Experimental histoplasmosis in the mouse. Immunoglobulin class response, total immunoglobulin A levels, immune complex formation, and occurrence of antigen in serum and urine.

Biotin-avidin enzyme-linked immunosorbent assays (ELISA) were developed to study the appearance of free antibody of the IgM, G, and A classes of immune complexes (IC) containing those classes and of antigen in the serum and urine of mice with experimentally induced, disseminated histoplasmosis (histo) over a period ranging from 4 to 64 days after infection. Free IgM was detected 4 days after infection, remained at low levels, and disappeared on Day 64, whereas free IgG was first detected on Day 7 and rose to very high levels before declining on Day 64. Free IgA was detected only once, on Day 21. IC containing IgM were seen first on Day 4, remained at low levels, and became undetectable on Day 64. IC containing IgM were detected on Day 7, peaked on Day 14, and declined through Day 64. IC containing IgA wee seen at low levels on Days 7, 14, and 21. Estimation of total IgA levels, done by single radial immunodiffusion, showed a statistically significant decrease on Day 14, followed by a slightly significant increase on Days 21 and 30. Antigen was detected in as much as 80% of serum specimens and 100% of urine samples during the first 2 wk postinfection but rarely afterwards. We conclude that ELISA provides a highly sensitive way to study antibody, IC, and antigen in host body fluids during histo infection and that IgM and antigen detection can be very early indices of infection. Measurements of IC and total IgA seem to be of relatively less importance in detection of infection.

Early and late antigens of human cytomegalovirus: electroimmunodiffusion assay of numbers, relationships, and reactivities with donor sera.

Early antigens (EA) of human cytomegalovirus extracted from cytosine arabinoside-blocked cells infected with 0.01-20 infectious units (IU)/cell were assayed with human serum by electroimmunodiffusion (EID). The number of detectable EA types increased from one to eight as the IU/cell was raised from 0.01 to 10. There was no increase in the number of EA with further increases in IU/cell, with prolonged culture, or when detergent was included in the extraction buffer. At least five of the eight EA gave reactions of identity with late-time antigens (LTA) extracted from unblocked cells at late times postinfection. In studies on a panel of sera from donors who were excreting virus and donors who were not, EID was as sensitive as conventional techniques (complement fixation and indirect hemagglutination for LTA, indirect immunofluorescence for EA) in detection of both types of antibodies from excretors but less sensitive in not detecting low levels of the antibodies in some of the sera from nonexcretors. No consistent relationships were observed between donor virological status and the numbers or types of antibodies to EA and LTA.

Detection of antibodies to cytomegalovirus by an improved counterimmunoelectrophoretic procedure.

An improved counterimmunoelectrophoretic (CIE) procedure for detection of antibodies to human cytomegalovirus (CMV) is described. The procedure, which uses high ionic strength buffer and concentrated antigen, is as sensitive and specific as complement fixation (CF), indirect hemagglutination (IHA), and anti-complement immunofluorescence (ACIF) but requires concentration of some sera. In studies on sera from 118 normal blood donors, the percent correlation of CIE results with those by CF, IHA, and ACIF were 97.5, 98.3, and 98.3, respectively, and exceeded slightly the correlations among the latter three procedures. The appearance of precipitation immediately or early after a CIE run and of more than one line of precipitate was indicative of high titers by the other procedures, while single lines of late precipitation occurred with most low titer sera but also with many of those of high titer.

Antigens of human cytomegalovirus: electroimmunodiffusion assay and comparison among strains.

The antigens of strain AD169 of human cytomegalovirus (CMV) were extracted by various methods and at different times following the appearance of cytopathic effects (c.p.e.) in infected fibroblasts. Assay with a pooled human serum in electroimmunodiffusion (EID) revealed that the most reactive preparations were obtained by shell-freeze (SF) extraction on the fourth day after 4+c.p.e. As many as 20 antigens could be detected in the original gels, most of which were stable upon storage at 4 degrees C for up to 4 weeks; of these, about 14 can be reproducibly seen on photographs. EID runs on day 4 SF preparations from high-passage CMV strains C87 and Davis and low passage recent isolates VD14, 1694 and 1723 resolved, respectively, 15, 15, 13, 11 and 11 antigens in the original gels (11, 9, 11,8 and 9 are visible in photographs). Strains 1694 and 1723 shared fewer antigens with one another and with high passage strains than were shared among the latter, whereas VD14 had relatively large numbers of antigens common to both low and high passage strains. At least six antigens were common to all strains.

