Örjan Ouchterlony

A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells

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.

Reverse ELISPOT assay for clonal analysis of cytokine production I. Enumeration of gamma-interferon-secreting cells

A reverse modification of ELISPOT assay using nitrocellulose membranes and epitope-specific monoclonal antibodies is described for the detection of single lymphokine-secreting cells. As a model, the production of gamma-interferon by mitogen stimulated human peripheral blood lymphocytes has been examined. The assay can also be modified to permit microscopic examination of spot-forming cells.

A simple spot technique for thin layer immunoassays (TIA) on plastic surfaces

A simple and sensitive technique for visualization of antigen--antibody reactions is described. The property of many antigens to become adsorbed firmly on to a hydrophobic polystyrene surface while retaining their serological reactivity is taken advantage of. On a surface with adsorbed antigen the corresponding immune serum is applied spot-wise. The antigen--antibody reaction areas on the surface are characterized by a distinct hydrophilic condensation pattern when exposed to water vapour. The results obtained by the immunoassay technique described can be reproduced with great accuracy. The method is well suited for quantitative determination of a wide range of antigens as well as their corresponding antibodies. Antigen concentrations of 0.2--0.8 mg/l and antibody concentrations about 1 mg/l can be detected. By employing an antiimmunoglobulin serum subsequent to the primary antigen--antibody reaction, an increase in sensitivity can be obtained.

Visualization Principles in Thin-Layer Immunoassays (TIA) on Plastic Surfaces

Macromolecules may adsorb firmly as a monolayer to plastic surfaces and still retain their property to react specifically with antibodies. In the present communication four different principles for visualization of such antigenantibody interactions on plastic surfaces are described. In addition, a diffusion-ingel method for quantitation of antibodies is presented. The described methods offer simple, sensitive and accurate means of assaying antigen-antibody reactions.

Solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of IgG rheumatoid factor-secreting cells

Although IgG rheumatoid factor may play an essential role in the pathogenesis of rheumatoid arthritis, there is no precise method for its specific detection at the cellular level. A modification of the recently developed enzyme-linked immunospot assay has been devised for enumeration of cells secreting IgG rheumatoid factor (IgG RF) and simultaneous quantitation of the IgG RF secreted. Specific, sensitive and simple, this new assay should provide a valuable tool for study of isotype-specific RF secretion by single cells.

A precipitate adsorption on surface technique: a combination of immunodiffusion and thin-layer immunoassay.

A new technique--precipitate adsorption on surface--is described. The technique implies a combination of immunoprecipitation in gel and diffusion-in-gel-thin-layer immunoassay. The principles of the technique are illustrated by a series of model experiments.

Paper discs impregnated with capillary blood. A sampling technique for immunoassays by means of DIG-ELISA and DIG-TIA

A modification of the diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) and the diffusion-in-gel thin layer immunoassay (DIG-TIA) using paper discs impregnated with blood or serum as source of diffusion is described. Using a BSA-anti-BSA model system the suitability of this sampling and assaying technique for simple and accurate quantitation of antibodies has been demonstrated. Paper discs impregnated with blood or serum and subsequently dried could be stored at temperatures between +4 degrees C and +37 degrees C for at least six weeks without significant loss of antibody activity. Application of paper disc DIG-ELISA was exemplified by assaying anti-BSA antibodies in rabbits immunized with BSA as well as antibodies against both soluble egg antigen and adult worm extract in mice experimentally infected with Schistosoma mansoni cercariae. The simplicity and good reproducibility of this technique should make it an attractive alternative in serological screening of large population groups. Definite advantages of this technique are the possibilities to analyse quantitatively antibodies in capillary blood, rather than serum from venous blood samples, and to store sampled material for a considerable time even under such climatic conditions as often prevail in tropical countries.

Application of thin layer immunoassay (TIA) as a serodiagnostic tool in schistosomiasis. A preliminary report

Thin layer immunoassay (TIA) is a recently developed simple serological technique which, in a preliminary study, has been applied to the serodiagnosis of schistosomiasis. 64 of a total of 69 sera from patients with schistosomiasis were positive in TIA when a Schistosoma mansoni worm antigen preparation was used as coating material. A trial of the diagnostic specificity of the assay was made by cross-testing sera from patients with different parasitic diseases, using TIA plates coated with extracts from the relevant parasites. All sera from patients with filariasis, fascioliasis and echinococcosis were positive in homologous TIA systems. In heterologous systems, cross-reactivity was noted for some sera in all groups except fascioliasis. The largest proportion of cross-reacting sera was registered in the echinococcosis group. It is suggested that, after further evaluation, the TIA technique might supplement or be used as an alternative to other serological tests already in use for the diagnosis of schistosomiasis. In favour of the TIA technique are its simplicity and its low cost.

Determination of Rheumatoid Factor by Means of Thin Layer Immunoassay

Thin layer immunoassay (TIA), a solid phase immunoassay based on a technically simple visualization method, was adapted for quantitative determination of rheumatoid factor (RF) in blood serum. The influence of various factors of possible significance for accurate quantitation of RF by TIA was studied. Comparison between TIA and the Waaler-Rose test for determination of RF was performed on 335 patient sera. The results obtained by the two methods agreed reasonably well. Preliminary experiments were also performed concerning the use of TIA for differentiation of RF with regard to the immunoglobulin class. It is concluded that, because of its simplicity and screening capacity, TIA could be an attractive method for detection and quantiation of RF.

Kinetics and Isotype Distribution of Parasite-Specific Antibody Responses in the Spleen of Mice during Primary Infection with Schistosoma mansoni

The solid-phase enzyme-linked immunospot assay has been adapted for enumerating cells secreting antibodies to crude antigenic extracts from the main developmental stages of Schistosoma mansoni. The frequencies of splenocytes secreting antibodies reactive with soluble antigenic extracts from cercariae, adult worms, and eggs, were examined in C57BL/6 mice during the first 12 weeks of a primary infection with S. mansoni. Despite the existence of cross-reactive moieties present in these antigenic preparations, the characteristics of the responses monitored differed with regard to kinetics, magnitude, and/or isotype distribution. The responses peaked between 8 and 10 weeks of infection, were characterized by a predominance of cells secreting IgM antibodies against all three antigens, and followed the patterns IgM greater than IgA greater than IgG for cercariae, IgM greater than IgG greater than IgA for adult worms and IgM greater than IgG greater than IgA for egg extracts. On the other hand, these patterns were not always reflected in serum, especially for cercariae-reactive circulating IgG and IgA responses. When examined for numbers of total IgM-, IgG- and IgA-producing cells, the spleen of S. mansoni-infected mice was shown to display considerable numbers of immunoglobulin-producing cells in all three isotypes studied. The most spectacular increases were noted for IgG-secreting cells (more than 10-fold) and for IgM-secreting cells (up to 6-fold) 4 weeks after initial cercarial exposure. Taken together, these observations indicate that primary infection with S. mansoni results in early immunoregulatory alterations which may contribute to the maintenance of specific as well as nonspecific B cell hyperactivity.(ABSTRACT TRUNCATED AT 250 WORDS)