Comparison of dot immunobinding assay, enzyme-linked immunosorbent assay and immunodiffusion for serodiagnosis of paratuberculosis.

A rapid, simple and inexpensive dot immunobinding assay (DIA) was evaluated for the serodiagnosis of paratuberculosis in cattle. The assay was performed on nitrocellulose strips which were dotted with purified protoplasmic antigen of Mycobacterium paratuberculosis. After incubation with test serum samples, the bound antibodies were detected using an enzyme-amplified immunostaining procedure. The efficacy of DIA as a screening test for paratuberculosis was compared to that of an enzyme-linked immunosorbent assay (ELISA), a modified agar gel immunodiffusion (mAGID) test, and an AGID test using 329 serum samples from cattle which were examined for M. paratuberculosis infection by a sensitive fecal culture technique. The DIA and ELISA had comparable results and both of the enzyme immunoassays had higher sensitivity than tests based on AGID. The sensitivity of all four tests was influenced by the intensity of fecal bacterial shedding. Preabsorption of sera with Mycobacterium phlei increased the sensitivity of both enzyme immunoassays. the specificity but reduced the sensitivity of both enzyme immunoassays.     

Use of the dot enzyme-linked immunosorbent assay with isolated Anaplasma marginale initial bodies for serodiagnosis of anaplasmosis in cattle

Isolated Anaplasma marginale initial bodies were successfully used in a dot ELISA for rapid detection of antibodies to Anaplasma organisms. The enzyme immunoassay used only 25 ng of antigen dotted onto nitrocellulose disks. Antigen-antibody complexes were detected by use of alkaline phosphatase-conjugated protein A, and reactions were read visually after addition of a precipitable, chromogenic substrate. The test allowed the processing of multiple sera, either for screening or for titer determination, in less than 3 hours and was found to be as sensitive as the indirect fluorescent antibody test. The overall performance of the dot ELISA, using isolated A marginale initial bodies, for 580 bovine serum samples was as follows: sensitivity, 93%; specificity, 96%; and predictive value, 95%. Cross-reactivity was not observed with sera positive to Babesia bovis and B bigemina, Trypanosoma vivax, or common bacteria or viruses infecting cattle. The antigen dotted onto nitrocellulose disks was stable when stored at -20, 4, or 25 C. Compared with the indirect fluorescent antibody test, the dot ELISA allowed easier, faster, and more objective interpretation of results. Its simplicity and low cost combined with high sensitivity and specificity indicate that this assay could effectively replace serologic assays currently used for diagnosis of anaplasmosis in cattle.     

Serodiagnosis of bovine paratuberculosis by use of a dot enzyme-linked immunosorbent assay

A dot ELISA was developed for detection of antibodies to Mycobacterium paratuberculosis. The assay was evaluated by testing sera from cattle that were determined, by bacteriologic culturing of feces, to be infected with M paratuberculosis and were suspected of having clinical disease. Further evaluation involved testing sera from cattle in which M paratuberculosis had not been isolated from feces on several attempts. Results of the dot ELISA were positive for sera from 86 of 101 infected cattle, and results were negative for sera from 64 of 64 noninfected cattle. Results of conventional ELISA and agar gel immunodiffusion (AGID) tests were positive for 79 of 99 and for 51 of 101 infected cattle, respectively. The dot ELISA also was evaluated by comparing results of testing 708 sera with results of bacteriologic culturing of matched fecal samples from 262 cattle in 3 central Ohio dairy herds known to include cattle infected with M paratuberculosis. Results of the dot ELISA were positive for 25 of 39 sera from cattle with positive results on culturing of concurrently obtained fecal specimens. The dot ELISA results were negative for 661 of 669 sera from cattle with negative results to culturing of concurrently obtained fecal specimens. The 39 sera from cattle with positive results on bacteriologic culturing of matched fecal specimens had positive results for ELISA and the AGID test 25 and 14 times, respectively. The 669 sera from cattle with concurrently negative results on bacteriologic culturing of feces had negative results to ELISA and the AGID test 559 and 668 times, respectively.     

Identification of ruminant mycoplasmas by dot immunobinding on membrane filtration (MF dot).

