Rapid COVID-19 Antibody Detection Tests: Principles and Methods

Rapid COVID-19 Antibody Detection Tests: Principles and Methods

What does the COVID-19 Rapid POC CE-IVD Test detect?

The test detects the presence of patient-generated antibodies against SARS-CoV-2, the virus which causes the disease COVID-19. The test can detect two types of antibody isotypes: IgG and IgM.

IgM antibodies are the first antibodies to appear in response to a novel antigen. They imply a more recently initiated infection.

IgG antibodies have a higher affinity for the target antigen, meaning they are more specifically able to bind the substance which caused the immune response. IgG antibodies are generated later in the course of infection.

IgM and IgG antibodies can both be present in a sample. This implies that the conversion from a primarily IgM to IgG humoral response is underway.

A sample can be positive if there are IgM, IgG, or both IgM and IgG antibodies present.

COVID-19 Detection Cassette Details

Fig 1.  COVID-19 Detection Test Strip, labelled with major functional areas.

How does the COVID-19 Rapid POC CE-IVD Test work?

Overview

The COVID-19 Rapid POC CE-IVD test is a lateral flow immunoassay. A lateral flow immunoassay is a way to qualitatively assess the presence of an analyte from a patient sample or specimen. In this case, the analytes being detected are IgG and IgM antibodies specific for SARS-CoV-2.  

The IgG/IgM test cassette is a qualitative membrane-based immunoassay for the detection of IgG and IgM antibodies to 2019-nCoV in whole blood, serum or plasma specimens.

The principle of this test is similar to that of a hCG pregnancy test, which also uses a rapid chromatographic immunoassay for qualitative detection of a human glycoprotein.

Components

Included:

  • Test cassette: the functional unit of the test which holds the immunoassay strip inside.
  • Droppers: used to transfer the samples to the specimen well.
  • Package insert: contains the full technical manual with instructions and further specifications.
  • PBS buffer solution: added to the specimen well to facilitate the test.

Other:

  • Specimen collection containers: used to hold samples after collection.
  • A centrifuge (for plasma only) and lancets (for fingerstick whole blood only)
  • Capillary tube: necessary if a fingerstick whole blood specimen is to be used.
  • Pipettes: can be used in place of droppers.  
  • Timer: to track the 10 minute wait time.

COVID-19 Rapid POC CE-IVD Test: Lateral Flow Detection Steps

Fig 2. A schematic of the COVID-19 lateral flow test.  

Steps:

  1. The whole blood, serum, or plasma clinical specimen is added to the well. Then, the dilution buffer (10 mM phosphate-buffered saline) is added.

2. The combined sample flows down to the sample pad.

3. Capillary action/lateral flow will move the sample across the test.

4. The sample will hit the conjugation pad. The conjugation pad contains the COVID-19 antigen conjugated to 40 nm gold nanoparticle (AuNP) colloid. During this stage, any antibodies in the sample with specificity for COVID-19 will bind the antigen and its conjugated gold nanoparticle. (The kit control, rabbit IgG, is conjugated to the same gold nanoparticle and travels with the rest of the sample from this step.)

5. Next, the sample/conjugate complex moves to the nitrocellulose membrane. Here, it comes in contact with the three test lines: IgG, IgM and control.

6. First is 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 line.

7. 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.

8. The control line is the last line the sample will encounter. The control line contains an immobilised antibody that recognises Rabbit IgG, the control antibody. To serve as a procedural control, a coloured line should always appear in the control line region, indicating that the proper volume of specimen has been added and membrane wicking has occurred.

9. Finally, any excess will flow through to the absorption pad.

10. After 10 minutes, the results of the test can be read.

COVID-19 Rapid POC CE-IVD Test Principle

This COVID-19 Rapid Point of Contact CE-IVD test consists of two components, an IgG and an IgM. In the IgG component, anti-human IgG coats the G test line region. In the IgM component, anti-human IgM coats the M test line region.

During testing, the specimen reacts with SARS-CoV-2 antigen-coated gold nanoparticles (AuNP) in the conjugation pad of the test cassette. Any antibody in the patient sample that recognises the MK201027 SARS-CoV-2 antigen binds to the Antigen-AuNP complex. The mixture then migrates laterally across the membrane by capillary action/lateral flow.

As these human antibody/antigen/AuNP complexes move across the test lines, they are captured at the anti-human IgM 'M' Line, the anti-human IgG 'G' Line, or both, depending on the antibody contents of the specimen.

