ABSTRACT
A reverse modification of the recently described enzyme-linked immunospot assay (ELISPOT), based on localized enzyme-substrate reactions in gel, is described for the enumeration of antigen-secreting cells using petri dishes coated with specific antibodies. As a model the detection of mouse and human immunoglobulin-secreting cells has been evaluated. Simple and sensitive, this new method, termed RELISPOT, can be adapted for the quantitation of secreted antigen thus providing additional information on the metabolic state of the population of cells tested.
Subject(s)
Antibody-Producing Cells/metabolism , Enzyme-Linked Immunosorbent Assay , Immunoenzyme Techniques , Spleen/cytology , Adult , Animals , Antibody-Producing Cells/immunology , Cell Count , Cell Transformation, Viral , Hemolytic Plaque Technique , Herpesvirus 4, Human , Humans , Immunoglobulins/biosynthesis , Mice , Mice, Inbred BALB C , RabbitsABSTRACT
A recently described solid phase immunoenzyme procedure (ELISPOT) has been adapted for the detection of individual cells secreting fibronectin. Simple and sensitive, this technique should find useful application for studying fibronectin production at the cellular level.
Subject(s)
Fibronectins/metabolism , Immunoenzyme Techniques , Cell Line , Embryo, Mammalian , Fibroblasts/metabolism , HumansABSTRACT
A solid-phase enzyme-linked immunosorbent assay (ELISPOT) is described for enumeration of cells secreting specific antibody. Spleen cells from immunized mice are incubated in antigen-coated polystyrene plates. After removal of the cells, bound antibodies are demonstrated by means of an immunoenzyme procedure in which enzyme-substrate reactions are performed in agarose. Dark-brown circular zones (spots), localized in areas of the dish where antibody production has occurred, are enumerated with the naked eye. Spectrophotometric estimation of enzyme-bound activity may be performed by substituting the gel for a liquid buffer, allowing accurate estimation of the total amount of secreted antibody. Versatile, sensitive and very easy to perform, this new assay provides a useful alternative to conventional plaque-forming cell assays.
Subject(s)
Antibody-Producing Cells , Enzyme-Linked Immunosorbent Assay , Hemocyanins , Immunoenzyme Techniques , Animals , Antibody Specificity , Antibody-Producing Cells/immunology , Antigens/immunology , Cell Count , Culture Media , Female , Fetal Blood/immunology , Hemolytic Plaque Technique , Kinetics , Male , Mice , Mice, Inbred C57BL , Ovalbumin/immunology , Spleen/cytology , Spleen/immunologyABSTRACT
A new method has been developed for demonstration of heat-labile (LT) enterotoxin produced by Escherichia coli. This method is based upon the release of LT from bacteria grown directly onto agar plates which have been coated with ganglioside GM1. Toxin bound to the GM1 solid state is subsequently demonstrated by means of a three-step immunoenzymatic procedure in which enzyme-substrate reactions are visualized as dark spots in agarose. When analyzing LT production from 105 E. coli strains, results obtained by this procedure (GM1-ELISPOT) correlated well with those of the GM1 enzyme-linked immunosorbent assay (GM1-ELISA); in no instance were any false-positive reactions observed when highly specific monoclonal antibodies against LT were used. Easy to perform, the GM1-ELISPOT allows demonstration of LT within 24 h after inoculation of the plates, and large numbers of specimens can be screened at the same time without the need of any special equipment. Thus, this new method should meet the requirements of any diagnostic laboratory.
