Method Description

IgG:

Diluted sera are incubated in antigen-coated microwells. Any antigen-specific antibody in the samples will bind to the immobilized antigen. The plate is washed to remove unbound antibody and other serum components. Peroxidase conjugated goat-antihuman IgG is added to the wells and incubated. The conjugate will react with the IgG antibody/antigen on the solid phase. The wells are washed to remove unreacted conjugate. The microwells containing immobilized conjugate are incubated with peroxidase substrate solution. Hydrolysis of the substrate by peroxidase produces a color change. After a period of time, the reaction is stopped by the addition of diluted acid, and the color changes are measured photometrically. The color intensity of the solution depends on the antibody concentration in the serum sample.(Package insert: M pneumoniae IgG Test System. Zeus Scientific Inc; Revision Date 12/2017)

IgM Enzyme Immunoassay:

Test sera are diluted with the sample diluent provided. The sample diluent contains antihuman IgG that precipitates and removes IgG and rheumatoid factor from the sample, leaving IgM free to react with immobilized antigen. Diluted sera are incubated in antigen-coated microwells. Any antigen-specific antibody in the samples will bind to the immobilized antigen. The plate is washed to remove unbound antibody and other serum components. Peroxidase-conjugated goat-antihuman IgM (chain specific) is added to the wells and incubated. The conjugate will react with the IgM antibody/antigen on the solid phase. The wells are washed to remove unbound conjugate. The microwells containing immobilized conjugate are incubated with peroxidase substrate solution. Hydrolysis of the substrate by peroxidase produces a color change. After a period of time, the reaction is stopped by the addition of diluted acid, and the color changes are measured photometrically. The color intensity of the solution depends on the antibody concentration in the serum sample.(Package insert: M pneumoniae IgM Test System. Zeus Scientific, Inc; Revision Date 9/22/2016)

IgM Immunofluorescence Assay:

Mycoplasma pneumoniae antigenic substrate is fixed onto microscope slide wells. Serum that has been pretreated to remove IgG antibodies is incubated with the substrate. If IgM antibody to M pneumoniae is present, it will bind to the substrate. Fluorescein-labeled antihuman-IgM conjugate is added to the slide wells, and the slide is incubated. If antibody is present, it can be observed as a characteristic positive, bright, apple-green fluorescent reaction when the slide is read on a fluorescence microscope.(Package insert: Mycoplasma pneumoniae IgM IFA Antibody Test System. Zeus Scientific, Inc; 09/2019)