Higher Absorbance Values Research Articles

Efficacy of different immunogens for raising monoclonal antibodies to Botrytis cinerea

A panel of monoclonal antibodies (MAbs) was raised to Botrytis cinerea using four different immunogens. Co-immunization with cross-reactive monoclonal antibodies helped to increase the percentage of specific hybridoma cell lines produced. The abilities of the MAbs to detect fungal antigens in extracts from infected tissue was correlated with their taxonomic specificities and with the localization of the antigens on the surfaces of the hyphae and conidia. Four distinct groups were identified: taxonomically unrelated, near-genus-, genus- and near-isolate-specific. Genus-specific antibodies gave the highest absorbance values in ELISAs with extracts from infected plant tissues. MAbs from this group all recognized heat-stable epitopes on antigens expressed along the length of the extracellular matrix of the hyphae and surface of the conidia. Antibodies from this group were all IgG1 antibodies.

Correlations between symptoms and DAS - Elisa values in two sources of resistance against tomato spotted wilt virus

The commercial tomato, Lycopersicon esculentum, cultivars Ângela Gigante I-5100 and Santa Clara (tospovirus susceptible), the Stevens cultivar, lines and experimental hybrids (tospovirus resistant), were used: a) to evaluate two sources of resistance to tospoviruses in tomato (one from L. esculentum/Rey de Los Tempranos, and another one from L. peruvianum/Stevens); and b) to verify statistically significant correlation between virus concentration determined through DAS-ELISA and symptomatology. In the Ângela Gigante I-5100 and the Santa Clara cultivars, the correlation coefficient was highly significant and the symptom average level was high, showing that in susceptible cultivars the symptom evolution is related to the virus multiplication in the plant. As for the TOM 547 and TOM 556 lines (background of Ângela Gigante I-5100 and Santa Clara, respectively), there was no statistical correlation between the symptoms and the viral particle concentration. Plants with light virus symptoms showed high absorbance values. Consequently we may assume that the resistance with `Rey de Los Tempranos' background may be of the tolerant kind because the high virus concentration found does not necessarily relate to a high level of symptoms. In the lines BPX320E 3902-01, BPX320E 3905 and BPX320F 7902 (both with Santa Clara background) only few plants showed symptoms, which can be explained by incomplete penetrance of Sw-5 gene. In the case of non-symptomatic plants, the diagnosis using DAS-ELISA revealed negative results. That outcome indicates that in some materials, where resistance is obtained from L. peruvianum, the virus multiplication in the tissues seemed not be present.

Monovalent binding of autoantibodies to beta2-glycoprotein I, detected using surface plasmon resonance at low antigen density.

The precise mechanism of interaction between autoantibodies and beta2-glycoprotein I (beta2GPI) and the experimental conditions to be used for their detection are still under debate. Until now, these interactions have been studied under static conditions. We have investigated the interactions of purified IgG from 25 lupus anticoagulant-positive patients with immobilized beta2GPI under flow conditions by real-time analysis based on surface plasmon resonance technology. Sensor chips were coated with purified human beta2GPI coupled to dextran via amino groups at low densities (1.4, 1.8 or 2. 4 ng beta2GPI/mm2). Four patients' IgG displayed efficient binding and had the highest so-called antiphospholipid IgG levels by enzyme-linked immunosorbent assay (ELISA) and the highest absorbance values in an anti- beta2GPI ELISA at a beta2GPI density reported to be around 12 ng/mm2. Binding of antibodies to the beta2GPI sensor chips proved to be dependent upon the IgG concentration and beta2GPI density and was inhibited by a rabbit antibody against beta2GPI. Similar association and dissociation profiles were observed for the four efficient binders. The fast rate of dissociation limited the binding of autoantibodies to beta2GPI and was highly suggestive of a monovalent association, confirmed by binding of Fab fragments under similar experimental conditions. In conclusion, monovalent binding of low-affinity antibodies to beta2GPI immobilized at a density as low as 1.8 ng/mm2 could be detected using surface plasmon resonance.

