Comparison of 44/107L one-step immunocapture enzyme-immunoassay and time-resolved fluoroimmunoassay for influenza A diagnosis

One-step immunocapture enzyme-immunoassay (EIA) was compared with time-resolved fluoroimmunoassay (TR-FIA) for rapid diagnosis of influenza A infection by antigen detection. The high-affinity monoclonal antibodies (MAbs) recognising two independent epitopes on the conservative nucleoprotein were used for capture (MAb 44) and detection (MAb 107L) of antigen by both assays. The detection limit for purified recombinant influenza A virus nucleoprotein was approximately 10 pg by EIA and 5 pg by TR-FIA. The performance of the methods was evaluated by testing 43 known positive and 50 negative clinical specimens (nasopharyngeal washes and aspirates). The sensitivity and specificity was 93% and 92% for EIA and 100% and 98% for TR-FIA, respectively, in comparison to the reference A3/A1 TR-FIA. The relationship of 44/107L immunoassays has been evaluated: in comparison to 44/107L TR-FIA (100%), EIA confirmed 93% of positive and 94% of negative samples. In conclusion, the capture-detector pair of MAbs 44 and 107L can be used for the sensitive detection of influenza A viral antigen in clinical samples by both immunocapture methods. Despite the slightly lower accuracy of the EIA, widespread availability and economy of the EIA methodology makes it an advantageous alternative for the laboratory diagnosis of influenza A virus infections.     

Direct enzyme immunoassay for detection of specific IgG antibody to rubella virus by use of labelled antigen

A new solid phase enzyme immunoassay (EIA) for detection of rubella-specific immunoglobulin G (IgG) antibody was developed. The test uses polystyrene microtiter strips coated with rabbit anti-human IgG immunoglobulins as the solid phase and an enzyme-labelled semipurified rubella antigen as indicator. The direct EIA was compared with hemagglutination inhibition (HI), single radial hemolysis (SRH), radioimmunoassay (RIA) and time-resolved fluoroimmunoassay (TR-FIA) using 52 serum specimens from patients with remote rubella infection. The overall agreement of direct EIA with HI was 96.1%, with SRH and RIA 98.1% and with TR-FIA 100%. The linear regression coefficient varied from 0.77 to 0.91, the best being obtained with direct EIA and SRH. The direct EIA was also suitable for diagnosis of acute infections, as a significant increase in antibody levels was detected in all paired specimens tested from patients with acute rubella infection. The sensitivity and were comparable to those of the assays employed. An advantage of the present assay is that the same method and same labelled antigen can be used to test for different classes of antibody using simply a solid phase with capture antibodies of different chain specificity.     

Study of the One-Step Growth Curve of Equine Infectious Anemia Virus by Immunofluorescence

Primary horse leukocyte cultures were inoculated with 2 or 10 50% tissue culture infective doses (TCID(50)) of equine infectious anemia (EIA) virus per cell, and the titer of cell-associated and fluid-phase virus was determined from 1 to 72 hr postinoculation (PI). Cover slips were collected from 4 to 72 hr PI and stained for EIA viral antigen by the indirect immunofluorescent (FA) technique. Viral replication was detected after a latent period of approximately 18 to 24 hr and reached peak titers of approximately 10(4.5) to 10(6) TCID(50)/0.5 ml from 48 to 72 hr PI. The fluid phase contained 10(1) to 10(2) TCID(50)/0.5 ml more virus than the cells. Viral antigen was first detected by FA from 18 to 24 hr PI. Approximately 75% of the cells contained antigen in their cytoplasm 72 hr PI. The FA technique is a sensitive method for detecting EIA virus in horse leukocyte cultures.     

Detection of adenovirus by enzyme-linked immunosorbent assay.

A solid-phase direct enzyme-linked immunosorbent assay (ELISA) was developed for the detection of adenovirus antigen in extracts of infected cells by using antihexon serum. Results with simulated clinical specimens consisting of normal nasal wash specimens seeded with varying concentrations of adenovirus type 5 showed that antigen could be detected in extracts of HEp-2 cell cultures inoculated with 10(2.5) 50% tissue culture infective doses (TCID50) and 10(1.5) TCID50 after 2 and 4 days of incubation, respectively. Fifty-three clinical nasal wash specimens containing adenovirus type 5 (stored for 5 years at -70 degrees C) were used to evaluate antigen detection by ELISA in HEp-2 cell extracts and by manifestation of cytopathic effect in human embryonic kidney cells. After 2 days of incubation, 62% were positive by ELISA, whereas none was positive for cytopathic effect. After 4 days of incubation, 76% were ELISA positive and 47% were positive for cytopathic effect. The results according to infectivity titers indicated that clinical specimens containing 10(3.0) TCID50 or greater were all positive by ELISA after 2 days of incubation in HEp-2 cells, and by 4 days all but one specimen containing 10(2.0) TCID50 or greater were ELISA positive. ELISA and immunofluorescent methods for antigen detection were compared using 24 of the 53 clinical specimens containing adenovirus type 5. Nearly equivalent sensitivities were demonstrated. These results suggest that ELISA may provide an alternative method of detecting and identifying adenoviral infections in humans.     

