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1.
This document is the consensus of the American Association of Veterinary Laboratory Diagnosticians (AAVLD) Subcommittee on Standardization of Immunohistochemistry on a set of guidelines for immunohistochemistry (IHC) testing in veterinary laboratories. Immunohistochemistry is a powerful ancillary methodology frequently used in many veterinary laboratories for both diagnostic and research purposes. However, neither standardization nor validation of IHC tests has been completely achieved in veterinary medicine. This document addresses both issues. Topics covered include antibody selection, fixation, antigen retrieval, antibody incubation, antibody dilutions, tissue and reagent controls, buffers, and detection systems. The validation of an IHC test is addressed for both infectious diseases and neoplastic processes. In addition, storage and handling of IHC reagents, interpretation, quality control and assurance, and troubleshooting are also discussed. Proper standardization and validation of IHC will improve the quality of diagnostics in veterinary laboratories.  相似文献   

2.
Serological responses of Icelandic sheep experimentally infected with visna virus (vv) were contrasted with responses in American Targhee sheep naturally infected with progressive pneumonia virus (PPV). Precipitating antibodies assayed by immunodiffusion were compared with the neutralising and complementing fixing antibody response. In experimental infections with vv, complement fixing and neutralising antibodies appeared early after infection and rose to high levels in all sheep, while precipitating antibodies were detected only at minimal titre. In natural infections with PPV, immune responses were less consistent and precipitating antibodies were detected more frequently than complement fixing or neutralising antibodies against PPV. These results may suggest important biological differences between the lytic fibroblast-tropic virus strains used for experimental infection of Icelandic sheep and the nonlytic macrophage-tropic strains of PPV circulating in nature. Lytic strains evoke a brisk response against the viral glycoprotein with high titre neutralising antibody while nonlytic strains induce a less consistent response to the glycoprotein.  相似文献   

3.
Six monoclonal antibodies and a polyclonal antibody (CM1) were used to investigate the overexpression of p53 protein by immunohistochemistry (IHC) in six sarcomas and 21 mammary carcinomas from 27 dogs. IHC was compared with p53 gene mutation analysis performed on the same samples. Only the monoclonal PAb240, PAb421 and the CM1 antibodies were able to detect expression of canine p53 protein. CM1 was found to give the highest concordance (8/11) between positive expression of the p53 protein by IHC and the presence of a gene mutation. In the samples that were negative for p53 expression by IHC, but contained a p53 gene mutation according to DNA analysis, the mutation often affected the epitopes that could have been recognized by these antibodies. Only one out of 16 tumours without a p53 gene mutation had a weakly positive IHC result. These findings indicate that in these two types of canine tumours, IHC – particularly with CM1 – can detect many alterations in p53 expression owing to a gene mutation. False‐positive results were very infrequent.  相似文献   

4.
This report describes an experimental infection with porcine circovirus type 2 (PCV2) in combination with porcine parvovirus (PPV) in 3-week-old conventional colostrum-fed pigs with maternal antibodies to both viruses. Two groups of four pigs each were inoculated with PCV2 and PPV. One of the groups received also a commercial inactivated vaccine against porcine pleuropneumonia to evaluate possible effects of the stimulation of the immune system of pigs on the infection. Another group of four pigs was kept as uninfected control. Clinical signs, rectal temperatures and body weights were recorded. Serum antibody titers to PCV2 and PPV were determined at weekly intervals. Pigs were killed 42 days after inoculation and tissue samples were examined for the presence of gross and microscopic lesions. Tissues were also analyzed for the presence of PCV2 and PPV DNA by PCR, and for the presence of PCV2 antigen by immunohistochemistry (IHC). All the pigs had serum antibodies to PCV2 and PPV at the beginning of the trial. None of them developed clinical symptoms or pathological lesions typical of post-weaning multisystemic wasting syndrome (PMWS), a disease associated to PCV2 infection. However, IHC and/or PCR analyses showed that clinically silent PCV2 infection developed in five of the eight inoculated pigs, regardless of the administration of the vaccine. In particular, PCV2 DNA and/or antigen were detected in most of the tissues examined in the two pigs with the lowest titer of maternal PCV2 antibodies at the beginning of the trial. PPV DNA was not detected in any of the samples examined. The five pigs with PCR and/or IHC evidence of PCV2 infection had a mean weight gain during the experiment lower than that of the inoculated PCR-negative pigs considered together and that of the control pigs. In conclusion, it would appear that passive immunity against PCV2 can play a role in preventing the development of PMWS, but is not able to prevent the establishing of clinically silent PCV2 infections. The dissemination and persistence of the virus in the tissues may depend on the level of PCV2 antibodies at the time of inoculation.  相似文献   

