DOI: 10.5061/dryad.n5tb2rc4t
title: SARS-CoV-2 spike protein ELISA calculations and summary data

description: Soon after commencement of the SARS-CoV-2 disease outbreak of 2019
 (COVID-19), it became evident that the receptor-binding domain of the
 viral spike protein is the target of neutralizing antibodies that comprise
 a critical element of protective immunity to the virus.  This
 study addresses the relative lack of information regarding actual antibody
 concentrations and binding affinities in convalescent plasma (CP) samples
 from COVID-19 patients and extends these analyses to post-vaccination (PV)
 samples to estimate protective IgG antibody (Ab) levels.  A
 direct enzyme-linked immunosorbent assay (ELISA) was used to measure IgG
 anti-spike protein (SP) antibodies (Abs) relative to human chimeric spike
 S1 Ab standards.  Microplate wells were coated with recombinant
 SP.  Affinities of Ab binding to SP were determined by previously
 described methods.  Binding affinities were also determined in an
 RBD-specific sandwich ELISA.  Two indices of protective immunity
 were determined as permutations of Ab molar concentration divided by
 affinity as dissociation constant (KD). The range and geometric means of
 Ab concentrations in 21 CP and 21 PV samples were similar and a protective
 Ab level of 7.5 µg/ml was determined for the latter population, based on
 95% of the normal distribution of the PV population.  A
 population (n = 21) of plasma samples from individuals receiving only one
 vaccination with the BNT162b2 or mRNA-1273 vaccines (PtV) exhibited a
 geometric mean Ab concentration significantly (p &lt; 0.03) lower than
 the PV population.  The results of this study have implications
 for future vaccine development, projection of protective efficacy
 duration, and understanding of the immune response to SARS-CoV-2
 infection.
description: Convalescent plasma (CP) samples were obtained from the
 University of Texas Health Science Center at Houston (UTHealth)/Memorial
 Hermann COVID-19 Convalescent Plasma Program under the direction of Dr.
 Henry E. Wang.  Convalescent plasma (CP) donors previously tested positive
 for COVID-19, were symptom-free for &gt;14 days, tested negative for
 COVID-19 antigen prior to donation, and tested positive for CP antibodies.
  Donor plasma was collected by the Gulf Coast Regional Blood Center before
 August 2020, which created a general stock of CP units that were
 distributed among therapeutic CP programs in the greater Houston area.
 Each plasma sample derived from a single donor.
 Post-vaccination (PV) plasma samples were obtained from Dr. Luis
 Ostrosky’s laboratory at the Division of Infectious Diseases, Department
 of Internal Medicine, McGovern School of Medicine of UTHealth.  Two groups
 of 21 samples, each collected 15-29 days after administration of the first
 or second dose of the Pfizer or Moderna COVID-19 vaccine between January 4
 and February 5, 2021, were studied.  Analysis of de-identified
 convalescent and post-vaccination plasma samples was approved by the
 UTHealth Committee for Protection of Human Subjects.