Dual-label time-resolved fluoroimmunoassay for the simultaneous detection of adenovirus and rotavirus in faeces

Dual-label time-resolved fluoroimmunoassay (TR-FIA) was used for the simultaneous detection of adenovirus and rotavirus in stool specimens. A one-incubation 1 h assay was used. Altogether 169 stool specimens collected from hospitalized children with acute gastroenteritis were tested. The specimens were dispensed with europium and terbium labelled antibodies against adenovirus and rotavirus, respectively, to coated microtitration strip wells. The 11 specimens positive for adenovirus in the EIA were also found positive in the TR-FIA. Fifty-six specimens positive for rotavirus with the reference EIA and one additional specimen were positive with the dual-label TR-FIA. Although the simultaneous assay using terbium and europium chelates gave lower assay sensitivities for both adenovirus and rotavirus when compared to separate assays using only europium chelates, the sensitivities were found to be at least equal to those of conventional standard antispecies EIA. With this one-incubation dual-label TR-FIA the results were obtained in less than 1.5 h for two viruses.     

Control of antigen mass transfer via capture substrate rotation: an absolute method for the determination of viral pathogen concentration and reduction of heterogeneous immunoassay incubation times

Immunosorbent assays are commonly employed as diagnostic tests in human healthcare, veterinary medicine and bioterrorism prevention. These assays, however, often require long incubation times, limiting sample throughput. As an approach to overcome this weakness, this paper examines the use of rotating capture substrates to increase the flux of antigen to the surface, thereby reducing the incubation time. To assess the capability of this approach, porcine parvovirus (PPV) was selectively extracted from solution by systematically varying the rotation rate of a gold substrate modified with a layer of anti-PPV monoclonal antibodies. The captured PPV were then directly imaged and quantified by atomic force microscopy. The benefits of substrate rotation are demonstrated by comparing an assay performed under stagnant conditions to one carried out with substrate rotation at 800 rpm, both for 10 min incubations at 25 degrees C. The use of rotation lowered the limit of detection to 3.4x10(4)TCID50/mL (approximately 80 fM) from 3.2x10(5)TCID50/mL (approximately 800 fM) under stagnant conditions. Results are also presented that show this strategy can be used: (1) to determine antigen concentrations without standards and (2) to establish the numerical relationship between quantal concentration units (e.g., 50% tissue culture infective dose (TCID50)) and quantitative concentration units (e.g., viruses/mL) The potential to broadly apply this technique to heterogeneous immunoassays is also briefly discussed.     

Detection of specific immunoglobulin M antibody to rubella virus by use of an enzyme-labeled antigen.

A direct antibody capture enzyme immunoassay (EIA) for the detection of rubella-specific immunoglobulin M (IgM) antibody was developed. Polystyrene microtiter strips coated with rabbit anti-human IgM were used as solid phase, and a semipurified rubella antigen labeled with horseradish peroxidase was used as indicator. By testing a panel of 238 serum specimens (not including sera from newborns with congenital rubella), the direct EIA was compared with the indirect EIA used routinely in our diagnostic unit and with a commercial IgM capture EIA (RubEnz M II) that employs a horseradish peroxidase-labeled anti-rubella monoclonal antibody as indicator. Overall agreement of direct EIA with indirect EIA and RubEnz M II was 95%, whereas agreement between direct and indirect EIA was 96.2%, and agreement between direct EIA and RubEnz M II was 97.8%. Sensitivity of the direct assay was higher than that of the indirect EIA and similar to that of the RubEnz M II assay. Specific IgM antibody could always be detected in serum specimens taken from patients with primary acute rubella infection between days 4 and 56 after the onset of rash. The assay was highly specific, and it was not affected either by rheumatoid factor or by high levels of specific IgG in sera. Another important advantage that the direct EIA has over the indirect EIA is that it requires 10 to 20 times less antigen per serum specimen tested.     

