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Serious about Serology: Understanding COVID19 Antibody Tests

Serious about serology: Understanding COVID19 Antibody Tests


As the COVID-19 has spread in the US, policymakers and public health experts such as former FDA chief Scott Gottlieb have been calling for 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.

Hundreds of companies have developed serology tests of varying quality, but the FDA recently released performance data on 12 tests. There are many types of tests in this FDA document, but how do they work and what are the limitations of each?

Major COVID-19 Antibody Test Types

Type

Advantages

Disadvantages

ELISA

-          Simpler to develop than newer technologies

-          Well-established in diagnostics

-          Low sensitivity

-          Slow

-          Low throughput

CMIA

-          High sensitivity

-          Low background

-          Can be automated and high-throughput

-          Rapid results

-          High false positive rate

Lateral Flow

-          Simple to run and interpret

-          Room temperature stability

-          High false positive/negative rate

-          Qualitative rather than quantitative

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.

As an older technology, 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. 

However, ELISAs tend to have lower sensitivity compared to newer technologies. They also require manual labor and significant time investment. Consequently, they are low-throughput.

An example on the market is Bio-Rad’s Platelia SARS-CoV-2 Total Ab, which tests for antibodies against the nucleocapsid proteins. This test uses plates coated with recombinant nucleocapsid and then uses recombinant nucleocapsid with peroxidase for the detecting agent. Some of the top performing serology tests are CLIAs (chemiluminescent immunoassays). The principle behind them is very similar to ELISAs, but these use a chemiluminescent, rather than colorimetric, reaction for the read-out. Chemiluminescent systems have a higher dynamic range and higher sensitivity than colorimetric systems (PMID: 20201791).

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.

This method has several advantages over ELISAs. First, it has higher sensitivity than ELISAs since the available surface area for binding to microparticles is greater than that of microplate wells. Second, there is lower background. Since the particles are paramagnetic, application of a magnetic field isolates the immunocomplex efficiently from the unwanted molecules in the sample. Third, the whole test can be automated and has a shorter incubation time than ELISA.

Unfortunately, as sensitivity increases, the possibility of false positives increases and this can be an issue with this method in certain circumstances (PMID: 28614239).

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.

Proteintech’s IgG/IgM/IgA Antibodies

Many of the serology tests use conjugated immunoglobulin antibodies as the detecting agent as these are easy to produce on a large scale and can identify a diverse set of antibodies.

Proteintech offers IgG/IgM/IgA antibodies in bulk quantities and in carrier-free formulations to enable conjugation and plate-coating.

Proteintech’s IgM, IgA & IgG Portfolio

Catalog number

Product name

Applications

Species reactivity

Publications

60099-1-Ig

Human IgA Monoclonal Antibody

WB, IHC, IF, ELISA

human

1

66484-1-Ig

human IgM Monoclonal antibody

WB, IHC, IF

human

 

CL488-66484

CL488-human IgM Monoclonal antibody

WB, IHC, IF

human

 

CL594-66484

CL594-human IgM Monoclonal antibody

WB, IHC, IF

human

 

11016-1-AP

human IgM Polyclonal antibody

WB, IHC

human

 

10284-1-AP

Rabbit Anti-Human IgG

WB, IHC

human

 

HRP-66484

HRP-Conjugated Human IgM Antibody

WB, ELISA

human

 

Request bulk and custom formulations here

Blog

Posted:
2 June, 2020

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