If the seeds didn't sprout, then no children were expected. Somewhat surprisingly, scientists have since speculated that there may in fact be some truth to this old wives tale, as higher than normal levels of oestrogen, may have stimulated the germination of the seeds. However, this ancient method certainly wasn't one you could rely on. Today, home test pregnancy kits take on a very different form and test sticks contain proteins called antibodies which detect certain hormones elevated during pregnancy. However, false positives and false negatives can occur, and so scientists are interested in looking at the reliability of these test kits, and how their efficiency could be further improved.​

If you’re an antibody and you know it, raise your hands.

Antibodies are 'Y' shaped proteins with two arms that have a unique structure and can specifically recognise another structure (antigen) on a foreign particle. It’s this outer weapon which they take into battle when viruses and bacteria evade our immune system. They grasp invading particles with their outer arms and bind to them specifically, tagging them for destruction.  And it’s this feature, of site specific structural recognition binding, which is exploited in many biomedical devices, including pregnancy tests, detection of viruses and the diagnosis of cancers.

Home pregnancy tests use antibodies to check for the Human Chorionic gonadotropin hormone, whose presence in urine over a certain level indicates a pregnancy. The test strip contains two antibodies which capture the hCG antigen in a “sandwich” formation. One of the antibodies (anti-b-hCG) is attached in a line on the white nitrocellulose strip whilst the other (anti-a-hCG) is bound to coloured blue latex particle and held in a dry state. If hCG is present in the urine sample, it binds the anti-a-hCG, and diffuses down to the test strip, where it is captured by immobilised anti-b-hCG. This creates the blue line seen on positive pregnancy tests.

The test strip contains two antibodies which capture the hCG antigen in a “sandwich” formation. One of the antibodies (anti-b-hCG) is attached in a line on the white nitrocellulose strip whilst the other (anti-a-hCG) is bound to coloured blue latex particle and held in a dry state.
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Scientists investigate the structural arrangement of the proteins on a model pregnancy test surface

Scientists from the University of Manchester, the University of Sheffield, and Keele University have been using neutron reflection at ISIS and the ILL in collaboration with Swiss Precision Diagnostics, a leading supplier in Home pregnancy and fertility tests, to investigate the structural arrangement of the proteins on a model pregnancy test surface in order to better understand how false positives occur and improve the reliability of pregnancy tests. Their research is published in the Journal Langmuir.

Dr Jian Lu, University of Manchester, explains “The past few decades have seen huge progress in antibody research and the maturing of many biotechnologies. Antibody based pregnancy test is one of the few examples that has gained global success. It hinges on how the antibodies interact with their antigen targets in real working environments. However, antibodies are fragile and many factors could cause unreliable performance, resulting in false positive and false negative readings. Our work of studying how antibodies interacting with antigens at model interfaces could help improve the reliability of such biotechnologies.”

Some of the factors which might cause unreliable performance in pregnancy tests are non-specific absorption of the blue coloured antibodies to the test strip resulting in false positives. One of the main reasons for this, is binding of the hCG-a-antibody to protein free areas on the test strip and so blocking agents which are proteins, such as Human serum albumin (HAS), are often used to try and counteract this.

Immobilised antibodies also bind antigen at a much lower capacity than those free in solution and its thought that the packing of antibodies on the surface and unfavourable orientation of the antibody may restrict access of the hCG antigen to the antibody.

In order to produce more sensitive tests it’s important to understand the interfacial behaviour of the immobilised antibodies and so the team of scientists have used neutron reflection to help determine the amount and location of antigen hCG bound to the surface confined antibody. Furthermore by using a hydrogenated and deuterated form of the HSA protein blocking agent they have been able to unravel how the antibody and antigen interacted and the role of the HSA.

“The results of this study, showing the molecular interactions, are critical to the control of specificity and consistency of the test devices”, explains Jian. “The results are also of importance for a full understanding of similar systems that are being developed for clinical diagnostic tests and in the detection of environmental contaminants”

“What’s striking is that we have shown that the antibodies do not stand up on the test surface, as they are often depicted in other literature. In addition we have shown how the antigen hCG binds to the surface confined antibodies, how the blocking agent molecules are packed at the interface and how their amount and location may vary with the total surface packing density.”

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Neutrons revealed that the antibodies do not stand up on the test surface and shown how the antigen hCG binds to the surface confined antibodies.
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This information has given clues into how to manipulate the bioactivity of surface confined antibody and how to minimise the non-specific binding in order to avoid false positive and false negative tests and better the performance of pregnancy tests and similar diagnostic tools based on antibody technology.

Having seen the full advantage neutron reflection can bring to this kind of research the team now would like to apply it to antibody drug research.

“This is a new horizon in which neutron refection and scattering could help understand how stable these new protein drugs are under different drug, and physiological environments, and how their bioactivity and specificity are controlled in binding to their targets”

Felice Laake

Research date: June 2014

Further Information

For further information please contact Dr Jian Lu

References

This research is published in the Journal Langmuir​