Rapid Mycobacterial Liquid Culture-Screening Method for Mycobacterium avium Complex Based on Secreted Antigen-Capture Enzyme-Linked Immunosorbent Assay

ABSTRACT Sensors in automated liquid culture systems for mycobacteria, such as MGIT, BacT/Alert 3D, and Trek ESP II, flag growth of any type of bacteria; a positive signal does not mean that the target mycobacteria are present. All signal-positive cultures thus require additional and often laborious testing. An immunoassay was developed to screen liquid mycobacterial cultures for evidence of Mycobacterium avium complex (MAC). The method, called the MAC-enzyme-linked immunosorbent assay (ELISA), relies on detection of MAC-specific secreted antigens in liquid culture. Secreted MAC antigens were captured by the MAC-ELISA with polyclonal anti- Mycobacterium avium subsp. paratuberculosis chicken immunoglobulin Y (IgY), detected using rabbit anti-MAC IgG, and then revealed using horseradish peroxidase-conjugated goat anti-rabbit IgG. When the MAC-ELISA was evaluated using pure cultures of known mycobacterial (n = 75) and nonmycobacterial (n = 17) organisms, no false-positive or false-negative MAC-ELISA results were found. By receiver operator characteristic (ROC) analysis of 1,275 previously identified clinical isolates, at the assay optimal cutoff the diagnostic sensitivity and specificity of the MAC-ELISA were 92.6% (95% confidence interval [95% CI], 90.3 to 94.5) and 99.9% (95% CI, 99.2 to 100), respectively, with an area under the ROC curve of 0.992. Prospective evaluation of the MAC-ELISA with an additional 652 clinical samples inoculated into MGIT ParaTB medium and signaling positive per the manufacturer's instructions found that the MAC-ELISA was effective in determining those cultures that actually contained MAC species and warranting the resources required to identify the organism by PCR. Of these 652 MGIT-positive cultures, the MAC-ELISA correctly identified 96.8% (of 219 MAC-ELISA-positive cultures) as truly containing MAC mycobacteria, based on PCR or high-performance liquid chromatography (HPLC) as reference tests. Only 6 of 433 MGIT signal-positive cultures (1.4%) were MAC-ELISA false negative, and only 7 of 219 MGIT signal-negative cultures (3.2%) were false positive. The MAC-ELISA is a low-cost, rapid, sensitive, and specific test for MAC in liquid cultures. It could be used in conjunction with or independent of automated culture reading instrumentation. For maximal accuracy and subspecies-specific identification, use of a confirmatory multiplex MAC PCR is recommended.

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Comparison of an ELISA-based Screening Test with Liquid Chromatography for the Determination of Aflatoxins in Corn

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/74.5.827 ◽

1991 ◽

Vol 74 (5) ◽

pp. 827-829

Author(s):

Rodney W Beaver ◽

Mary A James ◽

Tay Y Lin

Keyword(s):

Liquid Chromatography ◽

Immunosorbent Assay ◽

False Positive ◽

Screening Test ◽

False Negative ◽

Screening Method ◽

Incorrect Response ◽

False Positives ◽

Enzyme Linked Immunosorbent Assay

Abstract An enzyme-linked immunosorbent assay (ELISA) screening test (CITE PROBE) was compared to liquid chromatography (LC) for the determination of aflatoxins in naturally contaminated corn samples. The CITE PROBE, with a positive/negative cutoff of 5 ng/g aflatoxin Bi, was correct (based on LC results) on 47 of 51 samples. Two of the Incorrect responses by the CITE PROBE were false positives on samples containing 4.4 ng/g and 4.1 ng/g aflatoxins by LC. Another incorrect response was a false negative on a sample containing 5.5 ng/g aflatoxins by LC. The fourth Incorrect response was a false positive on a sample containing 1.9 ng/g aflatoxins by LC. On the basis of these results, the CITE PROBE was determined to be a reliable screening method for the detection of % 5 ng/g aflatoxins in corn.

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An Enzyme-Linked Immunosorbent Assay for the Detection of Aspergillus parasiticus and Aspergillus flavus

Journal of Food Protection ◽

10.4315/0362-028x-60.8.978 ◽

1997 ◽

Vol 60 (8) ◽

pp. 978-984 ◽

Cited By ~ 15

Author(s):

GUO-JANE TSAI ◽

SHOU-CHIN YU

Keyword(s):

Aspergillus Flavus ◽

Immunosorbent Assay ◽

False Positive ◽

Aspergillus Parasiticus ◽

False Negative ◽

Zealand White Rabbit ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Molecular Weights ◽