5.
In 290 Q fever positive cattle from three 2000 head dairy farms in the former district of Erfurt (Thüringen) the course of titers was examined serologically over several months by means of the complement fixation test (CFT). In 47.2% of the cows serologically observed for 2 up to 28 months complement fixing antibodies against Coxiella burnetii could be demonstrated til the end of the investigation period. Repeated tests during pregnancy showed increase of antibody titers in the first 4 months and after a short decrease again from the 5. til the 7. month. By observing the antibody titers during several pregnancies each time a new increase comparable to a booster immunisation could be found. This may explain the persistence of Q fever antibodies in cows for several years. The results of this investigation suggest that from a high antibody titer it can not be concluded an abortion in a positive cow being caused by a Coxiella burnetii infection.  相似文献   

6.
Antisera from rabbits immunized with acetone-killed whole cells and with lipopolysaccharides from Brucella abortus (B.a.) and Yersinia enterocolitica type IX (Y.e.) were gel-filtered on Sephadex G-200 columns.All the antisera had serologically cross-reacting agglutinins against B.a. and Y.e. both in the 19 S and in the 7 S antibody fractions.The homologous and the heterologous agglutinating activity of 19 S and of 7 S antibodies was tested before and after treatment with dithiothreitol (DTT) and subsequent alkylation. Unlike 19 S antibodies, 7 S antibodies were resistant to the DTT reduction.The results from heterologous absorptions of the respective 19 S and 7 S antibody fractions indicated that 19 S antibodies both to B.a. and to Y.e. had a greater tendency towards being absorbed to the cross-reacting antigenic determinants than had the corresponding 7 S antibodies. The agglutination titres for 19 as well as 7 S antibody fractions derived from the immunizations with B.a. (both whole cells and LPS) fell more markedly after heterologous absorptions than did the analogous titres for corresponding Y.e. antibody fractions. Possible explanations of these differences are discussed.  相似文献   

7.
Monoclonal antibodies were used to develop a double antibody enzyme-linked immunosorbent assay for the detection of canine parvovirus (CPV) antigen in fecal samples. The assay was specific for the hemagglutinating protein of CPV and detected as little as 1.5 ng of virus within a 15-minute incubation period. The use of monoclonal antibodies against 2 epitopes on the CPV antigen permitted the simultaneous addition of test sample and enzyme-conjugated antibody, thus considerably simplifying the manipulations required for the assay. Results were visually determined without special instrumentation. Clinical studies revealed greater than 95% correlation between enzyme-linked immunosorbent assay results and hemagglutination titers.  相似文献   

8.
This paper describes the development of an indirect immunoperoxidase assay (IIP) and an indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to chicken anemia virus (VAC). The IIP assay developed used CAV-infected MDCC-MSB1 cells for detecting antibody to CAV, whereas the ELISA utilized gradient-purified immunoadsorbed CAV as the target antigen. The IIP and ELISA were compared with the standard indirect immunofluorescent antibody (IFA) assay, which is more conventionally used to screen chicken serum for antibodies against CAV. Comparative test results of 185 field samples of chicken serum by these three methods were in agreement 84% of the time. Both IFA and IIP assays yielded fewer positive tests than did the ELISA. IFA and IIP assays were in agreement 93% of the time, as compared with 91% agreement of IIP and ELISA results, or 84% agreement for comparative IFA and ELISA results.  相似文献   

