Evaluation of a nonradioactive colorimetric assay for analysis of lymphocyte proliferation in healthy cats

OBJECTIVES: To compare results of a nonradioactive colorimetric microplate assay with results of a traditional radioactive proliferation assay for determination of its use as a reliable and accurate alternative method for determination of proliferative activity of feline lymphocytes. SAMPLE POPULATION: Blood samples from 10 clinically normal domestic shorthair cats. PROCEDURE: Double-density gradient separation was used to isolate mononuclear cells. Isolated cells were stimulated with various concentrations of concanavalin A (Con-A) and cultured for 72 hours. Lymphocyte proliferation was measured by radioactive ([3H]thymidine) and nonradioactive (colorimetric) techniques. Immunophenotypic analysis with feline-specific CD4+ and CD8+ monoclonal antibody was performed, using flow cytometry. RESULTS: Mononuclear cells were successfully isolated (97 to 99% purity and viability) from blood samples. A similar dose-dependent proliferative response to Con-A stimulation was measured with [3H]thymidine incorporation and the colorimetric assay. For both techniques, concentrations of 0.1 and 1.0 microg of Con-A/ml were submitogenic, and 100 microg/ml was toxic to cultured cells. For both techniques, maximal proliferation was observed with 5 microg of Con-A/ml. CONCLUSIONS AND CLINICAL RELEVANCE: These results indicate that the nonradioactive colorimetric technique is a reliable and accurate method for measuring proliferative activity of feline lymphocytes. Clinically, this assay can be used as part of a screening process to determine immunocompetence of at-risk cats and to evaluate treatments for cats with immune-mediated or T-cell-dependent diseases.     

Evaluation of canine lymphocyte proliferation: comparison of three different colorimetric methods with the 3H-thymidine incorporation assay.

To evaluate canine lymphocyte stimulation the radioactive thymidine incorporation assay is still the method of choice. In order to find a suitable non-radioactive alternative to the standard 3H-thymidine incorporation assay, proliferation of canine peripheral blood lymphocytes (PBL) was measured with three different colorimetric assays, using the two tetrazolium salts MTT and XTT and 5-bromo-deoxyuridine (BrdU). Isolated canine PBL were stimulated with two different mitogens, Concanavalin A (Con A) and Phytohemagglutinin (PHA), using different culture conditions. Applying statistical analysis we found that BrdU and MTT showed a high correlation to the 3H-thymidine incorporation assay, although the BrdU assay proved to be more sensitive than the MTT assay. No significant correlation between the XTT assay and the radioactive method was demonstrated. Consequently, the BrdU assay is the most suitable alternative to the radioactive method.     

Comparison of assays for the detection of West Nile virus antibodies in chicken serum

Six tests for the detection of West Nile virus (WNV) antibodies in the serum of experimentally infected chickens were compared. The tests included the hemagglutination-inhibition test (HIT), immunoglobulin M (IgM)-capture enzyme-linked immunosorbent assay (ELISA) with WNV-infected mouse brain antigen, immunoglobulin G (IgG) indirect ELISA with tickborne encephalitis viral antigen, the microtitre virus neutralization test, the standard plaque reduction neutralization test (PRNT), and the microtitre PRNT (micro-PRNT). Thirty adult chickens, intravenously and intramuscularly inoculated with 10⁷ plaque-forming units (PFU) of WNV strain Egypt 101, were bled and given a booster of 10⁷ PFU at 7, 15, and 21 d postinoculation; the final blood collection was on day 28. Although the micro-PRNT is capable of detecting the highest antibody titres during both early and late infection, because of the technical complexity and time requirements of this test a combination of IgM and IgG ELISAs is recommended for serologic screening. Serum samples that give positive results in the ELISAs can then be tested by the micro-PRNT to determine the specificity of antibodies to WNV.     

Comparison of intradermal injections of bromodeoxyuridine and tritiated thymidine in the in vivo measurement of epidermal cell turnover time in goats.

