Particle concentration fluorescence immunoassay (PCFIA): a new, rapid immunoassay technique with high sensitivity

A new solid-phase fluorescence immunoassay technique is described and is exemplified by the detection of murine monoclonal antibodies to human IgG in hybridoma culture supernatants and the detection of murine IgG. The assay is performed in a specially designed 96-well plate. For antibody detection, antigen bound to submicron polystyrene particles is bound to its specific antibody, which is in turn reacted with fluorescein-labeled affinity-purified goat anti-mouse IgG. The reaction is complete in 10 min at ambient temperature. The solid phase is separated from the reaction mixture by filtration, washed and the total particle-bound fluorescence is determined by front-surface fluorimetry. The sensitivity of the technique for antibody detection is equivalent to enzyme-linked immunoabsorbent assay and 2-4 ng/ml for murine IgG detection. It is readily amenable to automation.     

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, automated colorimetric assay for measuring antibody binding to cell surface antigens

An automated, colorimetric procedure is described for detecting antibodies specific for cell surface antigens. The procedure entails (a) coating the wells of 96-well microplates with either protein A or anti-immunoglobulin antibodies and (b) preincubating either the microplate or target cells with the test antibody. Target cells which react with the test antibody bind to the wells of the microplate and bound cells are quantitated by staining with the dye Rose Bengal. A microplate spectrophotometer is used to measure absorbance in each well of the plate, providing a rapid, automated measure of antibody titre. The assay is simple to perform, uses readily available reagents and gives comparable sensitivity to rosetting assays. With these features, and the capacity for handling large numbers of trays quickly, this method has obvious advantages in screening for antibody activity in culture supernatants of hybridoma clones.     

Purification of polymeric immunoglobulin from cell culture supernatants by affinity chromatography using secretory component

Human secretory component bound covalently to Sepharose 4B has been used as an affinity adsorbent to isolate and purify polymeric immunoglobulin from cell culture supernatants. The method was used to isolate murine IgM isotype anti-trinitrophenol antibody and rat IgM isotype anti-lymphocyte antibody from hybridoma cell culture supernatants. Gel filtration of the eluted antibodies followed by enzyme immunoassay showed that all recovered IgM was of pentameric molecular size. Murine IgA isotype anti-dinitrophenol antibody and murine IgA anti-human rotavirus antibody were isolated from cell culture supernatants of a plasmacytoma and a hybridoma respectively. Most of the IgA recovered following affinity adsorption with secretory component was of greater molecular size than dimer. Murine IgG was not adsorbed by secretory component bound to Sepharose. Eluted antibody retained antigen binding activity. Affinity chromatography using human secretory component bound covalently to a solid phase provides an antigen-independent technique for purification of murine and rat IgA and IgM polymeric immunoglobulin from cell cultures.     

An enzyme immunofiltration assay useful for detecting human monoclonal antibody

A microenzyme-linked immunoassay (EIA) utilizing an immunofiltration manifold has been developed which provides a rapid, simple, and sensitive method of detecting human monoclonal antibody class, concentration, and specificity. In this assay either whole cells or soluble antigens were immobilized on glass fiber filters followed by incubating with the test human hybridoma supernatant with subsequent analysis by EIA. A specially designed 96-chamber immunofiltration plate is employed which serves as both an incubation chamber and as a filtration manifold. The assay described is unique in that small volumes of human hybridoma supernatant are required, crude preparation of only a few target cells are needed, labile cell surface antigens are preserved and it can be completed in 3 h. This assay is well suited for the rapid screening of large numbers of human hybridoma supernatants.     

The adaptation of the plaque reduction assay for measuring specific cytotoxic cells in limiting dilution cultures

The use of the original haemolytic plaque reduction technique to measure cytotoxic T lymphocytes (CTL) has been developed further as a rapid screening assay, particularly suitable for limiting dilution analyses. Using hybridoma cells as targets, the cytotoxicity has been measured by the loss of haemolytic plaque formation and by the reduction of the amount of haemolytic monoclonal antibody secreted from viable target cells into the assay supernatants. The assessment of large numbers of cytotoxic samples has been greatly facilitated by quantitating the amount of haemoglobin released in the assay with an automated microELISA multiscanner and by scoring visually using a modification of the spot test. Using these new techniques, relatively high frequency estimates of cytotoxic cell precursors in an allogeneic response (1 in 462 spleen cells) and an anti-fluorescein response (1 in 3970 spleen cells) were obtained.     

