Development and validation of an indirect enzyme immunoassay for detection of antibody to Brucella abortus in milk

Nielsen, Klaus; Smith, Phillip D.; Gall, D.; Pérez, Bárbaro Agustín Armas; Cosma, Chiara; Mueller, Philipp; Trottier, J.; Côté, Geneviève; Boag, L; Bossé, Janine T.

doi:10.1016/0378-1135(96)00059-4

Cited by 36 publications

(22 citation statements)

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“…2. Using the cutoff of 140 mP, the bmFPA in theory could detect one animal with B. abortus antibodies milk in more than 2,000 animals negative for B. abortus, which exceeded the mELISA (one infected animal in 100 animals) and the MRT (one infected animal in 5 or 10 animals) using similar methodology (2,6,13 Twenty-three milk samples banked from animals from which B. abortus had been isolated were used in this study. It was apparent from the data in this study that antibody activity in the stored milk samples could still be detected after 20 years of storage (samples were collected in 1981 and 1982).…”

Section: Discussionmentioning

confidence: 99%

“…The indirect enzyme immunoassay for milk (mELISA) was done as described by Nielsen et al (13). Briefly, the mELISA used B. abortus smooth lipopolysaccharide as the antigen immobilized on 96-well polystyrene plates followed by sequential addition of test samples (diluted 1:2), controls, diluted murine monoclonal antibody specific for bovine immunoglobulin G1 (conjugated with horseradish peroxidase), and substrate or chromogen, with appropriate wash procedures and incubation periods.…”

Section: Preparationmentioning

confidence: 99%

“…The subjectivity inherent in interpreting the MRT was removed and sensitivity of the ELISAs was adequate to detect a single infected cow in bulk milk samples from herds of 100 or more cattle (2,6,13). However, problems associated with nonspecific reactions (12) and detection of residual antibodies due to vaccination (11,15,20) remained using these ELISAs.…”

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confidence: 99%

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Fluorescence Polarization Assay for Detection of Brucella abortus Antibodies in Bulk Tank Bovine Milk Samples

Gall

Nielsen

Bermudez

et al. 2002

Clin Vaccine Immunol

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A simple, rapid, inexpensive fluorescence polarization assay for the detection of antibodies to Brucella abortus in bulk tank milk samples at the farm level or at dairies with a sensitivity and specificity of 100 and 95.9%, respectively, is described. The assay detects antibodies to B. abortus in 15 min by testing undiluted whey produced by chemical and physical manipulation of milk from bulk tanks. This sampling is noninvasive and therefore costs less and is less stressful than blood-based tests. The assay is specific and can detect antibodies at levels below that of the indirect enzyme immunoassay for milk and the fluorescence polarization assay for individual milk samples. Use of this test would make programs for surveillance of dairy animals and eradication of B. abortus more cost-effective. [527][528] 1937). At the time, this test was considered highly sensitive (G. Fleischhauer and G. Hermann, Berl. Tieraerztl. Wochenschr. 54:333, 1938) due to its ability to detect antibodies in milk from one infected animal mixed with milk from 5 to 10 cows negative for this pathogen. However, there were many shortcomings, including false-positive reactions associated with the MRT, as listed in Table 1. Nicoletti (10) showed that the MRT correctly identified 88.5% of animals in which B. abortus was isolated and 77.4% of animals in which B. abortus was not isolated. Similar sensitivity and specificity (89 and 86%, respectively) based on culture status were obtained by Hunter and Allen (8).Using undefined antigens and polyclonal anti species immunoglobulin enzyme conjugates for detection (2,7,19), indirect enzyme immunoassays (indirect ELISA) attempted to eliminate some difficulties inherent in the MRT and to improve on detection of antibodies to B. abortus. The subjectivity inherent in interpreting the MRT was removed and sensitivity of the ELISAs was adequate to detect a single infected cow in bulk milk samples from herds of 100 or more cattle (2, 6, 13). However, problems associated with nonspecific reactions (12) and detection of residual antibodies due to vaccination (11,15,20) remained using these ELISAs.An improved indirect ELISA using purified smooth lipopolysaccharide, a monoclonal antibody specific for the epitope of bovine immunoglobulin G1 and divalent cation chelating agents (EDTA and EGTA) to reduce nonspecific reactions (13) was developed. The sensitivity (based on samples from herds infected with B. abortus) and specificity (based on samples from brucellosis-free herds) obtained by this assay were 96.5 and 99.9%, respectively. Evaluation of this assay under Argentinian field conditions resulted in relative sensitivity (based on matched serum samples that were positive on the complement fixation test) and specificity (based on samples from herds free of brucellosis for 5 years) values of 99.6 and 99.1%, respectively (22). Although the assay had improved specificity, tolerated poor quality samples, and allowed batch processing, it was relatively expensive, time-consuming, and labor-intensive and could only b...

