Evaluation and reporting of enzyme immunoassay determinations of antibody to herpes simplex virus in sera and cerebrospinal fluid

Competition between proteins and other macromolecules for adsorption sites on plastic was studied with the enzyme-linked immunosorbent assay (ELISA) to determine effects of the use of antigenic mixtures or extracts of organisms on assays of antibodies and antigens by ELISA. A comparison of a number of different polystyrene microplates with bovine albumin and human immunoglobulin G (IgG) as antigens showed two major classes of plates: those which adsorbed albumin poorly and those which adsorbed albumin well. IgG adsorbed well on all plates, but plates which adsorbed albumin best also gave significant background levels of nonspecific binding of conjugate. When mixtures of IgG and bovine serum albumin were used as coating antigens, significant competition was observed; the component present at 1% or less in the mixture was essentially undetectable unless excessive amounts of conjugate were used. The important factor was the ratio of competitor to antigen, not the absolute amount. Other proteins (ovalbumin, rabbit albumin, human albumin, and gelatin) were equally effective competitors for adsorption sites on plastic. Nonionic detergents (Tween 20, and Triton X-100) were strong competitors even at 10:1 competitor-to-antigen ratios. In antigen capture assays, normal serum components blocked attachment of antigen-specific IgG, but this competition could be lessened to a degree by the use of strongly binding polystyrene plates. In indirect ELISA for measurement of serum antibody, the use of antigenic mixtures gave significantly lower antibody titers when the desired antigen was less than 1% of the total protein coated. Therefore, the use of mixed or crude antigens in ELISA presents significant problems concerning the sensitivity and specificity of tests. The enzyme-linked immunosorbent assay (ELISA) (9) is a major tool for detecting antigens and antibodies in a wide variety of diseases (20, 21). Although the method was initially devised for measurement of antibodies to individual proteins, a large number of assays for individual disease agents have been devised using partially purified or even unpurified antigens. Such assays are not uniformly successful, and when they are it is not possible to define the reacting antigen(s). If we were to wait until suitably purified antigens were available, a number of currently useful assays would not be available for serodiagnosis. It has been recognized that components of antigenic mixtures (i.e., an extract of a microorganism) likely compete with each other for the limited sites on plastic surfaces (4, 5, 13, 16). Thus, studies to determine the nature of competition are necessary to define conditions under which ELISAs can best be performed with partially purified or unpurified antigens. Proteins apparently attach to plastic surfaces by hydrophobic interactions (12, 19), and the

Section: Polysaccharidesmentioning

confidence: 99%

Principles, problems, and strategies in the use of antigenic mixtures for the enzyme-linked immunosorbent assay

Kenny¹,

Dunsmoor²

1983

“…The standard curve method has also been applied to estimation of viral antibodies by ELISA using influenza A, cytomegalovirus, mumps virus, respiratory syncytial virus (14), herpes simplex virus (6), and the hexon antigen from adenoviruses (16) as antigens. The assay has been validated for each of these viruses and has been shown to be more sensitive than endpoint titration in detecting significant changes in antibody concentration (6). The major disadvantage of the standard curve method with the ELISA is a disadvantage that is shared by all assays based on ELISA, i.e., the fact that the technique is influenced by avidity of antibody (4,5) and underestimates antibody of low avidity present in early specimens of convalescent-phase serum (21 to 40 days postinfection).…”

Section: Discussionmentioning

confidence: 99%

“…successfully to the assay of IgG antibodies against influenza A virus, respiratory syncitial virus, cytomegalovirus, mumps virus, adenovirus, and herpes simplex virus (6,14,16). Test serum samples are assayed at a single dilution, and results are converted to units by reference to a standard serum titrated during each assay.…”

mentioning

confidence: 99%

Estimation of rotavirus immunoglobulin G antibodies in human serum samples by enzyme-linked immunosorbent assay: expression of results as units derived from a standard curve

Bishop¹,

Cipriani²,

Lund³

et al. 1984

A method for estimating rotavirus immunoglobulin G (IgG) antibodies by assay of human serum samples at a single serum dilution was studied. Antibody was measured by enzyme-linked immunosorbent assay (ELISA). The optical density of the reaction with a 1:100 dilution of each serum was expressed as ELISA units of antirotavirus IgG by reference to a standard curve. This standard curve was obtained by incorporation in each assay of five dilutions of a serum containing an aribitrary number of units of antirotavirus IgG. Test serum samples found to contain high amounts of antirotavirus IgG were reassayed at a 1:1,000 dilution. There was good correlation between antirotavirus IgG ELISA units in 45 serum samples and endpoint titers of the same samples (Spearman rank correlation coefficient rs, 0.95). Seroconversion during rotavirus infection was defined as an increase in antirotavirus IgG ELISA units per milliliter of greater than 28% (2 x intra-run coefficient of variation of the assay) in consecutive serum samples from the same child. Paired serum samples from nine children with diarrhea not due to rotavirus infection showed no seroconversions. Paired samples from eight children with rotavirus infection showed seroconversions. Estimation of antirotavirus IgG ELISA units in serum is simple, rapid, reproducible, and economical of serum samples. Standardization of results could be achieved by worldwide distribution of a standard serum. Its use would facilitate epidemiological surveys to evaluate potential rotavirus vaccines.

“…CMV was isolated from the urine of patient 1, and HSV was isolated from the throat of patient 9 ( Table 2). Slides of skin lesions were positive for HSV in patient 8 and negative in patient 12. Tissues for virus isolation or identification from the other patients were not submitted.…”

Section: Methodsmentioning

confidence: 98%

Anomalous antibody responses in viral infection: specific stimulation or polyclonal activation?

Cremer¹,

Devlin²,

Riggs³

et al. 1984

Self Cite

of current infection showed anomalous antibody results by complement fixation test when tested with a battery of agents (viruses, Mycoplasma pneumoniae, and chlamydia) selected for testing on the basis of the symptoms of the patient. Seventeen serum pairs showed a fourfold or greater rise in titer of antibody to two agents in the battery, and one showed only a twofold rise in titer of antibody to the identified causative agent but an eightfold rise in titer of antibody to a heterologous agent. The 18 serum pairs were tested for IgM antibody to the two involved agents to determine whether IgM antibody tests would better distinguish the probable cause of the current infection. The serum pairs were separated into three groups based on their IgM responses. Group I consisted of six serum pairs with IgM antibody to both agents, four pairs of which showed a fourfold or greater rise in titer of IgM antibody to both agents, and two of which showed a rise in titer of IgM antibody to only one of the two agents. Group II consisted of 10 serum pairs with IgM antibody to one of the two agents, 7 pairs of which showed a fourfold or greater rise in titer of IgM antibody to the agent. Group III consisted of two serum pairs with no IgM antibody to either agent. Results show that determination of presence or absence of IgM antibody per se or demonstration of a fourfold or greater rise in specific IgM antibody titer does not always help in distinguishing the causative agent in current infections.