Original Basic Science-General Background. Transplant recipients who develop COVID-19 may be at increased risk for morbidity and mortality. Determining the status of antibodies against SARS-CoV-2 in both candidates and recipients will be important to understand the epidemiology and clinical course of COVID-19 in this population. While there are multiple tests to detect antibodies to SARS-CoV-2, their performance is variable. Tests vary according to their platforms and the antigenic targets which make interpretation of the results challenging. Furthermore, for some assays, sensitivity and specificity are less than optimal. Additionally, currently available serological tests do not exclude the possibility that positive responses are due to cross reactive antibodies to community coronaviruses rather than SARS-CoV-2. Methods. This study describes the development and validation of a high-throughput multiplex antibody detection assay. Results. The multiplex assay has the capacity to identify, simultaneously, patient responses to 5 SARS-CoV-2 proteins, namely, the full spike protein, 3 individual domains of the spike protein (S1, S2, and receptor binding domain), and the nucleocapsid protein. The antibody response to the above proteins are SARS-CoV-2-specific, as antibodies against 4 common coronaviruses do not cross-react. Conclusions. This new assay provides a novel tool to interrogate the spectrum of immune responses to SAR-CoV-2 and is uniquely suitable for use in the transplant setting. Test configuration is essentially identical to the single antigen bead assays used in the majority of histocompatibility laboratories around the world and could easily be implemented into routine screening of transplant candidates and recipients.
Thaumatin is a plant protein that contains 8 disulfides and 207 amino acids in the mature form. The protein is of potential commercial interest since microgram quantities elicit an intense sweetness sensation. Two major variants of thaumatin have been identified in our laboratory by using sequence data obtained from thaumatin tryptic peptides. These differ by one amino acid at position 46 (asparagine or lysine), and both proteins differ from previously published sequences. We have synthesized DNA-coding sequences for three of these thaumatin variants using yeast preferred codons. The genes were inserted into an expression vector that contained a yeast 3-phosphoglycerate kinase promoter and terminator, and the vectors were transformed into yeast for expression of the recombinant protein. Upon lysis of the yeast cells, all thaumatin was localized in the insoluble cell fraction. Analysis of the sodium dodecyl sulfate solubilized yeast extracts by gel electrophoresis and Western blotting showed that thaumatin represented about 20% of the insoluble yeast protein. Although expressed at high levels, none of the thaumatins was biologically active (sweet). Preliminary protein folding experiments showed that two of three thaumatin variants could be folded to the sweet conformation.
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