Techniques used for antigen-antibody detection

MethodDescriptionExamples of tests or target detected
Latex agglutination testingLatex beads coated with antibodies are mixed with the patient sample. If the antigen is present in the sample, the antibodies will attach to the antigen and agglutination will occur. Testing of serial dilutions of the sample can give a quantitative measure of the amount of antigen present.Cryptococcal antigen detection.
Enzyme immunoassay
    DirectThe patient's sample is spread on a plate, and time is allowed for the antigen to adhere to the plastic through charge interactions. An antibody with an enzyme conjugate that changes color after addition of a substrate is added to the patient's sample. If the antigen is present, the antibodies attach and a color change is detected through optical density measurement. Coccidioides sp. and Blastomyces sp. antibody detection; Histoplasma sp., Blastomyces sp., and Cryptococcus sp. antigen detection. Also, the galactomannan antigen test uses sandwich immunoassay technology.
    IndirectThe patient's sample is added to a mixture that contains a specific antigen. If antibodies are present in the sample, they will attach to the antigen. Subsequently, antibodies with an enzyme conjugate that bind the primary antibodies are added to the mixture. Color change is detected by optical density measurement.
    SandwichSame as direct EIA, with the exception that the plate already contains a capture antibody that binds the target antigen, so no charge interactions are necessary.
ImmunodiffusionAn agarose gel is prepared with wells cut into the gel. The patient's sample is placed in the center well, and the control antigens or antibodies are added to the outside wells. If the target antibodies or antigens are present in the tested sample, they will form precipitin lines by interacting with the control antigens or antibodies, respectively. Histoplasma sp. and Blastomyces sp. antibody detection.
Complement fixationThe patient's sample is isolated and heated to destroy all existing complement proteins. Standardized complement proteins and a specific antigen or antibody are added to the sample. Sheep red blood cells (sRBCs) prebound with anti-sRBC antibodies are added to the mix. If the target antibodies or antigens are present in the sample, they will bind the added antigen or antibody and form complexes, which will react with and deplete the complement proteins and salvage the sRBCs. If not, the complement proteins will lyse the sRBCs, thus changing the color of the mix. Histoplasma sp., Blastomyces sp., and Coccidioides sp. antibody detection.
Lateral-flow assayThe technology is based on a series of capillary beds on an element that can spontaneously transport fluid. The patient's sample is added to the first bed. This soaks up all the extra fluid. The remains are transferred to the second bed, which contains antibodies with an enzyme conjugate that bind antigens or antibodies in the tested sample. The antigen-antibody complexes then move to a third bed, which has capture antibodies that bind the complexes. An additional bed binds only the control antibodies without the antigen, thus serving as a control to ensure that the method worked properly.Point-of-care diagnostic tests for Cryptococcus sp. and Aspergillus sp. antigen detection.
Immunofluorescence assay (IFA)The methodology is very similar to that for enzyme immunoassay, but instead of antibodies with an enzyme conjugate, this assay utilizes fluorescein-labeled antibodies which can then be visualized under a fluorescence microscope. It can be performed as both direct and indirect assays.CAGTA a assay is an indirect IFA.
G testThe G test is specific to beta-glucan detection. Factor G is a proclotting factor that is highly sensitive to beta-glucan. When a patient's sample containing beta-glucan is added to a mix containing factor G, it activates the factor, thus initiating an enzymatic cascade that results in a color or optical density change of the mixture, which can be detected with colorimetric or turbidimetric methods.Beta-glucan detection.
  • a CAGTA, Candida albicans germ tube antibody.