An enzyme-linked immunosorbert assay (ELISA) for the detection of monoclonal antibodies recognizing surface antigens expressed on viable cells

Feit, Carl; Bartal, Arie H.; Tauber, Gerald; Dymbort, Giny; Hirshaut, Yashar

doi:10.1016/0022-1759(83)90358-7

Cited by 37 publications

(14 citation statements)

References 10 publications

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“…The Selection of an appropriate method for preparation of the monolayer appears to be an important factor for success in development of an in situ ELISA. Several methods have been described for in situ ELISAs on viable cells (8,19), glutaraldehyde-fixed cells (1,2,11,15,20), Formalin-fixed cells (7), and methanol-fixed cells (6,13 The assay system described here may have applications beyond evaluation of intracellular development. For example, substitution of a polyclonal or monoclonal anti-sporozoite antibody in the ELISA could make it possible to detect inhibitors of sporozoite invasion in a short-incubation (e.g., (14).…”

Section: Methodsmentioning

confidence: 99%

In situ enzyme-linked immunosorbent assay to quantitate in vitro development of Eimeria tenella

1990

Antimicrob Agents Chemother

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An in situ enzyme-linked immunosorbent assay was developed to measure in vitro development of Eimeria tenella. The assay used a polyclonal, anti-merozoite serum produced by immunization with culture-derived, chromatographically purified merozoites. Although this antiserum cross-reacted with sporozoite-infected cultures (by indirect immunofluorescence and in situ enzyme-linked immunosorbent assay), it clearly distinguished the increase in antigen synthesized throughout intracellular growth. The assay can be used for high-throughput, anticoccidial drug screening, for which it gives quantitative results that are comparable to the published radiometric ([3H]uracil incorporation) endpoint.The demonstration that asexual stages of Eimeria tenella could be cultivated in vitro (18) made possible the design of cell culture screens to identify anticoccidial drugs. The first screens described (24) used microscopic observation to score parasite development, a method that was both laborious and qualitative. A more efficient screen endpoint has been described by Schmatz et al. (23), who measured parasite-specific incorporation of [5,6-3H]uracil to quantitate parasite growth. The most labor-intensive step in this radiometric scoring method is the processing of samples for liquid scintillation counting. I describe an alternative scoring method using a colorimetric, in situ enzyme-linked immunosorbent assay (ELISA) which is less laborious and should further improve the efficiency of anticoccidial screening in vitro.Although ELISAs have been applied to studies of E. tenella (3,9,16,17,21), the methods are not adapted to measuring parasite growth. Instead, they are designed to quantitate the antibody produced in response to infection by using a fixed amount of parasite extract as an antigen. In situ ELISAs have been developed to measure in vitro growth and drug inhibition in virus-infected (1, 2, 11, 20) and protozoan-infected (6, 7, 13) monolayers. These assays detect growth of the infectious agent by measuring the antigen with a fixed amount of antibody specific for the infectious agent. This study was designed to adapt in situ ELISA methods to an E. tenella in vitro model. MATERIALS AND METHODSAbbreviations. BSA, Bovine serum albumin; IC50, drug concentration producing 50% inhibition of growth; MDBK, Madin-Darby bovine kidney; OD405, optical density at 405 nm; PBS, phosphate-buffered saline.In vitro production of merozoites. MDBK cells (ATCC CCL 22) were grown in continuous culture at 40°C in 3% CO2 in nutrient mixture F12 (no. 320-1765, GIBCO Diagnostics) with 10% (vol/vol) fetal bovine serum (no. 110-1120, J. R. Scientific) and penicillin-streptomycin (no. 600-5140, GIBCO). Prior to infection, cells were subcultured into 175-cm2 flasks in RPMI 1640 (no. 320-1875, GIBCO) with 5% fetal bovine serum (RPSS). When MDBK cells had reached confluence, purified E. tenella sporozoites (22) were added at 2.5 x 105/cm2 in RP5S. After 24 h of incubation at 40°C to allow invasion, uninvaded sporozoites were removed by agitating the flask, r...

