Why the CDC recommends using two different antibody tests.

Read time: 3.5 min.

In its Interim Guidelines for Antibody Testing, the CDC recommends using two different antibody tests to improve accuracy, especially when confirming a positive test result. This article breaks down what sensitivity and specificity actually mean and explains the math behind the CDC’s recommendation.

What do Sensitivity and Specificity mean?

Autobio and Healgen antibody tests. The CDC says two is better than one.

The CDC and FDA urge labs and clinics to perform antibody testing with tests that have high sensitivity and specificity. But what do these terms mean in the real world?

Sensitivity is how good a test is at correctly identifying COVID-19 positive samples. For antibody testing, 100% sensitivity means that a test will detect antibodies in individuals that have been infected with coronavirus 100% of the time. If there are 50 positive samples, it will detect 50 out of 50. In other words, it is highly sensitive. If the test detects 48 out of 50, it would have 96% sensitivity. Another way of saying this is the test will give a false negative result only 4% of the time.

In the real world, no test will give the correct result 100% of the time if enough samples are used, but a really good test can achieve 99% or higher. 96% sensitivity is still very good, but below we’ll explain why it can be problematic if the infection rate is low in a population. The other crucial aspect of antibody testing is that most people don’t immediately develop antibodies to coronavirus. According to a recent clinical study, the average time it took for infected individuals was 13 days. One hundred percent of patients developed antibodies by Day 19. So antibody tests become most sensitive around 2-3 weeks after exposure, and would be much less sensitive during the first week.

Sensitivity is how good a test is at correctly identifying COVID-19 positive samples. 100% sensitivity means if there are 50 positive samples, the test will correctly detect 50 out of 50. If it detects 48 out of 50, that would be 96% sensitivity.

In some clinical studies and Instructions for Use, sensitivity is referred to as “Positive Percent Agreement.” They mean the exact same thing and are both reporting what percent of the time the test correctly identifies samples known to be positive for coronavirus.

Specificity is how good a test is at correctly identifying COVID-19 negative samples. 100% specificity means that a test will correctly detect no antibodies in a person who has never been infected with coronavirus 100% of the time. If there are 50 negative samples, it will detect 50 out of 50. In other words, it is highly specific because it does not detect COVID-19 when a person has not been exposed to coronavirus, or has had a different type of illness, like a cold or flu. If a test correctly identifies 49 out of 50 negative samples, it would have 98% specificity. Another way of saying this is the test will give a false positive result just 2% of the time.

Specificity is how good a test is at correctly identifying COVID-19 negative samples. 100% specificity means that a test will correctly detect a negative patient 100% of the time. If there are 50 negative samples, it will detect 50 out of 50. If it detects 49 out of 50, that would be 98% specificity.

Some clinical studies and Instructions for Use refer to specificity as “Negative Percent Agreement”, but they mean the same thing — reporting what percent of the time the test correctly identifies samples known to be negative for coronavirus. Using tests with a high specificity becomes very important when the infection rate is relatively low in a population, especially under 5%, and is the reason why the CDC recommends using two different antibody tests, which we explain next.

Why should two different antibody tests be used, especially to confirm a positive result?

Let’s use the two antibody tests we carry as an example — Autobio and Healgen. Both are EUA authorized and were clinically validated by the FDA’s serology testing program in conjunction with the National Cancer Institute. In other words, they passed the FDA’s criteria for sensitivity and specificity. So far only four lateral flow assays have been granted EUA.

As reported in the FDA’s test results, Autobio has a Sensitivity of 99.0% and a Specificity of 99.0%. Healgen has a Sensitivity of 100.0% and a Specificity of 97.5%.

This means if 100 individuals known to never have been exposed to coronavirus were tested with Autobio, it would correctly identify 99 out of 100 people, and would only give one false positive result. Fantastic, right?

Well, let’s say that only 5% of the population has been infected with coronaviurs. That means Autobio would detect the 5 patients out of 100 that have been infected, but would also give that 1 false positive result. That means it would only correctly detect 5 out of the 6 people who were actually exposed to coronavirus, meaning the test it is correct about 85% of the time. This is called the Positive Predictive Value, or PPV. So even 99.0% specificity means that about 15% of patients will be given a false positive result.

PPV depends on the percentage of the total population that has been infected. If 20% of the population had been infected, then the PPV would increase to around 95%, so the test becomes more reliable. But there is another solution.

The predictive value of antibody testing increases greatly if two really good tests are used together, especially when the infection rates are relatively low in a population.

Using two tests, called “orthogonal testing”, each with high sensitivity and specificity greatly increases the predictive value so long as the two tests were independently developed and have different formulations. Optimally, you would choose a test that targets two different antigens. In the case of coronavirus, one that targets the spike protein, and one that targets the nucleocapsid protein would be ideal. But even using two tests that both target the spike protein is valuable as long as the tests were independently developed because they likely target and detect the spike protein in slightly different ways.

Using Autobio and Healgen together increases the PPV to around 99.5%, which means if both tests give a positive result, there is a 99.5% probability it is the correct result. Performing orthogonal testing greatly increases the predictive value of the test, especially when infection rates are relatively low in a population.

The combined Negative Predictive Value of Autobio and Healgen, or NPV, is 99.9%. This means that if both tests return a negative result, there is a 99.9% probability it is the correct result.

You can download the CDC’s PPV calculator here and enter the sensitivity and specificity of two tests and get the PPV and NPV back.

We hope this has helped you understand why using two antibody tests is ideal. If you have any questions, or are interested in setting up testing at your lab or clinic, we would be happy to provide more information or supply your test kits. Just reach out and let us know how we can help.