A simple method of species identification of ruminant mycoplasmas by dot immunobinding (MF dot) is described. Mycoplasmas from broth cultures were directly trapped onto the surface of low protein-binding affinity membranes by vacuum filtration. Specific polyclonal anti-mycoplasma sera were then applied. Mycoplasma-antibody complexes were then revealed by an enzyme conjugated anti-immunoglobulin. A positive reaction was indicated by the development of a color reaction when substrate was added. The method was tested on 22 type-strains representative of species, subspecies, types or yet unnamed serogroups of mycoplasmas isolated from ruminants. MF dot presented a specificity close to that which could be expected from the reference serological tests. The advantages of the MF dot over the other tests are utility, rapidity, ready standardization and it allows many samples to be treated simultaneously.     

Comparison of blocking dot ELISA and competitive ELISA, using a monoclonal antibody for detection of bluetongue virus antibodies in cattle.

A blocking (B) dot enzyme-linked immunosorbent assay (ELISA), using a monoclonal antibody (mAb) against a group specific antigen of bluetongue virus (BTV) is described for the detection of BTV antibodies to BTV in cattle sera. Dots of BTV antigens were adsorbed to nitrocellulose (NC) strips and/or NC mounted in the windows of dipsticks. After blocking the remaining sites of the NC paper with milk powder solution and immersion in the test sample, the NC strips and dipsticks were exposed to mAb. Bound mAb was detected with peroxidase conjugated anti-mouse IgG (H and L). In the absence of anti-BTV antibody in the test sample, BTV antigen sites were reactive with mAb as indicated by a brown colored dot in the presence of the enzyme substrate, hydrogen peroxide and diaminobenzidine. In the presence of sufficient anti-BTV antibodies no color reaction was observed. The performance of these assays in detecting anti-BTV antibody in field blood eluate samples, prepared from whole blood dried on filter paper, from 395 bluetongue-free cattle in Canada and 635 sentinel cattle in Florida, USA, was evaluated and compared with the standard competitive (C) ELISA. The specificity of the dipstick B-dot ELISA was identical to that of the C-ELISA in testing of BT-free Canadian cattle but not in the testing of samples from the sentinel cattle in Florida, resulting in values of 100% diagnostic and 88.9% relative specificity, respectively. Based on the C-ELISA, the specificity of the NC strip B-dot ELISA was low and in the same order as that of the dipstick assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of a dot ELISA kit for measuring immunoglobulin M antibodies to canine parvovirus and distemper virus

A dot ELISA for the detection of immunoglobulin M (IgM) antibodies to canine distemper virus (CDC) and canine parvovirus (CPV) was assessed. The titres of IgM antibodies to CDV and CPV in 100 dogs were measured by the Immunocomb ELISA kit and compared with the results derived from the immunofluorescence assay (IFA). There was a strong correlation between the results of the dot ELISA technique and the IFA (P < 0.001). The dot ELISA kit was also used to assess the changes in the levels of immunoglobulin G (IgG) and IgM antibodies to CPV and CDV in 10 puppies vaccinated with a polyvalent vaccine. High levels of IgM antibodies to CPV were first detected seven days after they were vaccinated, and after nine days all the pups had high titres of IgG antibodies to CPV. High levels of IgM antibodies to CDV were detected after nine days and the highest average titres were recorded after 12 days. IgG antibodies to CDV were present from nine days after vaccination.     

Development of an avidin-biotin dot enzyme-linked immunosorbent assay and its comparison with other serological tests for diagnosis of glanders in equines

A dot enzyme-linked immunosorbent assay (dot ELISA) was developed for diagnosis of glanders in equines. The test was based on the detection of IgG antibodies to Pseudomonas mallei antigens bound to nitrocellulose coated on plastic strips (dipsticks), the reaction being amplified by an avidin-biotin system with biotinylated anti-horse IgG and horseradish peroxidase-avidin D. Sera from 810 normal, six naturally infected and 48 sensitized equines were tested by this assay, and results were compared with complement fixation, indirect haemagglutination and counter-immunoelectrophoresis tests. Dot ELISA had the highest sensitivity, and was superior to other tests in that it was rapid and easy to perform, the results were easy to interpret, the assay was not influenced by anti-complement activity, and it was able to detect antibodies at an early stage. Testing of serum at 1:200 dilution is proposed for epidemiological screening.     

