuracy of immunoglobulin class detection. RIA also compares favourably with ELISA in sensitivity and sample size required. Affinity-purified standards may also be used to quantify the ELISA test.     

Isolation of the human myelin basic protein by immunoaffinity chromatography with a monoclonal antibody

Immunoaffinity chromatography has been developed for the isolation of the human myelin basic protein (MBP). The method is based on the use of a monoclonal antibody which was produced to bovine MBP, cross-reacting with human MBP. The protein isolated from acidic extracts of the brain proteins was shown to be native MBP by its immunochemical reactivity, by its ability to elicit experimental allergic encephalomyelitis and by its mol. wt (18,600 ± 400). It represented a single-band purity after hypersensitive silver staining. The MBP isolated by the method described represents a higher purity than that of the MBP purified by conventional multistep biochemical separation techniques.     

Antibody production by human X human hybridomas in serum-free medium

Four human X human hybridomas were adapted to growth in serum-free medium consisting of RPMI 1640 supplemented with bovine serum albumin and transferrin (BSA/Tf medium). Production of specific monoclonal antibodies was maintained for more than 2 months. Although the maximal cell density achieved was lower than that in serum-supplemented medium, immunoglobulin production was similar or higher when results were expressed on a per viable cell basis. Thus it is feasible to grow human X human hybridomas in serum-free culture and it is possible that this will become the method of choice for large scale production of human monoclonal antibodies.     

An enzyme immunoassay for the detection of staphylococcal protein A in affinity-purified products

Rabbit antiserum, specific for protein A from Staphylococcus aureus, was conjugated to alkaline phosphatase and used in a double antibody solid-phase enzyme immunoassay. The assay was developed to monitor eluate from a large-scale protein A-Sepharose affinity column used to purify monoclonal antibodies for human clinical trials. The assay detected soluble protein A in the presence of immunoglobulin at concentrations as low as 4 ng/ml. Analysis of the product purified by affinity chromatography revealed the presence of protein A at ng/ml concentrations. The assay developed here can provide a reliable and convenient method for detecting soluble protein A.     

A rapid and efficient immunization protocol for production of monoclonal antibodies reactive with autoantigens

A simple, time-saving and efficient immunization method suitable for the production of mouse monoclonal antibody secreting hybridomas is described. Draining lymph nodes isolated 9 days after a primary immunization were used as the source of antibody producing cells. No systemic spread of antibody producing cells or specific antibodies could be detected. The present protocol was employed to generate a panel of collagen type II reactive monoclonal antibodies. Most of the monoclonals so generated were found to be of the IgG class.     

Cross-reactivity between solid-phase immunoassay plates and intermediate filaments demonstrated by human monoclonal antibodies

While screening supernatants of human-human hybridomas for rheumatoid factor and anti-cellular activity we found that a significant number of supernatants which react with the Falcon-polyvinyl chloride immunoassay plate used in an enzyme-linked immunosorbent rheumatoid factor assay also react with intracellular intermediate filaments.     

Immunodetection of cell-bound antigens using both mouse and human monoclonal antibodies

A micro enzyme-linked immunoassay (EIA) has been developed for the rapid and sensitive detection of either human or mouse monoclonal antibodies reactive with cell bound antigens. Whole intact cells are immobilized onto 96-well flat bottom microtiter plates by drying in an oven at 37 degrees C overnight prior to the start of the assay. This method of attachment was suitable for all cell types tested, regardless of origin, size and chromosomal content. The dried cells were then rehydrated, incubated with the appropriate test hybridoma supernatant, followed with subsequent analysis by EIA. The plates can be stored at 4 degrees C up to 1 month for future EIA analysis. This assay offers high sensitivity, requires only small amounts of target cells and test hybridoma supernatant, and can be completed within 3 h. This EIA is well suited for the rapid screening of large numbers of hybridoma supernatants and can also be adapted to include cells of any species, providing the appropriate antibody reagents are available.     

