Epizootic Hemorrhagic Disease Agar Gel Immunodiffusion Assay.

Agar gel immunodiffusion assay (AGID) is a laboratory test which detects specific antigen-antibody interactions by the development of visible precipitation lines in a semisolid matrix. Here we describe the preparation of agar gel plates, the method to test serum samples by AGID for the presence of EHDV antibodies, and the interpretation of test results. This test has known cross-reactivity to bluetongue antibodies; therefore positive samples by this assay require additional confirmatory testing; generally, its use should be limited to healthy animal attestations where required.

Deciphering immunodiffusion: In silico optimization for faster protein diagnostics.

Immunodiffusion tests offer a simple yet powerful method for detecting protein antigens, but their long assay times hinder clinical utility. We unveil the complex interplay of parameters governing this process using finite element simulations. By meticulously validating our model against real-world data, we elucidate how initial concentrations and diffusivities of antigen and antibody shape the intensity, size, and formation time of the precipitin ring. Our key innovation lies in employing phase diagram analysis to map the combined effects of these parameters on assay performance. This framework enables rapid in silico parameter estimation, paving the way for the design of novel immunodiffusion assays with drastically reduced assay times. The presented approach holds immense potential for optimizing protein diagnostics for fast and reliable diagnostics.

Diagnosis of Coccidioidomycosis with the Second-Generation Miravista IgG and IgM Enzyme Immunoassay and the Role of Adding Miravista Coccidioides Antigen Detection to Immunodiagnostic Assays.

In the present study, we validate and compare the second-generation Miravista Coccidioides IgG and IgM enzyme immunoassays (EIA) (MiraVista Diagnostics [MVD] Ab EIA) to Meridian Diagnostics Coccidioides IgG and IgM EIA (Meridian Ab EIA), immunodiffusion (ID) and complement fixation (CF). We also evaluated whether the addition of Coccidioides antigen testing to anti-Coccidioides antibody testing increased the sensitivity for the diagnosis of currently active coccidioidomycosis. We retrospectively studied 555 patients evaluated at Valleywise Health Medical Center between January 2013 and May 2017 for whom coccidioidomycosis was suspected and samples were submitted to MVD for testing. Specimens were tested for antigen in the MVD antigen enzyme immunoassay (MVD Ag EIA) and for IgG and IgM antibodies with MVD and Meridian Diagnostics EIAs. ID and CF were obtained from medical records. Sensitivity and specificity were 83.0% and 91.1% or MVD Ab EIA, 69.3% and 99.7% for Meridian Ab EIA, 85.4% and 100% for ID and 65.5% and 100% for CF. Combined MVD antigen and antibody detection by EIA and ID resulted in increased sensitivity in disseminated and pulmonary disease (MVD Ag/MVD Ab: 100%, 88.3%; MVD Ag/Meridian Ab: 98.2%, 78.6%; and MVD Ag/ID: 100%, 91.7%). The detection of antibodies by MVD EIA was more sensitive than Meridian EIA or CF but similar to ID. This study supports the use of antigen testing in immunocompromised patients and those with suspected disseminated disease. Furthermore, the addition of antigen detection by EIA to antibody detection resulted in higher sensitivity of all serological tests.

The most common methods for the diagnosis of moderate or severe coccidioidomycosis rely on the detection of antibodies or antigens. Here we present the validation of a new Miravista Coccidioides antibody detection test combined with antigen detection and compare it to other immunodiagnostics.

Evaluation of commercial immunodiffusion reagents for detecting serum anti-Paracoccidioides antibodies.

BACKGROUND: Accurate diagnosis of paracoccidioidomycosis is crucial for improving patient outcomes. Paracoccidioides antibody detection by double immunodiffusion (DID) is a convenient diagnostic tool, but testing performance can vary based on certain factors. METHODS: We assessed DID performance using a commercially prepared Paracoccidioides reagents (IMMY, USA), involving 40 serum specimens, including 20 from patients with proven paracoccidioidomycosis and 20 from patients without the disease. The DID test demonstrated a sensitivity of 90% (95% CI=68%-99%) and a specificity of 100% (95% CI=83%-100%). CONCLUSIONS: Our findings suggest that DID using commercial reagents may provide a feasible tool with satisfactory testing performance for anti-Paracoccidioides antibody detection.

Histoplasma antigens as novel players for the development of new enzyme immunoassays for the serodiagnosis of histoplasmosis: A comparative study of their analytical performance.