Development of a rapid microELISA assay for screening hybridoma supernatants for murine monoclonal antibodies

A micro enzyme-linked immunosorbent assay (ELISA) utilizing a filtration method has been developed which allows the rapid, simple, and sensitive detection of monoclonal antibodies that recognize either soluble or cell surface antigens. This assay involves the immobilization of target cells (or soluble antigen) onto glass fiber filter discs followed by an incubation with the test hybridoma supernatant and subsequent analysis by ELISA. A specially designed 96-well filtration device is employed which serves both as an incubation chamber and as a filtration manifold. This microELISA requires small volumes of antiserum, few target cells, and can be completed in less than 2 h. This assay is well suited for the rapid screening of murine hybridoma supernatants and can be adapted to detect monoclonal antibodies from other species.     

High-throughput homogeneous immunoassay readily identifies monoclonal antibody to serovariant clostridial neurotoxins

High-throughput screening can create the potential ability to screen large numbers of monoclonal antibodies (mAb) in a short time period. A major bottleneck in the hybridoma method for mAb development has historically been the inability to sift through large numbers of hybridoma culture supernatants to identify clones secreting mAbs of the desired specificity. Herein, we develop a homogeneous fluorometric microvolume assay technology (FMAT) and compare it to conventional ELISA screening techniques for monoclonal antibody against soluble protein toxin fragments of the Clostridium botulinum types A, B and E neurotoxin (BoNT) proteins. In total 8,744 hybridoma clones were screened to identify 29 stable hybridomas to neurotoxin binding domain; six of these would have been missed by ELISA alone. Screening of hybridoma supernatants on days 1 and 4 following cloning from semi-solid HAT agarose reveals that FMAT provides a reliable method for screening hybridoma clones to purified protein toxins. The homogeneous FMAT utilizes far less reagent (antigen and hybridoma supernatant) allowing for simultaneous screening against multiple serovariant antigens early in the hybridoma cloning cycle. This reduces costs for reagents and labour by lowering numbers of clones being maintained with undesired specificity. Furthermore, this assay easily accommodates replicate screening which facilitates identification of cross-reactivity to neurotoxin serotypes, thus readily identifying mAb to serovariant antigens. These findings have broad application in accelerating mAb development to serovariant cell-surface or bead bound targets without arraying devices. In summary, FMAT provides a reliable method for the screening of mAbs against C. botulinum neurotoxins.     

Production and use of rat monoclonal antibodies to the human myeloid cell nuclear differentiation antigen

A dot immunoblot screening assay was used to identify rat monoclonal antibodies to a human myeloid cell differentiation-specific nuclear antigen (MNDA). The selection was based on the positive reaction of hybridoma cell supernatants with a concentrated nuclear protein extract prepared from late stage human myeloid leukemia cells that express MNDA (HL-60) coincident with a negative reaction with the same extract prepared from a non-expressing more immature human myeloid leukemia cell line. The approach provided an efficient method for obtaining monoclonal antibodies to a specific low abundance nuclear antigen that has not been purified. Sixteen wells from three fusions contained antibody displaying a specific reaction with the nuclear protein fraction obtained from the HL-60 cells. Immunoblotting analysis revealed that all of the sixteen specific hybridoma cell lines produced antibody against the same Mr 55,000 nuclear antigen. Selecting hybridoma cells that produce antibody reactive with the native antigen provided antibody suitable for detecting MNDA in immunocytochemical tests. The rat monoclonal antibodies were purified and coupled to CNBr-activated agarose and carbonyldiimidazole-activated agarose. Although both antibody affinity matrices exhibited the same antigen binding capacities, the matrix prepared using carbonyldiimidazole-activated agarose bound the MNDA with a high level of specificity while the matrix prepared from CNBr-activated agarose bound numerous other nuclear proteins.     

一种狂犬病实验室诊断用单克隆抗体制备新路线的探索

目的 探索采用合成肽作为免疫原制备狂犬病实验室诊断用单克隆抗体的可行性.方法 以狂犬病病毒CVS-11核蛋白355-369位B细胞线性抗原表位合成肽与钥孔戚血蓝蛋白（Keyhole Limpe hemocyanin,KLH）大分子耦联后免疫BALB/c小鼠,利用经典杂交瘤细胞技术制备单克隆抗体.采用间接酶联免疫吸附试验（enzyme-linked immunosorbent assay,ELISA）和间接荧光试验（indirect fluorescent assay,IFA）筛选和鉴定杂交瘤细胞株.结果 经过对杂交瘤细胞株上清的间接ELISA和IFA筛选获得阳性杂交瘤细胞株2B1D11,该杂交瘤细胞株产生的抗体经纯化后在IFA中可以有效检出感染犬脑组织和BHK-21细胞的狂犬病病毒.结论 采用合成肽作为免疫原制备狂犬病实验室诊断用抗体在技术上是可行的.     

