We investigated the effect of multiple freeze-thaw cycles on mumps, measles, and rubella virus serum antibody levels with whole-virus immunoglobulin G enzyme-linked immunoassays. Fresh serum samples from nine healthy adult volunteers were divided into six sets of five aliquots each. Samples were taken through a total of 10 freeze-thaw cycles and stored at 4°C until assayed. Each assay measurement was done in replicates of five, and the mean value was reported. After completing 10 freeze-thaw cycles, we found no clinically or statistically significant effect on measured antibody levels and found no discernible detrimental effect on the ability to measure these antibodies by enzyme-linked immunoassays.Despite the widespread use of banked serum specimens that have undergone multiple freeze-thaw cycles, there is a paucity of data available regarding the effect of numerous freeze-thaw cycles on the measured antibody result. This information is particularly relevant for sensitive assays, such as enzyme-linked immunoassays (EIAs), which measure protein structures (antibodies) prone to denaturation. Because these biologic specimens may be used for multiple investigations over a period of time, concern may exist that repeated freeze-thaw cycles might affect the results of a particular assay by physically damaging the antibody of interest.The limited data available regarding the effect of multiple freeze-thaw cycles on stored serum samples have focused on serum chemistry determinations (1, 2) or measurement of apolipoproteins (3). Each of these investigations suggested a decline in the absolute value from baseline, but the decline was not deemed clinically significant. Petrakis (5) reports that antibodies are stable when stored at Ϫ70°C, but repeated freezethaw cycles significantly reduced detectable immunoglobulin G (IgG) and IgM activity. Unfortunately, details regarding the number of freeze-thaw cycles and the conditions of storage and testing of specimens in these studies were not specified. Because of this, we designed an experiment to investigate the effect of multiple freeze-thaw cycles on measles, mumps, and rubella virus (MMR) antibody measurements by utilizing whole-virus EIAs. MATERIALS AND METHODSAfter obtaining informed consent from nine adult volunteers (designated A to I), approximately 30 ml of whole blood was obtained by a standard venipuncture technique. Serum was separated by centrifugation of the sample and divided into six sets of five 0.5-ml aliquots each, for a total of 30 aliquots from each volunteer. One set of aliquots, designated as the baseline, was immediately stored at 4°C for the remainder of the study. The remaining five sets of aliquots were frozen at Ϫ80°C. Six hours after the initial freezing, the aliquot sets were removed from the freezer and allowed to stand at room temperature for approximately 2 h until completely thawed; they were then refrozen. After another 6-h freeze and 2-h thaw, one set of aliquots was stored at 4°C for the remainder of the study, and the remaining four sets we...

The ELISPOT assay is a highly sensitive technique used for the detection of individual cytokine releasing cells. We have developed an IFN-gamma ELISPOT assay utilizing unfractionated frozen peripheral blood mononuclear cells (PBMC) to quantify the frequency of measles virus (MV)-specific IFN-gamma-secreting T cells in 117 healthy children who had been previously immunized with two doses of the measles-mumps-rubella vaccine. We have also estimated the variability associated with the quantification of ELISPOT plates and compared the number of MV-specific IFN-gamma-secreting T cells for each subject as determined by two different operators of an ELISPOT reader. The median frequency of MV-specific IFN-gamma-producing memory T cells detected by this assay was 0.005 % and 0.01 % as determined by an in-house and commercial operator, respectively. Although we found a significant correlation (r = 0.83, p<0.0001) between the number of spots counted by the commercial and in-house operators of an ELISPOT reader, the median number of spots counted by the commercial operator was twice the number of spots counted by an in-house operator (p<0.001). This demonstrates the importance of using a common ELISPOT reader and operator, among other parameters, to quantify the number of spots when a large volume of plates are being scanned and analyzed.

High-throughput in vitro assays, which rapidly and succinctly assess the immune status of large cohorts of individuals, are essential tools for conducting population-based studies, including vaccine research. The enzyme-linked immunospot (ELISPOT) assay has emerged as a sensitive, reliable high-throughput tool to measure functional recall immunity by assessing the frequency of antigen-specific cytokine-secreting lymphocytes present in peripheral blood mononuclear cells (PBMCs). For the past 10 years, ELISPOT method has been the dominant platform and a standard for the cell-mediated immune (CMI) assays. ELISPOT assays are used extensively as a measure of CMI response to vaccines, including smallpox (vaccinia), following primary or secondary vaccination. Here, we present detailed methodology for using ELISPOT assays to detect the frequency of cytokine secreting vaccinia-specific lymphocytes including optimized protocols for growing, titrating, and inactivating vaccinia virus; isolating, cryopreserving, and thawing human PBMCs; and finally, detecting vaccinia-specific IL-10 and IFNγ secreting lymphocytes, as well as CD8(+) IFNγ T cells following in vitro stimulation of PBMCs with vaccinia virus. The methods presented below, although optimized for vaccinia virus, emphasize principles that can be generally applied to create ELISPOT assays capable of assessing the immune status as well as antiviral CD8(+) T cell response of individuals following primary or secondary vaccination with other licensed or novel vaccines.

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