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Section: Methodsmentioning

confidence: 99%

In situ enzyme-linked immunosorbent assay to quantitate in vitro development of Eimeria tenella

1990

Antimicrob Agents Chemother

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“…The disadvantage of using fluorogenic substrates is that they require a microplate fluorometer costing twice as much as a high-quality microtiter plate spectrophotometer. Cellular ELISAs have been shown to be as sensitive as flow cytometry analysis in detecting some cell-surface antigens (Bartlett and Noelle, 1987) and are potentially of great value in rapidly screening hybridoma supernatants for antibodies against surface molecules (Feit et al, 1983). Using ELISAs for screening large numbers of hybridoma supernatants has been hindered by the large number of cells required and high background signal.…”

Section: Background Informationmentioning

confidence: 99%

Enzyme‐Linked Immunosorbent Assays

Hornbeck

2015

CP in Immunology

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This unit describes six different ELISA systems for the detection of specific antibodies, soluble antigens, or cell-surface antigens. In all six systems, soluble reactants are removed from solution after specifically binding to solid-phase reactants. In the first four protocols, solid-phase reactants are prepared by adsorbing an antigen or antibody onto plastic microtiter plates; in the next two protocols, the solid-phase reactants are cell-associated molecules. In all protocols, the solid-phase reagents are incubated with secondary or tertiary reactants covalently coupled to an enzyme. Unbound conjugates are washed out and a chromogenic or fluorogenic substrate is added. As the substrate is hydrolyzed by the bound enzyme conjugate, a colored or fluorescent product is generated. Finally, the product is detected visually or with a microtiter plate reader. The amount of product generated is proportional to the amount of analysate in the test mixture. One of the support protocols can be used to optimize the different ELISAs. A second support protocol presents a method for preparing alkaline phosphatase conjugates.

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“…Cellular ELISAs have been shown to be as sensitive as flow cytometry analysis in detecting some cell-surface antigens (Bartlett and Noelle, 1987) and are potentially of great value in rapidly screening hybridoma supernatants for antibodies against surface molecules (Feit et al, 1983). Using ELISAs for screening large numbers of hybridoma supernatants has been hindered by the large number of cells required and high background signal.…”

Section: Commentary Background Informationmentioning

confidence: 99%

Enzyme‐Linked Immunosorbent Assays (ELISA)

Hornbeck¹,

Winston

Fuller

1991

CP Molecular Biology

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This unit describes six different ELISA systems for the detection of specific antibodies, soluble antigens, or cell-surface antigens. In all six systems, soluble reactants are removed from solution after specifically binding to solid-phase reactants. In the first four protocols, solid-phase reactants are prepared by adsorbing an antigen or antibody onto plastic microtiter plates; in the next two protocols, the solid-phase reactants are cell-associated molecules. In all protocols, the solid-phase reagents are incubated with secondary or tertiary reactants covalently coupled to an enzyme. Unbound conjugates are washed out and a chromogenic or fluorogenic substrate is added. As the substrate is hydrolyzed by the bound enzyme conjugate, a colored or fluorescent product is generated. Finally, the product is detected visually or with a microtiter plate reader. The amount of product generated is proportional to the amount of analysate in the test mixture. Support protocols are provided for optimizing the different ELISAs and preparing lysates for use as test antigen from bacterial cultures containing expressed protein.

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An enzyme-linked immunosorbert assay (ELISA) for the detection of monoclonal antibodies recognizing surface antigens expressed on viable cells

Cited by 37 publications

References 10 publications

In situ enzyme-linked immunosorbent assay to quantitate in vitro development of Eimeria tenella

In situ enzyme-linked immunosorbent assay to quantitate in vitro development of Eimeria tenella

Enzyme‐Linked Immunosorbent Assays

Enzyme‐Linked Immunosorbent Assays (ELISA)

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