Performance of virus isolation and Directigen Flu A to detect influenza A virus in experimental human infection.

BACKGROUND: Few data exist to assess the sensitivity of different specimen types for viral detection during the course of influenza virus infection. OBJECTIVES: This study assessed the relationships between quantitative influenza A virus replication and antigen detectability by the enzyme immunosorbent assay (EIA) Directigen Flu A in different type of samples during experimental human infection. STUDY DESIGN: Fourteen volunteers were inoculated with influenza A virus A/Texas/36/91 (H1N1). Four specimens types were collected in sequence for quantitative isolation in cell culture and antigen testing from days 1 to 8 after inoculation. RESULTS: Seventy-one (63%) of nasopharyngeal wash specimens were culture positive, compared to 51 (46%) of throat gargles, 51 (46%) of nasal swabs, and 27 (24%) of throat swabs. All subjects shed virus in their nasopharyngeal wash at least one day and 86% of subjects had a positive nasopharyngeal wash culture on day 2 after inoculation. The mean viral titers were highest on day 2 post inoculation for all specimen types and averaged 3.6 log10 TCID50/ml for nasal washes, 1.2 log10 TCID50/ml for throat gargles, 1.8 log10 TCID50/ml for the nasopharyngeal swabs, and 0.6 log10 TCID50/ml for the throat swabs. Mean viral titers in the nasal washes were significantly different (P<0.05) compared to other specimen types. The peak of sensitivity of EIA (compared to culture) was the second day after inoculation. Nasopharyngeal and throat swab results were combined for this analysis and considered positive by culture if positive in either or both samples. Thus, on day 2 the number of EIA positive samples relative to the number culture positive was 9/12 (75%) for nasopharyngeal wash specimens, 2/9 (22%) for throat gargles, and 7/11 (64%) for the combined throat and nasal swabs specimens. CONCLUSIONS: Nasopharyngeal washes are the most sensitive sample type detecting influenza A virus in adults. For rapid diagnosis the Directigen Flu A is an alternative with a sensitivity compared to culture ranging between 64 and 78% if performed on nasopharyngeal specimens on day two or three after experimental infection in adults. However, if performed on other specimens or later in the course of infection the sensitivity is lower.     

Diagnostic potential of baculovirus-expressed rubella virus envelope proteins.

The envelope glycoproteins E1 and E2 of rubella virus were abundantly expressed in Spodoptera frugiperda Sf9 insect cells by using a baculovirus expression vector. The recombinant protein products were purified by immunoaffinity chromatography and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and enzyme immunoassay (EIA). The purified recombinant antigen consisted of the envelope polypeptides, corresponding to the viral E1 and E2 proteins, and a polyprotein precursor (molecular mass, 90 to 95 kDa). The antigen was reactive with human convalescent-phase sera in immunoblot analysis, and the reactivity correlated well (r = 0.861) with that of a whole-virus antigen when tested by EIA by using a total of 106 rubella virus immunoglobulin G-positive and -negative serum specimens. When the sera from patients with recent rubella virus infection were tested with the recombinant glycoproteins by EIA, the correlation was not as close (r = 0.690). However, all of the 26 serum specimens were reactive with the recombinant antigen. The results demonstrate that these bioengineered antigens have a potential for use in routine diagnostic assays of rubella virus immunity and recent infection.     

Inactivation of 12 viruses by heating steps applied during manufacture of a hepatitis B vaccine

The efficacy of two heating cycles (90 sec at 103 degrees C and 10 hr at 65 degrees C) used during manufacture of a plasma-derived hepatitis-B vaccine was validated for the inactivation of 12 virus families. A period of 15 min warming up to 65 degrees C had already completely inactivated representatives of nine virus families, ie, poxvirus (vaccinia), picornavirus (encephalomyocarditis virus), togavirus (sindbis virus), coronavirus (mouse hepatitis virus), orthomyxovirus (influenza virus), rhabdovirus (vesicular stomatitis virus), herpes virus (cytomegalovirus), lentivirus (human immunodeficiency virus), and retrovirus (murine leukemia virus). After prolonged heating at 65 degrees C or heating for 90 sec at 103 degrees C, parvovirus (canine parvovirus) and the phage phiX174 were also completely inactivated. Papovavirus represented by simian virus 40 (SV-40) was the most heat-resistant virus evaluated. The infectivity of SV-40 was reduced by 10(4) Tissue Culture Infectious Doses (TCID50) per ml after 90 sec at 103 degrees C, but a marginal residual activity (less than 1.5 TCID50 per ml) was observed. Subsequent pasteurization for 10 h at 65 degrees C did not further reduce the infectivity of SV-40. This study shows that the two heat-inactivation steps used during the production of this vaccine kill a wide variety of viruses that might be present in human blood.     

