A solid phase radioimmunoassay on hydrophobic membrane filters: Detection of antibodies to gonoccccal surface antigens

A solid phase radioimmunoassay (SPRIA) has been developed for detection of IgG antibodies to gonococcal outer membrane components. Gonococcal antigens were immobilised on a solid support by covalent coupling to CNBr-activated Sepharose in the presence of the detergent Triton X-100. Binding of specific antibody to the Sepharose-antigen complex was detected using radiolabelled Protein A as the antiglobulin. Protein A was labelled by radioacetylation with tritiated acetic anhydride, yielding a product of high specific activity and high stability. No detectable loss of activity was observed over a ten month period. The entire assay was performed on Mitex teflon hydrophobic membrane filters which held the Sepharose beads and aqueous supernatant as a discrete drop of liquid. The supernatants and incubation media were easily and rapidly removed from the beads by suction on a specially designed manifold system. This procedure removed the need for repeated and time-consuming centrifugations. Titres were obtained graphically from double log plots of cpm bound versus antiserum dilution by extrapolation of the straight line to a point corresponding to twice the control level of radioactivity binding. The assay proved to be a very reliable and simple procedure for the detection of IgG antibodies to gonococcal surface antigens.     

A sensitive assay for the detection cytotoxic antibodies to mammalian cell surface antigens

A microcytotoxicity assay for the detection of complement-dependent serum toxicity against adherent cells has been modified to include post-labeling of the target cells with [3H]leucine or [3H]thymidine. The adaptation represents a significant improvement over the visual counting of residual cells. The technique is more objective and less tedious than the original procedure, while retaining the features of economy of cells and sera, reproducibility, and sensitivity. Examples are given which demonstrate the suitability of the modified assay for the analysis of human and mouse tumor antigens using autologous, allogeneic, and syngeneic sera.     

A rapid method for detection of flavivirus antigens: staphylococcal co-agglutination test using monoclonal antibodies to Japanese encephalitis virus

Staphylococcus aureus rich in protein A when coated with monoclonal antibodies (MoAb) to Japanese encephalitis virus (JEV) gave a highly specific reaction with flavivirus antigens. The bacteria coated with JEV species-specific MoAb gave a strong co-agglutination with fifty-six JEV isolates from various parts of China, but no co-agglutination with Murray Valley encephalitis (MVE) and Kunjin (Kun) virus antigens. The flavivirus- and subgroup-specific MoAbs were reactive with MVE and Kun, as well as with the majority of the JEV strains. Blocking test with homologous MoAbs abolished co-agglutination further confirming its specificity. Numerous virus particles were observed on the surface of MoAb-coated staphylococci under the electron microscope after co-agglutination. The test appeared rapid, specific, simple to perform, and useful for rapid detection and identification of flaviviruses.     

The detection and specificity of class specific antibodies to whole bacterial cells using a solid phase radioimmunoassay.

A solid phase radioimmunoassay has been developed which can be used for the detection of isotype specific antibodies to whole bacteria and other particulate antigens, and is applicable to a variety of species. Bacteria are bound to the solid phase by the use either of antibodies, or of methyl glyoxal. Both methods result in a sensitive and reproducible assay, and bacteria do not appear to desorb from the solid phase. The specificity of antibodies to whole bacteria was examined by absorption of antisera with various species of bacteria and retesting, or by determining the binding of antisera to various bacteria bound to the solid phase. Both methods revealed specificity for the bacteria examined. Inhibition studies showed that antibodies to Streptococcus mutans whole cells could be inhibited by purified cell surface antigens glucosyltransferase and antigen I/II, but only minimally by lipoteichoic acid, c polysaccharide or dextran. In murine antisera antibodies of the IgG, IgM, and IgA classes could be detected at amounts of less than 1 ng/ml.     

A simple assay for the detection of antibodies to endocrine islet cell surface antigens

A simple and sensitive immunoradiometric assay for the detection of islet cell surface antibodies (CIRMA) has been developed. Live, transformed islet cells derived from a liver metastasis of a transplantable islet cell tumor were grown in removable microtiter wells and incubated with antibody. Cell-bound antibodies were quantitated using 125I-labelled second antibodies. The assay was used to detect islet cell antibodies present in sera from non-diabetic and diabetic BB rats and proved to be particularly effective for screening hybridoma supernatants in order to identify monoclonal antibodies recognizing islet cell surface antigens.     