Study of keratinisation defects such as seborrhoea may require investigation of epidermal cell kinetics in clinical cases. For this to be practically useful a safe, reliable and quick method is essential. This study compared epidermal cell kinetics in the goat following the use of intradermal injection of tritiated thymidine ([3H]TdR) and bromodeoxyuridine (BURD) to label S phase nuclei. Turnover time for goat epithelial cells was not significantly different for the two agents; 26.3 +/- 6.0 days for thymidine and 21.8 +/- 5.2 days for BURD. It was concluded that intradermal injection of BURD will be a suitable technique for investigating epidermal cell kinetics in vivo and could have applications in a clinical situation because it is quicker and safer than [3H]TdR.     

Estimation of S phase duration in goat epidermis by an in vivo intradermal double labeling technique using bromodeoxyuridine and tritiated thymidine.

When studying skin diseases resulting from alterations in the rate of epidermal cell turnover it is useful to be able to quantify parts of the epidermal cell cycle. An in vivo intradermal technique is described which uses tritiated thymidine followed by bromodeoxyuridine to label cells in the S phase of the cell cycle. Combined autoradiographic and immunocytochemical techniques were used to quantify the flux of cells into and out of S phase. These results were then used to estimate the length of S phase. The technique was found to provide clear distinctive labelling of S phase nuclei with both reagents. This avoided many of the problems encountered with double labelling techniques using two radioactive labels. S phase was calculated to be 7.7 hours for goat skin.     

硫酸化人参总皂苷的制备及其对鸡外周血淋巴细胞增殖的影响

用氯磺酸-吡啶试剂制备了硫酸化人参总皂苷衍生物，采用红外光谱仪检测到制备的硫酸化人参总皂苷衍生物中在1230cm^-1和810cm^-1有硫酸酯键的特征吸收峰。为了初步探讨硫酸化人参总皂苷的免疫学活性．以MTT法测定了人参总皂苷及其衍生物对鸡外周血淋巴细胞增殖的影响。结果表明，150μg／m1人参总皂苷和衍生物均能显著抑制鸡外周血淋巴细胞的增殖（P〈0．05），10μg／ml的衍生物能极显著促进淋巴细胞增殖，与同浓度人参总皂苷相比活性极显著增强（P〈0．01）。实验结果提示，对人参总皂苷进行硫酸化修饰是可行的，经硫酸化后人参总皂苷衍生物的免疫活性明显增强。     

A routine diagnostic assay for the detection of benzimidazole resistance in parasitic nematodes using tritiated benzimidazole carbamates

Resistance to benzimidazoles (BZs) in parasitic nematodes has recently been shown to be due to a reduction in the ability of BZs to bind the structural protein, tubulin, in resistant isolates. Based on these observations the development and standardisation of a routine diagnostic assay has been undertaken by measuring the binding of tritiated mebendazole to crude supernatants of L3 larvae. The assay is rapid, requiring less than 2 h, and is robust, highly reproducible and sensitive to minor changes in the resistance status of parasite populations. Investigation of the routine application and validity of this assay has been documented using 24 isolates of known resistance status from the species Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta: In all cases the observed binding and calculated susceptibility factors were in accordance with their respective resistance status.     

Determination of BHV1 specific immune reactivity in naturally infected and vaccinated animals by lymphocyte proliferation assays

The in vitro BHV1-specific lymphocyte stimulation assay was used to investigate immune reactivity of cattle after natural infection or vaccination with BHV1. Proliferative responses to live virus were shown in tests with peripheral blood lymphocytes of seropositive field virus-infected animals and of vaccinated animals. Nineteen out of 36 seropositive field virus-infected animals did not show in vitro responses. Nine out of 12 animals showed, at least transient, responsiveness after vaccination. Antibody titers were maintained throughout the observation period. T cell activity is believed to play a role in protection against BHV1 infection. The in vitro proliferative assay, however, can not discriminate between BHV1 seropositive and seronegative field virus-infected animals. After vaccination, the BHV1-specific lymphocyte responses of at least one animal disappeared. Both observations may point to the fact that T cell memory is generated, or at least systemically present, to a limited extent.     

A rapid and simplified technique for the assay of chicken hemolytic complement.

