Sep 08, 2020
SARS-CoV-2 Milk ELISA
- Powell RL1
- 1Icahn School of Medicine at Mount Sinai
- XPRIZE Rapid Covid Testing
- Powell Lab/Lactiga Collab
Protocol Citation: Powell RL 2020. SARS-CoV-2 Milk ELISA. protocols.io https://dx.doi.org/10.17504/protocols.io.bk2skyee
Manuscript citation:
https://www.medrxiv.org/content/10.1101/2020.05.04.20089995v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 08, 2020
Last Modified: September 08, 2020
Protocol Integer ID: 41778
Keywords: SARS-CoV-2, ELISA, milk, IgA, sIgA,
Abstract
Currently, the COVID-19 diagnostics sector includes Point-of-Care and ELISA tests for IgG and IgM that can assess saliva or blood as described by the CDC [Centers for Disease Control and Prevention]. Similarly, the FDA webpage for EUA Authorized Serology Test Performance makes no reference to IgA COVID-19 assays [Food and Drug Administration]. Taken together, there are no direct competitors in the mucosal IgA quantitative diagnostics space.
To address this gap in diagnostic coverage we have generated a novel proof-of-concept dataset on the use of COVID-specific IgA antibodies as a marker of infection. As described below, our assay dataset demonstrates utility across several parameters:
-Highly specific
-Highly sensitive
-Works with undiluted human milk, simplifying sample collection and processing
-Defines a novel correlation between COVID-specific IgA and Secretory Component (SC), where SC is a known surrogate for dimeric IgA, the predominant IgA type in mucosal secretions
-Establishes that dimeric IgA antibodies detected by the assay are capable of neutralizing SARS-CoV-2
Guidelines
keep milk at room temperature for no more than 5 hours, or else keep at 4C for up to 5 days or freeze.
Materials
MATERIALS
Sulfuric acidFisher ScientificCatalog #A300C-212
Corning® 96-Well High-Binding Flat-Bottom Microplates 6 Plates/Pack
Stemcell TechnologiesCatalog #38019
Gibco™ (Phosphate Buffered Saline) Solution, pH 7.4 (PBS)Fisher ScientificCatalog # 10010-049
Lab Vision™ Tween™ 20 DetergentThermo Fisher ScientificCatalog #cat# TA-125-TW
BSAFitzgeraldCatalog #30-AB79
Skim Milk Powder
1-Step™ Ultra TMB-ELISA Substrate SolutionThermo FisherCatalog #34028
Normal Goat SerumThermo FisherCatalog #PCN5000
Falcon 50mL Conical Centrifuge TubesFisher ScientificCatalog #14-432-22
MilliporeSigma™ Goat anti-Human IgA alpha-Chain Specific Peroxidase Secondary AntibodyMillipore SigmaCatalog #40-113-52ML
Spike proteinGenscriptCatalog #Z03501
STEP MATERIALS
Tween 20Sigma AldrichCatalog #P7949
PBS pH 7.4
Normal Goat SerumThermo FisherCatalog #PCN5000
Skim Milk Powder
Tween 20Sigma AldrichCatalog #P7949
BSASigma Aldrich
MilliporeSigma™ Goat anti-Human IgA alpha-Chain Specific Peroxidase Secondary AntibodyMillipore SigmaCatalog #40-113-52ML
BSASigma Aldrich
1-Step™ Ultra TMB-ELISA Substrate SolutionThermo FisherCatalog #34028
Sulfuric acidSigma – AldrichCatalog #258105
| Reagent or Consumable | Supplier | Catalog # | |
| Text | Text | Numeric | |
| Thermo Scientific 50mL Conical Sterile Polypropylene Centrifuge Tubes | Fisher | 12-565-270 | |
| Corning™ 96-Well, High Binding, Flat-Bottom, Half-Area Microplate | Fisher | 07-200-37 | |
| Gibco™ PBS, pH 7.4 | Fisher | 10-010-023 | |
| Tween™ 20, Fisher BioReagents | Fisher | BP337-100 | |
| MilliporeSigma™ Calbiochem™BSA, 30% Sterile-Filtered Aqueous Solution, Preservative-Free | Fisher | 12-662-650ML | |
| Gibco™ Normal Goat Serum | Fisher | PCN5000 | |
| MilliporeSigma™ Calbiochem™ OmniPur™ Nonfat Powdered Milk | Fisher | 62-501-0GM | |
| Pierce 1-Step Ultra TMB-ELISA Substrate Solution | Fisher | PI34028 | |
| Sulfuric Acid, Fisher Chemical | Fisher | A510-P500 | |
| MilliporeSigma™ Goat anti-Human IgA, alpha-Chain Specific, Peroxidase, Secondary Antibody | Fisher | 40-113-52ML | |
| Spike protein (1mg = 1000000ng) | Genscript | Z03501 |
Protocol materials
Skim Milk Powder
Tween 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P7949