Definitive diagnosis of histoplasmosis relies on culture and/or cytology/histopathology; however, these procedures have limited sensitivity and cultures are time-consuming. Antibodies detection by immunodiffusion has low sensitivity in immunocompromised individuals and uses histoplasmin (HMN), a crude antigenic extract, as reagent. Novel protein antigen candidates have been recently identified and produced by DNA-recombinant techniques to obtain standardized and specific reagents for diagnosing histoplasmosis. To compare the analytical performance of novel enzyme-linked immunosorbent assays (ELISAs) for antibodies testing for diagnosing histoplasmosis using different Histoplasma capsulatum antigens as reagents. The H. capsulatum 100 kDa protein (Hcp100), the M antigen and its immunoreactive fragment F1 were produced by DNA-recombinant techniques. Galactomannan was purified from both the yeast and mycelial cell walls (yGM and mGM, respectively). The analytical performance of the ELISA tests for the serological detection of antibodies against these antigens was evaluated and compared with those obtained using HMN as reagent. Antibodies detection by the Hcp100 ELISA demonstrated 90.0% sensitivity and 92.0% specificity, versus 43.3% sensitivity and 95.0% specificity of the M ELISA, 33.3% sensitivity and 84.0% specificity of the F1 ELISA, 96.7% sensitivity and 94.0% specificity of the yGM ELISA, 83.3% sensitivity and 88.0% specificity of the mGM ELISA, and 70.0% sensitivity and 86.0% specificity for the HMN ELISA. In summary, Hcp100 is proposed as the most promising candidate for the serodiagnosis of histoplasmosis. The primary immunoreactive element in HMN proved to be GM rather than the M antigen. Nevertheless, a higher incidence of cross-reactions was noted with GM compared to M.

Hcp100 is a promising serodiagnostic candidate for histoplasmosis, boasting high sensitivity and specificity. Notably, GM, rather than M antigen, emerged as the primary immunoreactive element in HMN, despite a higher incidence of cross-reactions with GM compared to M.

Two lateral flow assays for detection of anti-coccidioidal antibodies show similar performance to immunodiffusion in dogs with coccidioidomycosis.

OBJECTIVE: To compare 2 point-of-care lateral flow assays (LFAs) with immunodiffusion (ID) IgG results for anti-coccidioidal antibody detection in dogs with coccidioidomycosis. A further aim was to compare the quantifiable output of 1 of the LFAs to ID antibody titers. SAMPLE: Serum banked from 73 client-owned dogs diagnosed with pulmonary or disseminated coccidioidomycosis. METHODS: ID was used to determine antibody presence and titer against a coccidioidal antigen preparation. All sera were subsequently tested on an LFA based on recombinant chitinase 1 (CTS1) and the commercially available sona LFA. LFA results were analyzed and compared to ID IgG results and clinical diagnosis. RESULTS: All assays showed similar sensitivities in detecting anti-coccidioidal antibodies (83.6% to 89.0%). When compared with ID IgG, the CTS1 LFA had a positive percent agreement of 100%, while the sona LFA had a positive percent agreement of 91.4%. Since the CTS1 LFA is semiquantitative, we were able to compare test line densities with ID titers and found a strong correlation between the 2 assays (Spearman ρ = 0.82). CLINICAL RELEVANCE: This is the first side-by-side evaluation of a commercially available LFA (sona) and a newer more rapid anti-CTS1 antibody LFA using serum from dogs with coccidioidomycosis. Both LFAs tested have similar sensitivity to ID IgG results. The CTS1 LFA can be read after 10 minutes and is semiquantitative, while the sona LFA is read after 30 minutes, and the results are subject to interpretation. Accurate and fast detection of anti-coccidioidal antibodies allows clinicians to initiate appropriate treatment without diagnostic delay.

Assessing colostral and serum immunoglobulin G in alpacas using Brix refractometry and total serum protein.

The adequate transfer of passive immunity is a critical factor in neonatal development and survivability. Although well documented in the dairy and equine industries, the recognition of inadequate immunoglobulin transfer on-farm and its impact on the ability of alpaca cria to thrive is largely unknown. Colostrum samples were collected from female alpaca within 24 h of parturition by the owners and whole blood collected from cria by the investigators between 1 and 7 days of age. Direct IgG concentration of milk and serum was determined using radial immunodiffusion assay (RID) and was indirectly estimated using optical and digital Brix refractometry for total solids and clinical refractometry for total serum protein. There was a strong correlation between optical and digital Brix refractometry, and colostral IgG concentration determined by RID. There was a moderate correlation between serum IgG concentration determined by RID and total serum protein in crias. Optical and digital Brix refractometry for colostral IgG estimation and total serum protein for serum IgG estimation are reliable, accurate and easy-to-use tools that can be used on-farm by trained, competent technicians to assess a failure of passive transfer in alpacas. A pilot study at one property only was performed, due to COVID-19 travel restriction interference. Further research is required to determine the reference intervals for these tools to be practical.

