The first pregnancy tests required injecting a woman’s urine into rodents or frogs and waiting for the animals to ovulate to confirm a positive result. The advent of antigen detection technology and rapid antibody production eliminated animal-dependent testing and made it possible for women to test for pregnancy early and at home.
Download this Drug Discovery News Science Milestone article to explore the major developments that led to the modern pregnancy test and laid the foundation for other types of rapid antigen-based diagnostic applications, such as COVID-19 tests.
Decades of endocrinology and immunology research predated the home test that allowed women to detect pregnancy quickly and reliably.
BY LUISA TORRES, PHD
To diagnose pregnancy, a woman once had to inspect her urine for color and cloudiness, mix her urine with wine, or urinate on bags of barley or wheat to see if they would sprout. Today, a more sophisticated method exists thanks to a key hormone produced during pregnancy.
Late 1800s- Early 1900s Hormone Science started it all
One of the first indications that internal secretions regulated human bodily functions came in 1849 when Arnold Adolph Berthold, a physician scientist at the University of Göttingen, removed the testes of immature male chickens and observed abnormal male behavior, including a lack of aggression toward other males, disinterest in hens, and a lack of crowing (1). When he transplanted the testes back into the castrated chickens, they regained their normal male characteristics. Berthold concluded that the testes released a chemical messenger into the blood, later known as testosterone, that governed male behavior.
In a similar experiment in 1900, Josef Halban, a gynecologist at the University of Vienna, removed the ovaries and parts of uteri from guinea pig pups and transplanted them under the animals’ skins (2). Even though the reproductive organs were in a new location, they still secreted a substance, later known as estrogen, that stimulated puberty. In 1905, Ernest Starling, a physiologist at the University College London, used the word “hormone” to describe secretions that traveled in the bloodstream to cause physiological effects (3). Starling’s colleague, Edward Sharpey-Schafer, used Starling’s new word two years later to postulate that the testicles and ovaries produced hormones that helped in the development of secondary sexual characteristics, such as body size and behavior (4).
1927-1931 Pregnancy testing in mice and rabbits
The discovery of hormones led to a boom in endocrine research. In 1927, endocrinologist Selmar Aschheim at the Universitäts-Frauenklinik and gynecologist Bernhard Zondek at the Hebrew University-Hadassah Medical School and Hospital found that only pregnant women had high concentrations of human chorionic gonadotropin (hCG), a hormone that mimicked the effect of the ovulation inducing luteinizing hormone (LH), in their urine (5). They observed that when they injected an immature female mouse with the urine of a pregnant woman, the mouse’s ovaries released an egg (6).
That same year, Aschheim and Zondek developed the “A-Z Test,” the first laboratory test for pregnancy, which involved injecting mice or rats with a woman’s urine (7). If the rodent went into estrus, it meant that the urine came from a pregnant woman.
In 1931, reproductive physiologists Maurice Friedman and Maxwell Lapham at the University of Pennsylvania developed a similar test called the “Friedman Test” using rabbits (8). Unlike mice, rabbits didn’t require microscopic examination to confirm a positive result, which reduced the waiting time from five days to 48 hours.
Despite the availability of laboratory pregnancy tests, women rarely visited a doctor before the second or third trimester, and doctors reserved the A-Z test and the Friedman test for urgent cases, such as distinguishing pregnancy from a malignant tumor that also produced hCG (9).
1935-1959 From mice and rabbits to frogs
By 1935, technicians at the Pregnancy Diagnosis Laboratory performed 6,000 mouse and rabbit pregnancy tests every year (10). However, testing on rodents was expensive, time consuming, and required many animals. In 1937, biologist Lancelot Hogben at the University of Cape Town sent his friend, Francis Albert Eley Crew, an animal geneticist at the Pregnancy Diagnosis Laboratory, some Xenopus frogs to determine if they could become appropriate experimental models for pregnancy testing (10). Two years later, Hogben found that Xenopus females laid eggs within 12 hours after injection with a pregnant woman’s urine, making the Hogben test using frogs the fastest available animal-based pregnancy test (11,12).
Frogs increased the availability of pregnancy testing, but women still relied on their doctors to send their urine for sampling, and doctors only ordered this testing for urgent medical reasons. Depending on frogs for pregnancy testing also meant that the tests remained expensive and time consuming. “It wasn’t until immunoassays came along that you could measure hormone levels quantitatively without using live animals,” said George Bousfield, a reproductive biologist at Wichita State University, whose early training in Xenopus egg fertilization sparked his interest in reproductive science.
