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.     

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.     

Characterization of a method using viable human target cells as the solid phase in a cell concentration fluorescence immunoassay (CCFIA) for screening of monoclonal antibodies and hybridoma supernatants

A new method has been characterized for the use of viable target cells as the solid phase for screening of hybridoma supernatants in a cell concentration fluorescence immunoassay (CCFIA). Briefly, the specific target antigen on the cells is bound by the monoclonal antibodies and revealed by use of a fluoresceinated second antibody. Separation of free from bound antibody is accomplished by filtration in the 0.2 micron filter-bottom wells of specialized assay plates. Processing is automated in a Pandex screen machine, resulting in numerical fluorescence values for each well. This method is rapid (under 1 h per 96-well plate), highly sensitive (down to 0.2 ng/ml) and sparing of target cells (0.3-2.5 X 10(4) cells per assay well). It has been applied to 37 different varieties of human solid tumor cells, as well as to human peripheral blood mononuclear cells. The cells used as targets for the characterization of this method were still capable of attachment and growth when recovered post-assay. This method was compared with a viable cell enzyme-linked immunosorbent assay (ELISA) method, showing similar sensitivity and greatly shortened assay time. Comparison of the results from this method with those obtained from flow cytometric analysis performed on viable cells showed close correlation, whereas a lower correlation was seen with immunohistochemical methods using acetone-fixed cells. Development of this method made it possible to rapidly screen many thousands of hybridoma supernatants and successfully select those which were specific for surface antigens on viable cells.     

A rapid solution-phase screening technique for hybridoma culture supernatants using radiolabeled antigen and a solid-phase immunoadsorbent

The solid-phase immunoassay commonly used to screen hybridoma supernatants at times gave falsely positive results. A solution-phase screening technique, which uses radiolabeled antigen and a solid-phase immunoadsorbent, is described. This technique overcomes the problem of false positives and can be easily adopted for other soluble antigens.     

A dot screening procedure on nitrocellulose for detecting anti-idiotopes in hybridoma supernatants

A dot assay on nitrocellulose was developed for screening hybridoma fusions for the presence of anti-idiotopes. The technique involves spotting of hybridoma supernatants onto nitrocellulose filters, and detection of anti-idiotope with an alkaline phosphatase-coupled idiotype.     

An improved dot immunobinding assay for screening hybridoma supernatants. Non-purified antigen immobilized on nitrocellulose paper discs

This report describes a modified dot immunobinding assay (DIA) in microplates using a crude mixture of non-purified antigen. Nitrocellulose filter paper discs exposed to the antigen mixture were inserted into the wells and kept in place by a specially constructed device. To test the efficiency of the modification a set of monoclonal antibodies from a mouse immunized with 58 kDa trpE-Bmyc fusion protein were screened. The advantage of this modified method over conventional ELISA is that it permits the use of non-purified antigen for screening large numbers of monoclonal antibodies.     

A fluorescent stain for viable rosette-forming cells

A rapid fluorescent staining technique for visualising viable rosette-forming cells is described.     

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.     

Inadequacy of traditional ELISA for screening hybridoma supernatants for murine monoclonal antibodies

Hybridomas producing anti-creatine kinase (CK) and anti-lactate dehydrogenase (LDH) antibodies were screened by enzyme linked immunosorbant assay (ELISA) with antigen coated onto plastic wells. Out of seven antibodies positive for each isoenzyme of CK, four antibodies failed to bind to the radiolabeled antigen in solution phase radioimmunoassay (RIA) or native antigen in competitive ELISA. Moreover, out of nine antibodies shown reactive with LDH-1 and seven antibodies binding to LDH-5 by ELISA, not a single antibody bound to either radiolabeled or native antigen in solution. Our results along with other recent studies strongly suggest that antigen conformation on solid phase may frequently be different from in solution and that screening by ELISA in which antigen is attached to solid phase is often inappropriate for determining the presence of useful monoclonal antibodies.     

A fast and simple dot-immunobinding assay for quantification of mouse immunoglobulins in hybridoma culture supernatants

Mouse monoclonal antibodies of IgG subclasses and IgM class in hybridoma culture supernatants were quantified using a dot-immunobinding assay. Immunoglobulins were bound to nitrocellulose (NC) membrane and, after blocking, the membrane was incubated with anti-mouse antibody conjugated to horseradish peroxidase (HRP). Binding was revealed by incubation with a sensitive chemiluminiscence reagent. Quantitation was achieved by densitometric comparison with standard curves produced by purified monoclonal antibodies of the same subclass or purified antibodies of the same clone as the antibody to be quantified. These quantitative results were compared with those obtained using purified IgG from mouse serum or purified mouse myeloma IgM as standards. The dot-immunobinding assay requires 1 microl of hybridoma culture sample and takes about 1 h in total. Good linearity between the staining intensity and the amount of immobilized immunoglobulins was observed over the range of 0.05-5 ng/spot. The assay is simple, reproducible and can process simultaneously a large number of samples.     

