Serology: Interpretation COVID-19 Antibody tests
As the COVID-19 has spread in the world, an increased testing to trace and control outbreaks. Besides increased RT-PCR testing for acute infections, there is a need for serology tests to assay who may have acquired immunity to future SARS-CoV-2 infections. These tests identify individuals with neutralizing antibodies against SARS-CoV-2 by using coronavirus proteins as bait.
- 1) Major COVID-19 Antibody Test Types
- 2) COVID-19 detection by ELISA antibody test
- 3) CMIA-based COVID-19 Antibody test
- 4) Lateral Flow Test for COVID-19 antibodies
- 5) Genetex’s Lateral Flow Recommended Pairs
1) Major COVID-19 Antibody Test Types
Hundreds of companies have developed serology tests of varying quality. There are many types of tests, but how do they work and what are the limitations of each?
– Simpler to develop than newer technologies
– Well-established in diagnostics
– Low sensitivity
– High sensitivity
– Low background
– Can be automated and high-throughput
– Rapid results
|– High false positive rate|
– Simple to run and interpret
– Room temperature stability
– High false positive/negative rate
– Qualitative rather than quantitative
2) COVID-19 detection by ELISA antibody test
ELISAs (Enzyme-linked immunosorbent assays) are the simplest technologies for quantifying an analyte in a sample. These are typically 96-well plates that are coated with a capturing agent. In most cases, these are polyclonal antibodies for the analyte. For COVID-19 serology tests, these are fragments or full recombinant viral proteins. As the samples are incubated, the capturing agent binds and isolates the analyte from the sample. After washing to remove unwanted molecules, a detecting agent is added. The detecting agent is typically a monoclonal antibody towards the analyte conjugated to an enzyme such as horseradish peroxidase. For COVID-19 serology tests, these can be another recombinant protein, or an anti-immunoglobulin antibody conjugated to an enzyme. Adding the enzyme’s substrate produces a colorimetric signal proportional to the amount of substrate, enabling quantification.
The major advantage of this method is that there are well-established protocols for development and quality control. It is also a common type used for medical diagnostics, so it is compatible with most clinical laboratories. They also require manual labor and significant time investment.
3) CMIA-based COVID-19 Antibody Test
CMIAs (chemiluminescent microparticle immunoassays) are another method for serology tests. In this assay, the capturing agent is coupled to a paramagnetic microparticle rather than coated on a plate. After washing, the detecting agent with a chemiluminescent conjugate is added. After formation of the immunocomplex and washing, the other component of the chemiluminescent conjugate is added and the resulting signal is read and used for quantification.
Unfortunately, as sensitivity increases, the possibility of false positives increases and this can be an issue with this method in certain circumstances.
4) Lateral Flow Test for COVID-19 antibodies
The lateral flow assay is a chromatographic test. In this assay, samples flow through a specialized strip with a simple yes/no read-out. Samples first pass a conjugate pad containing the bait conjugated to a fluorophore, gold, or another tag. Afterward, the immunocomplexes travel past a line of capture antibodies that immobilize them. The concentration of the immunocomplex in one line produces a readable signal. The rest of the sample then passes through a final line of antibodies to produce an internal positive control. The major advantage of this test is the simplicity. Depending upon the readout, it could be used at home or point-of-care. Additionally, lateral flow tests are often safe at room temperature.
This simplicity is a double-edged sword. At this moment, the level of binding needed to reasonably conclude the presence of neutralizing antibodies is unknown. This makes the test prone to false positives or negatives.
An example of this type is Cellex’s qSARS-CoV-2 IgG/IgM Rapid Test. The conjugate pad contains spike and nucleocapsid recombinant proteins conjugated to colloidal gold. This test has two sets of capture antibodies. The first set is IgG antibodies and the second set is IgM antibodies. Neutralizing antibodies bound to recombinant protein conjugated to colloidal gold produce a burgundy color when immobilized and concentrated at the capture antibody lines.
5) Genetex’s Lateral Flow recommended pairs
|Pair||Product Name||Clonality||EC50||Cat. No.|
|Pair I||Nucleocapsid antibody [HL5511]||Rb recAb||14 pM||GTX635689|
|Nucleocapsid antibody [HL448]||Rb recAb||11 pM||GTX635686|
|Pair II||Nucleocapsid antibody [HL5410]||Rb recAb||9 pM||GTX635685|
|Nucleocapsid antibody [HL455]||Rb recAb||6 pM||GTX635688|