Criteria for Preparing, Evaluating, and Standardizing Iodinated Globulins for Radioimmunoassay Procedures

Abstract

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.

Detection by radioimmunoassay of antibodies in human smallpox patients and vaccinees

“…Guinea pig anti-human globulin. Sodium sulfateprecipitated guinea pig anti-human globulin was iodinated by the chloramine-T procedure of Greenwood et al (6) as described previously (9). The only alteration in the procedures was an increase in the concentration of 125iodine (Na125I) to 1,000 ,Ci per mg of protein.…”

Radioimmunoassay for detection of antibodies to Epstein-Barr virus in human infectious mononucleosis serum specimens

“…The lot of I2'l-labeled guinea pig anti-human globulin used in this study was diluted to contain 0.65 Ag of protein per 0.025 ml in PBS containing 1% fetal calf serum. This dilution of '25I-labeled anti-human globulin was the optimal concentration for maximal differentiation of sera of different antibody potencies (10). Aliquots (0.025 ml) were added to each of the microtiter wells, and each plate was again covered and incubated at 37 C for 1 h. After incubation the excess labeled globulin was drained and the plates were washed five times with PBS and dehydrated with 95% ethanol.…”

Local antibody response in chickens: analysis of antibody synthesis to Newcastle disease virus by solid-phase radioimmunoassay and immunofluorescence with class-specific antibody for chicken immunoglobulins

“…Radioimmunoassay. The solid-phase radioimmunoassay described previously (7,(14)(15)(16)32) was modified for quantitation of class-specific anti-NDV immunoglobulins in chickens (technique to be reported elsewhere). Briefly, stocks of NDV antigen were prepared in cell culture.…”

“…Each section was covered with a drop of fluoresceinor rhodamineconjugated antibody to y, ,u, or a chains and incubated in a humidified chamber for 1 h at 37°C. Unbound antibody was rinsed off in a staining dish with three changes of PBS, 15 min each, and the slides were air dried. Specimens were mounted with a 10% Elvanol mounting medium and examined with a series 10 Microstar microscope (American Optical Corp., Buffalo, N.Y.) equipped with an incident-light fluorescent illuminator.…”