In a perfect world, protein hormones would be expressed, quickly fold into their bioactive conformation and execute their signaling before being rapidly cleared from the body in more or less their native conformation. Although we don’t live in a perfect world, the process described is often the dogma associated with protein hormones. Take human chorionic gonadotropin (hCG) for example. hCG is the hormone best known for its role in pregnancy, but is also known to be expressed by certain tumors. It is the hormone that most, if not all, physicians will choose to detect to rule pregnancy in or out, either by home pregnancy, point-of-care, or automated hCG assays.; Since it’s so widely used, hCG must be as simple as protein hormones come, right? Wrong.

hCG a heterodimeric glycoprotein produced by the placenta following fertilization. The hormone is composed of an alpha and beta subunit and undergoes complex post-translational modification and degradation pathways. This leads to multiple forms of hCG present in the placenta, the serum, and the urine. These hCG derivatives are referred to as hCG variants. hCG in its native conformation is generally referred to as intact hCG. Variants of hCG variants include nicked hCG (hCGn), the free beta subunit of hCG (hCGβ), nicked hCGβ (hCGβn), hyperglycosylated hCG (hCG-h), and the beta core fragment (hCGβcf). hCGβcf is the terminal degradation product of hCG and is present in urine only.

In 2009 Gronowski et al (1) published an alarming message that elegantly demonstrated how elevated concentrations of hCGβcf in urine could lead to false negative point-of-care hCG tests. The phenomenon was seen in three independent laboratories.The mechanism governing the false negative results was termed the “variant hook effect.” In these cases, the hCGβcf binds to either the solid phase or the detection antibody used in the assay. Since only one of the antibodies recognizes hCGβcf, while the other does not, the antibody-antigen sandwich cannot form. At threshold concentrations of hCGβcf, when the variant is typically present in at least two-fold molar excess to intact hCG, the hCGβcf blocks intact hCG from binding, leading to an erroneously negative result on the point-of-care device.

Naturally, the next question was: does this phenomenon occur in the quantitative assays? In 2010 Grenache et al (2) found that, indeed, the variant hook effect can also occur in some of the most widely used commercially available quantitative assays. In support of the proposed mechanism, the work demonstrated that the assays that produced false negative results when excess hCGβcf was present did not detect purified hCGβcf.

Although understanding these limitations in the context of hCG and the hCG assay(s) utilized by your laboratory is important, it is equally as important to take the effect of hCG variants as a lesson: biological systems are complex. In most cases, the proteins and molecules utilized for diagnostics will have metabolites and modifications. The presence of such variants should always be kept in mind whenever a questionable result emerges.


  1. Gronowski AM, Cervinski M, Stenman UH, Woodworth A, Ashby L, Scott MG. False-negative results in point-of-care qualitative human chorionic gonadotropin (hCG) devices due to excess hCGbeta core fragment. Clinical Chemistry 2009:55;1389–1394.
  2. Grenache DG, Greene DN, Dighe AS, Fantz CR, Hoefner D, McCudden C, Sokoll L, Wiley CL, Gronowski AM. Falsely decreased human chorionic gonadotropin (hCG) results due to increased concentrations of the free beta subunit and the beta core fragment in quantitative hCG assays. Clinical Chemistry 2010:56;1839–1844.