A rapid solid-phase enzyme-linked binding assay for screening monoclonal antibodies to cell surface antigens

A solid-phase enzyme-linked binding assay is described for screening monoclonal antibodies to cell surface antigens. E. coli β-galactosidase was coupled to rabbit anti-rat Ig and used to detect the binding of rat monoclonal antibodies to cells which had been fixed to the wells of microtitre plates using a combination of poly-l-lysine and glutaraldehyde. This method was found to be advantageous for the large scale screening of monoclonal antibodies with a panel of cell types, and has been useful in the selection of antibodies which would be candidates for differentiation markers within the human and mouse haemopoietic systems.     

A rapid and efficient method for the generation and screening of monoclonal antibodies specific for cell surface antigens

The generation of monoclonal antibodies (mAb) of desired specificity to cell surface antigens can serve as a valuable tool to study protein expression and function. However, traditional approaches to mAb generation usually involve large-scale protein purification and intensive screening, and may not result in mAb specificities to the native protein of interest. We describe a simple, inexpensive, high-throughput method for the generation and screening of hybridomas secreting mAb specific for cell surface receptors. Intact reporter cells expressing a CD3zeta-fusion receptor of the protein of interest are plated in 96-well arrays of captured, plate-bound hybridoma supernatants. A mAb to the protein of interest generates a signal leading to reporter-cell expression of beta-galactosidase, and enzyme activity can be screened in a single day using a non-radioactive substrate. Importantly, a single cell line can be used for immunization, screening, semi-quantitative affinity comparisons, and subsequent screening for physiological ligand expression, if the protein of interest is a receptor. We describe an application of this approach to generate mAb specific for a protein of previously unknown expression and undocumented function.     

A simple hemagglutination method for the detection of cell surface antigens

A rapid, easy and reproducible hemagglutination method is described for the detection of cell surface antigens. This method can be used for the determination of cell subpopulations and the screening of monoclonal antibody-secreting hybridomas. Fresh or paraformaldehyde-fixed cells, which have previously been labeled with an appropriate monoclonal antibody (of known or unknown specificity), are reacted with human erythrocytes coupled with the second antibody (rabbit anti-mouse immunoglobulins), in a round bottomed 96-well microtiter plate. The presence of specific agglutination and the end-point of serial dilutions of the labeled cells with unlabeled ones give an approximate estimation of the percentage of cells reacting with the monoclonal antibody under examination. The hemagglutination results correlate well with the results obtained using a conventional immunofluorescence method.     

A method for a radioimmunoassay using microtiter plates allowing simultaneous determination of antibodies to two non cross-reactive antigens

A triple isotype (²²Na,¹³¹I,¹²⁵I) radioimmunoassay method employing microtiter plates and specially designed disposable paperware has been devised. This procedure allows the determination of antibodies to two non cross-reactive antigens simultaneously on large numbers of samples.     

Bacteria as solid phase in a concentration fluorescence immunoassay analysis of antibodies to surface antigens

A modified solid-phase fluorescence immunoassay was developed using bacterial cells as the solid phase to screen antibodies produced against surface antigens from a clinical isolate of Escherichia coli, strain 1-149. The bacterial solid phase was used to analyze both polyclonal and monoclonal antibodies. The bacterial concentration fluorescence immunoassay (BCFIA) showed up to 50-fold greater sensitivity in bacterial cell detection as compared to ELISA (enzyme-linked immunosorbent assay). Moreover, BCFIA was considerably faster than ELISA with uniform reproducibility. This paper demonstrates the utility of using bacteria and their surface antigens as solid-phase matrices for antibody characterization in a FIA.     

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

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

A new radioimmunoassay using a commercially available solid support for the detection of IgE antibodies against muscle relaxants.

