P4Gold array

A Very Brief History of Milk Progesterone Measurement

With the shift from using bulls to primarily using artificial insemination to cover cattle, there has been a corresponding push to develop accurate heat detection methods.  A mistimed AI can be expensive and, in the worst case scenarios, cause infection or pregnancy loss. Progesterone tests emerged as one of the best methods of heat detection, due to their reliability and accuracy. Lateral flow tests like P4Gold, are now a common sight on dairy farms.

Progesterone testing has long been considered the ‘gold-standard’ of heat detection in cattle. Commercial applications of radio-immunoassay technology became viable in the 1970s, and now more methods are available. With a close relationship between milk and blood progesterone concentration, milk tests became the preferable option when assessing heat status. Milk samples are easy to take from lactating cows, and are far less invasive and stressful than blood samples.

In this article, we’ll take a look at the different methods of progesterone testing and cover a brief history of P4Gold.


Radio-immunoassay or RIA, is a technique which uses specific antibodies to measure the concentration of a substance in a sample. This form of testing uses radio-labelled molecules and an antibody which binds to the target molecule. It is widely used across the medical, pharmaceutical, and veterinary industries to detect many different target substances.

Initially, a known amount of the target molecule is made radioactive, and then this is mixed with the specific antibody. This mixture is then mixed with the samples. The target molecule in the sample will compete with the radioactive molecules to bind to the antibody. The target molecule in the samples is not radioactive. As a result, the radioactivity will change depending on how much of the target molecule was present in the sample. Once the test is complete, each sample will have a different amount of radioactivity. This determines how much of the target molecule is there.

While this technique is extremely accurate, it requires extensive experience with the technology and also specific equipment. As such, it is rarely, if ever, run outside of a laboratory setting. While it can be used to accurately measure progesterone in cows’ milk, the turn-around time is large. Farmers have to send samples to a lab for analysis and then wait for the results. This takes too long to make this a viable option for rapid decision making.

Enzyme-linked Immunosorbent Assay

ELISA and RIA testing is difficult to perform outside of a laboratory.
ELISA and RIA testing is difficult to perform outside of a laboratory.

Enzyme-linked immunosorbent assay or ELISA, is a very similar technique to RIA. The primary difference between these two techniques, is in the detection method. ELISA testing generally uses a colour change to indicate the presence of the target, rather than radioactivity.

Particular methods can vary greatly, but ELISA testing always relies on specific antibodies and their binding capacity to the target molecule. Some forms of this test are semi-quantitative and usable by laypersons. However, laboratories run the majority of these tests.

Lateral Flow Tests

Ridgeway Science perfected their progesterone ELISA plate back in the early 1990s. The next step was to develop a test that could give a result quickly and easily, without much technical know-how. Around this time, lateral flow tests were becoming more popular in the fields of medicine, veterinary science, and many others. These tests produce clear results in the form of lines developing on a stick to diagnose or indicate many things. In recent times, this technology has come into the spotlight with COVID-19 and its lateral flow detection test.

Lateral flow tests are common across many industries.
Lateral flow tests are common across many industries.

Since their first debut, there are now a multitude of tests which can detect all sorts of targets. From pregnancy tests and disease, to water quality and food safety, this technology has been implemented widely.

These tests work based on similar principles to ELISA and RIA technologies. Usually, they are impregnated with a specific antibody for the target molecule bound to a coloured particle. When the test is run, the stick can show lines or colour changes if the result is positive. Simultaneously, the vast majority of lateral flow tests also have a ‘Control’ line. This is a line that is designed to appear if the test has run properly.

The First Test: P4Rapid

Ridgeway Science released P4Rapid, the first generation of milk progesterone lateral flow test, in 2012. It used our progesterone antibody to accurately detect progesterone in milk samples. Using coloured latex beads, the test allowed farmers and AI technicians to check heat status with ease.

Since then, technology has moved on and paved the way for even more reliable testing. We developed P4Gold to keep up with the growing dairy industry while ensuring the highest quality of heat detection.

The Second Generation: P4Gold

A P4Gold test showing high levels of progesterone.
A P4Gold test showing high levels of progesterone.

P4Gold uses real gold nanoparticles to detect the level of progesterone in milk samples. Compared to the coloured latex used in P4 Rapid, it produces a clearer line on the test dipstick. The high binding affinity of P4Gold and small particle size means that the progesterone in the sample is reliably detected. The gold also produces a high-contrast red line on the dipstick, making it easier to read in poor light conditions. The extremely high sensitivity and specificity of the Ridgeway Science antibody provides an accurate and reliable test.

If there is no progesterone, the reagents bind to the test line to produce a strong red line. This indicates low progesterone. The less progesterone there is in the milk the stronger the test line will be. On the other hand, if there is progesterone in the milk, it will bind to the reagents first. So, as progesterone levels rise, the red in the test line will reduce. Ultimately, when progesterone levels in the milk are very high, there is no red in the test line at all.

The control line uses a reagent that is independent of progesterone. This is so a red line will develop regardless of the levels of progesterone in the milk. This shows the test is working.

P4Gold: Just the Beginning

All in all, P4Gold is the culmination of many years’ work. With input from dairy farmers, veterinarians, and AI technicians alike, we’ve created a real-time fertility tool that will suit farms big, and small. P4Gold is a quick, easy and accurate way to measure progesterone levels that can be used in the parlour. It allows vets and farmers to take immediate and appropriate action to maintain and improve the fertility and productivity of the dairy herds in their care.