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SARS-CoV-2 IgM ELISA Kit

Instructions for use.  For in vitro diagnostic use. For Research Use Only.  Not For Use in Clinical or Diagnostic Procedures.

Cat. No. E88-302 • Lot No. 200707

Intended Use

The Bethyl SARS-CoV-2 IgM ELISA is an Enzyme-Linked Immunosorbent Assay (ELISA) intended for semiquantitative detection of IgM antibodies to SARS-CoV-2 in human serum or plasma collected in Potassium EDTA, Sodium Citrate or Lithium Heparin.  The Bethyl SARS-CoV-2 IgM ELISA is intended for use as an aid in identifying individuals with an adaptive immune response to SARS-CoV-2, indicating recent or prior infection. Understanding the timing, duration and effectiveness of humoral immune responses in individuals previously infected with SARS-CoV-2 will be important for conducting vaccine and epidemiological research.  At this time, it is unknown for how long antibodies persist following infection and if the presence of antibodies confers protective immunity.

Results are for the detection of SARS-CoV-2 antibodies.  IgM antibodies to SARS-CoV-2 are generally detectable in blood several days after initial infection, although the duration of time antibodies are present post-infection is not well characterized. Individuals may have detectable virus present for several weeks following seroconversion.

The sensitivity of Bethyl SARS-CoV-2 IgM ELISA early after infection is unknown. Negative results do not preclude acute SARS-CoV-2 infection.

False positive results for Bethyl SARS-CoV-2 IgM ELISA may occur due to cross-reactivity from pre-existing antibodies to SARS-CoV-1 or other possible causes.

 

Background

Coronavirus Disease 2019 (COVID-19) is a systemic disease caused by the novel Coronavirus designated SARS-CoV-2.  The initial disease outbreak was first reported in Wuhan, Hubei province, China in December 2019.  Within months the World Health Organization declared a global pandemic.  COVID-19 can be a serious and life-threatening disease.  “Understanding the number of individuals who are exposed and whether they then develop immunity is absolutely key to managing the epidemic.”  (Ragon Institute of MGH, MIT and Harvard, n.d.)

 

Principle of the Assay

This kit provides for an indirect ELISA, in which a recombinant receptor binding domain (RBD) of the Spike1 protein of SARS-CoV-2 is coated on the wells of the microtiter plate.   Antibodies to SARS-CoV-2 RBD when present in the test sample bind specifically to the RBD protein. After sample binding, unbound proteins and molecules are washed off, and a HRP-conjugated detection antibody is added to the wells to bind to the captured anti-SARS-CoV-2 IgM antibodies.  The chromogenic substrate TMB (3,3’,5,5’-tetramethylbenzidine) is then added. This reaction produces a blue product, which turns yellow when the reaction is terminated by addition of dilute sulfuric acid. The absorbance of the yellow product at 450 nm, corrected for plate imperfections by subtracting the absorbance at 570 nm, is proportional to the amount of RBD-specific anti-SARS-CoV-2 IgM present in the sample. After determining that the values for the Positive Control and Negative Control are valid and acceptable by comparing them to the value for the Calibrator, values for samples are compared to the Calibrator to generate a ratio. Ratios above a cutoff indicate positive samples and values below a cutoff indicate negative samples.

 

Reagents:  Storage and Expiration

This kit should be refrigerated upon receipt and stored at 2-8oC.  Do not freeze. Expiration date is 6 months post receipt of the kit.

 

Materials Supplied

  1. 96 well Precoated SARS-CoV-2 S1-RBD plate (E88-S1-RBD-PC), 1 plate
  2. Positive Control: Recombinant Human anti-RBD IgM (ready to use) (E-PC88-202). 1 vial -0.3 ml
  3. Calibrator: Recombinant Human anti-RBD IgM antibody (ready to use) (E-RC88-202), 1 vial - 0.3 ml
  4. Negative Control: Dilute normal human serum (ready to use) (E-NC88-202), 1 vial - 0.3 ml
  5. Detection Antibody: Goat Anti-Human IgM-Fc antibody – HRP conjugated (ready to use) (E88-202P), 1 vial - 11 ml
  6. Assay Dilution Buffer (ready to use) (E-DBM60), 1 vial – 60 ml
  7. TNT Wash Buffer packet for reconstitution (E118) 1 packet.
  8. One Component TMB substrate: TMB/Hydrogen Peroxide (ready to use) (E-TMB12), 1 vial – 12 ml
  9. Stop solution (0.518 M Sulfuric Acid; ready to use) (E-SS12) 1 vial - 12 ml
  10. Sealing Tape:, 3 Sheets
  11. SDS
  12. Instructions for use
 