Establishment of Reference Reagents for Single-Radial-Immunodiffusion Assay on the 2022/23 Seasonal Influenza Vaccine in Japan and Their Quality Validation.

Potency tests for influenza vaccines are currently performed using a single-radial immunodiffusion (SRID) assay, which requires a reference antigen and anti-hemagglutinin (HA) serum as reference reagents. Reagents must be newly prepared each time a strain used for vaccine production is modified. Therefore, establishing reference reagents of consistent quality is crucial for conducting vaccine potency tests accurately and precisely. Here, we established reference reagents for the SRID assay to conduct lot release tests of quadrivalent influenza vaccines in Japan during the 2022/23 influenza season. The potency of reference antigens during storage was confirmed. Furthermore, we evaluated the cross-reactivity of each antiserum raised against the HA protein of the 2 lineages of influenza B virus toward different lineages of influenza B virus antigens to select a suitable procedure for the SRID assay for accurate measurement. Finally, the intralaboratory reproducibility of the SRID assay using the established reference reagents was validated, and the SRID reagents had sufficient consistent quality, comparable to that of the reagents used for testing vaccines during previous influenza seasons. Our study contributes to the quality control of influenza vaccines.

Comparison of coccidioidal complement fixation and quantitative immunodiffusion serology at a reference laboratory.

The incidence of coccidioidomycosis continues to increase. The diagnosis frequently relies on non-invasive diagnostic testing with immunodiffusion and complement fixation (CF) testing the current gold standard. A direct comparison of quantitative immunodiffusion and CF for IgG antibodies has not been previously reported. In a comparison of 368 samples, there was close concordance observed (360/368 = 97.8%) (P-value < .001). These tests can be considerably interchangeable in the reference laboratory setting.

There are several diagnostic methodologies available in coccidioidomycosis. Direct comparisons of these methods are limited. Prior studies have not compared quantitative immunodiffusion to complement fixation testing. Our results show these tests are highly concordant.

Estimating IgG concentration directly by radial immunodiffusion or indirectly by refractometry measure of serum total protein lack precision.

OBJECTIVE: To establish and compare the precision of serum total protein (STP) measured by an optical refractometer to the precision of IgG concentrations measured using radial immunodiffusion (RID), the reference test for quantifying IgG in neonatal calves. SAMPLE: 6 sera with previously measured IgG concentration using RID from neonatal beef calves were selected from 3 stratum: low-serum IgG stratum between >5.0 and <15.0g/L(n = 4); moderate-serum IgG stratum between 35.0-45.0g/L(n = 1); high-serum IgG stratum between 60.0-70.0g/L(n = 1). METHODS: STP was measured 13 times with an optical refractometer. IgG concentrations were measured 28 times with a commercial bovine IgG RID for each sera. The homogeneity of variance within the tests was evaluated with the Levene test (α = 0.10). Unrestricted random sampling bootstrapping (5,000 repetitions) was used to calculate the coefficient of variation (CV) for each serum and test. The homogeneity of variance between simulated test CVs by serum was evaluated (α = 0.10). Differences between simulated test CV by serum were assessed with the Kruskal-Wallis test (α = 0.05). RESULTS: No difference was observed in the variance for STP between sera (P = .39). The average CV for STP was 4.2%, 10.1% for the low IgG stratum, and 15.5% for the moderate/high IgG stratum. Variance differed in serum IgG concentration (P < .0001). Serum with higher IgG concentrations had more variance. Simulated CV for STP and IgG had homogeneity of variance for only 1 sera (P = .31). STP had a smaller CV compared to IgG for every serum (P < .0001). CLINICAL RELEVANCE: Estimating IgG concentration directly by RID or indirectly by STP lacks the precision that might affect diagnostic interpretation regarding a calf's absorption of maternal antibodies.

Evaluation of laboratory and on-farm tests to estimate colostrum quality for dairy cows.

