All human lives begin with a simple cell fusion… Following capacitation, sperm cells travel up the female reproductive tract and upon reaching the fallopian tubes encounter a newly released ovum or egg cell from one of the two ovaries. Cytoplasmic fusion, followed by nuclear fusion between the two haploid nuclei, results in a zygote or one cell embryo. Roughly thirty hours following the fusion event, the first cell division takes place and the cell count continues to double every couple of hours as the embryo undergoes cleavage (characterized by rapid cell division with limited growth in between). As the newly formed zygote continues to grow it travels down the fallopian tubes, and quickly transforms into a morula (ball of cells) and then into a blastula (hollow ball of cells). Within the blastula is the inner cell mass, which will develop into the growing fetus, and the trophoblast, which makes up the outer ring of cells and will transform into the placenta.

Five to six days following the initial fusion event, the blastocyst reaches the uterus. As the trophoblast cells attempt to attach to the uterine lining, another type of cell fusion takes place; this time resulting in a syncytium, where both mother and embryonic trophoblast nuclei remain intact within a single larger cell or syncytiotrophoblast. Failure of the multinucleated syncytiotrophoblasts to properly form has been linked to various gestational complications, such as pre-eclampsia. Following implantation, the placenta begins to form and becomes a vital organ (from the fetus’ perspective) where the exchange of oxygen and carbon dioxide, as well as the uptake of nutrients, continuously feed the growing cells.

At this point in development, the growing embryo begins producing copious amounts of the human chorionic gonadotropin (hCG) hormone, a heterodimeric glycoprotein composed of a highly conserved alpha and relatively unique beta subunit. The main role of the hormone is to prevent the dissolution of the corpus luteum, an endocrine structure that produces high levels of progesterone required to maintain the pregnancy state. Poor production of hCG by the growing embryo, or the presence of anti-hCG antibodies within the womb, can contribute to the loss of pregnancy.

The desire to know a woman’s pregnancy status as early as possible has spanned millennia, with the first well-known pregnancy tests dating back to ancient Egypt. Although more renowned for their

architectural feats, ancient Egyptians were no less impressive on the medical front. According to early papyri texts, Egyptian women were advised to pee on barley or wheat seeds, and if the seeds then sprouted, this reflected the sprouting of a human life in the womb. Although a relatively simple and primitive test, modern-day scientists have investigated this proceeding closer and have concluded that it was a relatively accurate experiment. Estrogen and other growth factors present in the urine would account for the growth stimulation of the seeds. Hopefully, one would have thought twice before trying a pint of beer or a piece of bread in 1,000 BC…

In the early twentieth century, scientists continued to investigate the pregnancy-predicting capability of urine. These early pregnancy tests required the injection of urine from a candidate pregnant woman into animals (such as rabbits or frogs) and the subsequent observation of ovulation in the experimental animal. The practicality of this test remained an inhibiting factor in its general application (as one would imagine). Luckily, we have come a long way since those times and since the 1970s at-home pregnancy tests that specifically detect hCG have become the norm. The hormone first appears in the maternal blood roughly six to eight days after the fertilization event and progressively rises before leveling off during weeks seven through ten. Nowadays, most at-home pregnancy tests are sensitive enough to detect hCG within the mother’s urine three to four days post-implantation, or roughly ten days post-fertilization.

Given the potential life-changing results of a pregnancy test, it is understandable that the science underlying the immunoassay is underappreciated as it is not necessarily a top priority at the time of use. The way that a modern at-home pregnancy test works is by harnessing the power of three distinct antibodies (some which were discovered via hybridoma technologies): one antibody type for capturing the excreted hCG, another for detecting the correct binding, and a third for confirming the integrity of the test and ultimately indicating the pregnancy state.

After applying urine to the sample pad or test strip, free-floating or mobile mouse-derived anti-hCG antibodies bind to the hCG (usually the beta subunit), if it is present, and move down the strip. Upon reaching the test region, the hCG-antibody complexes encounter another set of mouse anti-hCG antibodies, this time immobilized or secured to the test strip. If the original anti-hCG mouse antibodies are bound to hCG, then the secured antibodies will bind to another epitope on the hCG, usually the alpha subunit. If binding does occur, an enzymatic reaction ensues. The mobile antibodies are conjugated to an enzyme dye which will react with a dye substrate that is conjugated to the fixed antibodies when both are brought into physical proximity upon concurrent hCG binding. This dye reaction leads to the first blue bar appearing on the test strip. If no hCG is present in the urine, then neither the mobile nor the secured hCG antibodies are brought into significant proximity, and thus no blue bar should appear in the test region.

In order to make sure that the test is working properly, a control is included. Following the interaction at the test region, the remaining mobile antibodies that were not detected continue moving down the strip to the control region. There, the unbound antibodies are met by another secured antibody, this time around goat antibodies specifically targeting mouse antibodies. These secured goat anti-mouse antibodies are also conjugated to a dye substrate, and this step will confirm the actual presence of the original mobile anti-hCG antibodies. In every pregnancy test attempt, a blue bar should be present in the control region, regardless of the state of pregnancy, indicating that the immunoassay works properly.

Although there are various manufacturers, most of the at-home pregnancy tests today have an accuracy of approximately 99%. In fact, these tests now have a sensitivity and specificity that will unlikely be exceeded by other technology as they remain qualitative (positive or negative) and not quantitative (amount of hCG present); this of course does not replace the more formal blood tests that can be performed in a doctor’s office. Moreover, as we become more planet-conscious and come to accept that global warming is not something “Al Gore made up to sell books to Californians”, revamping other aspects of the technology, such as its biodegradability, are likely to drive improvements moving forward.

To think that such a simple three-minute-long assay can change the rest of your life… So, as we have once again reached this very special month of the year during which the most babies are born, I would like to wish all of them a very Happy Birthday, as I’m sure your Moms could hardly wait for this moment nine months ago!

Guillaume Trusz

Author Guillaume Trusz

Guillaume Trusz received his B.S. in Molecular, Cell, and Developmental Biology from the University of California, Los Angeles (UCLA) in 2015 and his M.S. in Biomedical Imaging from the University of California, San Francisco (UCSF) in 2018. Prior to working as an Associate Scientist in the Discovery Immunology Group at Curia, Guillaume contributed to various academic and industry related research projects pertaining to small molecules, nanoparticles, as well as biosimilars.

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