Procedures were examined for labeling immune globulins with radioactive iodine using chloramine-T as the oxidizing agent. The chloramine-T method was critically evaluated to establish the optimal conditions for preparing iodinated globulins with high specific radioactivities without impairing their immunospecificities for use in in vitro radioimmunoassays. The results showed that the use of 100 ,g of chloramine-T per ml, 500 to 1,000 ACi of Na 12"I per mg of protein, and a 10-min oxidation reaction time produced globulins of both high specific radioactivities and immunospecificities. Criteria were established for evaluating and determining optimal concentrations of iodine-labeled globulin for use in radioimmunoassays. The results of this investigation indicated that the amount of labeled indicator globulin used in radioimmunoassays should be based upon protein concentration rather than radioactivity.
A radioimmunoassay procedure was developed for determining smallpox and vaccinia antibodies in human sera. The test detected and measured both primary and secondary immune responses in persons infected with variola virus or vaccinia virus. The antibody titers obtained by complement fixation, hemagglutination inhibition, plaque reduction neutralization, and radioimmunoassay methods were compared. In sequential serum specimens, the radioimmunoassay test indicated fourfold or greater increases in all of the smallpox patients and in six of eight vaccinated persons. Both the complement fixation and the hemagglutination inhibition tests were less effective. In persons who had been vaccinated, radioimmunoassay and plaque reduction neutralization tests appeared to measure the same immune response. However, in smallpox patients the immune response was readily detected by radioimmunoassay, whereas an immune response was not detected by the plaque reduction neutralization test when vaccinia virus was the antigen in the test system. Radioimmunoassay is an operationally simple procedure which provides objective and quantitative end-point titers in serological determinations.
In the liver tissue of newborn mice, xanthine oxidase activity is very low during the first 7 to 14 days of life. Infection of mice with several different viruses prematurely induced xanthine oxidase activity 2- to 10-fold in the liver tissue. Generally, overt signs of illness appeared after xanthine oxidase induction; however, some viruses induced the enzyme activity without causing morbidity or deaths. The elevated enzyme activity could not be correlated with alteration of either lactate dehydrogenase or glutamate-pyruvate transaminase. Likewise, there were no histological changes in the livers of infected animals when xanthine oxidase levels were abnormally elevated. These observations suggest that measurement of xanthine oxidase may be an effective method for the detection of subclinical or inapparent viral infections in either naturally infected newborn mice or in newborn mice inoculated with suspected virus-containing materials.
A rapid microradioimmunoassay (RIA) technique was adapted for quantitatively measuring antibody titers to antigens occurring in Epstein-Barr virus (EBV)-infected lymphoid cells. In these experiments two EBV-infected cell lines, HR1K and EB-3, were used as antigen-positive cells and Molt-4 was used as the negative control cells. The antibody titers of sera from suspected infectious mononucleosis patients were compared by RIA and indirect fluorescent antibody (IFA) methods. As determined by each of the methods, 14 of 19 sera had positive antibody titers and the remainder of the sera had negative antibody titers. Thus, the two methods agreed completely in differentiating sera with antibodies to EBV antigens. To further evaluate the antibody specificity of the RIA, the antibody titers of paired sera, pre- or early infection and postinfection, from five confirmed infectious mononucleosis patients were determined by RIA and IFA. Seroconversion was demonstrated by both RIA and IFA for each of the patients. Thus, the sensitivity and specificity of the two procedures are about the same.
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