The objectives of this study were to evaluate different analytical methods to determine colostrum quality in dairy cattle, including one laboratory-based method (ELISA) and 4 on-farm tests. We hypothesized that the colostral IgG concentration using different analytical methods, such as ELISA (mg/mL), digital Brix refractometer (% Brix), colostrometer (specific gravity and mg/mL), an outflow funnel (seconds), and a lateral flow assay (mg/mL), were highly correlated with the reference method, radial immunodiffusion (RID; mg/mL) and would generate comparable results. Colostrum samples were collected from 209 Holstein Friesian cows on 2 commercial dairy farms in Germany. Colostrum weight and colostrum temperature were measured. Test characteristics, such as optimum thresholds, sensitivity, specificity, and area under the curve (AUC) were determined using a receiver operating characteristic curve analyses for each test. Out of 209 colostrum samples assessed by RID, 186 (89%) samples had high quality (≥50 mg IgG/mL), while 23 colostrum samples (11%) showed poor quality with IgG concentrations less than 50 mg/mL. The mean IgG concentration (±SD) was 101.3 ± 45.9 mg/mL and the range was 6.0 to 244.3 mg/mL. The Pearson correlation coefficient (r) between RID and ELISA was r = 0.78. In comparison to RID, Pearson correlation coefficients for the on-farm tests were: r = 0.79 (digital Brix refractometry), r = 0.58 (colostrometer: specific gravity), r = 0.61 (colostrometer: temperature corrected), r = 0.26 (outflow funnel) and r = 0.43 (lateral flow assay), respectively. The optimal threshold to identify high-quality colostrum using ELISA was 50.8 mg/mL with sensitivity 91.3%, specificity 92.3%, and AUC of 0.94. For the on-farm tests sensitivity ranged from 95.7% (Brix refractometry) to 60.9% (lateral flow assay). Specificity ranged from 88.6% (lateral flow assay) to 75.9% (colostrometer: temperature corrected). The AUC ranged from 0.93 (Brix refractometry) to 0.73 (outflow funnel). Based on the AUC, ELISA (0.94) and Brix refractometry (0.93) can be considered highly accurate. In conclusion, the ELISA is accurate to assess colostrum quality. Regarding the on-farm tests only the digital Brix refractometer and the colostrometer were adequate to determine colostrum quality.

Comparison of turbidometric immunoassay and brix refractometry to radial immunodiffusion for assessment of colostral immunoglobulin concentration in beef cattle.

BACKGROUND: Colostral immunoglobulin G (IgG) concentration is critical to the attainment of adequate transfer of passive immunity in cattle, however, studies comparing available tools for measurement of colostral IgG concentration in beef cattle are limited. OBJECTIVES: To report the agreement between 3 commercially available tests for evaluating IgG concentration in beef colostrum. ANIMALS: Two hundred six beef-breed cows hospitalized for calving management or dystocia. METHODS: Retrospective study to assess IgG of whole colostrum measured stall-side via turbidimetric immunoassay (TI) and brix refractometry (BRIX), compared to fat separated (FS) analysis via single radial-immunodiffusion (RID; reference standard), TI-FS and BRIX-FS. Test performance was assessed using Passing Bablock regression, Bland-Altman analysis, and area under the curve to determine optimal thresholds. RESULTS: Correlation between RID and TI-FS, BRIX-FS, or BRIX was similar (Spearman's ρ = 0.717, 0.715, 0.716, respectively) but correlation for TI was poor (ρ = 0.586). Regression analysis identified a substantial constant (-214.75 [CI: -272.03 to -178.07]) and proportional (13.24 [CI: 11.81-15.37]) bias between the RID and TI-FS which was similar for TI. TI-FS concentrations of 28.47, 38.75, and 50.62 g/L, BRIX-FS of ≤21.9%, ≤24.0%, and ≤27.4%, and BRIX of ≤21.3%, ≤23.8%, and ≤26.4% indicated IgG concentrations <50, <100, and <150 g/L, respectively; appropriate cutoffs for TI could not be generated. CONCLUSIONS AND CLINICAL IMPORTANCE: Both TI and TI-FS demonstrated a large constant and proportional bias compared to RID; BRIX and BRIX-FS were well correlated with RID and remain a reliable method for estimation of colostral IgG concentration in beef cattle.

Comparison of turbidometric immunoassay, refractometry, and gamma-glutamyl transferase to radial immunodiffusion for assessment of transfer of passive immunity in high-risk beef calves.