9.
Bovine viral diarrhea virus (BVDV) is one of the most relevant pathogens affecting today's cattle industries. Although great strides have been made in understanding this virus in cattle, little is known about the role of wildlife in the epidemiology of BVDV. While persistently infected cattle are the most important reservoir, free-ranging ungulates may become infected with BVDV as demonstrated by serosurveys and experimental infections. Therefore, free-ranging wildlife may maintain BVDV as the result of an independent cycle and may serve as a reservoir for the virus. Systematic studies on prevalence of BVDV-specific antibodies or frequency of persistent BVDV infection in North American wildlife are sparse, and no information is available from the southeastern United States. The objective of this study was to evaluate blood and skin samples from hunter-harvested white-tailed deer (Odocoileus virginianus) for evidence of BVDV infection. Virus-neutralizing antibodies were detected in 2 of 165 serum samples. Skin biopsy immunohistochemistry (IHC) was performed on samples from 406 deer using a BVDV-specific monoclonal antibody (MAb) (15c5), and BVDV antigen was detected in one sample. A similar IHC staining pattern was obtained using a second BVDV MAb (3.12F1). Viral antigen distribution in the skin sample of this deer resembled that found in persistently infected cattle and in a previously described persistently infected white-tailed deer; thus, the deer was presumed to be persistently infected. Evidence of BVDV infection in free-ranging white-tailed deer should encourage further systematic investigation of the prevalence of BVDV in wildlife.  相似文献   

10.
In an immunohistological/cytological study of canine bone marrow, the aim was to demonstrate canine erythroid cells with the help of various commercially available antibodies against human antigens (monoclonal antibody against glycophorin A, polyclonal antibodies against haemoglobin and spectrin). In order to preserve possible cross-reacting epitopes various fixation methods (cross-linking, precipitating and dehydrating fixing agents, partly in combination with unmasking measures), decalcification techniques [acid or ethylenediaminetetraacetic acid (EDTA) decalcification] and tissue-embedding methods (paraffin embedding, cryostat sectioning technique) were used. Alternative methods, such as the preparation of cell smears and immunoblotting, were also employed. The only result that was of use for routine diagnostic procedures (paraffin sections) was that obtained by using polyclonal antibodies against haemoglobin. Best results were achieved when tissue was fixed in a formaldehyde-glutaraldehyde mixture, decalcified in EDTA and treated with microwave irradiation. The primary antibody was used in a dilution of 1:500 and incubated for 16 h. With the exception of mature red blood cells and proerythroblasts, different stages of erythrocytopoietic cells in canine bone marrow were shown to be arranged in erythrons. The polyclonal antibody against spectrin also showed clear cross-reactivity, but was only employable in other systems (immunoblotting). The monoclonal antibody against glycophorin A reacted only when used on human tissue or cells.  相似文献   

11.
Six different serological tests were used to examine Kenyan cattle sera for antibodies to the herpesvirus of malignant catarrhal fever. Significantly higher levels of indirect immunofluorescent antibody to early and late virus antigens and of complement fixing antibody were found in the sera of 13 naturally infected cattle than in 482 sera collected from four different groups of normal cattle. Virus neutralising and immunoprecipitating antibodies were also found in some infected cattle sera but not in normal cattle sera. Many non-specific reactions occurred using counterimmunoelectrophoresis. These preliminary results indicate that the serological diagnosis of wildebeest-associated malignant catarrhal fever may be possible.  相似文献   

12.
Bovine viral diarrhea virus (BVDV) affects cattle populations causing clinical signs that range from subclinical immunosuppression to severe reproductive and respiratory problems. Detection and removal of persistently infected (PI) calves is the single most important factor for control and eradication of BVDV. Current testing strategies to detect PI calves rely heavily on immunohistochemistry (IHC) and a commercially available antigen capture ELISA (ACE) assay. These viral assays depend on 1 or 2 monoclonal antibodies which target the E(rns) glycoprotein of BVDV. The sensitivity and specificity of these two tests have been reported previously. The purpose of this research was to characterize a strain of BVDV (AU501) that was undetectable using IHC and ACE based on a single monoclonal antibody, but was consistently detected in samples from a Holstein steer using virus isolation and PCR testing. Sequencing of this AU501 viral isolate revealed a unique mutation in the portion of the genome coding for the E(rns) glycoprotein. This unique field strain of BVDV demonstrates the risk of relying on a single monoclonal antibody for detection of BVDV. Multiple testing strategies, including polyclonal or pooled monoclonal antibodies that detect more than one viral glycoprotein may be necessary to detect all PI calves and facilitate eradication of BVDV.  相似文献   

