key: cord-0978575-r4t8pdr4 authors: Yang, Xiaoqi; Fox, Alisa; Powell, Rebecca LR. title: QUALITATIVE IMMUNOASSAY FOR THE DETECTION OF ANTI-SARS-COV-2 SPIKE ANTIBODY IN HUMAN MILK SAMPLES date: 2022-02-14 journal: STAR Protoc DOI: 10.1016/j.xpro.2022.101203 sha: 2286573c97e2e7fac6a330e3091fa49a514b0b20 doc_id: 978575 cord_uid: r4t8pdr4 Antibodies in milk obtained from those previously SARS-CoV-2-infected or vaccinated against COVID-19 may provide passive immunity to the breastfed infant. Few assays have been established to measure antibodies in human milk, despite the public health importance of this topic. In the present protocol, we describe an optimized indirect ELISA assay aimed to measure SARS-CoV-2-reactive antibodies in human milk, which can be used as a rapid screen on undiluted samples or to designate samples as relatively low, moderate, or high titer. Lead contact *Xiaoqi.Yang@mssm.edu **Alisa.Fox@mssm.edu ***Rebecca.Powell@mssm.edu Summary Antibodies in milk obtained from those previously SARS-CoV-2-infected or vaccinated against COVID-19 may provide passive immunity to the breastfed infant. Few assays have been established to measure antibodies in human milk, despite the public health importance of this topic. In the present protocol, we describe an optimized indirect ELISA assay aimed to measure SARS-CoV-2-reactive antibodies in human milk, which can be used as a rapid screen on undiluted samples or to designate samples as relatively low, moderate, or high titer. For complete details on the use and execution of this protocol, please refer to (Fox et al., 2020) . Human milk samples used for this assay were collected from fully consented individuals under a protocol approved by the Institutional Review Board (IRB) at Mount Sinai Hospital (IRB 19-01243) . Use of this protocol may require specific institutional approval. 1. Produce or purchase SARS-CoV-2 Spike antigen. We recommend recombinant production following the published protocol (Stadlbauer et al., 2020) ; however, any commercially-available SARS-CoV-2 Spike antigen (including S1, Receptor Binding Domain (RBD)) can be used, as long as positive cutoffs are established using negative controls. Even other SARS-CoV-2 immunogens such as Nucleocapsid can be used. 2. Collect human milk at least 7 days after symptom onset or positive COVID-19 diagnostic test, or 14 days after the first dose of a COVID-19 vaccine (note these are minimal intervals based on (Pace et al., 2021) Step-by-step method details The experimental plates can be stored at -80C for up to a week. 1. Coat plates with 50 l of SARS-CoV-2 Spike protein at 0.5 ug/ml diluted in 1X PBS (pH 7.4). a. Cover plates using plate sealers. Day 2: ELISA Experiment J o u r n a l P r e -p r o o f For this step, an indirect ELISA is performed -the proteins reactive to SARS-CoV-2 Spike in human milk would bind to SARS-CoV-2 Spike coating, then the HRP-conjugated secondary antibody (Anti-IgG/IgA/IgM/SC) is added to target the human proteins, TMB and HCl (1N) is added for a colorimetric reaction that can be read on the plate reader. Timing: 6 h 2. Retrieve and thaw human milk samples from freezer. a. Fresh human milk samples can also be used in this protocol and all samples would be processed the same prior to storage as described in step 2 below. b. When choosing negative control samples, we recommend including at least 10 prepandemic (pre-Dec 2019) milk samples to determine cutoff values. If these samples are not available, samples obtained after Dec 2019 from participants with no history of confirmed or suspected infection or known exposure to close contacts, though in this case more control samples (20-30) may be needed in order to remove significant outliers from the control dataset as these may be due to unknown previous SARS-CoV-2 infection. i. Pre-pandemic control samples may be pooled to be used as a negative control in all experiments. ii. Similarly, positive samples can be pooled as a positive control to be used in each assay. iii. Screened controls should be included on every plate of each subsequent experiment to reduce plate-to-plate variation. 3. Centrifuge thawed samples in 50 ml conical tubes at 805 x g for 10 min at 20-25C. 4. Remove any remaining fat from the top of each sample. c. Remove fat by scooping with a sterile spatula or by pouring off fat layer. d. Samples can be frozen or used immediately. 5. Prepare 1X PBS/0.1% Tween-20 wash buffer. e. We recommend preparing at least 4 liters wash buffer in a bottle that can be connected to the Tecan HydroSpeed plate washer, or similar to uniformly wash all wells of each plate. 6. Prepare blocking buffer using 0.1% PBS-T/3% goat serum/0.5% milk powder. 7. Wash plates using the Tecan HydroSpeed plate washer using prepared wash buffer. f. Set up a program with 3 rounds of washing/aspiration filling to max capacity of your plate. g. Note, plate washing can be performed manually while ensuring proper aspiration of all liquid before moving forward. 8. Add 50 l blocking buffer to each well of your washed experimental plates. 9. Incubate experimental plates for 1 h at room temperature. 