BACKGROUND: Attainment of adequate transfer of passive immunity (TPI) is critical to health of calves; however, studies comparing available tools for measurement of TPI in individual beef animals are limited. OBJECTIVES: To report agreement between 4 tests evaluating individual TPI status in beef calves. ANIMALS: One hundred ninety-six beef calves born to cows and heifers presenting for calving management or dystocia. METHODS: Retrospective study to assess serum immunoglobulin (IgG) concentrations via turbidimetric immunoassay (TI), gamma-glutamyl transferase (GGT), serum total protein (TP), and single radial immunodiffusion (RID; reference standard). Test agreement was evaluated using Passing-Bablok regression, Bland-Altman analysis, Cohen's kappa, and receiver operating characteristic (ROC) curves with and without covariate adjustment to determine optimal thresholds. RESULTS: Correlation between RID and test results varied: TI, ρ = 0.757; TP, ρ = 0.715; GGT: ρ = 0.413. For the TI compared to RID, regression analysis identified a constant (intercept = -0.51 [CI: -2.63, 3.05]) and proportional (slope = 1.87 [CI: 1.69, 2.08]) bias. Based on ROC, TI concentrations of ≤9.89 and ≤13.76 g/L, and TP concentrations of ≤5.5 and ≤6.0 g/dL, indicated IgG concentrations <18.0 and <25.0 g/L, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Within this cohort of calves, TI demonstrated the best correlation with RID; however, significant bias was identified which led to frequent underestimation of IgG concentration. Serum total protein demonstrated less correlation with RID but had less misclassification than TI. Both TI and TP demonstrated less correlation for calves that received colostrum replacement prompting clinical awareness of colostrum type when evaluating individual TPI in beef calves.

Comparison of 4 agar gel immunodiffusion kits for serologic detection of equine infectious anemia virus antibodies.

Using 85 sera collected from horses that had been experimentally infected with equine infectious anemia virus (EIAV) and 200 field sera collected from racehorses in Japan, we compared 4 agar gel immunodiffusion (AGID) kits for serologic detection of EIAV antibodies from Idexx, VMRD, IDvet, and the National Engineering Research Center of Veterinary Biologics, China (NECVB). The positive control lines were sufficiently clear in all kits for evaluation to be made, with slight differences in sharpness: NECVB was the sharpest, followed by VMRD, IDvet, and Idexx. The test results for all 285 samples agreed among the 4 kits, with 62 positives and 223 negatives. The sensitivities and specificities of VMRD, IDvet, and NECVB compared with the Idexx kit were 100%, and the kappa coefficient values between the kits were 1.0 for all combinations. We concluded that the testing capacity of these 4 kits was virtually identical.

Evaluation of an enzyme immunoassay and immunodiffusion for detection of anti-Histoplasma antibodies in serum from cats and dogs.

BACKGROUND: Histoplasma antigen and anti-Histoplasma antibody detection are used to support the diagnosis of histoplasmosis. There is a paucity of published data on antibody assays. OBJECTIVES: Our primary hypothesis was that anti-Histoplasma immunoglobulin G (IgG) antibody detection using enzyme immunoassay (EIA) will be more sensitive as compared to immunodiffusion (ID). ANIMALS: Thirty-seven cats and 22 dogs with proven or probable histoplasmosis; 157 negative control animals. METHODS: Residual stored sera were tested for anti-Histoplasma antibodies using EIA and ID. Results of urine antigen EIA were reviewed retrospectively. Diagnostic sensitivity was calculated for all three assays and compared between immunoglobulin G (IgG) EIA and ID. The diagnostic sensitivity of urine antigen EIA and IgG EIA, interpreted in parallel, was reported. RESULTS: Sensitivity of IgG EIA was 30/37 (81.1%; 95% confidence interval [CI], 68.5%-93.4%) in cats and 17/22 (77.3%; 95% CI, 59.8%-94.8%) in dogs. Diagnostic sensitivity of ID was 0/37 (0%; 95% CI, 0%-9.5%) in cats and 3/22 (13.6%; 95% CI, 0%-28.0%) in dogs. Immunoglobulin G EIA was positive in all animals (2 cats and 2 dogs) with histoplasmosis but without detectable antigen in urine. Diagnostic specificity of IgG EIA was 18/19 (94.7%; 95% CI, 74.0%-99.9%) in cats and 128/138 (92.8%; 95% CI, 87.1%-96.5%) in dogs. CONCLUSION AND CLINICAL IMPORTANCE: Antibody detection by EIA can be used to support the diagnosis of histoplasmosis in cats and dogs. Immunodiffusion has an unacceptably low diagnostic sensitivity and is not recommended.

The impact of freezing and multiple freeze-thaw cycles on Brix refractometry estimates of immunoglobulin concentration in beef cattle colostrum.