The technique described is a modification of a qualitative hemolytic radial diffusion technique. The test involves the use of sensitized sheep erythrocytes that have been incorporated into agarose. Tube dilutions were made of chicken serum and samples of each dilution were placed into wells cut in the agarose. The test is quantitative for hemolytic complement in that the highest dilution showing visible hemolysis of sensitized erythrocytes in agarose is determined to be the endpoint for that serum sample. The test as compared with the standard tube assay was determined to be less sensitive by approximately one dilution. The advantages of speed, simplicity, and cost more than offset the decrease in sensitivity of the test.     

Use of a soluble tetrazolium/formazan assay for chicken cells.

We evaluated a soluble tetrazolium/formazan assay using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl ]-2H- tetrazolium hydroxide (XTT) for chicken cell growth. Fifty microliter of solution containing 1 mg/ml of XTT and 0.025 mM phenazine methosulfate was added to the cells in a well of 96-well microplate. After 4 hr incubation at 37 degrees C, the absorbance was measured at 490 nm. Under this condition, absorbances were well correlated with cell number of Marek's disease tumor cells and chicken embryo fibroblasts. Proliferation of chicken lymphocytes stimulated with mitogens was also effectively measured. The formazan of XTT is water-soluble and can be quantitated in culture medium without the necessity for extraction with organic solvents. Thus XTT assay is simple and useful for the quantity assay with chicken cells.     

Detection and differentiation of Mycoplasma gallisepticum and M. synoviae antibodies in chicken serum using enzyme-linked immunosorbent assay

Affinity-purified sheep IgG anti-chicken IgG horseradish peroxidase conjugate was utilized in an enzyme-linked immunosorbent assay (ELISA) to detect Mycoplasma gallisepticum- and M. synoviae-specific antibodies in chicken sera. Antigen, conjugate and substrate concentrations, and incubation times were adjusted to provide maximum differentiation between positive and negative sera. Use of phosphate-buffered saline containing 0.05% Tween 20 for washing and diluting steps and use of normal sheep serum to make the initial 1:10 serum dilution resulted in optimal differentiation between homologous and heterologous antisera. However, sera known to contain antibodies to M. gallisepticum or M. synoviae gave higher absorbance values with the heterologous antigen than did specific-pathogen-free sera. To reduce the frequency of nonspecific reactions to less than 2%, it was necessary to adjust the threshold absorbance for each antigen according to the known infectious status of the flock. Reproducibility of the assay was maintained by using positive and negative control sera on each plate. Results from 14.2% of the plates tested were rejected, because the endpoint of the positive control serum was more than one dilution from the most common value. Of four strains of M. gallisepticum used as antigens, none was clearly superior to the others in producing maximum titers with a range of M. gallisepticum antisera. However, nonspecific absorbance tended to be less with the S6 strain. The stability of M. gallisepticum-coated plates was maintained for up to 6 months at -8 C or below, whereas M. synoviae-coated plates were stored satisfactorily for 6 months at 4 C or below.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of an automated colorimetric assay for the measurement of lipase activity in canine sera.

An automated colorimetric method for determining lipase activity in canine sera was evaluated for precision, linearity and correlation to existing assay methods. The colorimetric method was a commercial reagent that used a series of enzymatic reactions based on the hydrolysis of 1,2 diglyceride by pancreatic lipase. Within-run and between-run coefficients of variation were < 6.8% and < 8.3%, respectively. Linearity was determined to be at least 1366 U/L. Canine serum lipase concentrations attained using the colorimetric method were compared to both titrimetric and dry-film methods for measuring serum lipase activity, resulting in significant (P < or = 0.05) correlation coefficients of 0.92 and 0.77, respectively. Canine serum lipase concentrations measured using the colorimetric assay on 2 different automated analyzers had a significant (P < or = 0.05) correlation coefficient of 0.92. A laboratory reference range using serum samples from 56 healthy dogs (0-561 U/L) was established. There were no significant (P < or = 0.05) differences in mean serum lipase concentrations comparing male and female dogs or comparing young dogs (< or = 3 y) to mature (4-7 y) and older (> 7 y) dogs using this assay. It was concluded that the automated colorimetric assay was a reliable indicator of canine serum lipase activity and offered several advantages, including small sample volume and short analysis time.     

