Evaluation of an enzyme immunoassay kit for the detection of Clostridium difficile enterotoxin

The Premier Clostridium difficile toxin A enzyme immunoassay (EIA) kit was evaluated for the detection of C. difficile enterotoxin in fecal samples. A total of 314 samples was tested by culture, cytotoxin detection and EIA kit. Compared to a combined culture/cytotoxin result the Premier EIA kit had a sensitivity of 88.3%, a specificity of 100%, a predictive value positive of 100% and a predictive value negative of 87.4%. Test results were available within 3 hrs providing a rapid and reliable means of detecting C. difficile enterotoxin.     

Single toxin detection is inadequate to diagnose Clostridium difficile diarrhea in pediatric patients

BACKGROUND & AIMS: Clostridium difficile is an important cause of symptomatic diarrhea in pediatric patients. The bacterium produces two toxins, although many laboratories assay for only one. We questioned this diagnostic approach when patients had positive results for C. difficile at our institution, but initially had tested negative at outside laboratories. METHODS: We retrospectively analyzed relative frequencies of C. difficile toxin A alone, toxin B alone, and toxins A and B from pediatric patients with diarrhea. Results were stratified according to toxin detection and patient age. RESULTS: Of 1061 specimens, 276 (26.8%) were positive for C. difficile toxin(s). Fifty-one (18.5%) were positive for toxin A alone, 133 (48.2%) for toxin B alone, and 92 (33.3%) for both toxins. Assaying for toxin B identified C. difficile infection more frequently than did assaying for toxin A (P < 0.0001). The frequency of toxin B detection was significantly higher for older children but not for infants. CONCLUSIONS: Testing for C. difficile toxin A or toxin B alone will result in more frequent misdiagnosis than testing for both toxins. This practice may lead to inappropriate further invasive investigations in children, although this finding may not be applicable to adults.     

Antigen retrieval: p53 staining in benign, pre-malignant and malignant tissues of the larynx

Verification of specimens positive for Chlamydia trachomatis by enzyme immunoassay (EIA) has been recommended when testing low prevalence populations. This study compared direct fluorescent antibody (DFA) and blocking antibody (BLA) verification assays in specimens presumptively positive for C. trachomatis by the Syva Microtrak II EIA. Of 1785 specimens originally tested by EIA, 96 were presumptively positive for C. trachomatis. Verification assays were concordant in 86 specimens (69 positive, 17 negative); nine of the remaining samples gave positive results in a second EIA and one was unresolved. Both verification assays gave some false-negative results. When initial EIA absorbance values were correlated with verified results, all EIA false positive results had absorbances in the low range (less than a three-fold increase over assay cut-off values). Verification of EIA results by both DFA and BLA was effective in detecting false positive results, but confining verification to low-value positive specimens could be considered for cost effective C. trachomatis testing.     

Effects of SL-probiotic preparation on the body weight and phagocytosis of white mice

Specimens from 15 young patients presenting with acute epididymitis were tested for the presence of Chlamydia trachomatis by an enzyme immunoassay (EIA), polymerase chain reaction (PCR), and for other bacteria by standard laboratory techniques. C. trachomatis urethral infection was detected in 3 patients by an EIA test of the urethral swabs (20%) and in 13 patients by the PCR (87%). This difference in detection rate was statistically significant (p < 0.005). Thirteen specimens were positive by the PCR, but only three of them were positive by the EIA method. These findings indicate that the PCR assay is a highly sensitive assay for the detection of C. trachomatis in male urine specimens and provides a noninvasive technique for routine screening of chlamydia infection in the patient with acute epididymitis.     

Upstream of Randle

Polymerase chain reaction is widely used in clinical practice as a reliable assay for the detection and quantitation of hepatitis C virus RNA in serum and tissue. Due to the high sensitivity of the test, both false positive and false negative results can occur. The presence of cryoglobulins in serum samples represents a variable that can influence the result of a polymerase chain reaction assay leading to false negative results. In cases of hepatitis C virus infection associated with cryoglobulinaemia, it is extremely important to very carefully process the samples to be tested in order to avoid cryoglobulin precipitation and the consequent removal of the virus from the sample.     