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.     

Enzyme-linked immunosorbent assay (ELISA) to detect antibodies in gonorrhea using whole cells

The choice of an antigen that will adequately differentiate between infected and non-infected patients has been a problem in detecting gonococcal antibodies for diagnosis. We have used the sensitive technique of ELISA to test various serotypes of Neisseria gonorrhoeae for their suitability as antigens. Whole cells of each serotype were attached to polystyrene plates using poly-L-lysine, N gonorrhoeae, strain H1 type 1 was used to detect antibodies in patients with known clinical history and then as a standard to evaluate the ability of different serotypes to differentiate between infected and non-infected groups.     

Use of an enzyme-linked immunosorbent assay (ELISA) for screening of hybridoma antibodies against cell surface antigens

A micro-ELISA for screening of antibodies from hybridoma cultures against surface antigens of human melanoma is described. The technique employs alkaline phosphatase-conjugated protein A and target cells attached to poly-l-lysine-coated microtiter plates. The micro-ELISA is equally sensitive as the radioimmunoassay. Mild glutaraldehyde treatment of cells did not lead to an appreciable loss of antigen activity. The fixed cells can be stored at 4°C for at least 6 weeks. It is concluded that the ELISA is superior to the radioimmunoassay in the following aspects: (1) exclusion of radioactive hazards, (2) speed of performance, and (3) lower costs.     

A solid-phase immunofluorescence assay (SIFA) for screening antigen-specific hybridomas

A solid-phase immunofluorescence assay (SIFA) is described for screening of monoclonal hybridoma antibodies against soluble antigens. The test is simple, needs a small amount of material (20-100 ng of labeled antigen per sample) and allows a large number of samples (500-1 000) to be tested in 2 days. The assay is performed in microtitration plates with small cellulose nitrate discs as the solid phase. Anti-mouse IgG, the sample to be tested and FITC-labeled antigen are successively incubated with the discs. The fluorescence intensity of the surface of the discs is then measured with a microfluorometer. The assay has been used to select hybridomas against human serum albumin. Comparison of the immunofluorescence assay with a radioimmunoassay using conventional antisera and hybridoma antibodies similar sensitivity for both assays.     

A six-hour in vitro virulence assay for Listeria monocytogenes using myeloma and hybridoma cells from murine and human sources

An in vitro cell culture assay using myeloma cells and hybrid lymphocytes was developed which detected pathogenic Listeria strains in just 6 h. Three separate hybridoma cell lines, murine Ped-2E9 and EM-7G1 and human RI.37 and murine myeloma NS1 cells, proved equally sensitive in responding to virulent Listeria species. Listeria monocytogenes along with other Listeria spp., collected from food and clinical sources, were inoculated at 10⁸ cfu/ml into a suspension of Ped-2E9 (10⁶/ml). Pathogenic Listeria spp. killed 80% of hybridoma cells by 4 h, as determined by trypan blue exclusion test, Conversely, none of all nonpathogenic Listeria spp. killed the hybridoma cells. Ped-2E9 cells exposed to three strains of L. monocytogenes strains showed 96-97.5% death in 6 h measured by trypan blue staining and release of 91-97% of lactate dehydrogenase (LDH) enzyme. RI.37 cells showed similar results. A multiplicity of exposure (MOE) of 100 L. monocytogenes to 1 hybridoma cell or of 10:1 killed about 80% of the hybridoma cells in 4 or 6 h respectively. The in vitro virulence assay of L. monocytogenes with hybridoma cells compared favorably with the immunocompromised mouse model, yielding results in 6 h instead of 3 days. Intracellular L. monocytogenes and L. innocua were not recovered from Ped-2E9 hybridoma cells after 2 or 4 h of exposure. However, attachment of both L. monocytogenes and L. innocua cells on Ped-2E9 cell surfaces were observed under epifluorescence microscopy. Direct contact of hemolysin positive L. monocytogenes with hybridoma cells is essential to cause death, since hybridoma cells were not killed when they were separated from the growing bacteria by a 0.45 μm filter.     

