Detection of specific anti-leptospiral immunoglobulins M and G in human serum by solid-phase enzyme-linked immunosorbent assay.

The enzyme-linked immunosorbent assay (ELISA) was used to detect leptospire-specific immunoglobulin M (IgM) and IgG in the sera of patients infected with leptospiral serovars hardjo, pomona, or copenhageni. All patients produced specific IgM and IgG detectable by ELISA. In contrast, only a few patients produced IgG agglutinins whereas all produced IgM agglutinins. The specificity and sensitivity of the test suggest that the ELISA anti-IgM technique is a suitable method for detecting leptospiral antibodies in human sera for diagnostic and epidemiological purposes.     

Detection of rinderpest antigen by latex agglutination test

A slightly modified latex agglutination test was applied for detection of rinderpest antigen. The antigen was added to sensitized latex particles in the presence of hyperimmune antiserum to facilitate agglutination. Out of 129 samples tested by latex agglutination (LA), solid phase aggregation of coated erythrocytes (SPACE), reverse phase passive haemagglutination (RPHA) and counter immunoelectrophoresis (CIE) test, 86.0, 86.8, 84.4 and 79.8 per cent, respectively, were found positive.     

Detection of Entamoeba histolytica immunoglobulins G and M to plasma membrane antigen by enzyme-linked immunosorbent assay.

Sixty-one serum specimens from 22 patients with clinically diagnosed amoebic liver abscess (ALA), 10 hospitalized patients with a variety of diseases other than amoebiasis, 12 normal healthy controls, and 17 subjects from an amoebiasis-endemic area were assayed by enzyme-linked immunosorbent assay (ELISA). The plasma membrane fraction of axenic cultures of Entamoeba histolytica HK9 separated from other subcellular fractions by differential centrifugation was used as the antigen to detect specific immunoglobulin G (IgG) and IgM antibodies. Using a single serum dilution of 1/100 and optical densities at 492 nm of 0.200 and 0.250 as the cutoff values for the IgM and IgG ELISAs, their respective sensitivities in 22 ALA patients were 91% (20 of 22) and 95% (21 of 22). In 22 patients (10 hospitalized and 12 normal healthy controls), the specificities of the IgM and IgG ELISAs were 95% (21 of 22) and 91% (20 of 22), respectively. All five asymptomatic carriers of pathogenic E. histolytica were seropositive by the IgG ELISA and the amoebic gel diffusion test (AGDT). The AGDT was positive for three of six culture-negative controls, while the IgG ELISA was positive for all six. For six asymptomatic carriers of nonpathogenic zymodemes, the AGDT was positive for two, and the IgG ELISA was positive for three. There was an excellent correlation (r = 0.96) between the IgG ELISA and the AGDT. Only one of six culture-negative controls, none of the asymptomatic carriers of pathogenic E. histolytica, and one of six carriers of nonpathogenic E. histolytica were seropositive by the IgM ELISA, thus highlighting the specificity of the IgM ELISA in the diagnosis of ALA. It is believed that the use of plasma membrane fractions has improved the diagnostic potential of the IgM ELISA.     

Detection of specific immunoglobulin E in patients with toxoplasmosis.

An immunocapture assay was developed to detect Toxoplasma gondii-specific immunoglobulin E (IgE) in sera from adults with acute acquired infection or reactivation and from babies with congenital toxoplasmosis. The components of this assay were monoclonal antibody to human IgE, samples from patients, and T. gondii tachyzoites treated with Formalin. When T. gondii-specific IgE antibodies were present, visually detectable agglutination occurred. Sera, umbilical cord blood, fetal blood, cerebrospinal fluid, and amniotic fluid were tested by this method. Specific IgE antibodies were detected in sera from 25 (86%) of 29 adults who developed specific IgG antibody during pregnancy or had specific IgA and IgM antibodies. Specific IgE was present early during infection, at the time that IgM antibodies were present, and slightly preceding the presence of specific IgA antibodies. In 23 patients tested serially, IgE antibodies never persisted for longer than 4 months. No nonspecific anti-T. gondii IgE was detected in sera from uninfected individuals. Maternal IgE antibodies did not cross the placenta. In sera of patients with congenital toxoplasmosis, specific IgE antibodies were found at birth, during the first year of life, and during immunologic recrudescence following discontinuation of pyrimethamine-sulfonamide therapy. The IgE immunocapture assay is simple to perform. It is especially useful for determining when T. gondii was acquired by recently infected pregnant women.     