Double immunochemical screening test (hemoglobin and albumin) for the detection of occult intestinal bleeding

BACKGROUND AND OBJECTIVES: Chlamydia trachomatis antigen testing of clinical specimens is replacing culture as the test of choice. Because of a potential for false positive results in low prevalence populations, there is an apparent need for confirming specimens positive by enzyme immunoassay (EIA). GOAL OF THIS STUDY: To examine specimens falsely positive in the Chlamydiazyme EIA assay according to gender and specimen type. STUDY DESIGN: Testing of genitourinary specimens from men and women consecutively enrolled from five health care delivery sources in an urban Canadian population. All specimens were initially tested in the Chlamydiazyme test and all positives repeated in a confirmatory blocking assay provided by the manufacturer. Additional confirmatory testing was performed using immunofluorescence (IF) staining for C. trachomatis elementary bodies (EB's) and polymerase chain reaction (PCR). RESULTS: From Jan. 1, 1990 to June 1, 1991, multiple specimens from 656 men and 5,628 women of varying population prevalences were screened. EIA-positive specimens from women had a repeat negative rate of 22% to 27% from cervical swabs and 29% from urethral swabs. Male urethral swabs had a high repeat negative rate of 22% when EIA was the only positive test, but 2.4% when the specimen was positive by EIA and culture. EIA-positive first void urine (FVU) specimens from men had a repeat negative rate of 8.7% as opposed to 17% to 32% from women. Only 1.7% (2/115) of male FVU did not block compared to rates of 47% (22/47) to 80% (4/5) in FVU from women. Analysis of EIA optical densities (OD's) and EB counts showed an association between the absorbance range 0.1 to 1.4 OD and 0-85 EB's. The greatest number of EB's and highest OD's were seen with cervical specimens, followed by urine and urethral specimens in women infected at all three specimens. All 55 specimens that did not confirm in the blocking test had no EB's and a convenience sample of seven were negative by PCR. All of a subset of 50 blocked specimens contained EB's or were positive by PCR. CONCLUSIONS: Although a variable proportion of specimens may not repeat positive in the EIA, use of the blocking reagent to confirm the repeat positives is highly recommended and the rate of blocking may be heavily influenced by gender and specimen type.     

Effects of tachykinin receptor antagonists, FK224 and FK888, in a guinea-pig model of nasal allergy

BACKGROUND: Since the development of the radioallergosorbent test (RAST) for quantification of allergen-specific IgE, numerous non-radoisotopic methods have been devised which combine the proven cellulose disc technology with enzyme-linked immunoassay methods. The HY.TEC EIA (Hycor Biomedical, Inc. Irvine, CA) was compared with Pharmacia CAP with respect to overall system features and assay performance characteristics. METHODS: The HY.TEC EIA and Pharmacia CAP were compared with respect to calibrator range, sensitivity, type of detection, type of solid phase, throughput, and mode of operation. To determine the assay sensitivity and specificity for a variety of allergens, a total of 2,447 tests were performed on both CAP and HY.TEC EIA. The samples were scored positive in both cases using a cutoff of 0.35 IU/mL. RESULTS: The general features of the HY.TEC EIA system are comparable to Pharmacia UniCAP, with the added advantage of higher throughput. Intra-assay precision was 7% and inter-assay precision was 9-15%. Using CAP as a comparative method, HY.TEC EIA has a sensitivity of 94.0% and a specificity of 94.4%. CONCLUSIONS: The HY.TEC EIA demonstrates excellent agreement with the Pharmacia CAP system in the determination of allergen-specific IgE. With the automation necessary in today's clinical laboratory, we conclude that the HY.TEC EIA is a state-of-the-art tool for the diagnosis of allergic disease.     

