Detection of cytomegalovirus antibodies by an enzyme-linked immunosorbent assay using recombinant polypeptides of the large phosphorylated tegument protein pp150.

Parts of the large phosphorylated tegument protein, pp150, of human cytomegalovirus (HCMV) were expressed in bacteria. The resulting fusion proteins were tested in a Western blot (immunoblot) assay for reactivity with a monoclonal antibody against pp150, with a polyspecific rabbit antiserum, and with human reconvalescent-phase sera. Those fusion proteins that performed well in the Western blot assay were used as antigens in enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against HCMV. Five different recombinant beta-galactosidase fusion proteins were evaluated by ELISA using 62 seropositive and 38 seronegative human serum samples. Of all the proteins tested, one peptide representing 162 amino acids of pp150 was superior to the others with regard to sensitivity and specificity. All sera known to be positive for antibodies against HCMV were identified by combining the results of the ELISAs with the different pp150 fusion proteins. Therefore, it appears that peptides from a single protein of HCMV might be sufficient to identify HCMV-seropositive individuals by recombinant ELISA.

Correlation of herpes simplex virus antibody titers and specific lymphocyte stimulation in adult blood donors.

Antibody titers to herpes simplex virus type 1 in sera from healthy adult donors were assayed by complement fixation, microneutralization, and an enzyme immunoassay (ELISA). This last test proved to be the most sensitive method for antibody detection. It was estimated that ELISA antibody titers were up to 40-fold higher than neutralizing antibody titers and up to 100-fold higher than complement fixation antibody titers. Due to the higher sensitivity of ELISA, only 3 of 36 blood donors tested in this assay were shown to be seronegative, whereas 6 additional persons of the same group were termed seronegative by the microneutralization assay. Furthermore, four of the latter also did not respond in the complement fixation test. In vitro stimulation of peripheral lymphocytes by using a partially purified herpes simplex virus type 1 particle antigen was achieved for all seropositive blood donors. Only those three donors who were ELISA negative reacted negatively in this stimulation assay. From these results it may be concluded that ELISA is an appropriate method not only for rapid and sensitive antibody determination but also for selecting herpes simplex virus-negative patients.

The enzyme-linked immunosorbent assay (ELISA) for determination of IgG and IgM antibodies after infection with mumps virus.

Under routine laboratory conditions ELISA was tested for suitability for serological demonstration of specific antibodies of the immunoglobulin classes G and M against mumps virus. Sera from patients with known clinical and virological data were used. The results of ELISA were compared with those of CFT. 45 paired sera were tested in ELISA IgG, 87 first sera in ELISA IgM. Both tests were highly sensitive, antibodies were detected earlier and with higher titers than with the CFT. The ELISA IgM is particularly suitable for early diagnosis of mumps infection with the first serum. In addition 23 paired sera from patients with acute parainfluenza virus infection were examined for cross-reacting antibodies. Low anti-mumps IgG antibody titers were found in some sera. These findings reduced the mumps specificity of the IgG test. In five serum samples from one patient--obtained before, during, and after an infection with mumps--the course of IgG and IgM antibodies could be demonstrated. Advantages and limitations of ELISA IgG and IgM are summarized.

A rapid microassay for detecting antibodies against poliovirus based on [14C]thymidine uptake of treated cell cultures.

DNA synthesis of mammalian cells propagated in microplates can easily be measured if cell cultures incubated with [14C]thymidine are harvested on the glass fibre filters by a semiautomatic harvesting technique. Soon after infection with poliovirus, [14C]thymidine uptake of U cells (established, human amniotic cell line) is inhibited. This inhibition can be prevented by previous virus neutralization with antibody. Based on this effect a rapid, precise assay method was set up to determine neutralizing antibody titres against poliovirus. There was a good correlation between titres obtained by this assay and those obtained by 50% end point titrations in cytopathogenic effect inhibition assays.