肌注狂犬病病毒糖蛋白cDNA表达载体诱导的小鼠体液免疫反应

将狂犬病病毒糖蛋白ｃＤＮＡＢｇｌⅡ（１．６７ｋｂ）片段分别正向插入ｐｍＭＴ－１ＢｇｌⅡ位点和ｐＭＴ０１０／Ａ＋ＢａｍＨＩ位点，构建重组质粒ｐｍＭＴ－Ｒｇｐ和ｐＭＴ０１０／Ａ＋－Ｒｇｐ，通过磷酸钙共沉淀法转染ＢＨＫ－２１细胞，经ＰＣＲ、打点杂交及ＥＬＩＳＡ检测，证明糖蛋白基因发生了整合并获得表达。以该表达载体给小鼠肌肉内注射２～３次，采其注射前、后双份血清，经ＥＬＩＳＡ检测证明，注射了表达载体的小鼠，血清中病毒特异性抗体明显升高，表明狂犬病病毒糖蛋白ｃＤＮＡ在小鼠体内表达后，刺激机体产生了抗该糖蛋白特异性抗体     

A sensitive dot immunobinding assay for serodiagnosis of African swine fever virus with application in field conditions.

The present work describes a simple dot immunobinding assay (DIA) for African swine fever virus (ASFV) antibody detection that can be used under field conditions. The assay uses nitrocellulose strips dotted with a cytoplasmic soluble antigen (CS-P) of ASFV. The nitrocellulose strips are adhered to a plastic handle. The test serum samples react with the CS-P, and antibodies are detected using a protein A-peroxidase conjugate. Both incubations are carried out at 20 C. The efficacy of the DIA as a screening test for ASFV was compared to an enzyme-linked immunosorbent assay (ELISA) and an immunoblotting (IB) test using 343 sera collected from natural African swine fever epizootics and from inapparent ASFV carriers. The DIA had comparable sensitivity to both reference techniques, and all samples positive in the ELISA and IB test were also positive in the DIA. False-positive reactions were not detected when whole blood or poorly preserved serum samples were tested by DIA. Some poorly preserved sera that were positive initially by the ELISA were no longer ELISA positive in a later run, although they were positive in IB and DIA. These positive DIA and IB test results could be caused by the differences in antibody epitope binding.     

Preparation of Anti-Rabies Virus N Protein IgYs by DNA Immunization of Hens Using Different Types of Adjuvants

DNA immunization has been used to study vaccination methods and for production of specific antibodies. The present study aimed to apply DNA immunization to prepare specific IgYs, which react against rabies virus N protein (RV-N) and can be used to research and diagnose rabies virus. The DNA sequence of RV-N was ligated into a pcDNA 3.1 plasmid for constructing pcDNA-N. Eight hens were divided into four groups. Group 1 comprised the control group (non-immunized). In Groups 2, 3, and 4, hens were injected intramuscularly with pcDNA-N (400 µg/hen). Eight injections were administered every other week. From the 4th week, an adjuvant was injected in addition to pcDNA-N. Freund''s complete adjuvant (FCA) and λ-carrageenan were administered to Groups 3 and 4, respectively. Eggs were collected daily, and the specific antibody activities of egg yolks were measured by ELISA. IgYs were purified from pooled egg yolks at 16–19 weeks post-administration in each group. The detection sensitivities of the RV-N were compared using purified IgY as the primary antibody for ELISA, dot blotting, and western blotting. Egg yolks from one of the two hens in Group 2 (pcDNA-N alone) and all hens in Groups 3 (pcDNA-N + FCA) and 4 (pcDNA-N + λCarra) had increased ELISA values. The combined use of λ-carrageen in DNA immunization resulted in an adjuvant effect comparable to that of FCA. Each purified specific IgY detected RV-N in the ELISA, western blotting, and dot blotting; however, the detection sensitivity differed. Higher detection sensitivity of the +λCarra IgY was observed by ELISA, whereas there was higher detection sensitivity of +FCA IgY in western blotting and dot blotting. In summary, anti-rabies virus N protein IgY was prepared through DNA immunization of hens using FCA or λ-carrageenan as adjuvants and can be used as a primary antibody to detect rabies viruses.     

Sandwich-dot enzyme-linked immunosorbent assay for the detection of canine distemper virus

A sandwich-dot enzyme-linked immunosorbent assay (dot ELISA) was developed for the detection of canine distemper virus (CDV). In 56 dogs suspected to have CD the rates of detection of CDV antigen in samples of blood lymphocytes and palpebral conjunctiva by dot ELISA and ELISA were, respectively, 91% (49/54) and 81% (44/54) for the lymphocyte samples and 88% (28/32) and 75% (24/32) for the conjunctival samples. The CDV detection limits were 10 ng/50 μL for dot ELISA and 40 ng/50 μL for ELISA. The reliability of dot ELISA relative to electron microscopy was 96% with 22 samples: all 21 samples in which CDV particles were observed by electron microscopy yielded positive results with dot ELISA; the single sample in which particles were not observed yielded false-positive results with dot ELISA. The results indicate that the dot ELISA developed can serve as a reliable rapid diagnostic test in suspected cases of CD and also be useful for epidemiologic surveillance of the disease.     