13.
This study was aimed to establish an indirect ELISA to detect antibodies against different strains of porcine epidemic diarrhea virus (PEDV). Puried nonstructural protein 7(Nsp7) was used as coating antigen, and the indirect ELISA was established by optimizing the ELISA reaction conditions. The results showed that the optimal reaction conditions were as follow:The amount of coating antigen was 0.20 μg/well, and the coating condition was at 37℃ incubation for 1 h then 4℃ overnight; The working dilution of serum samples and HRP-labelled secondary antibody were 1:300 and 1:10 000, and the incubation time were 2 and 1.5 h, respectively; TMB substrate incubation time was 15 min. Serum sample was determined as positive when its S/P>0.1694 and negative when its S/P<0.1398. The ELISA was specific, reproducible and sensitive. Forty samples of suspected PEDV serum samples were tested by the established ELISA, and the coincidence rate between the ELISA and the commercial kit was 95%. The ELISA established in this study could be used clinically to detect the antibody level of different strains of PEDV, and it also had the potential for early diagnosis of PEDV, providing a basis for the development of effective measures to control PEDV.  相似文献   

14.
The bluetongue virus (BTV) core particle contains 2 major polypeptides, P3 and P7, and is surrounded by an outer capsid layer that is composed of the 2 major polypeptides, P2 and P5. Analysis of the immune precipitates from soluble 14C-labelled BTV polypeptides and hyper-immune rabbit and guinea-pig sera indicated that polypeptide P2 precipitates only with homologous BTV sera. This would indicate that P2 is the main determinant of serotype specificity. It was also found that in sheep infected with BTV the P2-precipitating antibodies in the serum correlate with the neutralizing antibody titres, whereas the appearance and subsequent decline of P7-precipitating antibodies correspond well with those of the complement fixing antibodies. This suggests that BTV group specificity, as measured by a complement fixation tests, is determined by the core protein P7. This result was supported by the observation that mouse ascitic fluid, which contains a high titre of BTV-specific complement fixing antibodies and a very low titre of neutralizing antibodies, contains almost exclusively antibodies that precipitate P7.  相似文献   

15.
Four Neospora-seropositive pregnant cows (prebreeding indirect fluorescent antibody (IFA) titers between 1:400 and 1:1600) were confined and observed until parturition. All cows gave birth to normal calves. Selected tissues were tested for NC by histopathology, immunohistochemical (IHC) and polymerase chain reaction (PCR). Parasite isolation was attempted in vero cell cultures. At parturition, all cows were seronegative at 1:200 and two of four cows had a titer of 1:100 when further tested. Three of four calves were not infected, as determined by negative results of precolostral serology (1:25 cut-off), histopathology, IHC and PCR. One calf was congenitally infected, as shown by the presence of a thick-walled cyst labelled by IHC in its brain, positive PCR of brain and a precolostral IFA titer of 1:100. It was concluded that NC antibody titers may drop or convert to seronegative status in chronically infected cows by the time of parturition and this finding in four of four cows indicates that this could be a common occurrence. Similarly, the finding of an infected calf with a low antibody titer indicates that precolostral serology may not be a fool-proof means of identifying calves with congenital Neospora caninum infections. These findings call into question conclusions of other studies that have estimated rates of congenital transmission of this parasite based on serological tests at calving. This study is the first confirmed report of congenital NC infection in a calf in Thailand.  相似文献   

16.
The occurrence of group specific complement fixing antibodies was studied in random sera of cattle and reindeer in Finnish Lapland. Sixty-eight (40.5%) of the 168 cattle sera were positive. Sixty 4hree (21.6 %) of the 291 reindeer sera were positive. The difference is statistically nearly significant in the t-test. The antibody titer ≥ 1:16 was regarded as positive. The antibody frequency of cattle sera was statistically significantly higher and the antibody frequency of reindeer sera was nearly significantly higher than in earlier studies on cattle sera in South and Central Finland. The reasons are discussed.  相似文献   

17.
A seroepidemiological study of Ehrlichia canis was made in police dogs in Madrid (Spain). AntiEhrlichia canis antibodies were detected by indirect fluorescent antibody test for the detection of IgG.