description: # SARS-CoV-2 Spike Protein ELISA Calculations and Summary Data
 [https://doi.org/10.5061/dryad.n5tb2rc4t](https://doi.org/10.5061/dryad.n5tb2rc4t) ## Description of the data and file structure Data are protective IgG antibody levels against the SARS-CoV-2 spike protein (S1 + S2), determined by quantitative ELISA relative to a chimeric human standard.  Replicate values are the average of several re-assays of the same samples.  Antibody levels were also measured vs. the spike protein receptor binding domain (RBD).  These were corrected for high background levels in that assay.  SP antibody binding affinities were determined as described in the paper and the preprint.  These values were determined for COVID-19 convalescent plasma samples, samples from fully vaccinated individuals, and samples from partially vaccinated (one of two Moderna or Pfizer vaccinations). ### Files and variables #### File: UTTP34\_ELISA\_Affinity\_Calc\_2.xlsx **Description:** Calculated affinity values for standards and plasma samples. ##### Variables * Plot type - SigmaPlot hyperbolic regression (SPHR); Kd determined as b, the x-value at 1/2 maximal OD.  Reciprocal plot linear regression (RPLR); Kd determined as the regression slope of 1/[Ab] vs. Amax/A (ratio of maximal to individual ELISA OD). * Kd corrected for equilibrium perturbation as defined in Klegerman et al: Biochim Biophys Acta **1768**:1703-16, 2007. * Kassoc - reciprocal of Kd * ACE2-Fc - Affinity of spike protein binding to the ACE-2 receptor. * BADAS - Samples from the Texas A&M BADAS trial * IgG Smpls - Convalescent plasma samples * % x-rx - Measure of antibody reactivity with the RBD. * All blank columns and cells are no data/null #### File: UTTP34\_Conv\_Plasma\_Results.xlsx **Description:** Antibody concentration and affinity summaries. ##### Variables * S1 + S2 - Full spike protein specificity * RBD specificity corrected for high background * PrA - RBD assay using Protein A immobilization of capture antibody * LSA - data from Carterra-determined titers * RBD EC50 - Another measure of ACE-2 cross-reactivity * Indexes of antibody concentrations divided by binding affinities * Specific Index - indexes multiplied by x-rx. * Post Vax series includes protective levels calculated from log data. * Replicate Post Vax is precision data for one sample. * QC data is interassay variance for one sample and antibody values for negative plasma. * All blank columns and cells are no data/null #### File: Klegerman\_sample\_data.xlsx **Description:**  Collection information for vaccine recipients ##### Variables * Deidentified sample ID * Date of first vaccination * Date of second vaccination * Number of vaccinations * Type of vaccine administered * Date of sample collection * Time between vaccinations * All blank columns and cells are no data/null #### File: ELISA\_Development.xlsx **Description:**  Raw data of Spike Protein developmental steps  ##### Variables * Optical density (OD) at 405 nm wavelength in multiwell plate wells * \[ACE-2] - Concentration of ACE2-human Fc added to each well * \[L] - Concentration of human anti-spike protein antibody standard added to each well * Amax -  Maximum OD, from Sigmaplot-generated hyperbolic equation of dose-response curve * A - OD of corresponding well * F - Concentration of ACE2-Fc or antibody in incubation fluid (free) * B - Concentration of total ACE2-Fc or antibody minus free concentration = bound concentration * B/F - Ratio of bound to free * \[Ab] - Antibody concentration * Smpl. - sample * Dil. - dilution * undil. - undiluted value * Ave. - average value * % Rec. - percent recovered * Net OD - OD of antibody value minus ACE2-Fc alone value * % x-rx - Percent of antibody binding to the receptor-binding domain (RBD) * All blank columns and cells are no data/null #### File: Full\_Vax\_Series.xlsx **Description:**  Raw data of fully vaccinated plasma sample antibody measurements  ##### Variables * Optical density (OD) at 405 nm wavelength in multiwell plate wells * Net OD - OD of sample well minus OD of background well * \[L] - Concentration of human anti-spike protein antibody standard added to each well * Amax -  Maximum OD, from Sigmaplot-generated hyperbolic equation of dose-response curve * A - OD of corresponding well * Cor - Antibody concentration corrected for systematic background interference in the RBD ELISA * % Ab Only - Contribution of added ACE-2 * \[Ab] - Antibody concentration * Smpl. - sample * Dil. - dilution * undil. - undiluted value * Ave. - average value * % Rec. - percent recovered * Net OD - OD of antibody value minus ACE2-Fc alone value * % x-rx - Percent of antibody binding to the receptor-binding domain (RBD) * r - correlation coefficient of EC50 linear regression * b[1] - slope of EC50 linear regression * b[0] - y-intercept of EC50 linear regression * nM - nanomolar * EC50 - Measure of antibody neutralizing capacity * All blank columns and cells are no data/null ## Code/software Microsoft Excel ## Access information Other publicly accessible locations of the data: * MedRxiv, 2021; [https://www.medrxiv.org/content/10.1101/2021.11.19.21266547v1](https://www.medrxiv.org/content/10.1101/2021.11.19.21266547v1) Data was derived from the following sources: * MS Excel, SigmaPlot