Cellular pathway(s) of antigen processing in fish APC: effect of varying in vitro temperatures on antigen catabolism.

Studies were conducted to determine the effects of varying in vitro temperatures during cellular processing of T-dependent antigens by catfish antigen-presenting cells (APC). Strategy involved pulsing of long-term monocyte lines (used as APC) with antigen at different temperatures for various periods of time and then fixing and coculturing with autologous peripheral blood leukocytes (PBL) as responders at a permissive temperature (i.e., 27 degrees C). Results showed that APC incubated with antigen at low, nonpermissive, but physiologically relevant, temperatures (11 and 17 degrees C) elicited secondary proliferative responses by autologous PBL. However, responses elicited with APC pulsed at 11 and 17 degrees C required longer exposure to the antigen prior to fixation (i.e., greater than or equal to 8 h compared to 5 h, the optimal incubation time at 27 degrees C). Further, there was sufficient cell-associated antigen during a short-term pulsing period (1-2 h) at 11 and 17 degrees C to provide efficient presentation after subsequent incubation of the APC at 27 degrees C for an additional 10 h before fixation. In contrast, APC pulsed with antigen at an extremely low, physiologically irrelevant, temperature (4 degrees C) for extended periods of time did not elicit the proliferation of autologous responders unless antigen pulsing was carried out for 1-2 h and the APC subsequently shifted to 27 degrees C for an additional 10 hr. Antigen uptake assays revealed significant and similar levels of internalized antigen at 11, 17, and 27 degrees C, but not at 4 degrees C. However, intracellular degradation and formation of trichloroacetic acid-soluble antigen catabolites at 11 and 17 degrees C appeared to occur at a slower rate compared to APC at 27 degrees C. Significantly, primary anti-hapten plaque-forming cell responses were also observed with PBL cocultured with APC pulsed with hapten-carrier conjugates at 11, 17, and 27 degrees C. Consequently, the previously observed suppression of primary T-cell responses in fish at low, physiologically relevant, temperatures is not due to impaired antigen processing or presentation.     

Detection of St. Louis encephalitis virus antigen in mosquitoes by capture enzyme immunoassay.

Surveillance methods that measure St. Louis encephalitis (SLE) virus activity in nature may provide forewarning of its epidemic occurrence in humans. An antigen capture enzyme immunoassay was developed to detect SLE virus in infected mosquitoes. The assay detected purified SLE viral antigen at a concentration of 62 pg/0.1 ml when antigen was incubated overnight; 250 pg/0.1 ml was detected in a single-day assay (antigen incubated for 3 h). The assay detected 67.9 and 70.8% of laboratory-prepared pools of infected mosquitoes after 3 h and overnight incubation, respectively. The sensitivity of the procedure was 90.5% in identifying pools with infectious titers greater than dex 3.0. The specificity of the assay was controlled by retesting positive pools preincubated with SLE virus and normal antibodies, which led to a diminution of signal in the pools containing viral antigen. The procedure was suitably specific in discriminating between SLE and related flaviviruses, detecting only high infectious doses of heterologous antigens.     

Comparative detection of measles and rubella IgM and IgG derived from filter paper blood and serum samples.

We compared the use of serum and filter paper blood spots as specimen sources for the detection of measles- and rubella-specific IgM and IgG. We collected capillary blood into microtainer tubes and onto filter paper spots from 60 children and 60 healthy adults. The blood was collected from 12-15-month-old children approximately 3 weeks after primary vaccination with measles, mumps, rubella vaccine, and the sample-pairs were tested for measles-specific IgM and IgG antibodies by using a capture antibody EIA and an indirect EIA, respectively. We tested sample-pairs from a subset of participants for rubella- specific IgM and IgG antibodies by using commercially available capture IgM (Captia) and indirect IgG (Wampole) assays. The concordance of results from serum and filter paper blood spots was high for all assays: 98% for measles IgM, 93% for measles IgG, 94% for rubella IgM, and 93% for rubella IgG, and increased to between 96-100% for all four assays when indeterminate samples were excluded. The correlation coefficients for EIA signals were 0.99 and 0.77 for measles IgM and IgG, respectively, and 0.92 and 0.94 for rubella IgM and IgG, respectively. The cut-off values used for filter paper samples were the same as those used for serum samples for all tests except for the rubella IgM assay. The use of filter paper blood spots is a promising future option for the detection of measles- and rubella-specific antibodies.     