The results obtained (three positive dogs out of a population of 131 animals) represents a seroprevalence of canine ehrlichiosis of 2.29%. This seroprevalence is one of the lowest described for this type of population.

The seroprevalence obtained from hunting dogs kennelled in Madrid in 1993 was 66.7% (24 positive out of a population of 36). Environmental conditions (temperature, humidity, rainfall, etc.) in both populations were very similar. We suggest that these conflicting results are due to the different prophylactic programmes used in these two populations.  相似文献   


18.
本试验旨在建立特异检测牛奶蛋白质含量的ELISA方法。利用提纯酪蛋白免疫试验动物,获得针对酪蛋白的多克隆抗体;利用棋盘格试验确定抗原抗体的最佳作用浓度,并优化各步骤反应条件;根据标准样品建立牛奶中蛋白质含量的定量ELISA检测方法,并对该方法的特异性、敏感性、检测范围及重复性进行评价。通过试验确定封闭液为1% BSA,37 ℃孵育120 min;血清抗体工作浓度为1∶12000稀释,37 ℃孵育90 min;酶标二抗最适浓度为1∶3000稀释,37 ℃孵育30 min。牛奶酪蛋白检出范围为0.063~0.500 μg/mL;该方法具有良好的特异性、敏感性、重复性,回归率范围为97.6%~123%。成功建立了牛奶蛋白含量的定量ELISA检测方法。  相似文献   

19.
Ewes infected before gestation with chicken embryo or tissue culture adapted chlamydial strain B-577 were challenge inoculated with the homologous strain at four to 18 weeks of gestation. The ewes responsed with group specific complement fixing antibody titers of 1:8 to 1:256 by the second week after initial infection. A secondary antibody response in the surviving challenge inoculated ewes occurred at the time of lambing and reached titers of 1:32 to 1:256 by the second week after parturition. Group specific complement fixing antibodies did not appear to play a significant role in resistance to chlamydial infection. Ewes infected with the chicken embryo adapted strain B-577 excreted chlamydiae in their feces 60 days after inoculation. However, chlamydiae were not recovered from feces of ewes infected with the tissue culture adapted strain B-577. Placentas of ewes challenge inoculated by the intravenous route were consistently infected. Chlamydiae were recovered from placentas, some fetuses and lambs. In two instances when challenge inoculation was given by the intramuscular route, infection was detected only by the direct fluorescent antibody method.  相似文献   

20.
The prevalence of antibodies to Neospora caninum was examined in European bison (Bison bonasus bonasus L.) living in free and fenced areas in Poland. Sera of 320 European bison, different ages and sexes, from breeding areas in Poland were tested for N. caninum antibodies using ELISA test. Positive antibody responses were found in 23 bison (prevalence 7.3%). Additionally, all positive sera were tested by Western blot to verify the ELISA results. The Western blot results confirmed the presence of antibodies to Neospora tachyzoites antigens in all 23 sera tested. The antibodies were detected against a wide range of NC-1 tachyzoite antigens. The antibody responses were directed against proteins at: 9.5, 17, 21, 27, 31, 36.5, 38, 40, 43, 47, 48.5, 53.5 and 58 kDa. The most heavily stained bands had molecular weights of 9.5, 17, 27 and 58 kDa. The most important is that antibody to N. caninum was detected for the first time in sera from bison cow shot in 1988. It is the year of recognition of this protozoan parasite. Our results indicate strongly the presence of N. caninum in European bison in Poland and suggest that a sylvatic cycle of N. caninum can exist. However, further studies are needed to evaluate the existence of a sylvatic cycle of N. caninum. The study on the effect of the infection on the health status and conservation of European bison should be taken under consideration too.  相似文献   

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