A rapid and efficient strategy to generate allele-specific anti-HLA monoclonal antibodies

That generation of allele-specific anti-human leukocyte antigen (HLA) monoclonal antibodies (ASHmAb) is very difficult is well known. This is thought to be due to the unique epitope structure, an assemblage of amino acid residues that lie separately in the amino acid sequence of human HLA, and to its low antigenicity compared with that of common epitopes recognized as xenogeneic determinants by mice. Here we report a rapid and efficient strategy to generate ASHmAb. Different from usual immunization methods is that we suppressed the production of non-allele-specific anti-HLA antibodies against xenogeneic determinants of HLA molecules by immunizing human HLA-B51 transgenic mice against non-HLA-B51 HLA tetramers. In addition, HLA-coated beads enabled rapid and efficient screening for ASHmAb. ASHmAb generated by this strategy will be useful for HLA typing and for clinical diagnosis, such as flow cytometry-based chimerism analysis for early detection of graft failure and relapse of leukemia after HLA-mismatched hematopoietic stem cell transplantation.     

A sensitive enzyme-linked immunosorbent assay for the quantitation of antigen-specific murine immunoglobulin E

An enzyme-linked immunosorbent assay (ELISA) for the quantitation of 2,4-dinitrophenyl (DNP)-specific murine immunoglobulin (Ig) E is described. The assay uses beta-galactosidase, which is conjugated to goat anti-rabbit gamma-globulin (GARG) via a method using a mild heterobifunctional cross-linking reagent, m-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS). The assay has a sensitivity for detection of about 200 pg/ml of anti-DNP IgE. Analyses of murine serum samples using this ELISA correlate well with those obtained using the passive cutaneous anaphylaxis (PCA) reaction in rats. With the use of automated 96-well reader, data acquisition is rapid and, therefore, this ELISA is ideal for analyses of large numbers of samples. The assay can be easily modified for the measurement of other Ig classes and of IgE of other antigen specificities.     

Development of a specific monoclonal antibody assay and a rapid testing strip for the detection of apramycin residues in food samples

A specific monoclonal antibody (MAb) against apramycin (AP) was produced and used to develop an indirect competitive enzyme-linked immunosorbent assay (idcELISA) and a rapid testing strip for the detection of AP residues in foods. MAb exhibited negligible cross-reactivity with other aminoglycosides. Under optimized conditions in 0.01 M PBS, the half maximum inhibitory concentration (IC₅₀) of MAb was 0.41?ng/ml with a limit of detection (LOD) of 0.15?ng/ml. The ELISA results were obtained within 90?min. The mean recoveries from all the spiked food samples were within the range of 79.02–105.49%, with coefficients of variation in the range of 2.21–11.4%. The strip test results obtained within 5?min had visual LODs in the range 2.5–5?µg/kg (ng/ml) for all food samples tested. Therefore, the developed strip test represents a fast and convenient detection method of AP residues in foods.     

An ELISA assay for murine amyloid a and serum amyloid a utilizing monoclonal antibodies

An enzyme-linked immunoassay has been developed to quantitate the amyloid A (AA) and serum amyloid A (SAA) proteins of mice. The assay utilizes monoclonal rat anti-mouse AA as the antibody. The principle advantage of this method is that it avoids the need to denature the mouse sera before its SAA content can be measured.     

A rapid, sensitive two-site immunometric assay for TSH using monoclonal antibodies: investigation of factors affecting optimisation

Twenty-three monoclonal antibodies specific for human thyrotropin (TSH) have been prepared and characterised. Four different epitopes have been identified, 3 of which are sufficiently distinct to allow simultaneous binding of the corresponding antibodies. Only 1 epitope is expressed on free TSH beta-subunit. The best antibodies have affinities for TSH approaching 10(11) M-1 and cross-reactions with lutropin and chorionic gonadotropin of 0.2% or less. A 2-site immunoradiometric assay for TSH was established using 2 monoclonal antibodies, one of which was absorbed to plastic tubes and the other labelled with 125I. Several parameters affecting assay performance were investigated, including conditions for adsorption of antibodies to various plastics, selection of antibody combinations, concentration of labelled antibody and incubation protocol. The optimised assay covered a working range of 1-60 mU/1 (0.17-10 ng/ml) TSH in a 4 h, single incubation protocol, with no significant interference from other glycoprotein hormones at their maximum physiological or pathological concentrations.     