Detection of cucumber mosaic cucumovirus in weed species: a cautionary report on nonspecific reactions in ELISA

The reliability of enzyme-linked immunosorbent assay (ELISA) to detect cucumber mosaic cucumovirus (CMV) coat protein (CP) in three weed species (Taraxacum officinale, Gnaphalium obtusifolium, and Conyza canadensis) was compared with other detection methods, including Western blot analysis and a bioassay. Analysis of extracts prepared from these species by double antibody sandwich (DAS) ELISA yielded high absorbance values (A4O5nm) that were indicative of CMV concentrations of purified CMV standards of greater than 50 mg/mL. However, results from an ELISA procedure designed such that no viral antigen should be detected suggested that the strong ELISA absorbance values were nonspecific reactions with material(s) in the weed extracts. These findings were substantiated when no CMV CP was detected by Western blot analysis or infectious CMV was detected in bioassays involving inoculation of extracts onto an indicator host (Cucurbita pepo ‘Crookneck’ (squash)). In both DAS ELISA and indirect ELISA procedures, the nonspecific reaction was shown to result from the interaction of weed extract and alkaline phosphatase-conjugated immunoglobulin (including anti-CMV and goat anti-rabbit). Addition of bovine serum albumin to the extraction buffer for DAS ELISA or to the extraction buffer and at each antibody step for indirect ELISA dramatically reduced the nonspecific reactions.

Specificity of TAS-ELISA for Beet Necrotic Yellow Vein Virus and Its Application for Determining Rhizomania Resistance in Field-Grown Sugar Beets.

Levels of beet necrotic yellow vein virus (BNYVV), as measured by triple-antibody sandwich-enzyme-linked immunosorbent assay (TAS-ELISA), were compared with biological evaluations in representative commercial and experimental sugar beet cultivars developed for production in the United States and ranging in their reactions to rhizomania from uniformly susceptible to highly resistant. TAS-ELISA was specific for BNYVV and did not react with related soilborne sugar beet viruses. Differences in absorbance (A405nm) values measured in eight cultivars closely correlated with the dosage and frequency of the Rz allele, which conditions resistance to BNYVV. A diploid (Rzrz) hybrid had a significantly lower absorbance value (less virus) than a similar triploid (Rzrzrz) hybrid. Cultivars that segregated (Rzrz:rzrz) had higher absorbance values than uniformly resistant (Rzrz) hybrids, as was expected. For all cultivars, absorbance values decreased as the season progressed. Absorbance value was significantly positively correlated with rhizomania disease index score and negatively correlated with individual root weight, plot root weight, and sugar yield. This information should be useful in resistance-breeding and -evaluation programs and in the sugar industry when considering cultivar choice, inoculum production, and future crop rotations.

Isolation of Lyme disease Borrelia from puffins (Fratercula arctica) and seabird ticks (Ixodes uriae) on the Faeroe Islands.

This is the first report on the isolation of Lyme disease Borrelia from seabirds on the Faeroe Islands and the characteristics of its enzootic cycle. The major components of the Borrelia cycle include the puffin (Fratercula arctica) as the reservoir and Ixodes uriae as the vector. The importance of this cycle and its impact on the spread of human Lyme borreliosis have not yet been established. Borrelia spirochetes isolated from 2 of 102 sampled puffins were compared to the borreliae previously obtained from seabird ticks, I. uriae. The rrf-rrl intergenic spacer and the rrs and the ospC genes were sequenced and a series of phylogenetic trees were constructed. Sequence data and restriction fragment length polymorphism analysis grouped the strains together with Borrelia garinii. In a seroepidemiological survey performed with residents involved in puffin hunting on the Faeroe Islands, 3 of 81 serum samples were found to be positive by two commonly used clinical tests: a flagellin-based enzyme-linked immunosorbent assay (ELISA) and Western blotting. These three positive serum samples also had high optical density values in a whole-cell ELISA. The finding of seropositive Faeroe Islanders who are regularly exposed to I. uriae indicate that there may be a transfer of B. garinii by this tick species to humans.