Detection of Helicobacter pylori in stool specimens by PCR and antigen enzyme immunoassay

A highly sensitive seminested PCR assay to detect Helicobacter pylori DNA in feces was developed. PCR with stool specimens and a novel antigen enzyme immunoassay (EIA) for H. pylori detection in feces were evaluated as diagnostic tools and in follow-up with samples from 63 infected and 37 noninfected persons. Infected individuals received eradication therapy followed by endoscopic follow-up 35 days after the start of treatment. At that time, a second stool specimen was obtained from 55 of these patients. Before eradication, the sensitivity of PCR was 93.7% and that of EIA 88.9%. Specificities were 100 and 94.6%, respectively. Of the 55 follow-up specimens, 41 originated from patients from whom H. pylori had been eradicated. Of these, 21 were still positive by PCR and 13 were positive by EIA, indicating that 1 month may be too short a period for follow-up evaluation of stool specimens by these tests.     

Sensitivity and specificity of indirect immunofluorescence and Grocott-technique in comparison with immunocytology (alkaline phosphatase anti alkaline phosphatase = APAAP) for the diagnosis of Pneumocystis carinii in broncho-alveolar lavage (BAL)

The purpose of the study was to compare the sensitivity and specificity of the indirect method of immunofluorescence with the immunocytological technique of alkaline phosphatase anti alkaline phosphatase complex (APAAP) for the detection of Pneumocystis carinii by bronchoalveolar lavage (BAL) in HIV-1 positive patients. - 83 HIV-1 positive patients with clinical presentations suggestive of Pneumocystis carinii pneumonia (PcP) were included in the study. 28 samples were found Pc-positive by immunofluorescence (IFT), 26 by Grocott and 29 by APAAP. In comparison to the lab results 33 patients were diagnosed as PcP according to the clinical course (i.e. therapeutic outcome, drugs used, and therapy changes). Compared to the clinical diagnoses, the following lab tests proved to be false positive and false negative: false positive: IF = 1, Grocott = 0, APAAP = 4 (3F6). false negative: IF = 5, Grocott = 7, APAAP = 4 (3F6). - Grocott stain shows insufficient correlation to the clinical diagnoses (p = 0.0156, McNemar-Test, two-tailed). - The two different detection methods (IFT and APAAP) showed no significant statistical difference with regard to their sensitivity (p = 0.3438, McNemar-Test, two tailed) and specificity. Considering cost and time the immunofluorescence technique seems to be the most suitable for the diagnosis of PcP in HIV-1 positive patients.     

Comparison between enzyme-linked immunosorbent assay and cytotoxic cross-match procedures for detecting IgG anti-donor antibodies

BACKGROUND: Disadvantages inherent to complement-dependent cytotoxicity cross-match (CDC XM) methods are the requirements for complement and viable target cells, detection of antibodies (Abs) against non-HLA antigens, and subjective scoring. Cross-Stat (SangStat Medical Corp., Menlo Park, CA), a recently developed enzyme-linked immunosorbent assay XM procedure for the detection of IgG anti-donor HLA Abs, is theoretically devoid of these flaws. METHODS: We compared results of Cross-Stat and our standard anti-human globulin (AHG)-enhanced CDC XM procedure on 524 sera from 230 transplant candidates, which were evaluated against 51 cadaveric donors. RESULTS: There was a significant correlation between AHG-CDC IgG XM and Cross-Stat results (P<0.001). For false negative sera, repeat AHG-CDC IgG XMs were still positive after platelet absorption, indicating that the Abs present were either non-HLA Abs or anti-HLA class II. Flow cytometry testing of false positive sera usually (42/62) substantiated Cross-Stat results, indicating that the discrepancy with AHG-CDC IgG XM is caused by greater sensitivity of Cross-Stat. Relative to the AHG-CDC XM, the sensitivity of Cross-Stat was 100%, the specificity was 93%, the positive predictive value was 73%, and the negative predictive value was 100%. A technical shortcoming of the Cross-Stat assay is that the frequency of indeterminate samples in the assays was 15%. Among 49 Cross-Stat negative vs. 13 Cross-Stat positive primary cadaveric renal allograft recipients (all AHG-CDC IgG-XM negative), there was no statistical difference in overall graft survival. CONCLUSION: Given the important theoretical advantages of enzyme-linked immunosorbent assay-based XM methods over the CDC XM, however, further testing of the clinical relevance of the Cross-Stat is warranted.     

