Antibody Tests

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Hello it is Triska again. ProfB has asked me to talk about the anti-body tests that are out there. These are currently relevant to how people are being tested to see if they have had a COVID-19 infection, but the principles are the anti-drug antibody tests that we have been doing in the Lab

Lateral flow test

A lateral flow immunoassay, also referred to as a rapid diagnostic test is a simple to use, portable device used to confirm the presence or absence of a target that is being tested for. It produces qualitative results (positive or negative) through visually interpreted results. These tests can use samples such as blood from a finger prick, saliva, urine, and nasal swabs. Lateral flow immunoassays are similar to pregnancy tests, in that the test detects a certain hormone and indicates positive or negative results.

Protein A vs Protein G

The principle of a lateral flow test is that the liquid sample (e.g. blood) runs along the surface of the test strip that is coated with reactive molecules towards the test line. This works just like water wicking up a piece of paper after it is into water. Often in the tests the reactive molecules are the test protein attached to gold particles. The molecules then show a visual positive or negative result on the test line because there are molecules to catch the protein with the gold particles on the test line. They typically look red because when gold clusteres it gives a red colour. The red in stained-glass windows occurs because of gold in the glass. These tests are rapid and give results within 5-30 minutes. For COVID-19, these tests most often test for antibodies to IgG (G) and IgM (M). The antibodies in the blood bind to covid-19-gold and then they are captured on the test line that contains either antibodies against human IgM (which are the first type of antibodies made in an infection) and also human IgG (the second type of antibody made when IgM producting cells switch to produce IgG). In the COVID test there is an anti-covid antibody on the control line (C) and this captures the covid-protein-gold complex to shw the assay is working.

ELISA

This is a lab based test.

ELISA plate

ELISA (enzyme-linked immunosorbent assay) is an assay technique is done in a plastic plate that detects and quantifies target substances such as antibodies, hormones, peptides, and proteins in samples. An ELISA can be qualitative or quantitative. This test can be used to determine if you have antibodies or antigens related to medical conditions and can give results in around 24 hours. Blood, plasma or serum samples are usually used.

In ELISA, the antigen (target macromolecule) is immobilise on/stuck to on a microplate (via direct adsorption or indirectly by a capture antibody) and then complexed with an antibody that is linked to a reporter enzyme. The most commonly used enzymes are horseradish peroxidase (HRP) and alkaline phosphatase (AP); these cause a colour change in the solution if the target is present (SEE above). Detection is then accomplished by measuring the activity of the reporter enzyme through incubation with an appropriate substrate to create a measurable product. The substrate used depends on the instrument that will be used for detection (spectrophotometer [meaures the transmittance of a solution], luminometer [measures visible light], or fluorometer [measures visible spectrum of light after excitation of a spectrum of light suchas ultraviolet light). Detection can be accomplished directly with the use of a labelled primary antibody, or indirectly for example with labelled secondary antibodies.

Ag = antigen being detected for COVID assay the antigen is in afact an anti-COVID antibody

There are several ELISA techniques, which are: direct, indirect, or sandwich capture and detection methods. The most often used ELISA is in the sandwich format, this involves indirect immobilisation and indirect detection of the target antigen.

Chemiluminescence test

This is the lab based test

The chemiluminescent immunoassay (CLIA) is a variation of the standard ELISA, and combines chemiluminescence with immunochemical reactions. Instead of an enzyme, the method used to detect the target substance is a light (luminescence) emitting substance (e.g. luciferase = the molecule from a glow worm or fire fly). CLIA uses chemical probes that generate light emission via chemical reactions to label antibodies. The amount of radiance generated from each sample is measured and calculated to represent the number of antibodies present in the sample.

Unlike the ELISA, CLIA does not require long incubation times and the addition of stopping reagents which leads to a shorter turnaround time for results, and has wider dynamic ranges providing low detection limits and higher sensitivity.

This is what is currently used in lab based COVID-19 assays

The current lab based assays are robotic, so a magnetic particle pulls the antibody to an electrobe and this is used to excite the ruthenylated target antigen to emit a signal that is detected.

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The Globody that we use is a synthetic target. We synthesise the DNA of the target like the Variable heavy (VH) and light (VL) chains of antibodies and add luciferase molecules on either end. The target antibody binds to the globody and then we capture the complex using a molecule called protein G that binds to immunoglobulin (antibody moelcules) that is stuck to plastic abit like in an ELISA. So at the moment this assay is done by hand but we should be able to made it ready for robotic testing

ADA = Anti-drug antibodies

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2 comments

  • Good article!

    Tangentially related: do we have an idea yet if people on ocrelizumab would have a positive result on the antibody test after Sars Cov2 exposure ?

    I had something that could have been mild covid in March (or a pseudo exacerbatiion or a fairly persistent cold with mild fever on and off for 5 weeks) but never bothered to get the PCR test (figuring there is nothing anyone can do and assuming testing facilities to be among the worst risks) and am now wondering if there is any point in getting the antibody test….

    • Have the test and then you know…some people will make an anti covid response the question is how good will it be

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