Simultaneous screening for two different antibodies in ELISA by combining two solid phases: microtiter plate and Falcon assay screening test system lid

An enzyme-linked immunosorbent assay (ELISA) is described that used a combination of 2 solid phases: a microtiter plate covered with a Falcon assay screening test (F.A.S.T.) system lid. By coating each solid phase with a different antigen, it was possible to simultaneously detect 2 different antibodies. The results of the combined test were compared and found similar to those obtained in separate assays on the same solid phases by the usual ELISA method. Further, the combined test was more economical in time, work and materials than 2 separate tests for the 2 antibodies. This method was used in a serological survey for 2 turkey viral infections: hemorrhagic enteritis and paramyxovirus type 3.     

A double determinant sandwich immunoassay for quantitation of serum monoclonal anti-I-A antibody

A double-determinant sandwich radioimmunoassay (RIA) is described for the specific detection of anti-I-Ak monoclonal antibody (mAb) in the sera of non-Ighb murine hosts undergoing anti-Ia immunotherapy. This RIA utilizes 2 previously undescribed mAb reagents generated against an Ia.17-specific mAb secreted by the 10-3.6 hybridoma. The first reagent, 7.34, is specific for Ighb-linked allotypic determinants on the Fc portion of IgG2a immunoglobulins as defined by the pattern of reactivity with normal sera from a panel of inbred and Igh recombinant inbred strains. The second reagent, 58.3, is an anti-idiotypic mAb recognizing unique determinants in the combining site of 10-3.6 immunoglobulins, as determined by the specificity of the 58.3 mAb in solid-phase RIA and the capacity of this reagent to inhibit the binding of labeled 10-3.6 mAb to I-Ak-expressing spleen cells. In an RIA procedure using purified 58.3 mAb as substrate and 125I-labeled 7.34 as the detection reagent, serum concentrations of 10-3.6 as low as 1-5 ng/ml can be measured reproducibly after mathematical linearization of the sigmoid standard curve. In the present studies, the serum half-life of 10-3.6 mAb was calculated from assay data to be 3-5 h in I-Ak homo- or heterozygotes and 72 h in non-I-Ak mice. The serum level of 10-3.6 as a function of the mAb treatment protocol was also examined and results are considered with respect to the efficacy of different therapeutic regimens in prolonging transplant survival. Sandwich immunoassays of this type (RIA or ELISA) should provide a highly sensitive and specific means for monitoring serum mAb levels in individuals subjected to antibody immunotherapy for treatment of autoimmune disease, transplant rejection or tumor progression.     

A potential radioimmunoassay system for detection of Hanganutziu-Deicher type heterophile antigen(s) and antibodies in tissues and fluids

A relatively simple, specific and sensitive radioimmunoassay system has been developed for the detection of heterophile Hanganutziu-Deicher (H-D) antigen(s) and antibodies. The 125I-labeled H-D antigen-active molecule used for the assay is a bovine erythrocyte major glycoprotein previously found to have a strong H-D antigen potency. The antigen-antibody complex was precipitated with normal human serum as the carrier protein, followed by the addition of rabbit anti-human IgG F(ab')2 serum. With this method, different H-D antigen-active molecules were compared for heterophile H-D antigen potency with reasonable sensitivity detecting about 0.3 ng of cold glycoprotein. 8 different lung cancer tissues were assayed for H-D antigen. The sera from the 8 lung cancer patients were also screened by ELISA and RIA in an attempt to correlate expression of H-D antigen on tissues with elevation of H-D antibodies. The results showed that all patients' tissues expressed the antigen(s) but only 3 of them had abnormal levels of H-D antibodies. This could have been due to excess antigens in circulation or immune complexes.     

Murine hybridoma monoclonal antibodies against insulin: cross-reactivity with insulins of three species and blocking of insulin binding to its receptor

Six hybridoma cell lines secreting monoclonal antibodies against pig insulin and cross-reacting with human and bovine insulins were obtained. Five of these monoclonal antibodies were IgG1, kappa, one IgG2b, kappa; their pI values were in the range of pH 6.3-7.4 and dissociation constants of the insulin-antibody complexes were 0.3-2 X 10(-8) mol/l, as determined by an immunoradiometric inhibition assay. All of these antibodies reacted with sterically closely related determinants and blocked the binding of 125I-pig insulin to the receptor on human MOLT-4 cell line.