Physical and psychological effects of anogenital warts on female patients

To evaluate whether hepatitis C virus (HCV) infection is an occupational hazard in the dental environment, serum samples collected in 1990-1991 from 461 dentists were tested for the antibody to HCV (anti-HCV) with first- and second-generation HCV enzyme-linked immunoassays (EIAs). Five of the 363 (1.38%) serum samples were reactive by the first-generation (C100-3) HCV EIA. Of the same 363 samples and the other 98 samples, 3 (0.65%) were reactive by the second-generation test. Of the 5 first-generation EIA reactive samples, only the 2 samples showing an absorbance of greater than 2.0 were also reactive to the second-generation EIA. The other 3 low-absorbance samples became negative and were regarded as false positives. Among the 358 samples negative by the first-generation EIA, 1 was reacted by the second-generation EIA. Those samples positive by the first- and/or second-generation HCV EIA were analyzed further by cDNA/polymerase chain reaction (PCR) to detect the presence of HCV RNA. Only 1 of the 5 first-generation EIA reactive samples was positive by PCR, but 2 of the 3 second-generation EIA reactive samples were PCR positive. These results are comparable to the anti-HCV prevalence of healthy blood donors (0.95% by C100-3 assay) and pregnant women (0.63% by recombinant immunoblot assay). We conclude that the prevalence of HCV infection among dentists in Taiwan is low, and there is no increased risk of HCV infection through the practice of dentistry, at least in our area.     

Total knee arthroplasty without patellar resurfacing in active and overweight patients

Chlamydia trachomatis is a frequent sexually transmitted disease. The diagnosis of C. trachomatis infection by cytology is controversial. We compared the ability of Papanicolaou (Pap) smears to detect C. trachomatis infection with antigen detection (enzyme immunoassay; EIA) and polymerase chain reaction (PCR). One hundred sixty-seven women attending a therapeutic abortion clinic were enrolled in the study. Endocervical samples were first collected for EIA and PCR, and then Pap smears were prepared for cytologic evaluation. Eight patients were excluded from the study due to the lack of an endocervical component. The criteria established by Gupta and associates (Diagn Cytopathol 1988;4:224-229; Acta Cytol 1979;23:315-320) were used in this study to assess the specificity and sensitivity of the Pap smear in recognizing C. trachomatis infection. After EIA testing, the remaining sample was subjected to phenol-chloroform extraction to purify the DNA and then tested by PCR. Positive PCR samples were subjected to repeat phenol-chloroform and retested to confirm the positive result. Using a confirmed PCR or a blocked EIA as the extended gold standard, the incidence of C. trachomatis infection was 9.4%. Fifteen of the 159 cases reviewed were positive by extended gold standard. Thirteen (86.7%) of those 15 cases were interpreted as negative by cytology (false-negatives), and two (13.3%) cases were positive. Of the remaining 144 cases, 14 cases (9.7%) were interpreted as positive by cytology (false-positives) but were not confirmed by the extended gold standard. Ten (66.7%) of the 15 cases confirmed by the extended gold standard were interpreted as negative by EIA (false-negatives), and five (33.3%) were positive. There were no false-positives by EIA. In this study, the sensitivity and the specificity for cytology were 13.3% and 90.3%, respectively. The positive predictive value was 12.5%, and the negative predictive value for cytology was 90.9%. The sensitivity and the specificity for EIA were 33.3% and 100%, respectively. The positive predictive value was 100%, and the negative predictive value for EIA was 93.5%. Both EIA and cytology are insensitive methods compared with PCR. Based on these data, cytology should not be used to diagnose C. trachomatis infection in an asymptomatic female population with a moderate risk of C. trachomatis infection.     