10. During the one hour incubation, set up 4-fold titrations in separate round-bottom polypropylene dilution plates with samples and any relevant controls (Figure 1 ) at set concentrations in 1% BSA diluted in 1X PBS, pH 7.4. h. Start titrations with undiluted processed milk to maximize sensitivity. i. Include pre-pandemic/naïve and positive controls in the assay. j. We recommend leaving a few wells without any samples and a few wells without any secondary antibody to ensure reagents are performing as expected. 11. After incubating for 1 hr in blocking buffer, wash plates as described in step 6. 12. Transfer 50 l of samples prepared in dilution plates to washed experimental plates. 13. Incubate covered experimental plates containing relevant samples for 2 h at room temperature. 14. Prepare goat anti-human secondary antibodies in 1X PBS, pH 7.4 k. We recommend using the following ratios of antibody to 1X PBS if using the ones specified above: i. Goat anti-human In this step, the data is analyzed using GraphPad Prism, and the calculation of endpoint titer is determined. b. Using COVID-naïve control milk samples, ODs at the lowest titer tested (we recommend undiluted milk for this calculation, determine the mean and standard deviation values (can be done easily in excel). i. Positive cutoff should be set as two standard deviations above this mean, based on our published work (Fox et al., 2020) . 28. In the XY graph, double-click the Y axis, and insert a dotted line at the positive cutoff value. 29. If determination of endpoint titer is desired: c. On the XY table in the first empty row after the final OD value, add your endpoint OD value. We recommend using an OD of 1.0 based on our published work (Fox et al., 2020) . i. This represents the dilution of the highest analyte that provides a reading above the cutoff chosen at an absorbance best fit for these data. d. Using the 'Analysis' button, transform the XY table data as X=LogX. e. Using the 'Analysis' button on this transformed XY table, select non-linear regression and then 4-parameter (sigmoidal; X is log(concentration). i. Select 'interpolate unknowns from standard curve' at bottom of options window. f. In the nonlinear fit results sheet, select the 'Interpolated X mean values' tab. g. Using the 'Analysis' button, transform data as X=10 x to obtain final endpoint titer values. Limitations ELISA can be subject to changes in OD values due to temperature fluctuations, and it is best to perform this assay in a temperature-controlled environment. Positive and negative pooled control samples previously-aliquoted and stored as well as uncoated plate wells can serve as tools to ensure assay-to-assay consistency. It is important to develop all plates at the same temperature and to be stopped after the same amount of time each experiment for accurate comparison. This ELISA cannot determine if antibodies are neutralizing or capable of additional Fc-mediated function. Typically this is due to use of excess secondary antibody or accidental coating of excessive Spike protein. Improper washing can also lead to this issue, or omission of the blocking step. Be sure to stop the TMB reaction using 1N HCl in a timely manner to avoid over-developing plates. Problem 4: Plate well color and OD values exhibit high background (ELISA Experiment step 23/Data Analysis). This can be resolved by ensuring proper washing procedure to remove any residual buffer remaining in wells by forcefully tapping to absorb. Store experiment plates in dark areas away from light exposure during incubations and limit light exposure throughout. Adhere to recommended incubation times to prevent high background. Stadlbauer, D., Amanat, F., Chromikova, V., Jiang, K., Strohmeier, S., Arunkumar, G.A., Tan, J., Bhavsar, D., Capuano, C., Kirkpatrick, E., et al. (2020) . SARS-CoV-2 Seroconversion in Humans: A Detailed Protocol for a Serological Assay, Antigen Production, and Test Setup. Curr Protoc Microbiol 57, e100. 10.1002/cpmc.100. Robust and specific secretory IgA against SARS-CoV-2 detected in human milk Characterization of SARS-CoV-2 RNA, Antibodies, and Neutralizing Capacity in Milk Produced by Women with We are indebted to our milk donors. Spike protein was gifted by the Krammer Lab. This work was supported by the NIH/NIAID (R01 AI158214) and the Icahn School of Medicine at Mount Sinai. Alisa Fox performed all experiments described in the paper, assisted with data analysis, and drafted this manuscript. Xiaoqi Yang assisted with drafting this manuscript and creating figures. Rebecca Powell conceived of the protocol and its optimization, oversaw data collection, performed data analysis and interpretation, and revised this manuscript. Mount Sinai has filed for patent protection for this assay. After long-term storage (>6 months) at -20C, we have found that particularly the IgG in milk is not as reactive compared to when the milk was initially stored, thus yielding lower OD values. One solution is to keep milk at -80C. Another solution is to replace controls every 3 months with a fresh pool of samples. Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Rebecca Powell (Rebecca.Powell@mssm.edu). No novel materials were generated for this study. No code was generated for this study. Sample data from this protocol can be shared upon email request.