A method for the blocking of endogenous immunoglobulin on frozen tissue sections in the screening of human hybridoma antibody in culture supernatants

Endogenous immunoglobulin in tissue sections pose a problem in immuno-histochemical techniques employing homologous antibody as primary reagents and enzyme-labelled anti-immunoglobulin for the development. A method for the blocking of endogenous immunoglobulin in human tissue sections by incubation with monomeric pepsin fragments (Fab') of rabbit anti-human immunoglobulin before applying monoclonal antibody was evaluated for the screening of human monoclonal antibody. It was initially demonstrated that Fab' rabbit anti-human IgM and anti-IgG could block endogenous immunoglobulin in human IgM and IgG producing tumors thereby abolishing the binding of subsequently applied peroxidase-labelled anti-IgM or anti-IgG. Frozen sections of human colo-rectal adenocarcinomas show a variable background staining caused by the endogenous immunoglobulin. The background completely disappeared in the IgM system by preincubation with Fab' anti-IgM while the background was clearly reduced but not abolished in the IgG system. A human hybridoma supernatant containing IgM reactive with colo-rectal adenocarcinoma could easily be screened on frozen sections using this method. This approach should be generally useful for the screening of human antibody on human tissue sections.     

A novel cytolysis assay using fluorescent labeling and quantitative fluorescent scanning technology.

A novel cellular cytotoxicity assay using Calcein acetoxymethyl (Calcein-AM), a cytoplasmic fluorescent label, has been developed as an alternative to the standard 51Chromium (Cr)-release. Target cells were loaded with Calcein-AM and then co-incubated with effector cells. An additional reagent, FluoroQuench, is added to extinguish fluorescence of dying target cells and of the culture media. Assay plates are read on a quantitative fluorescent scanner for determination of viable target cells. Percent lysis is calculated as one minus the percent viable cells as compared to fluorescent-labeled targets-only wells. The assay was tested to demonstrate the lytic activity of cytotoxic T lymphocyte (CTL) cultures, lymphokine-activated killer (LAK), and natural killer (NK) cell line effectors against peptide-pulsed and melanoma targets. In addition to the acquisition of results comparable to the 51Cr-release assay, the Calcein assay reliably measures cell-mediated cytotoxicity with little variance among replicates. The fluorescent assay represents a simple and useful alternative to the use of radioactive materials and adds the additional benefit of digital images and analysis.     

A yellow fluorescent protein-based assay for high-throughput screening of glycine and GABAA receptor chloride channels

There is a significant clinical need to identify novel ligands with high selectivity and potency for GABA_A, GABA_C and glycine receptor Cl⁻ channels. Two recently developed, yellow fluorescent protein variants (YFP-I152L and YFP-V163S) are highly sensitive to quench by small anions and are thus suited to reporting anionic influx into cells. The aim of this study was to establish the optimal conditions for using these constructs for high-throughput screening of GABA_A, GABA_C and glycine receptors transiently expressed in HEK293 cells. We found that a 70% fluorescence reduction was achieved by quenching YFP-I152L with a 10 s influx of I⁻ ions, driven by an external I⁻ concentration of at least 50 mM. The fluorescence quench was rapid, with a mean time constant of 3 s. These responses were similar for all anion receptor types studied. We also show the assay is sufficiently sensitive to measure agonist and antagonist concentration–responses using either imaging- or photomultiplier-based detection systems. The robustness, sensitivity and low cost of this assay render it suited for high-throughput screening of transiently expressed anionic ligand-gated channels.     

Microwave fixation of whole peripheral nerve for rapid and efficient enzyme-linked immunosorbent assay (ELISA) screening of monoclonal antibodies

Testing hybridoma supernatants for antibodies of interest involves extensive screening, particularly when the immunogen comprises whole cells. The number of different screening procedures is often large and unmanageable and depends on whether one is interested in, for example, cell surface or intracellular binding. This paper describes an initial screening technique using whole tissue homogenate rather than the individual tissue components. The tissue is fixed to the surface of 96-well microtitre plates by microwaves using a conventional microwave oven. This technique provides a rapid and cost-effective means of screening large numbers of monoclonal antibodies.     

A hybrid poly(dimethylsiloxane) microsystem for on-chip whole blood filtration optimized for steroid screening

Miniaturized biochemical devices in glass, silicon and polymer materials are starting to find their way from the academic laboratories to real-life applications. However, most attention has been given to miniaturize the downstream functions of various microfluidic systems, leaving the sample introduction and preparation steps to more conventional, bulkier solutions. For point-of-care diagnostics in particular, it becomes crucial to be able to handle complex human samples in a miniaturized format. In this work, we report on a microsystem for on-chip sample preparation that is able to remove blood cells from whole blood. The hybrid system consists of a commercially available membrane filter incorporated into a poly(dimethylsiloxane) (PDMS) casted device. Membrane materials were evaluated on the bases of low nonspecific adsorption of free and protein-bound testosterone as analyte substance. The hybrid system including a hydrophilic polypropylene filter successfully removed blood cells from diluted human whole blood. Surface oxidation was sufficient to make the plasma filtrate flow through the membrane filter and the channel system by capillary force alone and thus no external pumping source was needed.