Comparison of the intravenous chicken challenge method with the embryo lethality assay for studies in avian colibacillosis

In previous studies, the embryo lethality assay (ELA) was able to discriminate between virulent and avirulent avian Escherichia coli isolates and to predict percent mortality of the embryos resulting from an isolate based on three traits. The abilities to resist host complement, presence of Colicin V activity, and presence of the increased serum survival gene cluster (iss), were used together in a logistic regression analysis to predict the percentage of embryo deaths resulting from each of 20 avian E. coli isolates used in the ELA. In the present study, the same 20 isolates are used in an intravenous chicken challenge model in an effort to determine whether the ELA could be used to replace chicken challenge studies. Correlations between the mortality and a combination of mortality and morbidity (the survivors at trial termination with lesions suggestive of colibacillosis) and the previous ELA results and with selected isolate traits were performed. Additionally, resulting body weights in surviving chickens were compared between groups. The highest positive correlations were observed between the ELA and the combined mortality/morbidity of the intravenous challenge (r = 0.861, P < 0.0001 for the first ELA challenge, and r = 0.830, P < 0.0001 for the second ELA challenge). The IV challenge combined mortality/morbidity results had the highest correlation coefficients with the presence of iss (r = 0.864, P < 0.0001) and the expression of ColV (r = 0.878, P < 0.0001). The presence of tsh was slightly correlated with mortality (r = 0.465, P = .0389) but demonstrated a higher correlation with the combined mortality and morbidity of the IV challenge (r = 0.558, P = 0.0106). Even though the ELA results in a higher number of nonspecific deaths, the two challenge methods exhibit similar results and a high correlation with each other. Interestingly, some of the isolates showed differences in their ability to cause mortality between the ELA and the IV challenge model. Furthermore, some isolates reflected significant differences in body weights of surviving birds at IV trial termination.     

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.     

Comparison of two enzyme-linked immunosorbent assays for diagnosis of Mycobacterium avium subsp. paratuberculosis.

Enzyme-linked immunosorbent assays (ELISAs) are used in Johne's disease (JD) control programs as a first screening for presence of the disease in a herd. A high sensitivity of the ELISA is therefore important, yet the commonly used ELISAs have relatively low sensitivity. The inclusion of an absorption phase, although improving specificity, potentially decreases sensitivity. Sera and feces of 383 adult dairy cows in 8 herds were used to compare the test characteristics of an absorbed and a nonabsorbed indirect ELISA for the detection of JD. The absorbed ELISA is based on a protoplasmic antigen, whereas the nonabsorbed uses a lipoarabinomannan-based antigen. The potential advantage of the nonabsorbed ELISA is that it may be less specific and more sensitive. Two herds certified free of JD were used to compare the specificity of the ELISAs. The other herds used to compare sensitivity were either infected with Mycobacterium avium subsp. paratuberculosis or had unknown status. Using fecal culture as a gold standard, the diagnostic specificity for the absorbed and nonabsorbed ELISAs were 98.4% and 87.9%, respectively. The diagnostic sensitivity was 72.4% and 65.5% for the absorbed and the nonabsorbed ELISA, respectively. Furthermore, a comparison using a fecal DNA probe as the comparison standard resulted in both ELISAs having a sensitivity of 61.9%. Agreement between the 2 ELISAs was moderate, with a kappa statistic of 0.58. The nonabsorbed ELISA did not have a higher sensitivity and had a lower specificity than the absorbed ELISA. Therefore, in this population, there was no advantage gained with using the nonabsorbed ELISA.     

Comparison of the dot blot hybridization assay with antigen detection assays for the diagnosis of infectious bursal disease virus infections

The use of cDNA probes as diagnostic tools for detecting infectious bursal disease virus (IBDV) antigens was compared with the agar-gel precipitin (AGP) and immunofluorescence (IF) assays. Specific-pathogen-free chickens were inoculated with the STC or IN strains of IBDV in three separate experiments. Tissue samples were collected at various intervals postinoculation (PI) and examined for viral antigens using the IF assay, the AGP assay, and the hybridization assay. Viral antigen was detected by the AGP assay for a period of approximately 3 days beginning 2 days PI and by the IF assay for approximately 4 days beginning 2 days PI. Virus was detected by the hybridization assay through the length of all the experiments (longest experiment was 24 days) beginning 1 day PI. These results suggested that the hybridization assay is more sensitive than the IF assay and the AGP assay.     

