Journal of Immunological Methods, 119 (1989) 117-125 117 Elsevier JIM 05142 Indirect double sandwich ELISA for the specific and quantitative measurement of mouse IgM, IgA and IgG subclasses Anne-Sophie Klein-Schneegans 1, Claire Gav&iaux 2, Paul Fonteneau 1 and Francis Loor 1 * Laboratory of Immunology, Louis Pasteur University Strasbourg 1, BP 24, F67401 lllkirch Cedex, France, and 2 Preclinical Research Department, Sandoz AG, Postfach, CH 4002 Basel, Switzerland (Received 7 October 1988, revised received 14 November 1988, accepted 5 December 1988) We have developed sensitive enzyme-linked immunosorbent assays (ELISA) which measure mouse serum heavy chain immunoglobulin isotypes in nanograms per milliliter. In each case the specific isotypic Ig is sandwiched between an isotype-specific antibody used for coating and another isotype-specific antibody coupled to biotin for detection (with alkaline phosphatase coupled to avidin). These methods are simple to perform, specific for each isotype, reproducible with an average coefficient of variation of 5% for IgG1, 3% for IgG2a, 7% for IgG2b, 10% for IgG3, 3% for IgA and 7% for IgM, and at least 100 times more sensitive than radial immunodiffusion. The assays have been used to determine the absolute concentrations of mouse serum heavy chain Ig isotypes. Key words: lg isotype, mouse; ELISA; Quantitation 1. Introduction The accurate measurement of mouse serum im- munoglobulins (Ig), particularly of the specific classes, is becoming increasingly important. It is crucial for a thorough analysis of normal and abnormal humoral immune responses, i.e., to un- derstand the mechanisms which control the Correspondence to: F. Loor, Laboratoire d'Immunologie, Universit6 Louis Pasteur Strasbourg 1, BP 24, F67401 Illkirch Cedex, France. Abbreviations: AP-Av, alkaline phosphatase-avidin con- jugate; B-GaMIg, biotin conjugate of goat anti-mouse Ig; B-mRaMlg, biotin conjugate of rat monoclonal antibody against mouse Ig; BSA, bovine serum albumin; CV, coefficient of variation; OD, optical density; ELISA, enzyme-linked im- munosorbent assay; GaMIg, goat anti-mouse Ig; Ig, immuno- globulin; PBS, phosphate-buffered saline; RaMIg, rabbit anti- mouse Ig; SaMIgG, sheep anti-mouse IgG; SD, standard devi- ation. synthesis of antibody of the different Ig classes, or the serum clearance of an Ig of a given isotype, and to better define the humoral parameters of various immunodeficiency and autoimmunity syn- dromes. The methods currently used include radial im- munodiffusion (Mancini et al., 1965), electroim- munoassay (Laurell, 1966) and radioimmunoassay (Izui et al., 1984). Each of these methods presents specific technical problems which limit their sensi- tivity, accuracy or efficiency for multiple analyses. The advantages of the enzyme-linked immuno- sorbent assay (ELISA) in terms of simplicity, safety and sensitivity are well established. The ELISA method is more sensitive and specific than radial immunodiffusion, and cheaper than radio- immunoassay. It also provides an efficient work- ing schedule for large scale screening, such as is required, for example, to compare the age-related evolution of Ig isotype levels in sera from large 0022-1759/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)