Immunoblotting and dot blotting

Stott, David I.

doi:10.1016/0022-1759(89)90394-3

Cited by 166 publications

(51 citation statements)

References 336 publications

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“…The major immunogenic components visualized on the immunoblot are of 8-12 (RLPS), 10,17,19,29,45, and 63 kD. Other workers 7 who also used SDS/ ME-extracted cells as antigen, found discrete zones of reactivity located at 55 and 60 kD (alpha), 27-29 kD (beta), 18.5-20 kD (gamma 1), and 17 and 18 kD (gam-ma 2).…”

Section: Discussionmentioning

confidence: 96%

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A Sensitive Immunoblotting Technique for the Serodiagnosis of Brucella Ovis Infections

Kittelberger¹,

Hansen²,

Ross³

et al. 1994

J VET Diagn Invest

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A sensitive immunoblotting technique for the serodiagnosis of Brucella ovis infectionsAbstract.Reinhold Kittelberger, Mike Hansen, Gail P. Ross, Frans Hilbink A simplified electrophoretic immunoblotting technique based on antigen extracted from Brucella ovis cells with sodium dodecyl sulfate/mercaptoethanol was compared with the complement fixation test (CFT), the enzyme-linked immunosorbent assay, and the gel diffusion test. Sera from 89 chronically infected, semen culture-positive rams, 378 sera from B. ovis-infected flocks, 300 sera from accredited disease-free flocks, and 29 sera from specific-pathogen-free sheep were used. The immunoblotting technique had sensitivity and specificity comparable to those of the standard tests and was able to identify several CFT-negative or -borderline sera as positive. The major immunoreactive antigens of B. ovis had molecular masses of 63,29, 19 kD (proteins) and 8-12 kD (rough lipopolysaccharide). Antibodies against these antigens were present in 96% of CFT-positive sera from infected flocks and in 100% of sera from semen culture-positive rams. However, immunoblotting also identified antibodies to components other than the major antigens in 1% of CFT-negative sera from infected flocks and in 7.7% of the sera from flocks with a history of freedom from the disease. These reactions probably represent cross-reactivities with other microorganisms and were distinguishable from truly positive reactions.Ovine contagious epididymitis is a disease of im-correlation was found between these methods, but beportance in most sheep raising countries. Its predom-cause of its labor intensiveness, EIB was ruled out for inant cause is the gram-negative bacterium Brucella use in larger scale testing. ovis. A wide range of serologic methods for the detecAn EIB technique more suitable for such an applition of antibodies against this microorganism have been cation is described here and compared with the CFT, evaluated. 3 Among these are the agglutination 4

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Section: Discussionmentioning

confidence: 96%

“…To a limited extent, it is used for diagnostic purposes. 15,29 In B. ovis serology, EIB studies have focused on antigenic composition rather than on diagnostic application. 7,8,11,14,24,25 In a recent study, 7 EIB was applied to sera from B. ovis-infected rams and compared with a whole cell ELISA (CELISA) and with the CFT.…”

mentioning

confidence: 99%

A Sensitive Immunoblotting Technique for the Serodiagnosis of Brucella Ovis Infections

Kittelberger¹,

Hansen²,

Ross³

et al. 1994

J VET Diagn Invest

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“…The dot immunobinding assay, using an NCP membrane as a test matrix, is becoming widely used in simple qualitative research applications (11,14). Colloidal gold-labeled antibodies are also used in dot blot assays to avoid use of the sometimes-problematic enzyme-labeled detecting antibodies (7,10).…”

Section: Discussionmentioning

confidence: 99%

Fast Dipstick Dye Immunoassay for Detection of Immunoglobulin G (IgG) and IgM Antibodies of Human Toxoplasmosis

Jin

Chang

et al. 2005

Clin Vaccine Immunol

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“…The conformational stability of rec-S protein, its reactivity with serum from TGEV/PRCV hyperimmunized animals, and its presence in infected supernatant fluids was confirmed by Western blot. 20,26 Approximately 0.05 g/ml (epitope A competition) and 1 g/ml (epitope D competition) of rec-S protein was used for coating the ELISA plates.…”

