Introduction
The human chorionic gonadotrophin (hCG) is a member of the heterodimeric glycoproteins hormones family which also includes the human follicle-stimulating hormone, the luteinizing hormone (LH), and the human thyroid-stimulating hormone (Dimiri and Kayisli 5). The subunit found in these hormones is identical but the b subunit varies from one hormone to another. HCG usually exists in three variants that are identified as regular HCG, the free b- subunit HCG, and the hyperglycosylated HCG (Gever 2). The three forms of HCG perform different biological functions. Apart from the three biological variants of HCG, other forms exist and include the secreted asubunit and eight other forms that are degraded following secretion. These degraded forms are found in urine or serum samples that are taken during pregnancy. These degraded forms are also found in patients suffering from a gestational trophoblastic disease (GTD) and nontrophoblastic malignancies (Honegger, Mann and Thleeruf 2). Several commercial tests have been developed to measure the levels of the different forms of HCG in the investigation of pregnancies and disease states in which they are elevated. This paper seeks to evaluate the different HCG tests with the aim of establishing the most selective and sensitive one. In order to correctly identify the best method, the paper will first address the various applications of HCG testing.
Applications HCG testing
Pregnancy testing
HCG is used to test for pregnancy as a medical examination for the use of certain steroids or following a request by a woman who wants to ascertain her pregnancy status. Hyperglycosylated HCG is predominant in early pregnancy but this often shifts to regular HCG as time progresses (Muller 8). A screening method should be able to detect the hypoglycosylated HCG, in addition to regular HCG if the pregnancy is to be detected early enough (Cole 2).
Gestational trophoblastic diseases (GTDs)
GTDs are disorders that are seen in pregnant women. Multiple forms of HCG are used to classify GTDS. Regular HCG is often used to identify partial and complete hydatidiform moles (Vareiro, Liu and Knoll 5). On the other hand, choriocarcinoma is identified using hyperglycosylated HCG. The enzymes produced by macrophages in these disease states initiate the degradation of regular and hypergycosylated HCG molecules (Willet 6). It’s therefore important to detect the different variants of HCG in GTDs.
Nontrophoblastic neoplasms
HCG variants form important markers for germ cell and other nontrophoblastic malignancies (Mehra, Huria and Gupta 7). Most germ cell tumors produce the free β-subunit. Normally, the free β-subunit and the β-core fragment are used as common markers for neoplasms (Honegger, Mann and Thleeruf 6).
Quantitative serum HCG assays (Immunometric assays)
Quantitative assay methods are based on the principle that a capture antibody binds on a single HCG site, immobilizes it, then a second (tracer) radioactive iodine or enzyme-labeled antibody binds to a distant HCG site forming a complex that can be quantified. The amount of the tracer antibody is directly proportional to the HCG concentration (Cole 3). All the available commercial immunoassay hCG tests are immunometric assays. This can further be categorized into two clear HCG test groups. The intact HCG tests that only detect the HCG dimer and the total HCG (β-hCG) tests further detect that detect the β- subunit, other variants and the degraded HCG, in addition to the intact HCG (Butler and Cole 2). The major limitation associated with the intact HCG test is the inability to evaluate dissociation in standards. Standards may dissociate as a result of transportation, storage and during usage in the automated platforms. The β-hCG test is able to detect the dissociated standard (Honegger, Mann and Thleeruf). The β-hCG or total assays are mostly utilized in the current commercial laboratory. The assays usually involve an antibody against the β-subunit folded core 1 or 2 in combination with an antibody to the β-subunit CTP natural (Breda 11). This eliminates the risk of a cross-reaction with LH, due to the fact that LH lacks the 30-amino acid CTP. The major limitation associated with the use of this combination of antibodies is their dependence on the O-linked oligosaccharide structure of the C- terminal segment (Kim, Josephson and Langer 17). The O-linked oligosaccharide accounts for most of the segments molecular weight. Thus most assays using this combination of antibodies will show a poor detection of the hyperglycosylated HCG and completely fail to detect HCG that lacks the C-terminal segment. The HCG produced in GTD and cancer usually lacks the C-terminal region. This limitation is overcome by using an alternative method that utilizes two to the HCG β-subunit folded core 1 or 2, as used by the Siemens Immulite HCG test (Cole 6). This combination is able to detect all the HCG forms and the majority of the dissociated variants.
The figure above shows the HCG epitopes that are commonly used.
POC & OTC HCG assays
The POC and the OTC rapid pregnancy tests also rely on dual antibody immunometric technologies (Muller 13). The functioning of these tests is based on the immobilization of one antibody in a translucent line and the other one that might be labeled with a red or blue or gold matrix dye is allowed to mix with serum or urine. The HCG dye antibody complex moves along the strip until it reaches the result window where it is stopped by the immobilized antibody. Positive results are indicated by a visible line that is formed by the immobilized antibody- HCG-dye antibody complex (Williams 25). There are four different types of POC and OTC test devices for HCG testing. This includes the nitrocellulose plastic stick devices that are directly immersed in urine and positive results indicated by the formation of a line (Willet 7). The nitrocellulose plastic sick devices form the most common form of OTC HCG testing devices (Huang and Huang 23). The second type of devices only differs by being positioned on a flat surface whereby the urine sample is added by a pipette. Positive results are indicated by a line in a second window. These are the most commonly used POC devices. The third type is basically in the form of an open nitrocellulose dipstick. The forth type is composed of digital devices that digitally display the results using words such as yes or No, and Pregnant or Not pregnant (Vareiro, Liu and Knoll 12). The digital devices are available for use as both POC and OTC products.
The specificities, sensitivities and the ability of POC and OTC devices to detect pregnancies vary depending on the company that makes the specific device. A study carried out in the United States established that devices manufactured by Church & Dwight Inc. are highly sensitive as they are able to detect regular HCG, free β-subunit and hyperglycosylated HCG on an equal basis (Mehra, Huria and Gupta 5). They however fail to detect the urine β- core fragment. Devices manufactured by other companies either fail to detect or show poor detection of the three variants.
Analysis and Conclusion
The analysis above shows that most commercial HCG assays detect the regular HCG but vary in the detection of other variants. It’s important to note that the detection of up to 11 HCG variants is crucial for the application of HCG in pregnancy testing, GTD and cancer tests.. In the US, the FDA has only approved the use of HCG tests in pregnancy testing. However, the specificity of the tests is highly required to distinguish between the different variants that manifest a given condition if HCG tests are to be used for other applications. For instance, only the hyperglycosylated HCG is the only HCG variant that is found in serum and urine samples during early pregnancy (Cole 13). Additionally, only the free β-subunit is produced in cancerous states. The above analysis has established that quantitative assays that utilize HCG β-subunit folded core 1 or 2, as used by the Siemens Immulite HCG test are able to detect all the HCG forms and the majority of the dissociated variants (Mehra, Huria and Gupta 9). Thus, in conclusion, the HCG β-subunit folded core 1 or 2 quantitative assays will be regarded as the most sensitive and selective HCG test. Such a test should be recommended for the most reliable HCG testing.
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