Evaluation of epidemiological and serological predictors of human immunodeficiency virus type-1 (HIV-1) infection among high risk professional blood donors with western blot indeterminate results.

Indeterminate pattern of results in Western blot (WBI) for human immunodeficiency virus type-1 (HIV-1) infection may represent early HIV-1 infection or may be non-specific in origin. This issue can be resolved by follow up testing upto at least 6 months resulting in psychological distress as well as in high drop out rates among those undergoing investigation pointing out the need for additional parameters that could help in determining the status of HIV-1 infection at the time of initial testing itself in individuals with WBI pattern. The objectives of the present study were: (i) to determine the frequency of HIV-1 infected individuals in a group of professional donors showing WBI patterns in initial testing on the basis of follow up serological studies; (ii) to find out if any HIV related epidemiological or serological characteristics recorded at the time of initial testing could be considered as predictor for HIV-1 infection in WBI specimens; and (iii) to evaluate two alternative serodiagnostic strategies for HIV-1 infection viz. multiple EIAs based on different antigen preparations/principles and a line immunoassay (LIA) employing recombinant antigens in resolving status of HIV-1 infection in specimens showing WBI results at initial testing. Professional donors with WBI patterns belonging to EIA reactive and EIA nonreactive groups were subjected to study of epidemiological profile, prevalence of sexually transmitted diseases (STD) markers and follow up serological testing for HIV-1 at 6, 12, 24 and 48 weeks intervals to record any seroconversion. The initial and follow up specimens from the donors with initial WBI results were subjected to two EIAs (one based on dot immunoassay using synthetic HIV-1 antigens and other based on microwell EIA using recombinant HIV-1 proteins) as well as to LIA. Professional donors with initial WBI results, from the EIA reactive group had higher proportion of unmarried individuals (90.3%), with history of heterosexual promiscuity (75%) and visit to STD clinics (36.1%) compared with the WBI donors from the EIA nonreactive group (72.7, 42.4 and 12.1%, respectively, P values < 0.001). Prevalence of antitreponemal antibodies was higher in the former group (16.7%) compared with the later group (1.5%, P value < 0.002). Seroconversion was recorded in 4 (7.3%) out of 55 EIA reactive WBI donors from the EIA reactive group that were characterised by high optical density (OD) values in EIA, 'p24 only' pattern of band in WB and positivity by LIA at the time of initial testing. LIA was found to be more reliable test compared with combination of EIAs to determine status of HIV-1 infection in WBI specimens at the time of initial testing. The present study points out that parameters like history of heterosexual promiscuity, prevalence of STD markers, high OD values in screening EIA, 'p24' only pattern of bands in WB and positivity by LIA could have individual predictive values for HIV-1 infection in specimens showing WBI pattern of results at initial testing.

Antibodies against human putamen in children with Tourette syndrome.

Similar to the model for Sydenham's chorea, antineuronal antibodies, which develop in response to a preceding streptococcal infection, have been speculated to have a role in the development of Tourette syndrome (TS). Serum antibodies against human caudate, putamen, and globus pallidus (interna and externa) were assayed by enzyme-linked immunosorbent assay (ELISA) and Western blot techniques and results were correlated with clinical characteristics and markers of streptococcal infection. A total of 41 children with TS (mean age, 11.3 years) and 39 controls (mean age, 12.1 years) were included. Compared with controls, TS subjects had a significant increase in the mean (p=0.006) and median (p=0.002) ELISA optical density (OD) levels of serum antibodies against putamen, but not caudate or globus pallidus. Western blots on 20 control and 20 TS serum samples showed that specific antibodies to caudate/putamen occurred more frequently in TS subjects at 83, 67, and 60 kDa; antigens were present in a synaptosomal fraction. TS subjects with a positive family history of tics had higher OD values (p < or = 0.04), but no association was shown with age of tic onset, tic severity, sudden onset of tics, or presence of attention-deficit hyperactivity disorder or obsessive-compulsive disorder. Risk ratio calculations in TS and control groups and in study subjects dichotomized for high and low putamen OD values were similar for titers of antistreptolysin O > or = 166 or antideoxyribonuclease B > or = 170. A subgroup analysis limited to subjects with elevated streptococcal titers, however, showed a significantly (p < or = 0.004) larger number of TS subjects with elevated OD levels. Children and adolescents with TS had significantly higher serum levels of antineuronal antibodies against putamen than did controls, but their relation to clinical characteristics and markers for streptococcal infection remains equivocal.