1960-1969 A game-changing immunoassay
In 1960, Leif Wide and Carl Gemzell, gynecologists at Uppsala University, published the first immunoassay that demonstrated the binding of hCG in human urine to anti-hCG antibodies (13). This led to the development of a laboratory pregnancy test in which red blood cells formed a doughnut pattern in the bottom of a test tube when hCG in a urine sample bound to red blood cells coated with anti-hCG antibodies (14).
In 1960, Rosalyn Yalow, a nuclear physicist at the Veterans Administration Hospital, developed the first radioimmunoassay to quantify antibody-antigen interactions using radioisotopes (15). Radioimmunoassays revolutionized the field of medical diagnostics and allowed the detection and quantification of very small amounts of analytes in blood and other bodily fluids.
In 1966, Rees Midgley, a reproductive endocrinologist at the University of Michigan, described the first radioimmunoassay for hCG, which allowed scientists to quantify hCG levels (16). This made it possible to determine how much hCG was in a urine sample, which became useful for diagnosing ectopic pregnancies and miscarriages (17). Although other researchers developed improved versions of Midgley’s radioimmunoassay (18), a new problem arose. Radioimmunoassays could not differentiate between hCG and the similarly structured ovulation-inducing hormone LH.
1969-1970 hCG stands out from LH
Several major advancements contributed to developing pregnancy tests specific for hCG. In 1969, Robert Canfield, a physician at Columbia University and Om Bahl, a molecular biologist at the State University in Buffalo, discovered that hCG had two subunits called alpha and beta (20). The alpha subunits of hCG and LH were almost identical, but their beta subunits harbored key differences: hCG had an additional 24 amino acid carboxy terminal tail and greater carbohydrate content (21).
In 1970, reproductive endocrinologists Judith Vaitukaitis, Glenn Braunstein, and Griff Ross developed an immunoassay specific for hCG at the National Institutes of Health. Vaitukaitis injected rabbits with the pure beta subunit of hCG, and Braunstein analyzed the resulting antibodies for their sensitivity and specificity. The sixth rabbit that Vaitukaitis injected produced high antibody titers with little cross reactivity with LH. “The surprise was that we found one very rapidly,” said Braunstein. “We realized right away that it was going to be a very useful asset.”
They used the new antibody to make a competitive radioimmunoassay that detected pregnancy as early as seven and a half days after conception (22). Although this assay became the gold standard for measuring hCG (14), it was not set up for home use.
1975-1977 The first home pregnancy test
Specifically detecting hCG required an efficient method for producing highspecificity antibodies. In 1975, Georges Köhler, a cell biologist at the Basel Institute for Immunology, and César Milstein, a biochemist at the Medical Research Council Laboratory of Molecular Biology, developed the hybridoma technology that sped up antibody production (23). They fused antibody making cells with tumor cells to create immortalized cell lines that generated monoclonal antibodies with predetermined specificity. “You now had the ability to make very specific antibodies very rapidly and fairly cheaply,” said Braunstein. These included antibodies against hCG and its subunits.
In 1977, the company Warner Chilcott released the “Early Pregnancy Test,” the first home pregnancy test in the United States. Although it gave a result within two hours, the test required high concentrations of hCG to keep the test’s hCG antibodies from cross-reacting with LH in the sample and giving a false positive result (21).
Bousfield, who was teaching at the University of Michigan at this time, made several preparations of human hCG and LH in his laboratory so that his students could test the available home ovulation and pregnancy tests. “It turned out that the LH test and the early pregnancy test measured both LH and hCG,” he said. “If you were ovulating, you might get a false positive. And if you were pregnant, an at home ovulation kit would probably detect this,” he said.
1980s-2000s The birth of the modern pregnancy test
In addition to monoclonal antibodies against hCG and its subunits, metal antibody labels such as gold and silver improved hCG antibody detectability and the reliability of pregnancy testing. James Hainfeld, a chemist at the Brookhaven National Laboratory, was the first to use metal nanoparticles as labels for biological structures and the first to introduce gold clusters as immunolabels in 1987 (24). With more powerful antibody technology, scientists developed materials that could hold urine or serum and onto which antibodies and antigens could bind. These technological advancements allowed Abbott Laboratories to create a prototype of the modern pregnancy test.
The home pregnancy test meant that women could diagnose pregnancy themselves. “To allow women to figure out what was going on in their own private space was tremendously useful,” said Braunstein.