Head-to-head evaluation of five chlamydia tests relative to a quality-assured culture standard

Nucleic acid amplification tests offer superior sensitivity for the detection of Chlamydia trachomatis infection, but many laboratories still use nonamplification methods because of the lower cost and ease of use. In spite of their availability for more than a decade, few studies have directly compared the nonamplification tests. Such comparisons are still needed in addition to studies that directly compare individual nonamplification and amplification tests. The purpose of this study was to evaluate and compare the performance characteristics relative to culture of five different tests for the detection of C. trachomatis with and without confirmation of positive results. The tests were applied to endocervical specimens from 4,980 women attending family planning clinics in the northwestern United States. The five nonculture tests included Chlamydiazyme (Abbott), MicroTrak direct fluorescent antibody (DFA) (Syva), MicroTrak enzyme immunoassay (EIA) (Syva), Pace 2 (Gen-Probe), and Pathfinder EIA (Sanofi/Kallestad). All positive results obtained with a nonculture test (except MicroTrak DFA) were confirmed by testing the original specimens with a blocking antibody test (Chlamydiazyme), a cytospin DFA (MicroTrak EIA and Pathfinder EIA), and a probe competition assay (Pace 2). The prevalence of culture-proven chlamydia was 3.9%. The sensitivities of the nonculture tests were in a range from 62 to 75%, and significant differences between tests in terms of sensitivity were observed. The positive predictive value for each test was 0.85 or higher. The specificities of the nonculture tests without performance of confirmations were greater than 99%. Performing confirmatory tests eliminated nearly all of the false positives.     

A study of nurses'' attitudes towards research: a factor analytic approach

Clostridium difficile diarrhea and colitis result from the actions of bacterial exotoxins on the colonic mucosa. This study examined the ability of hyperimmune bovine colostral antibodies to neutralize the biological effects of these toxins. Anti-C. difficile bovine immunoglobulin concentrate was prepared from the colostral milk of Holstein cows previously immunized with C. difficile toxoids. The anti-C. difficile bovine immunoglobulin concentrate contained high levels of bovine immunoglobulin G specific for C. difficile toxins A and B, as evaluated by enzyme-linked immunosorbent assay. Anti-C. difficile bovine immunoglobulin concentrate neutralized the cytotoxic effects of purified toxin A and toxin B on cultured human fibroblasts, whereas control bovine immunoglobulin concentrate had little toxin-neutralizing activity. Anti-C. difficile bovine immunoglobulin concentrate also blocked the binding of toxin A to its enterocyte receptor and inhibited the enterotoxic effects of C. difficile toxins on the rat ileum, as measured by an increased rat ileal loop weight/length ratio (63% inhibition; P < 0.01), increased mannitol permeability (92% inhibition; P < 0.01), and histologic grading of enteritis (P < 0.01 versus nonimmune bovine immunoglobulin concentrate). Thus, anti-C. difficile bovine immunoglobulin concentrate neutralizes the cytotoxic effects of C. difficile toxins in vitro and inhibits their enterotoxic effects in vivo. This agent may be clinically useful in the prevention and treatment of C. difficile diarrhea and colitis.     

Evaluation of an antigen-capture enzyme immunoassay for detection of Entamoeba histolytica in stool samples

In order to identify the prevalence of Entamoeba histolytica in tourists with diarrhoea returning from countries of the developing world, sensitivity and specificity of a commercially available enzyme immunoassay (EIA) kit for the detection of Entamoeba histolytica coproantigen in stool were evaluated. Five hundred seventy-seven specimens from 469 patients were examined by microscopy and EIA. Sixty-two specimens from 49 patients were considered positive for Entamoeba histolytica. Compared with microscopic examination of stool samples, the EIA was found to be slightly more sensitive (90.3% vs. 87.1%) and was 97.7% specific for Entamoeba histolytica.     