Use of the MAIEA assay to demonstrate that Fy3 is on the same glycoprotein as Fy6, Fya, and Fyb

Abstract The monoclonal-antibody immobilization of erythrocyte antigens (MAIEA) assay is a technique that detects trimolecular complexes formed by a human antibody and a mouse monoclonal antibody with specific red cell epitopes. This enzyme-linked immunoadsorbent assay test gives a positive reaction when two different epitopes on the same membrane protein are separately recognized by human and mouse antibodies. In this study, the MAIEA test was used to determine if the Duffy system antigen Fy3 is on the same membrane protein as Fy6. The well-established location and relationship of Duffy blood group antigens Fya, Fyb, and Fy6 were again confirmed by the MAIEA assay, and those facts were used to standardize a variation of the assay to establish the relationship between Fy3 and Fy6 using red cells with various Fy phenotypes. The MAIEA assay generated high absorbance values when Fy6 and Fya (or Fyb) antigens were evaluated. Similarly, high absorbance values were seen when Fy3 and Fy6 antigens were tested using Fy(a+b+), Fy(a+b–), and Fy(a–b+) red cells. The MAIEA assay is a valid technique for detecting Duffy system antigens. By this technique, it was shown that Fy3 and Fy6 antigens are carried on the Duffy glycoprotein D and by extension that Fy3 is also located on the Duffy glycoprotein D.

Monoclonal Antibodies for Detection of Neotyphodium coenophialum

A simple endophyte detection system is vital for breeding efforts designed to produce low alkaloid, endophyte‐infected tall rescue (Festuca arundinacea Schreb.) cultivars. The objectives of this research were to generate monoclonal antibodies specific to endophytes for immunochcmical detection purposes. Protein extracts from Neotyphodium coenophialum (Morgan‐Jones and Gains) Glenn, Bacon, and Hanlin comb. nov. isolate EDN11w ere injected into Balb/c mice to elicit an immuner esponse. Hybridomac ell lines were screened for antibody production to the Neotyphodium extract and for cross recognition of protein extracts from N. uncinatum Glen, Bacon, and Hanlin and N. starrii Glen, Bacon, and Hanlin, as well as other fungi in the genera Ciadosporlum, Penicilllum, Fusarium, and Aspergillus. Three hybridomace ll lines produceda ntibodies that reacted to Neotyphodium spp. proteins, but not with proteins isolated from the other fungi. Monoclonal antibody 5C7 was immunochemically tested for specificity to endophyte in leaf sheaths of plant genotype DN11. Fluorescent staining suggested the antibody preferentially bound to the cell wall of the endophyte. Antibodies were tested for affinity to protein extracts from 14 endophyte‐infected and endophyte‐free tall fescue genotypes by an enzyme‐linked immunosorbenta ssay (ELISA) method. Mean absorbance values for the protein extracts from the endophyte‐infected genotypcs differed and ranged from 0.317 to 0.583. Mean absorbance values for the endophyte‐free forms did not differ (P ≥ 0.05). All infected plants had higher absorbance values than non‐infected plants regardless of antibody tested. Monoclonal antibodies can be used in an ELISA to detect Neotyphodium spp. fungal mycelia present in tall fescue.

Identification and evaluation of a major cytotoxin of A. fumigatus.