Today, the antigen-detection technology used for home pregnancy tests has expanded to other useful applications. “The experience developing pregnancy tests allowed the rapid development of the COVID antigen tests. It’s all the same technology,” said Braunstein. “In the future, we’re going to see more and more of these things.”
REFERENCES
1. Soma, K. K. Testosterone and aggression: berthold, birds and beyond. J Neuroendo 18, 7 (2006).
2. Moscucci, O. & Clarke, A. Prophylactic oophorectomy: a historical perspective. J Epidemiol Community Health 61(3), 182-184 (2007).
3. Henderson, J. Ernest Starling and ‘hormones’: an historical commentary. J Endocrinol 184(1), 5-10 (2005).
4. Evans, B. & Jones, E. Organ extracts and the development of psychiatry: hormonal treatments at the Maudsley Hospital 1923-1938. J Hist Behav Sci 48(3), 251-276 (2012).
5. Choi, J. & Smitz, J. Luteinizing hormone and human chorionic gonadotropin: origins of difference. Mol Cell Endocrinol 383(1-2), 203-213 (2014).
6. Lunenfeld, B. Historical perspectives in gonadotrophin therapy. Hum Reprod Update 10(6), 453-467 (2004).
7. Aschheim, S. & Zondek, B. Hypophysenvorderlappenhormon und ovarialhormon im harn von schwangeren. Klin Wochenschr 6(13), 22 (1927).
8. Friedman, M. H. & Lapham, M. E. A simple, rapid procedure for the laboratory diagnosis of early pregnancies. Am J Obstet Gynecol 21, 405-410 (1931).
9. Olszynko-Gryn, J. The demand for pregnancy testing: the Aschheim-Zondek reaction, diagnostic versatility, and laboratory services in 1930s Britain. Stud Hist Philos Biol Biomed Sci 47, 233-247 (2014).
10. Gurdon, J. B. & Hopwood, N. The introduction of Xenopus laevis into developmental biology: of empire, pregnancy testing and ribosomal genes. Int J Dev Biol 44(1), 43-50 (2000).
11. Hogben, L. Xenopus test for pregnancy. BMJ 2(4095), 38-39 (1939).
12. Crew, F. A. E. Biological pregnancy diagnosis tests: a comparison of the rabbit, the mouse, and the ‘clawed toad’ (Xenopus laevis) as the experimental animal. Br Med J 1, 766-770 (1939).
13. Wide, L. & Gemzell, C. An immunological pregnancy test. Acta Endocrinol 35(2), 261-267 (1960).
14. Lamb, E. J. Immunologic pregnancy tests: evaluation of pregnosticon dri-dot and pregnosticon accuspheres. Obstet Gynecol 39(5), 665-672 (1972).
15. Yalow, R. S. & Berson, S. A. Immunoassay of endogenous plasma insulin in man. J Clin Invest 39(7), 1157-1175 (1960).
16. Midgley, A. R. Radioimmunoassay: a method for human chorionic gonadotropin and human luteinizing hormone. Endocrinology 79(1), 10-18 (1966).
17. Norman, R. J., Buck, R. H., & De Medeiros, S. F. Measurement of human chorionic gonadotrophin (hCG): indications and techniques for the clinical laboratory. Ann Clin Biochem 27(3), 183-194 (1990).
18. Saxena, B. B., Hasan, S. H., Haour, F. & Schmidt-Gollwitzer, M. Radioreceptor assay of human chorionic gonadotropin: detection of early pregnancy. Science 184(4138), 793-795 (1974).
19. The nobel prize in physiology or medicine 1977. https://www.nobelprize.org/prizes/medicine/1977/summary/
20. Bell, J. J., Canfield, R. E. & Sciarra, J. J. Purification and characterization of human chorionic gonadotropin. Endocrinology 84(2), 298- 307 (1969).
21. Braunstein, G. D. The long gestation of the modern home pregnancy test. Clin Chem 60(1), 18-21 (2014).
22. Vaitukaitis, J. L., Braunstein, G. D., & Ross, G. T. A radioimmunoassay which specifically measures human chorionic gonadotropin in the presence of human luteinizing hormone. Am J Obstet Gynecol 113, 751–8 (1972).
23. The nobel prize in physiology or medicine 1984. https://www.nobelprize.org/prizes/medicine/1984/press-release/
24. Hainfeld, J. F. A small gold-conjugated antibody label: improved resolution for electron microscopy. Science 236(4800), 450-453 (1987).