Sds Page

An enzyme-linked immunosorbent assay (ELISA) was established for the specific detection of Aspergillus parasiticus and Aspergillus flavus. A New Zealand white rabbit was immunized intravenously with 100 μg of A. parasiticus CCRC 30117 mycelial protein extracts. The antibodies were separated and purified. The optimal concentration of the antibody and antibody-peroxidase conjugate used in the established ELISA was 10 μg/ml with a detection limit of 1 μg/ml. Among the 126 strains tested (including 21 strains of A. parasiticus, 11 strains of A. flavus, 34 isolates of A. parasiticus/A. flavus from cereals, and 60 strains of non-A. parasiticus/A. flavus fungi), the false-negative and false-positive rates were 1.5 and 3.3%, respectively. Strains of Aspergillus flavofrucatis and Aspergillus sojae produced false-positive reactions. However, their antigens had much lower cross-reactivity with the antibodies raised against A. parasiticus, as shown from I50 values. The molecular weights of the main antigens of A. parasiticus were 94, 82, and 40 kDa. The two heavier antigens had higher sugar contents, as demonstrated by SDS-PAGE and immunoblotting. A good correlation (r = 0.97) was found between mycelium measurement by weighing and by ELISA for A. parasiticus grown in yeast extract sucrose broth (YESB) at 25°C.

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Screening Procedures for Clenbuterol Residue Determination in Raw Swine Livers Using Lateral-Flow Assay and Enzyme-Linked Immunosorbent Assay

Journal of Food Protection ◽

10.4315/0362-028x-71.4.865 ◽

2008 ◽

Vol 71 (4) ◽

pp. 865-869 ◽

Cited By ~ 13

Author(s):

WEI H. LAI ◽

DANIEL Y. C. FUNG ◽

YANG XU ◽

YONG H. XIONG

Keyword(s):

Immunosorbent Assay ◽

False Positive Rate ◽

False Negative ◽

Screening Method ◽

The United States ◽

Lateral Flow ◽

Enzyme Linked Immunosorbent Assay ◽

Surveillance Program ◽

The European Union ◽

Lateral Flow Strip

Clenbuterol, which may cause symptoms of increased heart rate, muscular tremors, headache, nausea, and muscular cramps in patients, has been prohibited for consumption in many countries including the European Union, the United States, and China. A rapid lateral-flow strip assay was developed in our laboratory, and results obtained with this assay were compared with those obtained with a commercial enzyme-linked immunosorbent assay (ELISA) kit for the screening of clenbuterol in raw swine liver. A total of 128 swine livers were acquired from five local markets and prepared for analysis by the lateral-flow strip assay and ELISA. Analysis was completed in 10 min with the lateral-flow strip assay and in 90 min with the ELISA. In parallel with the ELISA, the rapid detection strip produced no false-negative results but had a false-positive rate of 6.3%. Cross-reactivity of the strip was assessed and was negative after tests with clenbuterol analogues such as terbutaline, salbutamol, ractopamine, ritodrine, and fenoterol. These data suggest that a lateral-flow strip assay can be used safely as a screening method as part of a clenbuterol residue surveillance program and should be a valuable tool in the food safety field, especially in developing countries.

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Cross-Reactivity of Antibodies with Phenolic Compounds in Pistachios during Quantification of Ochratoxin A by Commercial Enzyme-Linked Immunosorbent Assay Kits

Journal of Food Protection ◽

10.4315/0362-028x.jfp-14-189 ◽

2014 ◽

Vol 77 (10) ◽

pp. 1754-1759 ◽

Cited By ~ 6

Author(s):

HYUN JUNG LEE ◽

ALEXANDER D. MELDRUM ◽

NICHOLAS RIVERA ◽

DOJIN RYU

Keyword(s):

Phenolic Compounds ◽

Ochratoxin A ◽

Immunosorbent Assay ◽

False Positive ◽

High Performance ◽

Cross Reactivity ◽

Total Phenolic ◽

Enzyme Linked Immunosorbent Assay ◽

Wide Range ◽

The Cross

Ochratoxin A (OTA), a nephrotoxic mycotoxin, naturally occurs in wide range of agricultural commodities. Typical screening of OTA involves various enzyme-linked immunosorbent assay (ELISA) methods. Pistachio (Pistacia vera L.) is a rich source of phenolic compounds that may result in a false positive due to structural similarities to OTA. The present study investigated the cross-reactivity profiles of phenolic compounds using two commercial ELISA test kits. High-performance liquid chromatography was used to confirm the concentration of OTA in the pistachio samples and compared with the results obtained from ELISA. When the degree of interaction and 50% inhibitory concentration of phenolic compounds were determined, the cross-reactivity showed a pattern similar to that observed with the commercial ELSIA kits, although quantitatively different. In addition, the degree of interaction increased with the increasing concentration of phenolic compounds. The ELISA value had stronger correlations with the content of total phenolic compound, gallic acid, and catechin (R2 = 0.757, 0.732, and 0.729, respectively) compared with epicatechin (R2 = 0.590). These results suggest that phenolic compounds in pistachio skins may cross-react with the OTA antibody and lead to a false positive or to an overestimation of OTA concentration in ELISA-based tests.