Aspergillus fumigatus is a highly pathogenic fungus causing a wide spectrum of diseases in immunocompromised as well as immunocompetent hosts. The present work was undertaken to evaluate the cytotoxic nature of fractionated antigens of A. fumigatus against the mammalian cell lines (J774, RAW, CHO and L929). An enriched protein antigenic fraction of A. fumigatus was subjected to con A Sepharose and phenyl Sepharose chromatography. Antigenic fractions, ConAub (conA unbound) and PSC III (fraction III of phenyl Sepharose column) containing low mw antigens showed higher cytotoxicity as compared to other antigenic fractions. PSC III was further purified on HPLC resulting in an 18 kDa homogeneous protein. The purified protein showed high ELISA absorbance values for specific IgG and IgE antibodies in sera of ABPA patients. Monoclonal antibody raised against Asp fl, a major allergen/antigen of A. fumigatus recognised the purified 18 kDa by ELISA and western blot. The 18 kDa allergen/antigen or Asp fl showed similar toxicity towards all the four cell lines (macrophage and fibroblast) with an IC50 of 75 ng/ml or 4.16 nM. Reduction in toxicity of 18 kDa at low temperatures and potentiation in presence of ammonium chloride and monensin indicates mechanism of internalisation of 18 kDa in eukaryotic cells is similar to alpha-sarcin. The present work shows that the 18 kDa allergen/antigen (Asp fl) is a major cytotoxin secreted by A. fumigatus which may play multiple roles in the pathogenesis of Aspergillosis through allergenicity, antigenicity and cytotoxicity.

Modeling Particle Size and Clumping Effects in the IR Absorbance Spectra of Dilute Powders

The calculation of the IR absorbance of a powdered crystalline, ionic material, diluted in an appropriate IR-transparent matrix in a known proportion, is in principle possible, provided that the size and shape distributions of the particles, as well as the optical parameters of the bulk material, and those of the matrix, are known, and, in the case of the FT-IR technique, whether absolute calibration is possible, particularly at high absorbance values. In practice, a close match between observed and calculated absorbance is hindered by several factors intrinsic to the sample that are difficult to quantify, such as the degree of crystallinity of the particles and the very common clumping and aggregation of these particles, which produce a serious problem in the determination of the real filling factor of the composite sample. Some remedies to these problems are proposed in this paper and are applied to several well-known materials.

Purification and characterisation of isocitrate dehydrogenase and malate dehydrogenase from Mycobacterium tuberculosis and evaluation of their potential as suitable antigens for the serodiagnosis of tuberculosis

Setting: Enzymes from Mycobacterium tuberculosis are potent antigens and might thus be of interest in the serodiagnosis of tuberculosis. Objective: The purpose of the study was to purify and characterize the two enzymes isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH) from, M. tuberculosis and to evaluate their potential in the serodiagnosis of tuberculosis. Design: The two enzymes were analysed for specificity by electrophoresis and then purified by means of affinity chromatography using reactive dyes and ion exchange chromatography. The two isolated enzyme fractions were analysed by ELISA, using antisera against related organisms. They were then tested as antigens in ELISA together with sera from tuberculous patients and controls. Results: The electrophoretical analyses showed that the two enzymes each differed markedly from the corresponding enzymes of other mycobacteria. The serological analyses, however, could not distinguish between either IDH or MDH from other mycobacteria, but organisms of other genera, such as Nocardia, gave much weaker responses. When IDH and MDH were tested with sera from tuberculous patients and controls the former gave clearly higher optical density values than the latter. Conclusion: The enzymes/antigens IDH and MDH may be of value in developing a serological test for tuberculosis. The latter fraction seemed particularly capable of discriminating patients from controls.

Budding Method Affects Transmission of Xylella fastidiosa in Plum

Detection of Xylella fastidiosa Wells et al. by enzyme-linked immunosorbent assay indicated that plums (Prunus hybrids) had higher absorbance values than peaches [Prunus persica (L.) Batsch]. The slip-budded trees had lower readings than those that were chip budded; however, the scion × method interaction was significant. Further comparison of slip vs. chip budding indicated that the lower absorbance value of slip budding occurred in plums only; there was no difference between budding methods in peach.

Contagious bovine pleuropneumonia in northern Tanzania, culture confirmation and serological studies.