Epitope binning of murine monoclonal antibodies by a multiplexed pairing assay

We describe an assay and data evaluation technique for sorting a panel of murine monoclonal antibodies according to epitope specificities. The assay analyzes the simultaneous binding (pairing) of antibodies to an antigen and groups together antibodies with similar pairing profiles. Similar profiles indicate that the antibodies bind to the same or closely related epitopes. The assay works well with crude hybridoma supernatants and can be multiplexed. These features make the assay particularly suitable for the early phase of hybridoma/antibody screening when antibodies are available only as low volume culture harvest samples.     

A rapid and sensitive chemiluminescence dot-immunobinding assay for screening hybridoma supernatants

The present report describes a simple and rapid dot-immunobinding assay combined with a chemiluminescence detection system for screening hybridoma supernatants for specific monoclonal antibodies (MAbs). Small rectangular nitrocellulose filters dotted with either crude mixtures of antigens, or with control samples, were placed in six well plates, incubated with hybridoma supernatants, then stained with peroxidase-conjugated anti-mouse IgG. The reaction was performed with a chemiluminescence detection system. We used this method to screen hybridoma supernatants for MAbs against a 354 amino acid polypeptide of hog cholera virus (HCV) gp33-gp55 protein expressed as a fusion protein. We also extended it for the screening of MAbs against foot-and-mouth disease virus (FMDV). The chemiluminescence dot-immunobinding assay (CDIA) was compared with neutralization (N) and immunofluorescence (IF) screening tests and some FMDV seroneutralizing MAbs were found to either poorly reactive or undetected by the IF test. The advantage of the present method is that it detects in only one step all MAbs detected in the IF and/or N tests together with some MAbs not detected by either of these methods. The present method is at least 356 times more sensitive than the IF test.     

A semi-automated fluorescent (SAF) assay using viable, whole cells for screening hybridoma supernatants

In the production of monoclonal antibodies, a rapid, sensitive, accurate assay is needed for the critical step of screening. We report the modification of an assay using viable whole cells for screening hybridoma supernatants. The modified assay uses fluorescent second antibodies for detection and has been adapted to an instrument capable of automating a number of assay steps. The modified assay is compared to a dot radioimmunoassay developed and used in our laboratory. The fluorescence assay is highly sensitive but shows more background effect, especially in samples with high protein content, such as ascites. The automated fluorescence assay is very rapid, capable of completing an assay in less than 90 min, and can be performed with minimal operator involvement. The assay was performed successfully with several different antibodies and cell types. This screening procedure should be especially useful for laboratories with large numbers of fusions to evaluate.     

Cryopreservation of newly formed hybridomas

A new freezing technique is described which permits time-consuming protocols as a first screening of newly formed hybridomas. In this procedure complete 96 well clustertrays with growing hybridomas are cryopreserved after programmed freezing. This procedure has been successfully applied to a number of fusion protocols for which the objective was to obtain monoclonal antibodies against tissue specific antigens. To this end hybridoma supernatants were screened by an immunoperoxidase technique on frozen sections. Freezing of the hybridoma containing clustertrays permitted extensive screening and partial characterization of the previously collected supernatants. After subsequent thawing of appropriate wells, the hybridoma clones proved to be viable and usually no loss of antibody production was observed.     

Production and detection of monoclonal anti-idiotype antibodies directed against a monoclonal anti-beta-adrenergic ligand antibody

A new method has been developed to raise monoclonal anti-idiotypic antibodies. Monoclonal anti-idiotypic antibodies were obtained by fusion of NS-1 myeloma cells with splenocytes of mice immunised by intravenous injections of fixed hybridoma cells bearing a monoclonal antibody specific for beta-adrenergic ligands. New screening tests were developed to analyse the resulting hybridoma supernatants for different anti-idiotypic properties. Among 23 hybridoma supernatants recognising the idiotype, 6 were found to inhibit hapten binding and 3 of these recognised beta-adrenergic receptors.     

Comparison of two GM1-erythrocyte assays to detect heat-labile Escherichia coli enterotoxin in stool specimens.

Two erythrocyte immunoassay techniques to detect the presence of Escherichia coli heat-labile enterotoxin (LTh) in stool supernatants and cell-free culture supernatants were compared. In the competitive assay, GM1 ganglioside was coated onto V-shaped-well microdilution plates and enterotoxin was coupled to sheep erythrocytes. As little as 0.8 ng of LTh per ml was detected by this method, which was based on the competition between the LTh of the test sample and the sensitized erythrocytes. The second assay made use of chimera antibody prepared by coupling polyclonal anti-LTh antibody to a monoclonal antibody specific for sheep erythrocytes. In this case, LTh, which was specifically bound to a GM1 ganglioside-coated plate, was detected by successively adding the chimera antibody and sheep erythrocytes. The limit of detection of the chimera antibody erythrocyte immunoassay was 0.2 ng/ml. Stool samples were collected from 167 infants hospitalized for diarrhea in the hospital of Noumea, New Caledonia. False-negative reactions due to proteases present in the stool samples were avoided by the addition of phenylmethylsulfonyl fluoride.     