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Section: Discussionmentioning

confidence: 99%

Section: Preparationmentioning

confidence: 99%

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Fluorescence Polarization Assay for Detection of Brucella abortus Antibodies in Bulk Tank Bovine Milk Samples

Gall

Nielsen

Bermudez

et al. 2002

Clin Vaccine Immunol

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“…La magnitud clí-nica usualmente se puede transformar en un caso binario, adoptando un punto de corte para separar un caso positivo de uno negativo (4). La prueba de ELISA-i en leche se realizó luego en todas las muestras de leche que se mantuvieron congeladas a -20 °C, mediante el protocolo de Nielsen y colaboradores (12 …”

Section: Materiales Y Métodosunclassified

Epidemiologic issues in the validation of veterinary diagnostic tests

Greiner

Gardner

2000

Preventive Veterinary Medicine

391

315

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“…In dairy cattle, bacteriological, serological and molecular methods have been carried out for the diagnosis of brucellosis 4-6 . Although isolation of the bacteria leads to the definitive diagnosis of the disease, bovine brucellosis diagnosis is essentially based on the serological methods using serum or milk samples [4][5][6] . As milk particularly reflects IgG based antibody response of the animal 7, 8 and is a non-invasive sampling method, its use instead of blood in serological detection represents an important advantage in lactating animals.…”

Section: İletişim (Correspondence)mentioning

confidence: 99%

İnek Sütünde Brucella Antikoru ve DNA’sının ELISA ve PCR Yöntemleri ile Tespiti

Terzi¹,

Büyüktanir²,

Genç³

et al. 2009

Kafkas Univ Vet Fak Derg

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SummaryThe aim of the present study was to determine comparatively the presence of anti-Brucella antibody and Brucella DNA in cow milk. Anti-Brucella antibody was detected by ELISA based on the lipopolysaccharide (LPS) as diagnostic antigen. Besides, the presence of Brucella DNA in milk samples was screened by eryCD gene-targeted PCR and B. abortus DNA was determined by amplification of alkB genes. For this purpose, 70 raw cow milk samples collected from open markets were used. Among these samples, 15 samples (21.4%) were found positive for anti-Brucella LPS antibody in ELISA. In contrast, only 5 milk samples (7.1%) were determined as positive by eryCD gene-targeted PCR. All of the eryCD positive samples giving an amplicon of 904 bp indicated the presence of wildtype Brucella DNA but not B. abortus S19 vaccine strain allowing amplification of only an amplicon of 202 bp. In addition, amplification of the alkB gene demonstrated the presence of B. abortus DNA in 5 eryCD positive samples. No statistical agreement was observed between ELISA and PCR results with 95% confidence interval. These results strongly suggest that use of both ELISA and PCR methods could lead to more reliable diagnosis of brucellosis from bovine milk samples.Keywords: Brucella, Bovine, Milk, ELISA, PCR İnek Sütünde Brucella Antikoru ve DNA'sının ELISA ve PCR Yöntemleri ile Tespiti ÖzetBu çalışmada inek sütünde anti-Brucella antikorları ve Brucella DNA'sının karşılaştırmalı yöntemlerle belirlenmesi amaçlanmıştır. Anti-Brucella antikorların tespitinde spesifik tanı antijeni olarak lipopolisakkaride (LPS) dayalı ELISA kullanıldı. Ayrıca, süt örneklerinde Brucella DNA varlığı ery genine dayalı PCR tekniği ile araştırıldı ve B. abortus DNA varlığı ise alkB geni amplifikasyonu ile belirlendi. Bu amaçla, 70 adet çiğ inek sütü örneği yerel pazarlardan toplandı. Bu örneklerin 15'i (% 21.4) anti-Brucella LPS antikoru yönünden ELISA'da pozitif bulundu. Buna karşın, 5 süt örneğinin (% 7.1) eryCD genine dayalı PCR'da pozitif olduğu belirlendi. Tüm eryCD pozitif örneklerin 904 bp boyutunda amplikon vermesi, Brucella DNA'sının saha suşuna ait olduğunu ancak yalnızca 202 bp büyüklüğünde amplikon çoğaltılabilenn B. abortus S19 aşı suşuna ait olmadığını gösterdi. Ayrıca, alkB geninin PCR'da çoğaltılması ile eryCD pozitif bulunan 5 örnekte de B. abortus DNA varlığı belirlendi. %95 güven aralığında, PCR ve ELISA sonuçları arasında istatistiksel uyumluluk gözlenmedi. Bu bulgular, inek süt örneklerinde brusellozis tanısının ELISA ve PCR yöntemlerinin birlikte kullanımı ile daha güvenilir olabileceğine kuvvetle işaret etmektedir.

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Development and validation of an indirect enzyme immunoassay for detection of antibody to Brucella abortus in milk

Cited by 36 publications

References 6 publications

Fluorescence Polarization Assay for Detection of Brucella abortus Antibodies in Bulk Tank Bovine Milk Samples

Fluorescence Polarization Assay for Detection of Brucella abortus Antibodies in Bulk Tank Bovine Milk Samples

Epidemiologic issues in the validation of veterinary diagnostic tests

İnek Sütünde Brucella Antikoru ve DNA’sının ELISA ve PCR Yöntemleri ile Tespiti

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