Effects of protein kinase inhibitors on heat-induced hsp72 gene expression in a human glioblastoma cell line

This study aims to evaluate the performance of a new diagnostic method (LCx Tuberculosis Assay, Abbott Laboratories) based on Ligase Chain Reaction (LCR) technology, for the detection of Mycobacterium tuberculosis in respiratory and non-respiratory specimens and compare it with standard microbiological data and the clinical diagnosis of tuberculosis. Nine hundred specimens were collected from patients with a high suspicion of tuberculosis (740 respiratory samples and 160 non-respiratory specimens). The study was divided into two separate groups: samples washed and distilled water (207 samples) and unwashed samples that were directly resuspended in phosphate buffer (693 samples). The overall sensitivity, specificity, positive and negative predictive values of samples washed with distilled water after decontamination with SDS-NaOH were: 54%, 100%, 100%, and 94%, respectively. If these results were divided according to origin of specimens, the sensitivity, specificity, positive and negative predictive values in respiratory and non-respiratory samples were 54.5%, 100%, 100%, 94% and 50 100%, 100%, 93%, respectively. In contrast, for the non-washed samples, values were 85%, 95%, 80% and 98%, respectively. Respiratory and non-respiratory samples gave values of 84%, 96%, 77%, and 97.5% versus 89%, 99%, 94%, and 98%. The LCx M. tuberculosis assay is a novel, semi-automated assay and a rapid and highly specific technique for screening all forms of tuberculosis, including non-respiratory forms.     

Present status and advances in detecting Shiga toxin-producing Escherichia coli O157

Currently, detection of Shiga toxin-producing Escherichia coli(STEC) in stool samples is based on the isolation method in most clinical laboratories. The procedures are as follows: i) isolation with selective agar plates, ii) biological test with differential media, iii) serological test of anti-O antisera, iv) detection of toxin or toxin gene. These procedures take 4 days, therefore more rapid method is required. In the near future, a rapid detection method that detects STEC directly from stool samples will be introduced. Polymerase-chain reaction (PCR), enzyme-linked immuno-sorbent assay (ELISA), detection of serum anti-O157 antibodies are now available in clinical laboratories. Result of PCR for detection Shiga toxin gene and serum anti-O157 antibodies are described. Fifteen stool and serum samples from patients suspected of STEC infection were examined. With the culture and PCR method, 2 patients were positive by both methods and the results were confirmed in both cases. Six patients were positive by the antibodies detection method. From these results, the PCR method using stool samples was useful as a rapid detection method in clinical laboratories. Detection of serum antibodies has been simplified and is not an expensive method. Therefore, the method is useful for clinical diagnosis of STEC infection, especially, for diagnosing HUS or after antimicrobial agents have been administered to patients.     

Rapid detection of Chlamydia pneumoniae by PCR-enzyme immunoassay

Chlamydia pneumoniae is an important human respiratory pathogen. Laboratory diagnosis of infection with this organism is difficult. To facilitate the detection of C. pneumoniae by PCR, an enzyme immunoassay (EIA) for analysis of PCR products was developed. Biotin-labeled PCR products generated from the 16S rRNA gene of C. pneumoniae were hybridized to a digoxigenin-labeled probe and then immobilized to streptavidin-coated microtiter plates. Bound PCR product-probe hybrids were detected with antidigoxigenin peroxidase conjugate and a colorimetric substrate. This EIA was as sensitive as Southern blot hybridization for the detection of PCR products and 100 times more sensitive than visualization of PCR products on agarose gels. The diagnostic value of the PCR-EIA in comparison to cell culture was assessed in throat swab specimens from children with respiratory tract infections. C. pneumoniae was isolated from only 1 of 368 specimens tested. In contrast, 15 patient specimens were repeatedly positive for C. pneumoniae by PCR and Southern analysis. All of these 15 specimens were also identified by PCR-EIA. Of the 15 specimens positive by 16S rRNA-based PCR, 13 specimens could be confirmed by omp1-based PCR or direct fluorescent-antibody assay. Results of this study demonstrate that PCR is more sensitive than cell culture for the detection of C. pneumoniae. The EIA described here is a rapid, sensitive, and simple method for detection of amplified C. pneumoniae DNA.     

