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The page below is a sample from the LabCE course Zika Virus: Overview and Laboratory Testing. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Enzyme-linked immunosorbent assay (ELISA) is a type of enzyme immunoassay (EIA) that is a plate-based assay technique designed for detecting and quantifying substances such as peptides, proteins, antibodies, and hormones. In an ELISA, an antigen is immobilized to a solid surface and then a specific antibody that is linked to an enzyme is applied over the surface where it binds to the antigen. The most commonly used enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (AP). In the final step, a substrate is added to produce a reaction which produces a detectable signal. The most common detectable signal is a color change in the substrate. The signal detection is typically accomplished using a spectrophotometer, fluorometer, or luminometer.
ELISAs can be performed with several modifications to the basic procedure. In addition, the assay can be designed to generate qualitative or quantitative results. (an overview of a simple, direct ELISA method is shown in the image).
IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) provides a useful alternative to immunofluorescence for documentation of a serologic response. This technique appears to be the method of choice for many specific and sensitive assays for IgM infectious disease antibodies. MAC-ELISA typically gives significant improvement in the specificity of an ELISA assay.
In the MAC-ELISA assay a solid phase support such as a microtitre plate is coated with anti-human IgM antibodies (the capture antibody) that can bind to IgM type antibodies present in the specimen. After binding occurs, an enzyme-labeled, antigen-specific antibody is added. If IgM antibodies specific for the antigen in question are present, a complex is then produced resulting in an enzymatic color change proportional to the concentration of IgM specific antibody present.
Typically, Zika virus-specific IgM and neutralizing antibodies develop toward the end of the first week of illness. Although IgM levels can vary, usually the levels are positive starting about four days post the onset of symptoms and continuing for about 12 weeks. If serum or urine specimens tested by rRT-PCR produce a negative result or no samples were collected <14 days after symptom onset, then the sample should be tested for the Zika IgM antibody. Therefore, the Zika MAC-ELISA was developed and is now available from the CDC for the qualitative detection of Zika virus IgM antibodies in human sera or CSF (submitted alongside a patient-matched serum specimen) collected from individuals meeting CDC's Zika virus clinical criteria and/or epidemiological criteria.
In the Zika MAC-ELISA, anti-IgM (the capture antibody) is coated onto microwell plates. Then patient serum is sequentially added to the wells followed by known non-infectious viral antigen. The presence of antigen is detected by using enzyme-conjugated anti-viral antibody. A colorimetric result is generated by the interaction of the enzyme and a chromogenic substrate. This colorimetric change is detected by a spectrophotometer or ELISA reader.
Before the result can be calculated using the Zika MAC-ELISA, the test must be validated. The validation process involves the determination of a positive control to normal serum optical density (OD) ratio. This ratio is called the P/N ratio and is defined as:

(P) Mean OD of the flavivirus IgM positive control reacted with Zika viral antigen ___________________________________________________________________________
(N) Mean OD of the normal human serum reacted with Zika viral antigen
For a test to be valid, the P/N ratio must be greater or equal to 2.0.