酶联免疫吸附试验夹心法检测梅花鹿狂犬病病毒抗原的研究

以马抗狂犬病固定毒抗体建立的酶联免疫吸附试验夹心法,检测了动物脑组织中的梅花鹿狂犬病病毒抗原。对已知阳性和阴性鼠脑抗原的检测表明,本法具有敏感、特异、快速,简便等特点。实验室检测了50份阳性鼠脑抗原及40份阴性鼠脑抗原,其消光值(OD值)均数(X)分别为0.928和0.209。对5只鹿、3头牛和4只羊脑组织的检测结果皆与其它诊断结果相符;对15只犬的检测发现,其敏感性远远高于补体结合试验的敏感性。     

Enzyme-linked immunosorbent assay (ELISA) using staphylococcal protein A for the measurement of rabies antibody in various species

An ELISA was developed using staphylococcal protein A linked with horseradish peroxidase for detecting IgG antibody of rabies virus in human and carnivore sera (80 human, 270 fox, 40 cat, 35 marten, 5 badger and 4 polecat sera were tested in the present work). In comparison with the serum neutralization (SN) test in cell culture, close overall agreement was obtained particularly in human and cat sera (97.5%). Two post-vaccination human sera were found positive with ELISA values of 2.16 and 2.65 IU/0.2 ml, but with SN titers less than 1:10. All prevaccination human sera were found negative by both tests. Regression analysis on 30 post-vaccination human sera revealed better correlation between ELISA and SN test at a serum dilution of 1:100 than at lower serum dilutions of 1:80 or 1:20. The correlation coefficient (r) was 0.73 (p less than or equal to 0.0001).     

应用夹心阻断ELISA测定血清中抗狂犬病抗体的研究

本研究建立的检测狂犬病抗体的夹心阻断ELISA,直接利用未经提纯的病毒悬液代替提纯的抗原,并仅用一种酶标抗体,测定了9种动物的血清.本方法敏感度的95%可信限为0.0013～0.0071IU/mL,比小鼠中和试验和微量免疫酶试验均敏感.按阻断50%判定血清ELISA的阴阳性,9种动物的1134份血清中有91份阳性,其中发病点的牛、猪、犬、家鼠血清的阳性率均在10%以上.根据对一定量的抗原阻断率相同时,抗体浓度一致的原理,测定了7种动物的185份血清效价,结果狂犬病抗体浓度在0.01IU/mL以上的为63份.利用夹心阻断ELISA和小鼠中和试验测定了7种动物的23份血清,阳性份数分别为12和11.两种方法均证明家鼠血清中有狂犬病抗体.本研究表明,测定狂犬病抗体的夹心阻断ELISA,简便、敏感、快速,可用于流行病学调查.     

A dot immunobinding assay on nitrocellulose for the detection of bovid herpesvirus-4 antibodies

A dot immunobinding assay (DIA) was developed for the detection of antibody against bovid herpesvirus-4 (BHV-4) in bovine sera. A semipurified virus preparation was used as the antigen and an antispecies horseradish peroxidase-labeled IgG and diaminobenzidine were employed as the detection system. The sensitivity and specificity of the DIA were similar to those of indirect fluorescent antibody test, indicating the suitability of DIA as a rapid field test for the detection of anti-BHV-4 antibodies in cattle.     

Comparison of enzyme-linked immunosorbent assay with dot immunobinding assay for detection of antibodies against Pasteurella multocida in turkeys

A dot immunobinding assay (DIA) was developed to detect antibodies against Pasteurella multocida in turkey serum. Five coating antigens, namely, whole-cell (WC) antigen, sonicated cell lysate (SCL), crude capsular extract (CE), formalin extract (FE), and heat-stable antigen (HSA), were compared by enzyme-linked immunosorbent assay (ELISA) and DIA using reference antisera against P. multocida organisms. WC and SCL antigens showed higher sensitivity, whereas FE and HSA antigens were more specific coating antigens in both assays. The specificity of DIA was greater than ELISA by comparing the P/N ratios of HSA against serum prepared from heterologous serotype of P. multocida. The DIA had also several distinct advantages over the ELISA, which included reduction of the manipulation time and more uniform binding of coating antigens onto the nitrocellulose membranes compared with binding of coating antigens to microtiter plates for ELISA.     

