MicroELISA

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The Total hCG EIAgen kit has been designed for the quantitative determination of human chorionic gonadotropin (hCG) in human serum.

Human chorionic gonadotropin (hCG) is a sialoglycoprotein of molecular weight about 40,000. It is composed of two subunits of unequal size, a and b. The a-subunit is virtually identical to that of the pituitary hormones hLH, hFSH, and hTSH. Biological specificity is conferred on these hormones by the differences in their b-subunits, which also allows them to be distinguished immunologically (1). The hCG b-subunit is similar to that of hLH, differing mainly in having extra C-terminal amino acids, and in being more heavily glycosylated. While the glycosylation has no effect on the action or immunological properties of hCG, it does affect its metabolism (2). Removal of the sugar residues greatly reduces the biological half-life of the molecule.

The physiological action of hCG reflects its structural similarity to hLH. It is produced in large amounts by the placental syncytiotrophoblast, and in early pregnancy acts to sustain the corpus luteum beyond its normal lifetime (3). In this way, estrogen and progestrone production, necessary for the maintenance of the endometrium, is continued, allowing pregnancy to proceed. Later in pregnancy, the placenta becomes able to produce adequate amounts of the steroid hormones itself, and hCG production is reduced. Another action in which hCG resembles hLH is its stimulation of testosterone production by the developing testes of a male fetus, necessary for normal masculinization. It has also been proposed that the production and secretion of hCG by the developing placenta protects it from attack by the maternal immune system (4).

hCG can also be produced in significant quantities by a variety of tumors, especially those of trophoblastic origin (choriocarcinoma, teratoma). Indeed, sensitive assay techniques have shown small amounts of hCG-like material to be present in virtually all tissues, both normal and abnormal. In most cases, only trophoblastic tumors release detectable amounts into the circulation. It has been found in these cases that the quantity of circulating hCG reflects the amount of tumor present. The first tests for the detection of hCG were bioassays which could confirm pregnancy about 2 months after a missed period. The introduction of hemagglutination and latex agglutination methods simplified testing and improved early pregnancy detection; however, sensitivity was limited to 150-1000 mIU/mL. Sensitive radioimmunoassay and immunoradiometric techniques now available allow the accurate and precise quantitation of hCG through the use of highly specific antibodies that reduce cross-reactivity with LH (5,6). These assays can detect pregnancy before the first missed menstrual period.

Immunoenzymometric assay:

In this method, calibrators, patient specimens and/or controls (containing the native hCG antigen) are first added to streptavidin coated wells. Biotinylated monoclonal and enzyme labeled antibodies are then added and the reactants mixed: these antibodies have high affinity and specificity and are directed against distinct and different epitopes of hCG.

Reaction between the various hCG antibodies and native hCG occurs in the microwells without competition or steric hindrance, forming a soluble sandwich complex.

Simultaneously, the complex is deposited to the well through the high affinity reaction of streptavidin and biotinylated antibody.  

After equilibrium is attained, the antibody-bound fraction is separated from unbound antigen by decantation or aspiration. The activity of the enzyme present on the surface of the

well is quantitated by reaction with a suitable substrate to produce color.

The enzyme activity in the antibody-bound fraction is directly proportional to the native antigen concentration.

By utilizing several different serum references of known antigen values, a dose response curve is generated from which the antigen concentration of an unknown is ascertained.

The kit contains reagents for 96 tests (code LI4012).

Bring reagents to room temperature before use.