Value of specific immunoglobulin a detection by two immunocapture assays in the diagnosis of toxoplasmosis

The diagnosis ofToxoplasma gondii infection is currently based on immunological tests, but tests for IgG and IgM antibodies alone are often insufficient to assess the risk of active disease, especially during pregnancy and in immunodeficient subjects. The supplementary diagnostic value of testing for antitoxoplasmic IgA in cases of acute, chronic, congenital and reactivated toxoplasmosis, relative to classical immunological tests, was evaluated using two immunocapture tests, one based on tachyzoite agglutination and the other on an immunoenzymatic complex recognizing the membrane protein P30 ofToxoplasma gondii. A total of 4,541 sera from 395 uninfected subjects, 468 immunized subjects with chronic infection, 117 subjects with acute infection and 403 children, 103 of whom had congenital toxoplasmosis, was tested. Specific IgA tests were negative in the nonimmune population, but tests for this immunoglobulin subtype became positive very rapidly during primary infection, and IgA disappeared more rapidly than IgM. In the children infected in utero, specific IgA was detected more frequently than IgM. In contrast, in a population of HIV-seropositive subjects with clinical toxoplasmosis, tests for IgA were poorly sensitive. The two tests for specific IgA produced similar results, except in the early stages of primary infection, in which immunoenzymatic testing for anti-P30 IgA was less sensitive than the agglutination method.     

Detection of Epstein-Barr virus antigens and antibodies by peroxidase-labeled specific immunoglobulins.

Detection of the Epstein-Barr (EBV) antigens, early antigen (EA), viral capsid antigen (VCA), and nuclear antigen (EBNA) by the indirect immunoperoxidase technique was highly sensitive. Antibody titers to EBNA, EA, and VCA were determined in more than 25 sera of patients with Burkitt lymphoma (BL), nasopharyngeal carcinoma (NPC), or normal persons. A good correlation between the titers of these antigens was obtained by the immunoperoxidase and immunofluorescence methods. The indirect (anti-IgG) immunoperoxidase technique for the detection of EBNA is, in contrast to the indirect immunofluorescence method, highly sensitive. EBNA was associated with the chromosomes in cells arrested in the metaphase with colchicine.     

Latex agglutination reaction for the diagnosis of toxoplasmosis

Agglutination of latex-particles sensitized with toxoplasma-antigen is a simple, fast, inexpensive test for toxoplasmosis-diagnosis. This test has been compared with passive-hemagglutination (1091 sera) and immunofluorescence (1093 sera). From the results, this new test is showed to be closer to passive-hemagglutination than indirect immunofluorescence. After studying possible false results and biological qualities it is thought that this test has good qualities to render great services for the toxoplasmosis diagnosis, and specially for epidemiological investigations.     

Simple, speedy, sensitive, and specific serodiagnosis of pertussis by using a particle agglutination test.

We developed a particle agglutination test (KPA) with poly(gamma-methyl L-glutamate) as the solid particle for measurement of pertussis toxin (PT) antibody. In this study, KPA was assessed as a means of serodiagnosing pertussis, and the results were compared with those of indirect enzyme-linked immunosorbent assay (indirect ELISA) and the microagglutination test. First, four serum samples were collected from each of 21 healthy children: before and 4 weeks after receiving three primary doses of acellular pertussis vaccines and before and 4 weeks after receiving a booster dose. In all 21 vaccinees, a significant rise in PT antibody titers was observed by KPA after each vaccination, and among all 84 serum samples collected, an excellent correlation was demonstrated between the values obtained by indirect ELISA and those obtained by KPA (r = 0.92). Second, paired serum samples were collected at intervals of approximately 2 weeks from 51 patients with culture-confirmed pertussis. A significant increase in titer (fourfold or more) was observed in 39 (76%) patients by KPA, 34 (67%) patients by indirect ELISA, and 23 (45%) patients by the microagglutination test. In acute- and convalescent-phase sera collected from 20 nonpertussis patients, there were no changes in titers by KPA. The KPA procedure was as simple as that of the microagglutination test, and the reaction time was only 2 h (or overnight). In this study, KPA was demonstrated to be a simple, speedy, sensitive, and specific serodiagnostic method for pertussis.     