Hyporesponsiveness to Virus Antigens in Rheumatoid Synovial and Blood Lymphocytes Using the Indirect Leucocyte Migration Inhibition Test

Mononuclear cells (MNC) from rheumatoid synovial tissue and peripheral blood were tested plasma pneumonia by the indirect leucocyte migration inhibition test. MNC from the eleven rheumatoid synovial tissues tested had deficient leucocyte inhibitory factor production against all antigens tested for, and this was also the case in the peripheral blood of seven juvenile rheumatoid arthritis patients (JRA). In the peripheral blood of eight rheumatoid arthritis (RA) patients there was also generally low reactivity. However, significant differences in migration indexes were found with rubella viral antigen and with PPD at 5 micrigram/ml when zero-hour and overnight incubations of the culture were compared. In contrast, MNC of peripheral blood of control donors had significant responses to PPD (19/19), mumps virus (7/11), rubella virus (10/19), cytomegalovirus (4/11), and herpes simplex type 1 virus (4/11) antigen after zero-hour culture, and no differences was seen after overnight incubation.     

Simultaneous use of poly- and monoclonal antibodies as enzyme tracers in a one-step enzyme immunoassay for the detection of hepatitis B surface antigen.

A one-step third generation enzyme immunoassay (EIA) was developed for the detection of hepatitis B surface antigen (HBsAg) in human serum or plasma using a polyclonal (Pab-HBsAg) and two monoclonal antibodies to HBsAg (Mab1-HBsAg and Mab2-HBsAg). In this assay, the solid phase is coated with Mab1-HBsAg and the specimen is incubated simultaneously with peroxidase labelled Pab-HBsAg and Mab2-HBsAg. If HBsAg is present in a specimen it will form sandwich complexes with capture and tracer antibodies. The hook effect, observed in some HBsAg detection tests when a high concentration of HBsAg is present, was minimized in this assay by increasing the concentration of the peroxidase-labelled Mab2-HBsAg. The sensitivity of this assay for HBsAg/ay and HBsAg/ad subtypes in a standard (2 h incubation) procedure was 0.6 and 0.3 ng/ml and in an overnight (16-22 h incubation) procedure 0.2 and 0.15 ng/ml, respectively. Strong elimination of the hook effect was observed with specimens containing high levels of HBsAg compared with test results using peroxidase-labelled Pab-HBsAg alone as enzyme tracer. This EIA offers a procedure, with a high specificity and wide range of sensitivity for the detection of HBsAg in human sera or plasma.     

Use of biotinylated 17beta-estradiol in enzyme-immunoassay development: spacer length and chemical structure of the bridge are the main determinants in simultaneous streptavidin-antibody binding

17beta-estradiol (E2) concentrations are in the low pg/ml range in plasma. To develop a sensitive enzyme immunoassay (EIA) for E2-determination a highly specific antibody raised against a 6-carboxymethyl (CMO)-E2-bovine serum albumine conjugate was used. Based on 6-CMO-E2 and 6-amino-E2, four biotinylated tracers with two different spacer lengths between E2 and biotin were synthesized using biotinylation reagents in one step reactions. All amino-based tracers were unsuitable for assay development because the antibody binding was too weak compared to the analyte E2. For 6-CMO-based tracers the simultaneous binding of the tracer to the antibody and streptavidin seems to be the determining step in the procedure depending on incubation temperature and spacer lengths. While a short spacer of 9 carbon atoms was susceptible to room temperature, a longer spacer of 16 carbon atoms showed nearly the same results for incubation at 4 degrees C or at room temperature. The absolute detection limit of this system was 0.63 pg/well. For sample clean-up, porcine plasma was solvent-extracted and depending on the initial plasma volume further purified by solvent partition. Determination of reproducibility resulted in intraassay coefficients of variation of 13% and 5.3% for samples with E2-levels of 15 pg/ml and 236 pg/ml, respectively. Measurement of E2-spiked blood plasma revealed recoveries of 83% up to 100% for E2 concentrations between 50 pg/ml and 1000 pg/ml. Only for the lowest concentration (20 pg/ml) a recovery of 58% was observed. Correlation of the EIA with an established radio immunoassay resulted in r=0.991 using the same antibody.     