Comparison of the ligase chain reaction with solid and liquid culture media for routine detection of Mycobacterium tuberculosis in nonrespiratory specimens

The aim of this study was to compare the results of a commercial assay based on the ligase chain reaction [(LCR) LCx Probe System MTB; Abbott, USA] with those of culture in liquid medium (Septi-Chek AFB; Becton-Dickinson, USA) and culture on the egg-based L?wenstein-Jensen solid medium for the direct detection of Mycobacterium tuberculosis complex in nonrespiratory specimens. The results were analyzed according to the standard definition of a true-positive result. Two hundred thirty-five nonrespiratory samples routinely submitted to rule out tuberculosis were analyzed. All samples were smear-negative. Mycobacterial growth in either culture medium was detected in 18 (7.6%) specimens: Mycobacterium tuberculosis was recovered from seven (38.9%) specimens cultured on L?wenstein-Jensen medium and from 18 (100%) specimens cultured in Septi-Chek AFB. The LCR protocol was positive in 22 specimens. None of the LCR-negative controls showed positive results. All samples positive by culture on L?wenstein-Jensen medium were positive by culture in liquid medium and by the LCR assay. However, Mycobacterium tuberculosis was detected by culture in liquid medium in two specimens that were negative by the LCR assay, whereas six specimens negative by culture in liquid medium were positive by the LCR protocol; three of these were identified as true-positive results of the LCR assay. The sensitivity, specificity, and positive and negative predictive values were 33.3%, 100%, 100%, and 93.8%, respectively, for L?wenstein-Jensen medium; 85.7%, 100%, 100%, and 98.6% for the liquid medium; and 90.4%, 98.5%, 86.3%, and 99% for the LCR assay. These findings indicate that the LCR assay may be a valid method of high diagnostic yield for direct detection of Mycobacterium tuberculosis complex in nonrespiratory specimens.     

Development and evaluation of a novel antigen capture assay for the detection of classical swine fever virus antigens

An antigen-capture enzyme immunoassay (EIA) was developed to detect classical swine fever virus (CSFV) antigen directly from 10% w/v tissue suspension. The assay, based on the sandwich principle, uses a biotinylated monoclonal antibody bound to streptavidin-coated microplates as the capture system and a swine anti-CSFV antibody and rabbit anti-swine HRPO-conjugate as the detector system. The antigen-capture EIA was compared with conventional virus isolation and polymerase chain reaction (PCR) for detection of CSFV in tissues. The ability of the antigen-capture EIA to discriminate classical swine fever (CSF) from bovine viral diarrhea and African swine fever viruses was also tested. The assay was shown to detect 21 different strains of CSFV and was unreactive with tissues from uninfected animals. Signal to noise (S/N) ratios were calculated from the EIA absorbance values. Readings from samples positive by virus isolation (n = 47) averaged a S/N ratio of 5.34. In contrast, samples negative by virus isolation (n = 96) demonstrated a mean S/N ratio of 0.16. At S/N cut-off value of 1.0, all samples that yield virus isolation and PCR negative result were negative in the antigen-capture EIA. Compared with virus propagation in tissue culture using PK15 cells (followed by indirect peroxidase assay detection) and PCR, the EIA had a specificity of 98.7% and a sensitivity of 91.4%. The EIA is simple, can be performed in 4 h and lends itself to automation for screening of tissues sample from pigs suspected of CSFV infection.