Evaluation of immunoglobulin G (IgG) concentration in colostrum is important to guide on-farm management. Studies have shown that digital Brix refractometry accurately estimates colostrum IgG concentration in both dairy and beef cattle colostrum. Colostrum is often frozen in both clinical and research settings. The implications of this freezing on the accuracy of Brix refractometry measurements are largely unknown. The first objective of this study was to evaluate the agreement between digital Brix percentage measurements of IgG in beef cattle colostrum taken before and after different durations of freezing. The second objective was to evaluate the effects of multiple freeze-thaw (FT) cycles on Brix percentage measurements of IgG in beef cattle colostrum. There was good agreement between Brix percentages in fresh colostrum and after short (2 to 8 d), medium (4 to 7 mo), and long (3 y) periods of freezing (concordance correlation coefficient: 0.95, 0.96, and 0.96, respectively). Although there was no significant change in mean Brix percentages over 2 FT cycles (P > 0.05), mean Brix percentages decreased with 3 FT cycles (P = 0.017). Samples from the fourth and fifth FT cycles were observably coagulated, and these measurements were therefore deemed inaccurate. Data from this study indicate that freezing had minimal impact on digital Brix refractometer estimates of IgG concentration in beef cattle colostrum, but that samples stored for future testing should not undergo more than 2 FT cycles.

L'évaluation de la concentration d'immunoglobuline G (IgG) dans le colostrum est importante pour guider la gestion à la ferme. Des études ont montré que la réfractométrie Brix numérique estime avec précision la concentration d'IgG du colostrum dans le colostrum des bovins laitiers et de boucherie. Le colostrum est souvent congelé dans les milieux cliniques et de recherche. Les implications de cette congélation sur la précision des mesures de réfractométrie Brix sont largement inconnues. Le premier objectif de cette étude était d'évaluer la concordance entre les mesures numériques du pourcentage de Brix d'IgG dans le colostrum de bovins de boucherie prises avant et après différentes durées de congélation. Le deuxième objectif était d'évaluer les effets de plusieurs cycles de congélation-décongélation (FT) sur les mesures du pourcentage Brix d'IgG dans le colostrum de bovins de boucherie. Il y avait un bon accord entre les pourcentages de Brix dans le colostrum frais et après des périodes de congélation courtes (2 à 8 jours), moyennes (4 à 7 mois) et longues (3 ans) (coefficient de corrélation de concordance : 0,95, 0,96 et 0,96, respectivement). Bien qu'il n'y ait pas eu de changement significatif dans les pourcentages moyens de Brix sur deux cycles FT (P > 0,05), les pourcentages moyens de Brix ont diminué avec trois cycles FT (P = 0,017). Les échantillons des quatrième et cinquième cycles FT étaient coagulés de manière observable, et ces mesures ont donc été jugées inexactes. Les données de cette étude indiquent que la congélation a eu un impact minimal sur les estimations du réfractomètre numérique Brix de la concentration d'IgG dans le colostrum de les bovins de boucherie, mais que les échantillons stockés pour les tests futurs ne doivent pas subir plus de deux cycles FT.(Traduit par Docteur Serge Messier).

Diagnostic characteristics of refractometry cut-off points for the estimation of immunoglobulin G concentration in mare colostrum.

BACKGROUND: Feeding foals with poor quality colostrum predisposes them to failure of passive transfer (FPT). FPT is a major risk factor for neonatal infections. OBJECTIVES: To assess the optimal cut-offs for the optical (OR) and digital (DR) refractometer and determine their accuracy for poor quality colostrum diagnosis. STUDY DESIGN: A diagnostic validation study. METHODS: Eighty-one colostrum samples and sera were collected from broodmares and their neonatal foals, respectively. Colostral and serum IgG concentrations were measured by radial immunodiffusion (RID), DR and OR. Correlation coefficients were calculated. ROC curves were generated to identify optimal cut-offs for the refractometers and their diagnostic characteristics were evaluated. RESULTS: The optimal cut-offs for DR and OR were ≤23.75% and 23.9%, respectively. The sensitivity and specificity of the DR were 93.3% (95% CI: 66.0-99.7) and 87.9% (95% CI: 77.0-94.3) to detect colostral IgG <60 g/L, respectively. The sensitivity and specificity of the OR were 93.3% (95% CI: 66.0-99.7) and 81.8% (95% CI: 70.0-89.9), respectively. DR and OR had negative predictive values of 98.3% (95% CI: 89.7-99.9) and 98.2% (95% CI: 89.0-99.9), respectively, whilst positive predictive values were lower. No maternal variable, including breed, significantly influenced colostral IgG concentrations. Fifteen out of 81 colostrum samples had IgG <60 g/L. FPT and PFPT were diagnosed in 4/81 and 10/81 foals, respectively. Nine out of 14 animals with FPT/PFPT suckled colostrum with IgG <60 g/L. A moderate correlation (rs 0.542; P = .01) was observed between IgG concentrations measured by RID in sera and colostrum. MAIN LIMITATIONS: A smaller number of samples than the size requirement based on a priori estimate of specificity and the low prevalence of poor quality colostrum. CONCLUSIONS: The method has the potential to reliably differentiate between good and poor quality colostrum. Assessing colostrum quality by refractometry may be an indicator of passive transfer of immunity.