Virus-like particles as a source for obtaining antigens for diagnosing rubella

Rubella diagnostic agents for hemagglutination inhibition (HI) and enzyme immunoassay (EIA) based on rubella virus-like particles (RVLP) have been developed. Noninfectious RVLPs containing three structural E1, E2, and C proteins were expressed in transfected CHO24S cell culture. HI titer in culture medium was 1:256. Tween-80 treatment and ether increased HI titer 4-6-fold and rendered the antigen higher stability. Immunogenic properties of RVLPs were similar to the native rubella virus in HI test with international reference human rubella serum and sera from convalescents after rubella. Serum of mice immunized with RVLP reacted similarly with RVLP antigens and native virus. Antigen for EIA from RVLP was prepared by concentrating RVLP from culture fluid and partial purification by ultracentrifugation. The results of human sera testing by HI and EIA with RVLP and native virus antigens coincided. RVLP are identical to antigenic structure of the virus, are stable and easily purified, and can therefore be used for commercial production of HI and EIA antigens.     

Enzyme-linked immunosorbent assay for detection of Salmonella typhi protein antigen.

A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was designed for the detection of Salmonella typhi protein antigen. The optimal concentration of antibody for coating the plate was found to be 50 micrograms/ml. The optimum conditions for antibody coating and antigen and conjugate incubation were 37 degrees C for 3 h, 37 degrees C for 2 h, and 4 degrees C overnight, respectively. The enzyme-substrate reaction was allowed to take place at 30 degrees C for 1 h. The established ELISA was found to be reproducible, with an inter-run coefficient of variation of less than 12% for the detection of an S. typhi protein antigen concentration of 0.5 to 50 micrograms/ml. The minimal detectable level of the antigen was 0.5 micrograms/ml. Cross-reactions were observed with the high level (50 micrograms/ml) of protein antigens obtained from Salmonella typhimurium, Escherichia coli, Salmonella paratyphi A, and Salmonella enteritidis. The ELISA established was used for the detection of S. typhi protein antigen in serum from 62 patients with typhoid, 30 patients with clinically diagnosed typhoid fever, 21 patients with paratyphoid, 17 patients with pyrexia caused by other bacteria, and 160 normal, healthy individuals. It was found that the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of this assay were 83.87, 89.04, 87.93, 67.53, and 95.31%, respectively.     

Rapid Immunofluorescence Staining by Microwave Incubation in Patients with IgA Nephropathy

Abstract

The effect of microwave incubation on the immunofluorescence staining findings in renal tissues in patients with IgA nephropathy is described. Ten patients with IgA nephropathy were examined. The renal sections were incubated with FITC-labeled rabbit anti-human IgA, IgM, IgG, or C3 antisera in a microwave oven that was operated at 2,450 MHz with an output of 500 W. There was no significant difference in the distribution of IgA or C3 deposition in the glomeruli between incubation in the microwave for 3–7 sec and 4°C overnight incubation. However, the immunofluorescence intensity of IgA after microwave incubation was less than that after incubation at 37°C for 1 min to 2 hr or 4°C overnight incubation. The intensity or distribution of IgG and IgM deposition in the glomeruli was markedly less after microwave incubation than that after incubation of 4°C overnight. We conclude that rapid immunofluorescence by microwave incubation is useful in the examination of renal biopsy specimens, especially IgA and C3 staining findings in patients with IgA nephropathy. (The J Histotechnol 13:175, 1990)     

Determination of prostaglandin metabolites in biological samples by competitive time-resolved fluoroimmunoassay.

We describe a time-resolved fluoroimmunoassay method for the determination of 13,14-dihydro-15-ketoprostaglandin E2 (PGEM) and 13,14-dihydro-15-ketoprostaglandin F2 alpha (PGFM) in femtomolar ranges. Polyclonal antibodies were raised in rabbits and the antigen was labelled with europium by the avidin-biotin technique. A second antibody, directed to rabbit IgG, was coated onto the solid phase. The IgG-PG-europium complex was bound by the second antibody, giving a rapid and complete separation of antibody-bound and free antigen. The detection limit was 0.2 pg/assay for PGFM and 1.2 pg/assay for PGEM. The intra-assay CV ranged from 4.6% to 9.3% and the interassay CV from 6.7% to 11.4%. A good correlation was obtained between the results from TR-FIA and RIA when the method was applied to the investigation of tissues from breast cancer. We conclude that the TR-FIA is more sensitive and much faster than the RIA and avoids the use of radioactive material.