Section: Competition Elisa Using the Baculovirus-expressed Tgev S Promentioning

confidence: 99%

Evaluation of the Baculovirus-Expressed S Glycoprotein of Transmissible Gastroenteritis Virus (TGEV) as Antigen in a Competition ELISA to Differentiate Porcine Respiratory Coronavirus from TGEV Antibodies in Pigs

Sestak¹,

Zhou²,

Shoup³

et al. 1999

J VET Diagn Invest

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Abstract. The spike (S) glycoprotein of the Miller strain of transmissible gastroenteritis virus (TGEV) was recently cloned and expressed in baculovirus. The recombinant S protein was used as the coating antigen in a competition (blocking) enzyme-linked immunosorbent assay (ELISA) in combination with monoclonal antibodies to the S protein epitope A (conserved on TGEV and porcine respiratory coronavirus [PRCV]) or epitope D (present on TGEV only) to differentiate PRCV-from TGEV-induced antibodies. One set (set A) of 125 serum samples were collected at different times after inoculation of caesarean-derived, colostrum-deprived (n ϭ 52) and conventional young pigs (n ϭ 73) with 1 of the 2 porcine coronaviruses or uninoculated negative controls (TGEV/PRCV/negative ϭ 75/30/20). A second set (set B) of 63 serum samples originated from adult sows inoculated with PRCV and the recombinant TGEV S protein or with mock-protein control and then exposed to virulent TGEV after challenge of their litters. Sera from set A were used to assess the accuracy indicators (sensitivity, specificity, accuracy) of the fixed-cell blocking ELISA, which uses swine testicular cells infected with the M6 strain of TGEV as the antigen source (ELISA 1) and the newly developed ELISA based on the recombinant S protein as antigen (ELISA 2). The sera from set B (adults) were tested for comparison. The plaque reduction virus neutralization test was used as a confirmatory test for the presence of antibodies to TGEV/PRCV in the test sera. The accuracy indicators for both ELISAs suggest that differential diagnosis can be of practical use at least 3 weeks after inoculation by testing the dual (acute/convalescent) samples from each individual in conjunction with another confirmatory (virus neutralization) antibody assay to provide valid and complete differentiation information. Moreover, whereas ELISA 1 had 10-20% false positive results to epitope D for PRCV-infected pigs (set A samples), no false-positive results to epitope D occurred using ELISA 2, indicating its greater specificity. The progression of seroresponses to the TGEV S protein epitopes A or D, as measured by the 2 ELISAs, was similar for both sets (A and B) of samples. Differentiation between TGEV and PRCV antibodies (based on seroresponses to epitope D) was consistently measured after the third week of inoculation.Transmissible gastroenteritis virus (TGEV), a prominent cause of neonatal diarrhea, causes death in 90-100% of seronegative pigs under 2 weeks of age and costs the US swine industry nearly $200 million/ year. 15,19 Surveys of TGEV antibodies in swine sera collected prior to the detection of porcine respiratory coronavirus (PRCV) indicated that 19-54% of swine herds in North America have antibodies, 17 but few differential serosurveys have been done since the detection of PRCV. 32 A variant of TGEV, referred to as PRCV, was isolated from pigs in Europe in 1986 and identified in the USA in 1989. 15,18,19 As reported from Iowa swine herds, a recent increase observed in TGEV/PRCV seropre...

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Immunoblotting and dot blotting

Cited by 166 publications

References 336 publications

A Sensitive Immunoblotting Technique for the Serodiagnosis of Brucella Ovis Infections

A Sensitive Immunoblotting Technique for the Serodiagnosis of Brucella Ovis Infections

Fast Dipstick Dye Immunoassay for Detection of Immunoglobulin G (IgG) and IgM Antibodies of Human Toxoplasmosis

Evaluation of the Baculovirus-Expressed S Glycoprotein of Transmissible Gastroenteritis Virus (TGEV) as Antigen in a Competition ELISA to Differentiate Porcine Respiratory Coronavirus from TGEV Antibodies in Pigs

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