INTRODUCTION/CONTEXTE: Nourrir les poulains avec du colostrum de mauvaise qualité prédispose à l'échec du transfert d'immunité passive (FPT). FPT constitue un risque majeur pour les infections néonatales. OBJECTIFS: Évaluer les valeurs limites optimales au réfractomètre optique (RO) et digital (RD) et déterminer leur précision pour le diagnostic du colostrum de pauvre qualité. TYPE D'ÉTUDE: Étude de validation diagnostique. MÉTHODES: Quatre-vingt-un colostrums et sérums ont été recueillis à partir de juments de reproduction et de leur poulains nouveaux-nés respectivement. Les concentrations d'IgG dans le sérum et le colostrum ont été mesurées par immunodiffusion radiale (IDR), RO et RD. Les coefficients de corrélation ont été calculés. Des graphes d'air sous la courbe (ASC) ont été générés afin d'identifier les valeurs limites optimales aux différents réfractomètres et leurs caractéristiques diagnostiques ont été évaluées. RÉSULTATS: Les valeurs limites optimales pour les RD et RO étaient ≤23.75% et 23.9% respectivement. La sensibilité et la spécificité du RD étaient 93.3% (95% IC: 66.0-99.7) et 87.9% (95% IC: 77.0-94.3) pour la détection des IgG colostraux <6000 mg/dl, respectivement. La sensibilité et spécificité du RO étaient de 93.3% (95% IC: 66.0-99.7) et 81.8% (95% IC: 70.0-89.9), respectivement. Les RD et RO avaient une valeur prédictive négative de 98.3% (95% IC: 89.7-99.9) et 98.2% (95% IC: 89.0-99.9) respectivement, alors que les valeurs prédictives positives étaient plus basses. Aucune variable maternelle, incluant la race, n'a influencé significativement les concentrations colostrales en IgG. Quinze des 81 échantillons colostraux avaient une valeur d'IgG <6000 mg/dl. FPT et PFPT ont été diagnostiqué chez 4/81 et 10/81 poulains respectivement. Neuf des 14 animaux avec FPT/PFPT ont reçu du colostrum ayant des valeurs d'IgG <6000 mg/dl. Une corrélation modérée (rs 0.542; p= 0.01) a été observée entre les concentrations d'IgG mesurées par IDR dans le sérum et le colostrum. LIMITES PRINCIPALES: Le nombre d'échantillon est inférieur à celui recommandé basé sur unestimé a priori de la spécificité et considérant la faible prévalence de colostrum de pauvre qualité. CONCLUSIONS: La méthodologie utilisée pourrait différencier de façon fiable les colostrums de pauvre et bonne qualité. L'évaluation de la qualité du colostrum par réfractométrie pourrait représenter un indice du transfert d'immunité passive.

Validation of an indirect in-house ELISA using synthetic peptides to detect antibodies anti-gp90 and gp45 of the equine infectious anaemia virus.