狂犬病毒G蛋白原核表达及间接ELISA检测方法的建立

目前狂犬病疫苗的效力检验采用NIH法,需要使用狂犬病毒CVS-24毒株进行攻毒试验,具有一定的生物安全风险。为寻求替代NIH法中脑内攻毒试验的方法,研究扩增狂犬病毒G蛋白基因,并将其克隆至大肠杆菌pET-32a载体上进行表达,以该蛋白作包被抗原,摸索试验条件,建立了检测小鼠血清抗体效价的间接ELISA方法。使用此方法与国际公认的荧光抗体病毒中和试验(FAVN)法比较,两者检测结果曲线相关系数为0.986,表明相关性较好,但ELSIA方法更加快捷、简便。本试验建立的间接ELISA方法可用于检测小鼠血清中狂犬病抗体,为狂犬病毒血清抗体测定和单克隆抗体筛选提供依据。     

Spill-over of European bat lyssavirus type 1 into a stone marten (Martes foina) in Germany

European bat lyssavirus type 1 (EBLV-1, genotype 5) is known to endemically circulate in insectivorous bat populations in Germany. In August 2001, a rabies suspect stone marten (Martes foina) was found in the city of Burg (Saxony-Anhalt, Germany) and was sent to the regional veterinary laboratory for routine rabies diagnosis. Whereas brain samples repeatedly tested negative in the fluorescent antibody test for classical rabies virus (genotype 1), the mouse inoculation test and the rabies tissue culture inoculation test yielded positive results. Rabies viral RNA was also detected in the stone marten brain sample both by nested and heminested RT-PCR specific for the nucleoprotein gene and for the nucleoprotein phosphoprotein junction of rabies virus. The amplification products were sequenced to genotype the isolate. Sequence data obtained from the first-round RT-PCR products were analysed and the suspect stone marten isolate was confirmed as a rabies related virus (EBLV-1a). Phylogenetic comparison with sequences from recent genotype five isolates from Germany and Denmark showed that it was closely related to a previous isolate of EBLV-1 from a serotine bat in Saxony-Anhalt obtained in the same year in an area adjacent to the place where the EBLV-1 infected stone marten was found. Both EBLV-1 isolates share a 99.5% identity. This is the first report of an EBLV-1a spill-over from an insectivorous bat into wildlife in Europe.     

Persistence of elevated rabies prevention costs following post-epizootic declines in rates of rabies among raccoons (Procyon lotor)

Determining the benefits to cost relationships among different approaches to rabies control and prevention has been hindered by the inherent temporal variability in the dynamics of disease among wildlife reservoir hosts and a tangible and objective measure of the cost of rabies prevention. A major and unavoidable component of rabies prevention programs involves diagnostic testing of animals and the subsequent initiation of appropriate public health responses. The unit cost per negative and positive diagnostic test outcome can be reasonably estimated. This metric when linked to methodologies subdividing the epizootic process into distinct temporal stages provided the requisite detail to estimate benefits derived from rabies control strategies. Oral rabies vaccine (ORV), for prevention of the raccoon-associated variant of rabies, has been distributed in Ohio and adjoining states in an effort to develop an immune barrier to the westward spread of epizootic raccoon rabies. The costs of ORV delivery have been quantified. Herein, the cost structures required to assess the benefits accrued by prevention were developed. A regression model was developed effectively predicting (r² = 0.70) the total number of rabies diagnostic tests performed by 53 counties in five northeastern (NE) states from 1992 to 2001. Five temporal stages sufficed to capture the range of variability in the raccoon rabies epizootic process. Unit costs, dollars per diagnostic test outcome, were calculated for negative and positive results from published reports. Ohio counties were matched to NE counties based on similar socioeconomic characters. A “pseudo-epizootic” of raccoon rabies was introduced into Ohio and the costs savings from ORV were derived as the excess costs imposed by epizootic spread throughout the state. At 46 km/year (range modeled, 30–60 km/year), the pseudo epizootic spread, and reached the enzootic stage, in all Ohio counties by year 13 (range modeled, 11–17 years). Cumulative excess costs for Ohio ranged between $11 and $21 million; counties of low socioeconomic status experienced the greatest relative excess costs. The costs for rabies prevention activities reached apices during the epizootic stage of raccoon rabies (2.7–10.8 times baseline) an unforeseen finding indicated elevated costs persisted (1.7–7.2 times baseline) into the enzootic stage.