Duration of specific immunoglobulin a antibody following acute toxoplasmosis as determined by enzyme immunoassay and immunosorbent agglutination assay

Modifications of an enzyme immunoassay (EIA) and an immunosorbent agglutination assay (ISAGA) for measuringToxoplasma gondii-specific IgM antibody were made to enable the measurement ofToxoplasma gondii-specific IgA antibody. It was shown that specific IgA could be measured by both assays but that the ISAGA was slightly more sensitive. IgA appears about two weeks after IgM and persists for 6 to 7 months. However, the IgA response varies considerably both in degree and duration, and demonstration of IgM antibody is at present the most suitable routine test for the diagnosis of recentToxoplasma gondii infection.     

Detection of antibodies to human immunodeficiency virus by latex agglutination with recombinant antigen.

Recombinant human immunodeficiency virus (HIV) env antigen was attached to polystyrene particles, and these complexes were used to develop the first latex agglutination assay for antibodies to HIV. A total of 95 positive and 116 negative human serum samples were assayed for antibodies to HIV by latex agglutination, and results were compared with those of a commercial enzyme immunoassay. Latex agglutination was also compared with, and found to be completely concordant with, Western blot (immunoblot) analysis with virion antigens.     

Detection of immunoglobulins G and M to rubella virus by time-resolved immunofluorometry.

We describe new methods for the detection of immunoglobulin G (IgG) and IgM rubella-specific antibodies in serum. The IgG assay was based on a solid-phase rubella antigen immobilization approach, and the IgM assay was based on the IgM capture assay principle. Both assays used biotinylated antibodies (anti-human IgG and antirubella monoclonal antibody, respectively). The tracer system was based on streptavidin labeled with a fluorescent europium chelate. The final measurements were done by using time-resolved fluorescence. Both assays were thoroughly evaluated with clinical samples and compared successfully with established techniques. We anticipate that these assays are suitable for routine clinical use.     

Diagnosis of postnatally acquired rubella by use of three enzyme-linked immunosorbent assays for specific immunoglobulins G and M and single radial hemolysis for specific immunoglobulin G.

Three commercial indirect enzyme-linked immunosorbent assays (ELISAs) (RUBELISA, Enzygnost-Rubella, and Rubazyme) and a commercial single radial hemolysis (SRH) test (Rubazone) were evaluated for the diagnosis of acute rubella by testing 41 acute- (less than 7 days postonset) and convalescent-phase (8 to 82 days postonset) serum pairs from cases of rubella previously confirmed by significant change in the hemagglutination inhibition test titer. Specificity was tested by using 10 acute- and convalescent-phase serum samples from patients with rash not confirmed as rubella (control group). In testing for rubella-specific immunoglobulin G (IgG) antibody, Enzygnost-Rubella and Rubazyme confirmed infection in 40 and RUBELISA in 39 of the 41 proven rubella patients. For one patient all three ELISAs failed to show a significant titer rise. No false-positive diagnoses occurred in the control group, although a suspected infection was shown by Rubazyme in one patient. No specific IgM could be detected in this case. Single radial hemolysis confirmed infection in 39 of the 41 proven rubella patients, and one false-positive diagnosis occurred in the control group patients. Of the 43 convalescent-phase serum samples, rubella-specific IgM was detected in 42 by Enzygnost-Rubella, in 41 by RUBELISA M, and in 39 by Rubazyme-M. For a rapid diagnosis with acute-phase sera, specific IgM detection by ELISA was most reliable in hemagglutination inhibition test-positive sera; of 18 such serum samples IgM was shown in 15 by Enzygnost-Rubella, in 13 by RUBELISA M, and in 11 by Rubazyme-M. False-positive specific IgM results were shown by Rubazyme-M in two serum samples from one patient in the control group. These serum samples were negative with the other two ELISA methods.     

Diagnosis of systemic candidiasis by latex agglutination for serum antigen.

Three latex agglutination test procedures for detecting Candida antigen in human serum were compared in a retrospective study of 69 patients and 20 normal volunteers. Untreated human serum was reacted with two different latex reagents; one reagent also was reacted with serum treated with protease and heat. The test procedure with treated serum was best, detecting serum antigen in 17 of 21 patients (81%) with disseminated candidiasis. Judging by autopsy-proven cases, there was an increase in positive test results in the last 2 weeks of life. When untreated sera were tested with this reagent, only 3 (14%) of the 21 patients with disseminated candidiasis had detectable antigen in serum. A subset of these same sera was tested by a commercial latex reagent (Candida Detection System lot C001; Ramco Laboratories, Inc., Houston, Tex.) and untreated serum. Of 18 patients with disseminated candidiasis, 5 (28%) had at least one positive serum. Sera from patients with less severe clinical forms of candidiasis were usually negative regardless of the test procedure used. With one exception, sera from control patients were negative or were positive only in sera containing rheumatoid factor. Latex agglutination tests for Candida spp. in treated serum may prove to be a useful procedure for the rapid diagnosis of severe disseminated candidiasis.     

