COVID-19: The Antibody Test

When a new infection enters the body, the immune system gets to work.  It produces antibodies that attack the antigen (virus or bacterium), but this takes time.  The first wave of antibodies is of a type called immunoglobulin M (IgM).  These bind with proteins on a virus’s surface, stopping it from working and marking it for destruction by other cells.  They do not last for long and are gone from the blood within 3 or 4 weeks.  The second wave of defence uses a more specific type of antibody called immunoglobulin G (IgG), which are better tailored to the antigen.  They are the basis for a much longer-term form of immunity, which can last for many years, or even a lifetime.

Immunoglobulins are glycoproteins with a molecular weight around 150,000 Daltons, their structure means that some parts are the same, but other parts are tailored to bind to specific antigens.  The structures of IgM and IgG are similar but sufficiently different that they have distinguishable binding sites – where the stereochemistry and hydrogen bonding can be used for docking another molecule.

In the early stages of infection, it is more insightful to test for the cause of the infection itself.  As the infection proceeds, the number of antibodies in the blood increase and they can be tested for.  Once the infection has been dealt with by the immune system, it is only the antibodies that can be seen.

There are various ways to test for the antibodies, but they all rely on having something to mimic the antigen in the test.  In the case of SARS-Cov-2 some groups are using the proteins that make up the surface “spike” as a representative of the actual virus, although some are using just the receptor binding part of it. 

The laboratory-based test is known as ELISA (Enzyme-Linked Immunosorbent Assay).  In it, the antigen (or its mimic) is immobilised in wells of a plastic plate by physisorption.  A sample of the patients’ blood is put into the well.  If the blood has antibodies for this antigen, they will bind to the antigen.  The blood is then removed, and the surface is then washed and treated with another biological material (bovine serum albumin or casein) to mask the underlying substrate.  Then the detection antibody is added. The detection antibody is designed to bind to the now bound target antibody and is labelled with an enzyme, such as horse radish peroxidase or alkaline phosphatase.  Finally a solution is added containing a molecule that reacts with horse radish peroxidase or alkaline phosphatase to give a colour ((2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) or 3,3’,5,5’-tetramethylbenzidine for horse radish peroxidase or the disodium salt of p-nitrophenyl phosphate for alkaline phosphatase).  The depth of colour developed scales with the amount of the antibody present in the original blood sample.

The rapid home tests everyone is getting excited about use the same underlying technology but use lateral flow to drive the process.  A sample of blood is placed at one end of the cassette and then the dilution buffer (10 mM phosphate-buffered saline) is added.  The sample then moves by capillary action/lateral flow and first passes a pad which contains the COVID-19 antigen conjugated to 20-60 nm gold nanoparticle. During this stage, any antibodies in the sample with specificity for COVID-19 will bind to the antigen and its conjugated gold nanoparticle. (although not important for the actual chemistry, there is also a control antibody, often rabbit IgG, similarly conjugated to gold nanoparticles which travels with the rest of the sample.)  From here, the sample/conjugate complex moves to a nitrocellulose membrane. Here, it comes in contact with the three test lines: IgM, IgG and control.  First comes the M line, which contains an immobilised antibody that recognises human IgM. Any IgM antibodies will bind here. However, only human IgM antibody/COVID-19 antigen/gold nanoparticle complexes will produce a visible coloured (pinky red) line.  Second is the G Line, which contains an immobilised antibody that recognises Human IgG. All IgG antibodies will bind here. However, only human IgG antibody/COVID-19 antigen/gold nanoparticle complexes will produce a visible coloured line.  Finally comes the control line, which contains an immobilised antibody that recognises Rabbit IgG, the control antibody. The appearance of this last coloured line shows that the proper volume of specimen has been added and membrane wicking has occurred – which means the test is valid.  If there is a pinky red M line, the patient is probably early in their infection.  If there is a pinky-red G line, the patient is probably late on in the infection, or has recovered.  Both lines indicate an ongoing infection.

Why use gold nano- particle, and why do they give a pink/red lines?

Gold is chemically quite inert and the colour of a colloidal dispersion changes with the particle size and shape.  This effect is caused by localised surface plasmon resonance (LSPR).  When gold nanoparticles are exposed to light of a certain wavelength, their conduction electrons are driven by the electric field so that they collectively oscillate relative to the lattice of the positive nuclei, creating intense extinction peaks at resonant wavelengths. They therefore absorb light at that wavelength, which results in the emission of light of a complementary wavelength. For example, around 40 nm gold nanospheres absorb green light at 520 nm and produce complementary red light.