It is well established that muscle-relaxant drugs may be responsible for anaphylactoid reactions during anesthesia. In this study, we developed an in vitro test with a commercially available solid phase for the detection of specific IgE directed to the tertiary or quaternary ammonium groups of neuromuscular-blocking drugs. The solid-phase complex was P-aminophenylphosphoryl-choline (PAPPC) immobilized on agarose, and an RIA was performed with an antihuman IgE labeled with 125I. The results, expressed as the percentage of 125I-labeled anti-IgE linked to the solid phase, were at 0.41 +/- 0.19 for 34 control sera from nonallergic healthy adults, with an upper limit estimated at 1%. The values obtained with the sera of 31 allergic patients ranged from 0.6% to 41% with a sensitivity of 97%. The specificity and the positive predictive value of the PAPPC RIA were 97% and 94%, respectively. These results were compared with results of other RIAs with morphine, trimethylamine, triethylamine immobilized on epoxy-activated Sepharose, and choline hydrochloride immobilized on Sepharose (quaternary ammonium Sepharose RIA) and with Phadebas RAST succinylcholine and Phadebas RAST alcuronium. The PAPPC RIA appears to be the most efficient test to screen sera for the presence of IgE antibodies directed to neuromuscular-blocking drugs. One major advantage is that this solid phase is commercially available and ready to use. This advantage will improve the accuracy in the comparison of the results with results from different laboratories.     

Detection of antibodies to mycobacterium tuberculosis by solid phase radioimmunoassay.

The sandwich solid phase radioimmunoassay procedure has been adapted to the serological diagnosis of tuberculosis by the use of disposable plastic haemagglutination trays in which the wells were sensitized with soluble mycobacterial antigen and the subsequent uptake of globulin from test sera detected with radioiodinated antiglobulin. The influence of time and temperature of the incubation stages, pH, washing times, drying and the efficiency of various non-specific blocking agents have been studied. In an initial survey carried out on 319 sera the results obtained showed a good correlation with the presence or absence of established tuberculous infection.     

A simple and rapid method for the detection of lymphocytotoxic antibodies using cell panels frozen on Terasaki plates

A 40 cell panel of lymphocytes selected for HLA-A,B and DR antigens, frozen in Terasaki microtest trays can be used routinely to identify the presence of specific HLA antibodies within two hours. Blind testing using well defined HLA typing sera showed that specificities could be identified to a high degree of significance using this method. The method has proved successful for screening for T cell, including HLA-A,B and B cell, including HLA-DR antibodies. The method is particularly useful for the routine tissue typing laboratory as the frozen panel can be used without the need for complicated and time-consuming cell washing procedures which have been the downfall of previously published methods.     

A rapid, objective method for the detection of lymphocytotoxic antibodies using flow cytometry

Whilst several centres have reported lymphocytotoxic antibody detection using single and dual fluorescent stains with analysis of the fluorescence emitted from the cell population present in a well of a multiwell plate, problems are encountered with cell concentration and light emission overlap. A method we have developed using flow cytometry produces similar values of percentage cell death using single or double staining techniques (correlation coefficient = 0.9896). This method is not influenced by slight variation in cell number or light emission overlap. The effect of introducing red cell impurities into the normal lymphocyte preparation is described.     

Bacterial cell microarrays for the detection and characterization of antibodies against surface antigens

Bacterial cell surface antigens interact with the host immune system resulting in the production of antibodies. Detection of antibodies against surface antigens has applications in diagnosis of many bacterial infections, assessment of immune status and epidemiological studies. We developed a microarray platform, for antibody detection, by printing Gram-negative and Gram-positive whole bacterial cells on nitrocellulose coated glass substrates. Antibody binding was detected using fluorophore labeled secondary antibodies. The sensitivity of antibody detection was found to be 0.1 microg/ml. Using bacterial cell microarrays it was also possible to successfully detect antibodies against Francisella tularensis in canine serum samples declared positive for tularemia based on microagglutination antibody titer. Use of bacterial cells as the antigen source in immunoassays has the advantages of simulating in vivo presentation of surface antigens and also eliminating the need for antigen purification. The microarray format gives the added advantage of simultaneous detection of antibodies against multiple bacteria employing only small amounts of samples and reagents.     

A modified method for glutaraldehyde fixation of cells to a solid phase surface

Summary A method is described for fixing cells to a solid phase surface for use in an antibody detection assay.