Additional Materials Required

 

Safety Precautions

Universal precautions using appropriate personal protective equipment should be observed when handling all reagents.  Dispose of all reagents and samples appropriately.  Avoid eye and skin contact with reagents and samples.  Stop solution contains dilute sulfuric acid. In case of eye or skin contact, rinse area with copious amounts of water.  Remove and wash any contaminated clothing.  Do not ingest. Individual components may contain preservatives.

 

Additional Precautions

 

Procedure Overview

  1. Allow all kit components to equilibrate to room temperature.
  2. Prepare TNT Wash Buffer.
  3. Prepare 1:100 patient serum or plasma sample dilutions by diluting 5 µl of sample into 495 µl Dilution Buffer. Upon adding sample to dilution buffer, a noticeable flocculence is common and expected.
  4. Once at room temperature, remove the pre-coated microtiter plate from its foil pouch.
  5. Add 100 µl each of Positive Control, Negative Control, Calibrator and diluted samples to designated wells on the pre-coated plate. Note: It is suggested to run each control or sample in duplicate.
  6. Cover the plate with sealing tape and incubate at room temperature (20-25oC) for 30 minutes.
  7. Wash the plate 5 times with TNT Wash Buffer. Wash volume 250 µl per well, each wash.
  8. Add 100 µl of anti-human IgM-Fc Detection Antibody (E88-202P) to each well.
  9. Cover the plate with fresh sealing tape and incubate at room temperature for 30 minutes.
  10. Wash the plate 5 times with TNT Wash Buffer. Wash volume 250 µl per well, each wash.
  11. Add 100 µl of TMB Substrate Solution to each well.
  12. Incubate the plate in the dark at room temperature for 15 minutes.
  13. Stop the reaction by adding 100 µl of Stop Solution to each well.
  14. Measure absorbance on a plate reader at 450 nm and at 570 nm.
 

Handling and Preparation of Reagents, Standards, and Samples

Sample Handling   Sample Preparation   Preparation of TNT Wash Buffer  

Assay Procedure

Sample Incubation
  1. Add 100 ml of controls and diluted samples to the appropriate wells. Run each control, and the samples in duplicate if appropriate.
  2. Carefully cover the wells with a new sealing tape and incubate for 30 minutes at room temperature, 20-25°
  3. Carefully remove the adhesive plate cover and wash 5 times with TNT Wash Buffer, as described in the Plate Washing section below.
  Plate Washing
  1. Rinse the tips of the plate washer by dispensing the TNT Wash Buffer into the wash trough and aspirating the solution. Repeat this step 5 times. For automated plate washers, program the rinse step accordingly. Note: This initial rinse step is necessary especially if the plate washer has been idle for several days or longer. Automated plate washers are susceptible to microbial growth in the fluid lines and cavities.
  1. Aspirate the solutions from the wells. Fill the wells to about 90% (250 µl) full with TNT Wash Buffer and then aspirate the wash solution. Repeat this wash step 4 more times. For automated plate washers, program 5 washes at 300 µl per wash, according to the manufacturer’s instructions.
  Incubation with Detection Antibody
  1. Add 100 µl of Detection Antibody to each well. Mix by gently tapping the plate several times.
  2. Carefully attach a new adhesive plate cover and incubate the plate for 30 minutes at room temperature, 20-25°
  3. Carefully remove the adhesive plate cover and wash 5 times with TNT Wash Buffer (see Plate Washing section above).
    Incubation with TMB Substrate and Stopping the Reaction
  1. Add 100 µl of TMB Substrate Solution into each well and incubate at room temperature (20-25ºC) in the dark for 15 minutes. Do NOT cover plate with a plate sealer.
  2. Stop the reaction by adding 100 µl of Stop Solution to each well. Tap plate gently to mix. The solution in the wells should immediately change color from blue to yellow.
 

