Comparison of enzyme-linked immunosorbent assay (ELISA) technique and complement-fixation test for estimation of cytomegalovirus IgG antibody.

Abstract

SUMMARY The ELISA technique has been found to be reliable for the detection and titration of cytomegalovirus-specific IgG antibody in serum. It is about six times more sensitive than the CF test although some discrepancies were found between the antibody titres determined by the two methods. (Dienstag et al., 1976), and radioimmunoassay (Forghani et al., 1976;Knez et al., 1976), have also been used and found to be satisfactory but have not been generally adopted.The latest technique is the enzyme-linked immunosorbent assay (ELISA) (Engvall and Perlmann, 1972;, which has been shown to be more sensitive than passive haemagglutination for detecting IgG antibody (Castellano et al., 1977) and comparable in sensitivity to immunofluorescence for detecting IgM antibody (Schmitz et al., 1977). The present report compares the ELISA technique with the CF test in examining for CMV-specific IgG antibody in sera from patients.Material and methods PREPARATION OF ANTIGENSConfluent monolayers of MRC-5 diploid human fibroblasts (Jacobs et al., 1970) were grown in Roux bottles. They were infected with CMV strain AD 169, at a multiplicity of 0-1 plaque-forming units per cell, and then incubated in Eagle's minimal Received for publication 19 July 1978 essential medium with 10% fetal calf serum at 37'C. After 7-10 days, when gross cytopathic effects had developed, the cells were scraped off the bottles into a small volume of phosphate-buffered saline (PBS), pH 7-2, washed three times in PBS, and resuspended in a volume of 0-1 M glycine-NaClNaOH buffer, pH 10, equivalent to a final concentration of 1 ml per culture bottle of cells. The concentrated cells were immediately disrupted by ultrasonication for 5 s and the cellular debris was removed by centrifugation at 800 g for 10 min. The final supernatant was stored, in 0 25 ml quantities, at -70'C. Control antigen was prepared in the same way from uninfected MRC-5 cells. These antigens were used for both the CF and ELISA tests. COMPLEMENT-FIXATION TESTThis was carried out by the microtitre technique (Sever, 1962) with 2 units of antigen and 3 MHD5o of complement. Antibody titres were read as the reciprocal of the highest dilution of serum giving 50% (2+) fixation. ELISA PROCEDUREThis was done by the method of . Flat-bottomed wells in polystyrene microtitre plates (Cooke Microtitre M29AR, Dynatech Ltd) were coated with viral or control antigen by the addition of 0 3 ml of the antigen, diluted to optimal concentration in 01 M carbonate-bicarbonate buffer, pH 9-6, and incubated overnight. The plates were then washed three times in PBS containing 0 05% Tween 20 and 0-02 % NaN3 (PBST) and shaken dry.

A Murine Model of Herpes Simplex Virus Recrudescence

“…Protein content was estimated by Lowry's method. The ELISA was based on that described by Booth et al (1979) with HSV or control antigen being used to coat immunoplates (Nunc) with 15 gg/ml protein at 4 °C overnight. Sera were pooled from at least three mice (five normally).…”

Lack of Association of Cytomegalovirus Antibody Level with Carcinoma of the Uterine Cervix

“…The serological surveys mentioned above were performed using a com plement fixation test and newer methods such as enzyme-linked immuno sorbent assays (ELISA) and anti-complement immunofluorescence (ACIF) known to be more sensitive [Booth et al, 1979], as well as by measuring different classes of antibody. Thus we decided to take a group of women with cervical disorders ranging from mild, moderate and severe dysplasia to carcinoma in situ and to measure their circulating antibody titre to CMV, using three methods, namely ELISA, ACIF and neutralisation.…”

Detection of Specific IgA Antibodies in Serum of Kidney Transplant Patients with Recurrent Cytomegalovirus Infection

“…The enzyme-linked immunosorbent assay (ELISA), a simple technique of high sensitivity [10], was recently adapted for determination of CMV-specific IgG, IgM and IgA anti bodies [11][12][13][14][15]. This technique was found to be a reliable means for rapid serological diag nosis of primary CMV infections by detection of specific IgM antibodies [13,15], In the pre sent study, the CMV-specific IgA antibody response was evaluated by the ELISA tech nique in 59 sera from 10 kidney transplant patients who experienced recurrent CMV in fection.…”