After an absence of about 25 years contagious bovine pleuropneumonia (CBPP) appeared again in 1990 in Tanzania. It was preceded by a spread in Kenya to an area bordering Tanzania. Due to the frequent cattle movements across the border it was soon introduced into Loliondo in northern Tanzania. One month after the first cases, CBPP was suspected in a total of 9 herds comprising 1,500 cattle. However, few animals showed clear clinical signs and frequent antibiotic treatment at an early stage further obscured the clinical picture. In one herd with acute cases, the diagnosis was confirmed by autopsy and Mycoplasma mycoides subsp. mycoides, SC type, was isolated. From this herd several serum samples were positive in the complement fixation test and gave high absorbance values in an ELISA with M. mycoides subsp. mycoides antigen. From 5 other herds with suspected cases blood samples were negative by the complement fixation test but in the enzyme-linked immunosorbent assay at least one in each herd was positive.

Extinction coefficient of polymerized diaminobenzidine complexed with cobalt as final reaction product of histochemical oxidase reactions.

The extinction coefficient is essential for the conversion of cytophotometric (mean integrated) absorbance values into absolute units of enzyme activity, for instance expressed in terms of moles of substrate converted per unit time and per unit wet weight of tissue. The extinction coefficient of polymerized diaminobenzidine (polyDAB) complexed with cobalt as the final reaction product of oxidase reactions was estimated at 575 nm by comparison of the amounts of final reaction products formed after incubation of serial unfixed cryostat sections of rat kidney to demonstrate D-amino acid oxidase activity with either the tetrazolium salt method or the cerium-DAB-cobalt-hydrogen peroxide method. Both procedures resulted in similar localization patterns of final reaction product in a granular form in epithelial cells of proximal tubules in rat kidney. The granules were peroxisomes. Linear relationships were found for both methods between the specific amounts of final reaction product generated by D-amino acid oxidase activity and incubation time. The cerium salt method gave rise to 7.4 times higher absorbance values of final reaction product generated per unit time and per unit wet weight of tissue than the tetrazolium salt procedure. The extinction coefficient of tetranitro BT-formazan is 19,000 at 557 nm. Therefore, the cytophotometric extinction coefficient of the polyDAB-cobalt complex as final reaction product of oxidase reactions was established to be 140,000.

Leishmania infantum: Sequence Homology within Minicircle Classes Regardless of Geographical Distance

An enzyme-linked immunosorbent assay (ELISA) is described utilizing the surface glycocalyx crude antigen of adult blood trematodes Learedius learedi, Hapalotrema dorsopora, and Carettacola hawaiiensis, for the detection of circulating antibodies (Ab) in Hawaiian green turtles (Chelonia mydas) naturally infected with the parasites and with or without green turtle fibropapillomatosis (GTFP). A concentration of 10.0 micrograms/ml of antigen was optimal in terms of test specificity and sensitivity. A direct ELISA with anti-reptilian/amphibian phosphatase-labeled IgG identified C. mydas Ab at a dilution of 1/12,800. Utilizing indirect ELISA, it was possible to detect Ab to blood flukes at a dilution of 1/3,200. The gross lesions and histopathology were typical for cardiovascular spirorchidiasis in C. mydas. No significant relationship was found between the size of turtles and the degree of GTFP severity. Forty-seven of 59 (80%) samples, from 5 sites, gave a positive ELISA reaction; 6 of the 47 (13%) specimens gave significantly (P < 0.001) higher absorbance values, with 5 from the same location. All 12 (20%) ELISA-negative turtles originated from 1 site, and the absorbance values of the 41 animals from this location were significantly lower (P < 0.015) when compared with the other 4 sites. The proposed assay is fast, has the feature of visual scoring, and can be used for determination of exposure of C. mydas to the spirorchid trematodes.

Examination by ELISA of sera obtained from chicken breeder and layer flocks showing evidence of fowl typhoid or pullorum disease

An indirect ELISA using soluble whole cell antigen was used to screen serum samples obtained from breeder and layer flocks some of which had shown clinical or bacteriological evidence of infection with Salmonella Gallinarum or S. Pullorum. There was good correlation between Salmonella infection and the presence of serum samples showing high optical density (OD) values. Sera from seven flocks showing high values were retested using group D (0-1, 9, 12) lipopolysaccharide (LPS) and g,m-H flagella as detecting antigens. Sera from six flocks produced high OD values with LPS and low values with flagella confirming infection with a non-flagellate, group D Salmonella while one produced high values with both antigens indicating mixed infection with another group D serotype.