The sample first reaches the anti-human IgM antibodies which coat the M line. If the specimen contains IgM antibodies to SARS-CoV-2, a coloured line will appear in the M test line region.

Next, the sample reaches the anti-human IgG antibodies which coat the G line. If a specimen contains IgG antibodies to SARS-CoV-2, the conjugate-specimen complex reacts with anti-human IgG. A coloured line appears in the G test line region as a result.

Only human antibody/SARS-CoV-2 Antigen/AuNP complexes will produce a visible red or pink line at the M or G Line. Other antibodies produce no colour.

The rabbit IgG-AuNP complexes are captured by the control line (which contains anti-rabbit-IgG). This visible line indicates that there has been successful lateral flow across the detection strip. It is last to ensure that the sample had sufficient volume to move across the entirety of the test cassette.

Excess antigen-AuNP complexes will not be captured by the M or G lines. If no anti-MK201027 antibodies are present in a patient sample, no Ag-AuNP complex will be captured at the M or G Lines, and thus no coloured line will appear.

To summarise, if the specimen contains SARS-CoV-2 IgG antibodies, a coloured line will appear in IgG test line region. If the specimen contains SARS-CoV-2 IgM antibodies, a coloured line will appear in IgM test line region. If the specimen does not contain SARS-CoV-2 antibodies, no coloured line will appear in either of the test line regions, indicating a negative result. In all cases, a coloured line should appear at the control, C line.

Is the COVID-19 Rapid POC CE-IVD Test accurate?

Accuracy is derived from two key metrics: sensitivity and specificity.

Sensitivity is defined as the proportion of true positives which are correctly identified by the test (eg. a person has IgG antibodies in their sample, and the test detects those antibodies). It is also sometimes called the probability of detection.

Specificity is defined as the proportion of true negatives that are correctly identified by a test.

The data for IgG and IgM specificity and sensitivity is below.

IgG SARS-CoV-2 sensitivity and specificity results.

Fig 3. IgG Relative Sensitivity: 100% (95%CI*: 86.0%-100%). Relative Specificity: 98.0% (95%CI*: 89.4%-99.9%). Accuracy: 98.6% (95%CI*: 92.3%-99.96%)         *Confidence Interval

IgM SARS-CoV-2 sensitivity and specificity results.

Fig 4. IgM Relative Sensitivity: 85.0% (95%CI*: 62.1%-96.8%). Relative Specificity: 96.0% (95%CI*: 86.3%-99.5%). Accuracy: 92.9% (95%CI*: 84.1%-97.6%)

Control

A control measurement is in place to ensure the test is functioning correctly. It confirms sufficient specimen volume and correct procedural technique. Any test that is missing the control line (‘C’) is invalid and should be performed again. If possible, it is always good laboratory practice to run a positive and negative control to verify proper test performance prior to diagnostic testing.  

