The principle of antigen and antibody interaction has been applied to pregnancy testing since the first agglutination tests were developed in the 1960s. These assays have replaced animal testing.

Human Chorionic Gonadotropin

 Pregnancy tests are designed to detect minute amounts of human chorionic gonadotropin (hCG), a glycoprotein hormone secreted by the trophoblast of the developing embryo that rapidly increases in the urine or serum during the early stages of pregnancy.

This glycoprotein hormone consists of two noncovalently linked subunits, alpha (α) and beta (β). The α unit is identical to that found in luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH). The β subunit has a unique carboxy-terminal region. Using antibodies made against the β subunit will cut down on cross-reactivity with the other three hormones. Accordingly, many pregnancy test kits contain monoclonal antibody (MAb) directed against the β subunit to increase the specificity of the reaction.

For the first 6 to 8 weeks after conception, hCG helps maintain the corpus luteum and stimulate the production of progesterone. As a general rule, the level of hCG should double every 2 to 3 days. Pregnant women usually attain serum concentrations of 10 to 50 mIU/mL of hCG in the week after conception. If a test is negative at this stage, the test should be repeated within a week. Peak levels are reached approximately 2 to 3 months after the last menstrual period (LMP).

Agglutination Inhibition

The determination of in vitro agglutination inhibition depends on the incubation of the patient’s specimen with anti hCG, followed by the addition of latex particles coated with hCG. If hCG is present, it neutralizes the antibody; thus, no agglutination of latex particles is seen. If no hCG is present, agglutination occurs between the anti-hCG and hCG-coated latex particles.

Pregnancy Latex Slide Agglutination

Principle

 The rapid, direct, monoclonal latex slide agglutination test for detection of hCG is based on the principle of agglutination between latex particles coated with anti-hCG antibodies and hCG, if present, in the test specimen.

Results

• Agglutination within 2 minutes represents a positive reaction.

• No agglutination represents a negative reaction.

Technical Sources of Error

 Reagents should never be expired; latex reagent must be well shaken and agglutination should be read within 3 minutes to avoid erroneous results caused by evaporation.

False-Positive Results

If a patient has been given an hCG injection (e.g., Pregnyl) to trigger ovulation or lengthen the luteal phase of the menstrual cycle, trace amounts can remain in the patient’s system for as long as 10 days after the last injection. This will produce a false positive result. Two consecutive quantitative hCG blood assays can circumvent this problem. If the hCG level increases by the second test, the patient is probably pregnant. Chorioepithelioma, hydatidiform mole, or excessive ingestion of aspirin may give false-positive results.

In men, a test identical to that used for pregnancy may be performed to detect the presence of a testicular tumor. If MAb against the β subunit is not used, other hormones with the same α unit may cross-react and cause a false-positive reaction.

False-Negative Results

Testing before reaching detectable levels of hCG will yield false-negative results.

Alternate Procedural Protocols

Latex agglutination slide tests have been replaced in many situations (e.g., home testing) by one-step chromatographic color-labeled immunoassays for the qualitative detection of hCG in urine (e.g., Clearview hCG II and Clearview hCG Easy, Wampole Laboratories, Princeton, NJ). Another variation is a one-step chromatographic color-labeled immunoassay for use with urine or serum (e.g., Wampole PreVue hCG Stick or Cassette, Status hCG).