BSAMerck MilliporeSigma (Sigma-Aldrich)
BSAMerck MilliporeSigma (Sigma-Aldrich)
1-Step™ Ultra TMB-ELISA Substrate SolutionThermo FisherCatalog #34028
Sulfuric acidFisher ScientificCatalog #A300C-212
Spike proteinGenscriptCatalog #Z03501
MilliporeSigma™ Goat anti-Human IgA alpha-Chain Specific Peroxidase Secondary AntibodyMerck MilliporeSigma (Sigma-Aldrich)Catalog #40-113-52ML
Normal Goat SerumThermo FisherCatalog #PCN5000
Falcon 50mL Conical Centrifuge TubesFisher ScientificCatalog #14-432-22
Corning® 96-Well High-Binding Flat-Bottom Microplates 6 Plates/Pack
STEMCELL Technologies Inc.Catalog #38019
Tween 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P7949
Normal Goat SerumThermo FisherCatalog #PCN5000
Skim Milk Powder
1-Step™ Ultra TMB-ELISA Substrate SolutionThermo FisherCatalog #34028
PBS pH 7.4
Gibco™ (Phosphate Buffered Saline) Solution, pH 7.4 (PBS)Fisher ScientificCatalog # 10010-049
Sulfuric acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #258105
Lab Vision™ Tween™ 20 DetergentThermo Fisher ScientificCatalog #cat# TA-125-TW
BSAFitzgeraldCatalog #30-AB79
MilliporeSigma™ Goat anti-Human IgA alpha-Chain Specific Peroxidase Secondary AntibodyMerck MilliporeSigma (Sigma-Aldrich)Catalog #40-113-52ML
Spike proteinGenscriptCatalog #Z03501
Tween 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P7949
PBS pH 7.4
Skim Milk Powder
Tween 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P7949
Normal Goat SerumThermo FisherCatalog #PCN5000
BSAMerck MilliporeSigma (Sigma-Aldrich)
MilliporeSigma™ Goat anti-Human IgA alpha-Chain Specific Peroxidase Secondary AntibodyMerck MilliporeSigma (Sigma-Aldrich)Catalog #40-113-52ML
BSAMerck MilliporeSigma (Sigma-Aldrich)
1-Step™ Ultra TMB-ELISA Substrate SolutionThermo FisherCatalog #34028
Sulfuric acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #258105
Safety warnings
Handle biological samples in a biological containment hood
Milk Processing
Milk Processing
1h
1h
00:15:00
800 x g, 22°C, 00:15:00
Pour fresh milk into 50mL conical tube and centrifuge at 800g for 15min at room temperature
Equipment
new equipment
NAME
Centrifuge
BRAND
-
SKU
remove fat using a sterile spatula
carefully pour skim milk into a clean 50mL tube
repeat centrifugation and fat removal 2x
Day 1
Day 1
5m
5m
Coat half-area 96-well plates with 50ng SARS-CoV-2 spike protein in 50ul PBS 0.001 mg/mL
50 µL
Spike proteinGenscriptCatalog #Z03501
over and store overnight at 4COvernight
4 °C
Day 2
Day 2
5h
5h
Wash plates with 100ul 0.1% Tween 20/PBS (PBS-T) 6x 100 µL
0.1 % volume
Equipment
405 TS Washer
NAME
plate washer
TYPE
Biotek
BRAND
405 TS
SKU
Tween 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P7949
PBS pH 7.4
Block plates with 100ul 3% goat serum/0.5% milk powder/3.5% PBS-T for 1h at room temperature100 µL
01:00:00
Room temperature
Normal Goat SerumThermo FisherCatalog #PCN5000
Skim Milk Powder
Tween 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P7949
Wash as above
Add 50ul milk undiluted or diluted with 1%BSA/PBS to each well in duplicate50 µL
1 % volume
BSAMerck MilliporeSigma (Sigma-Aldrich)
Incubate at room temperature for 2h02:00:00
Room temperature
Wash as above
Add 50ul 50 µL horseradish peroxidase (HRP)-conjugated secondary antibody: goat anti-human-IgA at 1/3000 in 1%BSA/PBS.
MilliporeSigma™ Goat anti-Human IgA alpha-Chain Specific Peroxidase Secondary AntibodyMerck MilliporeSigma (Sigma-Aldrich)Catalog #40-113-52ML
BSAMerck MilliporeSigma (Sigma-Aldrich)
Incubate at room temperature for 1h01:00:00 Room temperature
Wash as above
Add 50u Pierce 1-Step Ultra TMB-ELISA Substrate Solution
1-Step™ Ultra TMB-ELISA Substrate SolutionThermo FisherCatalog #34028
50 µL
Sulfuric acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #258105
followed by 50ul 2N hydrochloric acid (HCl)
Measure OD at 450nm on an ELISA plate reader
Equipment
800 TS Absorbance Reader
NAME
Plate reader
TYPE
BioTek
BRAND
800 TS
SKU