Rapid Antibody & Antigen tests available from Assay Genie

SKU Product Name Size

INCP-AC0502

20 Test cassettes

ICIC-ACO525

20 Test cassettes


SKU Product Name Antigen Supplier

5513C

Nucleocapsid

Cellex Inc

CG2057

Nucleocapsid and spike protein

Getein Inc

SARS-CoV-2 antibodies

SKU Product Name Conjugation Application

AGEL2109

Unconjugated

ELISA

AGEL2110

Unconjugated

WB,ELISA

AGEL2111

Unconjugated

WB,ELISA

AGEL2112

Unconjugated

IF/ELISA

AGEL2113

Unconjugated

WB,ELISA

AGEL2114

Unconjugated

ELISA

AGEL2115

Unconjugated

ELISA

AGEL2116

Unconjugated

WB,ELISA

AGEL2117

Unconjugated

WB,ELISA

AGEL2118

Unconjugated

IHC,ELISA

AGEL2119

Unconjugated

WB

PACOV0001

Unconjugated

ELISA, GICA

PACOV0002

Unconjugated

ELISA, GICA, Neutralising

PACOV0003

Unconjugated

ELISA, GICA, Neutralising

PACOV0004

HRP

ELISA  

PACOV0005

FITC

ELISA

PACOV0006

Biotin

ELISA

PACOV0007

Unconjugate

ELISA, GICA

PACOV0008

Unconjugated

ELISA, WB, GICS

PACOV0009

HRP

ELISA

PACOV00010

FITC

ELISA

PACOV00011

Biotin

ELISA

SARS-CoV-2 recombinant proteins

SKU Product Name Conjugation Region Species

CARP01258

His Tag

Arg319-Phe541

SARS-CoV-2

CARP01259

His Tag

Val11-Arg682

SARS-CoV-2

CARP01260

His Tag

Val11-Gln1208

SARS-CoV-2

CARP01261

His Tag

Gln14-Arg683

SARS-CoV-2

CARP01262

His Tag

Val16-Arg685

SARS-CoV-2

CARP01263

His Tag

Met1-Val75

SARS-CoV-2

CARP01264

His Tag

Met1-Ala419

SARS-CoV-2

RPES0001

His Tag

Ser1-Gln306

SARS-CoV-2

RPES0002

His Tag

Ala1-Gln601

SARS-CoV-2

RPES0003

His Tag

Met1-Gly180

SARS-CoV-2

RPES0004

His Tag

Ala1-Gly638

SARS-CoV-2

RPES0005

His Tag

Ser1-Gln83

SARS-CoV-2

RPES0006

His Tag

Ala1-Gln198

SARS-CoV-2

RPES0007

His Tag

Ala1-Gln139

SARS-CoV-2

RPES0008

Glu1564-Lys1878

SARS-CoV-2

RPES0009

His Tag

Ala1-Gln527

SARS-CoV-2

RPES0010

mFc Tag

Asn331-Val524

SARS-CoV-2

RPES0011

His Tag

Asn331-Val524

SARS-CoV-2

RPES0012

mFc Tag

Arg319-Pro589

SARS-CoV-2

RPES0013

His Tag

Arg319-Ser591

SARS-CoV-2

RPES0014

Avi-His Tag

Arg319-Ser591

SARS-CoV-2

RPES0015

mFc Tag

SARS-CoV-2

RPES0016

Fc Tag

SARS-CoV-2

RPES0017

SARS-CoV-2

RPES0018

His Tag

SARS-CoV-2

RPES0019

His Tag

SARS-CoV-2

RPES0020

His Tag

SARS-CoV-2

RPES0021

His Tag

Met1-Ala419

SARS-CoV-2

RPES0022

His Tag

SARS-Cov-2

RPES0023

SARS-Cov-2

RPES0024

His Tag

Met1-Val75

SARS-Cov-2

RPES0025

His Tag

SARS-Cov-2

RPES0026

Fc Tag

SARS-Cov-2

RPES0027

His Tag

Gln18-Ser740

SARS-Cov-2

RPES0028

Fc Tag

Gln18-Ser740

SARS-Cov-2

RPES0029

Avi-His Tag

Gln18-Ser740

SARS-Cov-2

RPES0030

mFc Tag

Gln18-Ser740

SARS-Cov-2

RPES0031

His Tag

Ala22-His205

SARS-Cov-2

RPES0032

His Tag

Ala22-Arg325

SARS-Cov-2

RPES0033

His Tag

Arg18-Ile339

SARS-Cov-2

RPES0034

His Tag

Met 1-Phe 339

SARS-Cov-2

RPES0035

His Tag

Thr18-Val333

SARS-Cov-2

RPES0036

His Tag

Thr18-Asn334

SARS-Cov-2

RPES0037

His Tag

Lys69­Lys967

SARS-Cov-2

RPES0038

His Tag

Lys69­Ser966

SARS-Cov-2

RPES0039

His Tag

Met1-Ala419

SARS-Cov-2

RPES0040

His Tag

Val16-Pro1213

SARS-Cov-2

RPES0041

His Tag

Arg319-Phe541

SARS-Cov-2

RPES0042

mFc Tag

Arg319-Phe541

SARS-Cov-2

RPES0043

His Tag

Arg319-Phe541

SARS-Cov-2

RPES0044

Fc Tag

Val16-Arg685

SARS-Cov-2

RPES0045

His Tag

Val16-Arg685

SARS-Cov-2

RPES0046

mFc Tag

Val16-Arg685

SARS-Cov-2

RPES0047

His Tag

Ser686-Pro1213

SARS-Cov-2

RPES0048

His Tag

Met1-Ala422

SARS-Cov-2

RPES0049

His Tag

Arg306-Phe527

SARS-Cov-2

RPES0050

His Tag

Met1-Ser740

SARS-Cov-2

RPES0051

His Tag

Glu1564-Val1880

SARS-Cov-2

6th Nov 2020 Paige Dougherty MSc.

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