Adaptation of a colorimetric microtitration assay for quantifying Pasteurella haemolytica A1 leukotoxin and antileukotoxin

A colorimetric microtitration assay was adapted to quantify the cytotoxicity of Pasteurella haemolytica A1 leukotoxin to bovine neutrophils used as target cells. The viability of leukotoxin-treated target cells was detected by use of a tetrazolium dye that living cells reduced to dark blue formazan. The amount of formazan formed (which was quantified by use of an ELISA plate reader) was directly proportional to the number of viable target cells. This assay system also was used to measure leukotoxin-neutralization antibody titers of bovine serum and lung lavage specimens obtained during vaccination experiments. The major advantages of this assay over other methods such as the 51Cr-release and trypan blue-exclusion assays are precision, rapidity, and low cost; it also does not use radioisotopes.     

Application of an MTT reduction assay for assessing sperm quality and predicting fertilising ability of domestic fowl semen

1. The ability of domestic fowl spermatozoa to reduce MTT tetrazolium to its coloured formazan was compared with other tests of sperm quality and fertilising ability. 2. MTT reduction was highly correlated with sperm ATP content (r2 = 0.85); sperm mobility (r2 = 0.62.); sperm:perivitelline layer interaction (r2 = 0.80) and fertilizing ability (r2 = 0.83). 3. The simple, robust, MTT-reduction assay may therefore be used to select male chickens on the basis of their sperm quality and thus potential fertilising ability.     

Comparison of a new gold-immunochromatographic assay for the detection of antibodies against avian influenza virus with hemagglutination inhibition and agar gel immunodiffusion assays

A gold-immunochromatographic test-strip kit is used for the detection of IgG antibodies against the nucleocapsid protein of Avian Influenza Virus (AIV). Compared with the "gold standard", i.e. hemagglutination inhibition (HI) and agar gel immunodiffusion (AGID) assays, the gold-immunochromatographic test strip has many advantages, such as high specificity, high sensitivity, convenience, is rapid and has low cost. The gold-immunochromatographic test strip provides a unique tool for the on-site surveillance and diagnosis of Avian Influenza.     

Comparative studies of mitogen- and antigen-induced lymphocyte proliferation in four captive rhinoceros species.

Cellular immune function in four rhinoceros species was evaluated by way of in vitro lymphocyte proliferation responses to mitogenic and antigenic stimuli to establish normative data on white blood cell activity for each species and to identify species-specific differences that might help explain the predisposition of black rhinoceroses (Diceros bicornis) to disease. A cross section of the U.S. rhinoceros population encompassing all four captive species was sampled, including the Sumatran rhinoceros (Dicerorhinus sumatrensis) (n = 3); Indian rhinoceros (Rhinoceros unicornis) (n = 4); African black rhinoceros (n = 16); and African white rhinoceros (Ceratotherium simum) (n = 10). Of the four species evaluated, African black rhinoceroses exhibited the weakest (P < 0.05) lymphocyte proliferative responses to the mitogens: pokeweed (0.1 microg/ml), phytohemagglutinin (0.3 microg/ml), and concanavalin A (5.0 microg/ml). Total cell density at the end of culture was only 70% of that achieved with lymphocytes isolated from African white rhinoceroses, Indian rhinoceroses, and Sumatran rhinoceroses. However, lymphocyte response to bacterial endotoxin lipopolysaccharide was similar (P > 0.05) across species. Antigenic stimulation produced much weaker responses than mitogenic stimulation. No differences (P > 0.05) were observed among rhinoceros species in response to 1 and 10 microg/ml of Leptospira icterohemorrhagiae or Leptospira gryppotyphosa. Lymphocytes from African white rhinoceroses proliferated weakly in the presence of Aspergillus fumigatus filtrate, whereas lymphocytes from the southern black rhinoceros subspecies appeared slightly suppressed in the presence of increasing doses (0.1, 1, and 10 microg/ml) of Aspergillus filtrate. This comparative data set characterizing lymphocyte proliferation in the rhinoceros reveals several differences in immune cell responses among rhinoceros species and provides some evidence that lymphocytes of captive African black rhinoceroses are less vigorous than those of the other rhinoceros species.