BACKGROUND: Equine infectious anaemia (EIA) is controlled by the identification of seropositive animals. The official diagnostic method is the agar gel immunodiffusion (AGID) test, which detects antibodies against a viral core protein (p26). Although AGID is inexpensive and specific, the report of results takes considerable time and the test has low analytical sensitivity. OBJECTIVE: To validate our in-house indirect ELISAgp90/45 , following the World Organization of Animal Health (OIE) criteria. STUDY DESIGN: Test validation. METHODS: Synthetic peptides gp90 and gp45 were used as antigens in ELISAgp90/45 . Tests used for validation, calibration and linear working operating range, analytical and diagnostic sensitivity and specificity, repeatability and reproducibility were assessed by comparing them with the AGID test and using 1844 equine sera grouped into five different panels. RESULTS: We were able to replace the National References Sera with our Internal Reference Sera. ELISAgp90/45 had acceptable repeatability and reproducibility. Analytical sensitivity of the ELISAgp90/45 was 800 times greater than that of AGID test for positive sera and 400 times greater for weak positive sera. ELISAgp90/45 also showed optimal analytical specificity, since no cross-reactivity was detected with antibodies against other equine viruses. One sample was positive by AGID test and negative by ELISAgp90/45. ELISAgp90/45 was performed using 243 EIA positive and 878 negative equid sera, and showed a diagnostic sensitivity of 99.59% [CI 97.73%-99.99%] and a diagnostic specificity of 90.32% [CI 88.17%-92.19%], compared to AGID test; thus, it was demonstrated to be a robust test. MAIN LIMITATIONS: Samples were derived from naturally infected equid populations showing heterogeneous clinical states: therefore, their status was uncertain and some horses were sampled more than once. The AGID test may not be the most useful gold standard. CONCLUSION: ELISAgp90/45 is a useful tool for the diagnosis of EIAV infection and meets validation requirements established by the OIE.

Diagnostic accuracy of refractometry methods for estimating passive immunity status in neonatal beef calves.

BACKGROUND: Assessing the inadequate transfer of passive immunity (ITPI) in beef calves is crucial because calves with ITPI are at high risk for morbidity and mortality. OBJECTIVES: The aim of this study was to determine the accuracy of digital Brix (D-BRIX) and digital serum total protein (D-STP) refractometers to estimate different passive immunity status in beef calves and to determine the robustness of thresholds. METHODS: Blood samples were collected from 202 (1-7 days old) beef calves. Serum total solid percentages, total protein concentrations, and IgG concentrations were measured with the D-BRIX refractometer, D-STP refractometer, and gold standard radial immunodiffusion (RID) assay, respectively. Data were analyzed using diagnostic test accuracy, areas under the receiver operating characteristics curve, Cohen's kappa coefficient, and misclassification costs analysis to estimate IgG concentrations <10, <16, and <24 mg/mL. RESULTS: For the prediction of serum IgG concentrations <10, <16 and <24 mg/mL, the optimal cut-off values were determined to be <8.5% (Se: 100.0% (95% CI: 87.9-100.0); Sp: 94.2% [95% CI: 89.6-97.2]), <8.5% (Se: 92.1% [95% CI: 78.6-98.2]; Sp: 97.6% [95% CI: 93.9-99.3]), and <10.1% (Se: 88.8% [95% CI: 79.7-94.7]; Sp: 67.2% [95% CI: 58.1-75.4]), respectively, for the D-BRIX refractometer; and <5.2 g/dL (Se: 100.0% [95% CI: 87.9-100.0]; Sp: 93.6% [95% CI: 88.9-96.8]), <5.2 g/dL (Se: 92.1% [95% CI: 78.6-98.2]; Sp: 97.0% [95% CI: 93.0-99.0]), and <6.4 g/dL (Se: 87.5% [95% CI: 78.2-93.8]; Sp: 69.7% [95% CI: 60.7-77.7]), respectively, for the D-STP refractometer. CONCLUSIONS: The digital Brix and digital serum total protein refractometers can be used as monitoring tools for assessing passive immunity transfer in neonatal beef calves.

Sources of variance in the results of a commercial bovine immunoglobulin G radial immunodiffusion assay.

Radial immunodiffusion (RID) is used to quantify IgG concentration in neonatal beef or dairy calf serum; variability has been noted that may affect the precision and accuracy of assay results. We determined the source, range, and homogeneity of variance in the results of a commercial bovine IgG RID assay (Triple J Farm). To estimate the variance in the precipitin ring diameter, we used 6 sera, measured 28 times across 8 plates and 4 lots, and 3 standards with known IgG concentrations, measured 75 times across 69 plates and 5 lots. The source of diameter variance was determined using variance partition coefficients for lot, plate, and repetition. We used 11 different methods to generate standard curves to convert RID precipitin ring diameters to IgG concentrations. The Levene test of homogeneity of variance (α = 0.1) was used to evaluate the equality of variance between the standards or serum precipitin ring diameters and calculated IgG concentrations. Lot and plate contributed minimally to the diameter variance. Precipitin ring diameters had equal variance. Calculated IgG concentrations for serum not requiring dilution had equal variance. A linear equation from aggregated standards, performed within the same day, had greater accuracy for the calculated IgG concentrations of the standards compared to other equation methods. Regardless of standard curve methodology or IgG concentration, variability inherent to the assay limits its clinical usefulness.