Absorbance Measurement

Note: Wipe the underside of the wells with a lint-free tissue.

Measure the absorbance on an ELISA plate reader set at 450 nm and 570 nm within 30 minutes of stopping the reaction.

  Calculations:
  1. For each well, derive the corrected A450 value by subtracting the value measured at 570 nm from the value measured at 450 nm.
  2. For calibrator, each control and each sample
    1. Calculate the mean of the corrected A450 values.
    2. Calculate the standard deviation (StdDev) of the corrected A450 values.
    3. Calculate the Percent Coefficient of Variation (%CV) of the corrected A450 values: %CV = StdDev ÷ mean × 100
  3. Derive the ratio of the Positive Control to the Calibrator by dividing the mean of the corrected A450 values of the Positive Control by the mean of the corrected A450 values of the Calibrator.
  4. Derive the ratio of the Negative Control to the Calibrator by dividing the mean of the corrected A450 values of the Negative Control by the mean of the corrected A450 values of the Calibrator.
  5. For each sample, calculate the ratio for the sample by dividing the mean of the corrected A450 values of the sample by the mean of the corrected A450 values of the Calibrator.
  Validity and Acceptability:
    1. The following conditions must be met for the assay to be valid and acceptable
      1. %CV for Positive control is less than 15.
      2. %CV for Calibrator is less than 15.
      3. The ratio of the Positive Control is between 3 and 8.
      4. The ratio of the Negative Control is less than 0.5.
If any condition is not met, repeat the assay.
    1. The following condition must be met for values for an individual sample to be valid and acceptable:
      1. %CV for the sample is less than 15. If the condition is not met, discard the values and repeat the assay of the sample.
  Interpretation of Results:
          • Samples for which the ratio is 1.0 or greater are positive.
          • Samples for which the ratio is 0.9 or less are negative.
          • Samples for which the ratio is between 0.9 and 1.0 are equivocal requiring repeating of the test. If the results of the subsequent assessment are also equivocal, no determination can be made.
  Limitations of the Assay
  1. This test is for the semiquantitative assessment of seroconversion. Higher absorbance values correlate with higher relative concentration of specific antibody.  This assay detects antibody specific to the RBD protein only and does not reflect total antibody response to other SARS-CoV-2 associated proteins.
  2. Microbial contamination of samples or reagents, cross contamination of samples and/or kit reagents or extreme temperature excursions may yield erroneous results.
  3. Previous infection with SARS-CoV-1 may result in a positive test result.
 

Performance Characteristics

Analytical Sensitivity, Specificity and Cross Reactivity

Samples procured from a commercial source from 34 distinct patients that had tested positive by molecular test (Roche Swab or Abbot RealTime) and tested as reactive by the Siemens ADVIA Centaur Sars-CoV-2 Total assay performed by an independent clinical laboratory were evaluated with the Bethyl SARS-CoV-2 IgM IVD ELISA.

Bethyl SARS-CoV-2 IgM ELISA Results
Days from Symptom Onset Number of Samples Tested IgM Positive results IgM PPA 95% CI
0-7 days 0 0 n/a n/a
8-14 days 0 0 n/a n/a
≥15 days 34 26 76.5% 62.2% - 90.7%

Serum samples collected prior to December 2019 from 161 distinct donors, from two commercial sources were evaluated. One hundred fifty-eight samples (98.1%) were negative and three (1.9%) were not negative for IgM to SARS-CoV-2.

Bethyl SARS-CoV-2 IgM ELISA Results
Number of Samples Tested IgM Negative results IgM NPA (95% CI)
161 158 98.1% (96.0% - 100.2%)
 

Class Specificity

Human serum samples previously identified as either Negative or Positive in the SARS-CoV-2 IgM ELISA were incubated with either diluent alone (Untreated), diluent + goat polyclonal anti-human IgM (Bethyl A80-100) to deplete IgM, or diluent + goat polyclonal anti-human IgG (Bethyl A80-104) to deplete IgG, and then RBD-specific IgM binding was measured in the SARS-CoV-2 IgM assay.  All samples were also evaluated in an assay for anti-SARS-CoV-2 IgG binding (Bethyl, E88-201).