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Detection of Listeria spp. in Meat and Environmental Samples by an Enzyme-linked Immunosorbent Assay (ELISA)

Journal of Food Protection ◽

10.4315/0362-028x-54.3.230 ◽

1991 ◽

Vol 54 (3) ◽

pp. 230-231 ◽

Cited By ~ 4

Author(s):

PAUL B. VANDERLINDE ◽

FREDERICK H. GRAU

Keyword(s):

Listeria Monocytogenes ◽

Immunosorbent Assay ◽

False Positive ◽

Environmental Samples ◽

False Negative ◽

Enzyme Linked Immunosorbent Assay ◽

Meat Processing ◽

Elisa Kit ◽

Processing Plants ◽

Selective Plating

An ELISA kit (TECRA™) for the detection of Listeria spp. was evaluated for its ability to detect these organisms in naturally contaminated meat and in environmental samples from meat processing plants. Of the 170 samples examined, Listeria monocytogenes and/or L. innocua were detected in 74 by enrichment and selective plating. Testing of the enrichment broths with the ELISA kit detected 72 of these positive samples and gave 2 false-negative and 2 false-positive reactions.

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Unreliable Measurement of Basophil Maximum Leukotriene Release with the Bühlmann CAST 2000 Enzyme-Linked Immunosorbent Assay Kit

Clinical and Vaccine Immunology ◽

10.1128/cvi.13.3.420-422.2006 ◽

2006 ◽

Vol 13 (3) ◽

pp. 420-422 ◽

Cited By ~ 6

Author(s):

S. E. Burastero ◽

C. Paolucci ◽

D. Breda ◽

G. Monasterolo ◽

R. E. Rossi ◽

...

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Immunosorbent Assay ◽

False Positive ◽

Positive Control ◽

Enzyme Linked Immunosorbent Assay ◽

Positive Results

ABSTRACT The Bühlmann CAST 2000 enzyme-linked immunosorbent assay is a potentially useful assay for measuring sulfidoleukotrienes released in vitro by allergen-challenged basophils. However, we observed that the positive-control reagent yielded positive signals in cell-free systems. These false-positive results depended on using a mouse anti-FcεRI monoclonal antibody and were prevented by degranulation-inducing reagents other than mouse monoclonal antibodies.

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Immunological discrimination between a sap-staining fungus and a biological control fungus

Canadian Journal of Botany ◽

10.1139/b90-203 ◽

1990 ◽

Vol 68 (7) ◽

pp. 1578-1588 ◽

Cited By ~ 4

Author(s):

Brian T. Luck ◽

Colette Breuil ◽

David L. Brown

Keyword(s):

Electron Microscopy ◽

Biological Control ◽

Immunosorbent Assay ◽

Liquid Culture ◽

Biological Control Agent ◽

Dry Weight ◽

Cross Reactivity ◽

Immunogold Labeling ◽

Enzyme Linked Immunosorbent Assay ◽

Polyclonal Serum

An enzyme-linked immunosorbent assay (ELISA) was used to detect a sap-staining fungus, Ophiostoma piceae, and a biological-control agent, Gliocladium roseum, grown in liquid culture and in wood. A polyclonal serum prepared against whole cell fragments from broken mycelia of O. piceae detected O. piceae in liquid culture at 0.25 μg dry weight/mL; however, there was moderate cross-reactivity with G. roseum. Antiserum adsorbed on G. roseum had almost no reactivity with G. roseum but still reacted strongly with O. piceae. The specificity of these sera was verified, and the antigenic sites were localized, by immunogold labeling and electron microscopy. These studies confirmed that the adsorbed serum could differentiate between G. roseum and O. piceae and showed that the cell wall was the most reactive cellular component. These results are discussed in relation to the development of immunological probes for the detection of sap-staining and biological control fungi. Key words: polyclonal serum, enzyme-linked immunosorbent assay, immunogold labeling, sap-staining and biological control fungi, electron microscopy.

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Identification of Streptococcus pyogenes in a Pediatric Outpatient Department: A Practical System Designed for Rapid Results and Resident Teaching

PEDIATRICS ◽

10.1542/peds.54.6.718 ◽

1974 ◽

Vol 54 (6) ◽

pp. 718-723

Author(s):

Katherine Sprunt ◽

Dorothea Vail ◽

Russell S. Asnes

Keyword(s):

False Positive ◽

Fluorescent Antibody ◽

False Negative ◽

Screening Method ◽

Rapid Screening ◽

Resident Teaching ◽

Antibody Uptake ◽

Busy Clinic ◽

Group A ◽

Laboratory Tool

A rapid screening method for identification of clinic patients with pharyngitis who are carrying group A beta-hemolytic streptococci and for teaching residents the values and limitations of the culture-disk approach to identification has been reviewed as developed for a busy clinic and a busy hospital laboratory. Identification of positive cultures in less than 24 hours, using Taxos A disk and specific fluorescent antibody uptake, resulted in 12% apparent false-positive and 3.6% false-negative reports. However, when viewed in the light of the techniques used for verifying results, there were probably 3% false-positive and 3% false-negative reports. The screening method is considered acceptably reliable and practical as a laboratory tool and a resident teaching device.

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