Studies by crossed electroimmunodiffusion on the individuality and sexual orgin of bloodstains.

Crossed electroimmunodiffusion (CEID) was evaluated as a means for individualizing human blood stains by studying variations within and among individuals in 22 serum antigens in ten subjects over a four-month period. The extent of variation within an individual was determined by making CEID runs on blood stains obtained on ten different occasions and measuring the precipitin peak heights produced by each of the 22 antigens. When the range in height of any particular peak was completely different in subject-subject comparisons, the peak was judged to be of value in individualization. By this criterion, each of the ten subjects could be distinguished from all others (65 subject-subject distinctions), but in most cases the distinction had to be based on difference in 5 or less of the 22 antigens. The antigens of value in distinguishing among males were largely different from those of value in distinguishing among females. Overall, the antigens of greatest value in individualization were 8, 9, 10, and 11. Only one of these (10, ceruloplasmin) could be identified as a particular serum proteins.

Human bloodstains: individualization by crossed electroimmunodiffusion.

Crossed electroimmunodiffusion was evaluated as a means for establishing the individuality ("fingerprinting") of human bloodstains. In ten separate examinations on stains from each of ten persons there was at least one peak with a unique range in height so that individualization was possible. The heights of certain peaks showed statistically significant female-male differences.

Immunochemical studies on burro antibodies to Histoplasma capsulatum.

Burro antiserum to Histoplasma capsulatum was studied from the viewpoint of precipitating antibody distribution among euglobulins, pseudoglobulins, and immunoglobulin classes. By immunodiffusion analysis it was determined that throughout immunization most of the antibody was euglobular, but there was an increase in pseudoglobular antibody as immunization progressed. By immunoelectrophoretic and immunodiffusion analyses of specifically purified antibody, and antibody non-specifically fractionated by column chromatography and ultracentrifugation, it was established that there was no demonstrable IgM antibody, that most of the antibody appeared to be IgG, and that a gamma1 immunoglobulin, probably IgA or T-globulin, may have been responsible for some of the activity. No major changes in the distribution of antibody among immunoglobulin classes seemed to occur during immunization.

Evaluation of fluorescein isothiocyanate-labeled whole antiserum in the immunofluorescent identification of microorganisms.

Portions of a whole antiserum to Histoplasma capsulatum were reacted with amounts of fluorescein isothiocyanate (FITC) that ranged from 50 to 400 mug/mg of protein. Portions of the globulin from the same antiserum were reacted with amounts of FITC that ranged from 12.5 to 50 mug of FITC per mg of protein. The globulin conjugates (postlabeled globulins), the whole serum conjugates, and the globulins from the whole serum conjugates (prelabeled globulins) were compared with respect to their fluorescein-protein (F:P) ratios and fluorescent-antibody (FA) activities. The whole serum sample treated with 50 mug of FITC per mg of protein was least reactive in FA tests, and its globulin had the lowest F:P. All other conjugates had globulins with F:P ratios that were considered to be adequate for high FA activity. It was found, however, that the prelabeled globulins were considerably less reactive than the postlabeled globulins or the whole serum conjugates. A larger amount of brightly staining reagent per milliliter of original serum could be obtained from labeled whole serum than from postlabeled globulin. Lissamine-rhodamine conjugated to bovine serum albumin (LRBSA) was evaluated as a counterstain to be used in conjunction with FITC-labeled whole antisera. The counterstain was effective in masking nonspecific FITC fluorescence in Formalin-fixed tissues and in culture smears of fungi. Masking was incomplete in culture smears of a bacterium and in blood smears containing a protozoan.