    % Untreated Ratio (95% CI)
IgM Ratio Range IgG Ratio Range IgM-Depleted IgG-Depleted
Neg (n=20) 0.15 - 0.80 0.18 - 5.58 14.8 (11.9 - 17.7) 86.7 (83.5 - 89.9)
Pos (n=11) 1.10 - 13.04 2.12 - 11.78 2.4 (2.0 - 2.7) 94.9 (92.4 - 97.4)
 
IgM-Depleted IgG-Depleted
IgM Ratio Range IgG Ratio Range Neg. Pos Neg. Pos
Neg (n=20) 0.15 - 0.80 0.18 - 5.58 20 0 20 0
Pos (n=11) 1.10 - 13.04 2.12 - 11.78 11 0 0 11

The results demonstrate that depletion of IgM (>97% reduction in IgM assay ratio) from Positive samples converts them to Negatives and that depletion of IgG (>99% reduction in IgG assay ratio, data not shown) from Positive samples has no effect on their disposition in the assay.  Similarly, the Negative samples saw no change in assay disposition with IgG depletion.

These data show that signal in the SARS-CoV-2 IgM ELISA is due specifically to the IgM component of a patient’s immune response to SARS-CoV-2 without significant interference from SARS-CoV-2-specific IgG.

 

Matrix Equivalency

Serum, citrate plasma, EDTA plasma, and heparin plasma were each collected from five individuals testing negative in both Bethyl’s SARS-CoV-2 IgM ELISA and SARS-CoV-2 IgG ELISA.  Convalescent serum from an individual who tested positive in Bethyl’s SARS-CoV-2 IgM ELISA was spiked into a 1:100 dilution of each negative matrix at four different concentrations intended to yield negative, low positive (two different concentrations), and moderate positive IgM ELISA outcomes.  1:100 negative matrices were also tested without any spike.  Each combination of negative matrix and spike was tested in duplicate.

Deming regression (α = 0.05) was performed for the comparison of plasma to serum and showed the following results:

Citrate EDTA Heparin
n 25 25 25
Assay Ratio Range (Serum) 0.10 to 2.66 0.11 to 2.42 0.11 to 2.55
Assay Ratio Range (Plasma) 0.10 to 2.46 0.10 to 2.46 0.10 to 2.46
Regression Equation (y = plasma, x = serum) -0.035 + 1.050 x -0.01 + 0.982 x 0.015 + 1.004 x
95% CI of intercept -0.07 to 0.001 -0.067 to 0.048 -0.028 to 0.058
95% CI of slope 0.997 to 1.104 0.894 to 1.071 0.94 to 1.069
    Ho: slope = 1, H1: slope ≠ 1; p-value 0.064 0.685 0.889
Variance ratio X/Y (λ)  1.238 0.973 1.370

For each spike amount, there was 100 % agreement of assay outcome across all matrices, all individuals, and all replicates, thus demonstrating robust matrix equivalency.

No Spike Negative Spike Low Positive Spike #1 Low Positive Spike #2 Moderate Positive Spike
Ratio Range 0.11 to 0.40 0.23 to 0.49 1.04 to 1.36 1.33 to 1.65 1.96 to 2.31
Assay Outcome NPA PPA
Serum, n=5 100% 100% 100% 100% 100%
Citrate Plasma n=5 100% 100% 100% 100% 100%
EDTA Plasma n=5 100% 100% 100% 100% 100%
Heparin Plasma n=5 100% 100% 100% 100% 100%
 

References

  1. Ragon Institute of MGH, MIT and Harvard. n.d. "The Ragon Institute of MGH, MIT and Harvard Receives Gift from Nancy Zimmerman to Help Fund Development of Early Response COVID-19 Diagnostic." March 2020. Ragon Institute of MGH, MIT and Harvard.