Detection of rabies virus antigen in dog saliva using a latex agglutination test

Dog bites are responsible for more than 90% of human rabies deaths in Asia. We developed a simple and inexpensive test based on latex agglutination (LA) for rabies virus antigen detection in dog saliva. Rabies virus antigen could be detected by agglutination on a glass slide using latex particles coated with gamma globulin. By evaluation of paired saliva-brain specimens from 238 dogs, the LA test using saliva was 99% specific and 95% sensitive compared to the fluorescent antibody test (FAT) on brain smears. The advantages of the LA test over the standard FAT are that it is comparatively simple and there is no need to kill the animal before examination.     

Use of antibody-coated red cells for the sensitive detection of antigen and in rosette tests for cells bearing surface immunoglobulins.

Conditions for an improved chromic chloride method for the attachment of antibody to red cells are described. The method, which is applicable to whole Ig fractions as well as affinity-purified antibodies, is reproducible and has a sensitivity for detection of antigen in the nanogram range. The coated cells may be used in a rosette assay for the detection of cell surface-bound antigens.     

Specificity and usefulness of an IgE immunosorbent agglutination assay for toxoplasmosis.

AIMS--To develop an immunosorbent agglutination assay for the detection of Toxoplasma gondii IgE antibodies (IgE-ISAGA); to assess its specificity; and to determine the role of specific IgE in the diagnosis of current toxoplasma infection. METHODS--Rabbit antihuman IgE capture antibody was adsorbed onto microtitre plates and formaldehyde fixed tachyzoites were used to identify specific antibody. Specificity was assessed in 513 serum samples (blood donor, potentially interfering and difficult, elevated and low total IgE and myeloma). Serum samples (n = 108) from 65 patients with documented toxoplasmosis were tested, as were 26 serum samples from nine pregnant women positive for specific IgM and 30 from 20 HIV positive patients. RESULTS--IgE-ISAGA was highly specific with only three of 513 (0.58%) positive reactions amongst the control groups, one of which (0.19%) was regarded as a false positive. Elevated total IgE did not influence specific IgE results nor did the presence of abnormal immunoglobulin concentrations. Sixty (92.3%) patients with toxoplasma associated lymphadenopathy had specific IgE in one or more samples. Positive or borderline results were obtained in 68 of 77 (88.3%) serum samples taken up to four months after onset and were also detected for up to 11 months in 21 of 31 (67.7%) sera. Of the nine pregnant women with detectable specific IgM, specific IgE was absent in five (12 specimens). Specific IgE was also detected in 10 of 30 (33.3%) serum samples from the 20 HIV positive patients, which was similar to the number with specific IgM. Neither the specific IgE nor IgM tests could distinguish symptomatic from asymptomatic HIV positive patients. CONCLUSIONS--IgE-ISAGA is sensitive, specific, and easy to perform. Although results suggest that specific IgE may be less helpful than previously claimed, specific IgE has a useful role in the diagnosis of current toxoplasma infection when used in conjunction with other tests.     

Demonstration of enterobacterial common antigen by bacterial agglutination.

Potent antisera against the enterobacterial common antigen (ECA) agglutinate R bacteria of the Enterobacteriaceae family that possess unimpaired R-core structures of the Escherichia coli R1 or E. coli R4 core type. In these strains, known to be ECA immunogenic, ECA is most probably linked to the lipopolysaccharide R core. R mutants of other core types (e.g., Salmonella Ra, E. coli R2 or R3) or R mutants with incomplete core structures of the E. coli R1 type, as well as an rfaL mutant deficient in the O-translocase system, agglutinate to a much lesser extent or not at all. All the later mutants are nonimmunogenic; they possess the ECA in a free form, not linked to the R core. None of the S forms tested from many different enterobacterial genera was found to be agglutinable with the ECA antiserum. The dynamics of the ECA agglutinin formation in rabbits parallels the ECA hemagglutinin formation, indicating that the same antibody class might be involved in bacterial agglutination and hemagglutination.