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 novel cytotoxic T lymphocyte activation assay. Optimized conditions for antigen receptor triggered granule enzyme secretion

A method is described for the quantitative studies of cytotoxic T lymphocyte (CTL) activation. This functional assay is based on the measurements of secreted granule-associated enzymatic activity (BLT esterase (BLT-E) ) after incubation of CTL with activating stimuli. Immobilized mAb against CTL's antigen receptor (anti-TcR mAb), concanavalin A or a combination of PMA and ionophore A23187, were able to trigger the secretion of enzyme in the absence of target cells. Soluble anti-TcR mAb alone did not activate CTL, but using their conjugate with immobilized rabbit anti-mouse Ig antibody (RAMIg) TcR-mediated secretion of BLT-E was detected. Use of non-ionic detergents Nonidet P-40 or Triton X-100 (0.0125-0.2%) did not affect measurements of BLT-E activity. The efficiency of CTL exocytosis triggering by anti-TcR mAb which were immobilized on the surface of different plasticware is compared and conditions for studies of small and large numbers of CTL are described. The intensity of CTL response varies markedly with changes in buffer system, culture medium, additions of proteins. The optimal conditions for TcR complex triggered activation of murine CTL are described. Intensity of secretion can be easily manipulated by changing the surface density of immobilized anti-TcR mAb, thereby providing the possibility to screen inhibiting or activating agents (drugs or mAb) at selected sub-optimal levels of CTL activation. The potential for the use of described assay in screening of hybridoma supernatants for the presence of activating or inhibitory mAb against CTL's surface proteins is discussed. Since BLT-E secretion reflects exocytosis of granules from CTL, the conditions described here could be used for the detection of secretion of other markers of granules in future modifications of granule exocytosis assay.     

Immunofluorescence for detection of antibodies against human cytomegalovirus-induced membrane antigens.

This paper describes an improved method for the in vitro detection of antibodies specifically directed against human cytomegalovirus (CMV)-induced membrane antigens present on the surface of CMV-infected fibroblasts (CMV-MA). Viable cells were found to be essential for specific visualization of CMV-MA staining. The addition of divalent cations (2.6 mM Ca2+ and 2.2 mM Mg2+) and glucose (180 mM) to the incubation and washing buffers improved the viability and morphology of the cells and increased the cell yield at the end of the assay. Clustering of antigen-antibody complexes on the surface of viable CMV-infected cells was prevented by low-temperature incubation (0 to 4 degrees C) rather than by the addition of agents which act on the metabolism of the cell. No interaction with the CMV-induced Fc receptor was observed at 0 degrees C with either human sera or murine monoclonal antibodies. The specificity of the CMV-MA reaction was confirmed by using monoclonal antibodies to CMV nuclear, cytoplasmic, and membrane-associated antigens. Furthermore, a microplate modification of the membrane fluorescence test is described which is suitable for multisample screening purposes. This method can be applied to the determination of anti-CMV-MA antibody titers in human sera and to the screening of hybridoma supernatants for the presence of antibodies with specificity for CMV-MA.     

Antibody-mediated activation of T cell clones as a method for screening hybridomas producing antibodies to the T cell receptor

In this study, supernatants (SN) of hybridomas established by fusing P3X63.Ag8.653 to spleen cells from C57L mice (Vß8 family of T cell receptor (TcR) gene negative) immunized with the H-Y specific Vß8 allotype positive helper T cell (HTL) clone OI6 were screened for the capacity to activate cloned T cells in the absence of interacting stimulator cells. In the first assay, SNs were mixed with Vß8⁺ H-Y specific CTL OH2 and ⁵¹Cr-labelled non-specific B lymphoma (L10.A). In this system, antibodies (Ab) which can bind to L10.A by Fc-Fc receptor interaction and recognize TcR can facilitate lysis of L10.A target cells by OH2 CTL. In the second assay, OI6 clone cells were cultured in microtiter well, previously coated with hybridoma supernatants (SN). In this assay, Ab recognizing OI6 TcR complex and bound to plastic plates can stimulate OI6 cells to proliferate in the absence of stimulator cells. Using these two screening methods, nine hybridomas were established. Analysis of these hybridoma SN using surface staining, inhibition of T cell function and immunoprecipitation of radiolabelled surface molecules followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) showed that five Ab were directed to the allotypic determinant (Vß8) of TcR and four Ab were specific to the clonotypic determinant of OI6 TcR. These results suggest that this Ab-mediated activation of T cell clone can be used for the screening of hybridomas secreting anti-TcR Ab and the immunogenicity of OI6 clonotypic determinants is similar to that of the Vß8 allotypic determinant even in strains which do not express the Vß8 TcR allotype.