Detection by ELISA of Circulating Anti-Blood Fluke (Carettacola, Hapalotrema, and Learedius) Immunoglobulins in Hawaiian Green Turtles (Chelonia mydas)

An enzyme-linked immunosorbent assay (ELISA) is described utilizing the surface glycocalyx crude antigen of adult blood trematodes Learedius learedi, Hapalotrema dorsopora, and Carettacola hawaiiensis, for the detection of circulating antibodies (Ab) in Hawaiian green turtles (Chelonia mydas) naturally infected with the parasites and with or without green turtle fibropapillomatosis (GTFP). A concentration of 10.0 micrograms/ml of antigen was optimal in terms of test specificity and sensitivity. A direct ELISA with anti-reptilian/amphibian phosphatase-labeled IgG identified C. mydas Ab at a dilution of 1/12,800. Utilizing indirect ELISA, it was possible to detect Ab to blood flukes at a dilution of 1/3,200. The gross lesions and histopathology were typical for cardiovascular spirorchidiasis in C. mydas. No significant relationship was found between the size of turtles and the degree of GTFP severity. Forty-seven of 59 (80%) samples, from 5 sites, gave a positive ELISA reaction; 6 of the 47 (13%) specimens gave significantly (P < 0.001) higher absorbance values, with 5 from the same location. All 12 (20%) ELISA-negative turtles originated from 1 site, and the absorbance values of the 41 animals from this location were significantly lower (P < 0.015) when compared with the other 4 sites. The proposed assay is fast, has the feature of visual scoring, and can be used for determination of exposure of C. mydas to the spirorchid trematodes.

Bovine vaginal antibody responses to immunoaffinity-purified surface antigen of Tritrichomonas foetus

Bovine trichomoniasis is a prevalent sexually transmitted disease of cattle caused by the protozoan Tritrichomonas foetus. Currently, diagnosis is most often made by culture. In order to provide a faster immunodiagnostic approach, a specific enzyme-linked immunosorbent assay (ELISA) was investigated. A protective surface antigen (TF1.17 antigen) of T. foetus was immunoaffinity purified and used in an ELISA to detect antibodies in vaginal mucus from heifers inoculated with T. foetus. In preliminary studies, antibodies of the immunoglobulin A (IgA) isotype were detected in mucus from all experimentally infected heifers which were tested at 6 weeks postinoculation, whereas IgG1 and IgG2 were not. In addition, IgA responses detected in postinoculation samples were all greater than those detected in preinoculation samples, unlike those detected by a whole-cell antigen ELISA. For these two reasons, IgA antibodies appeared to be useful diagnostically. Further investigation of IgA antibodies used vaginal mucus collected weekly from heifers inoculated intravaginally with 10(2), 10(4), or 10(6) T. foetus organisms. Heifers with positive cultures for T. foetus had similar IgA responses to TF1.17 antigen over the 10 weeks of infection regardless of the initial inoculum dose. This indicates that if the dose is sufficient to establish infection, the magnitude and duration of the immune response are no longer dependent on dose. All of the infected animals receiving all dosages responded with high absorbance values in the IgA anti-TF1.17 antigen ELISA by 6 weeks postinoculation, and all absorbance values remained high at 10 weeks. To determine the duration of the IgA response, four other heifers inoculated with 7 x 10(6) T. foetus organisms were studied through 24 weeks postinoculation. IgA antibody responses to TF1.17 antigen were still high at 24 weeks, even though some heifers cleared the infection several weeks earlier. These results indicate that experimentally infected heifers produced detectable long-lasting IgA responses to TF1.17 antigen in vaginal mucus, which may be useful in a herd test for the diagnosis of bovine trichomoniasis.