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.

[Determination of the immunoglobulin G supply in the newborn calf]. / Bestimmung der Immunglobulin-G-Versorgung beim neugeborenen Kalb.

A sufficient supply of colostral antibodies within the first hours of life is crucial for the development and the health status in young calves. It is rational to examine the immunoglobulin uptake of single animals, but particularly on a herd basis, during herd controls and consultations. This enables economical calf rearing in accordance with animal welfare. Because of the costly, laboratory-dependent and in part time-consuming direct measurement of the absorbed immunoglobulins using radial immunodiffusion (RID) or ELISA, multiple studies attempted to develop indirect methods, which would be affordable and operational in the field. These aim to draw an inference for the absorbed quantity of colostral antibodies based on other correlated parameters. Multiple validations showed in part significant differences between various methods concerning specificity and sensitivity in comparison to the direct methods. In addition to RID and ELISA, this article presents the measurement of the γ-glutamyltransferase (GGT) activity, the determination of the total serum protein concentration using refractometry and the zinc sulphate turbidity test, and describes the advantages and disadvantages of their application. Refractory measurement and determination of the GGT activity represent a valuable alternative to a laboratory-dependent immunoglobulin G measurement. Nevertheless, there is no ideal rapid test method, such that several influencing factors have to be considered.

Immunoelectrophoresis: A Method with Many Faces.

Immunoelectrophoresis (IEP) was the first practical method that combined electrophoresis and immunoprecipitation for identifying and characterizing proteins within complex mixtures. Over the years, IEP has been extended to include a variety of techniques and, as a general name, has been applied to virtually any technique that involves electrophoresis and antigen-antibody precipitin reaction for proteins. Because of the diversity in technical details of different IEP versions, the method described here deals only with classic IEP. Although it requires some manual expertise, IEP is versatile, relatively easy to customize, and economical with no need for expensive instrumentation. Further, it can discern identity, partial identity, and nonidentity of the proteins. Any low-viscosity body fluid specimen or, possibly, culture fluid and tissue extract could be tested with IEP if proper antibodies are available. With these attributes, classic IEP remains a valuable tool for clinical diagnostic testing, purity checking of biochemical and pharmaceutical products, and research.

Performance assessment of imported ELISA in the serodiagnosis of the enzootic bovine leukosis in herds of Pernambuco state, Brazil / Avaliação do desempenho de ELISA importado no sorodiagnóstico da leucose enzoótica bovina em rebanhos do estado de Pernambuco, Brasil

Enzootic bovine leukosis (EBL) is an infectious disease of cosmopolitan distribution and chronic character caused by a virus of the Retroviridae family, bovine leukemia virus (BLV). The epidemiological situation of EBL in Brazil has motivated studies to improve its diagnosis, based on the recommended serological techniques: agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA). This study was designed to evaluate the use of imported ELISA for the detection of BLV in dairy herds raised in Pernambuco, Brazil, comparing it to AGID. Blood serum samples from 327 dairy cattle from the state of Pernambuco were tested to AGID and the imported commercial ELISA CHEKIT-Leucose-serum, produced by the IDEXX® laboratory for the diagnosis of EBL. Discarding 25 inconclusive samples from one or both tests, 302 samples were analyzed, being 24.1% positive (73/302) in the AGID and 45% (136/302) in the ELISA, which compared to the AGID, a technique considered standard, presented sensitivity of 98.6%, specificity of 72% and Kappa coefficient of 0.55. The lack of agreement in the diagnostic methods was probably due to the high sensitivity of the ELISA, which makes it possible to detect antibodies even in situations with low serum levels. Although AGID has been shown to be an efficient test so far, in more advanced stages of an EBL control and eradication program, with low prevalence rates, ELISA will present better performance, due to its higher sensitivity, avoiding the permanence of animals that spread the disease in the herds.(AU)

A leucose enzoótica bovina (LEB) é uma doença infecciosa de distribuição cosmopolita e caráter crônico causada por um vírus da família Retroviridae, o vírus da leucemia bovina (VLB). A situação epidemiológica da LEB no Brasil vem motivando estudos para o aprimoramento do seu diagnóstico, tendo como base as técnicas sorológicas recomendadas: imunodifusão em gel de ágar (IDGA) e Enzyme-Linked Immunoabsorbent Assay (ELISA). Este estudo teve como objetivo avaliar o uso de ELISA importado para a detecção do VLB em rebanhos leiteiros criados em Pernambuco, Brasil, comparando-o ao IDGA. Amostras de soro sanguíneo de 327 bovinos leiteiros do estado de Pernambuco foram testadas para IDGA e ELISA comercial importado CHEKIT-Leucose-serum, produzido pelo laboratório IDEXX® para o diagnóstico da LEB. Descartadas 25 amostras inconclusivas de um ou ambos os testes, foram analisadas 302 amostras, sendo 24,1% positivas (73/302) na IDGA e 45% (136/302) no ELISA, que em relação à IDGA, técnica considerada padrão, apresentou sensibilidade de 98,6%, especificidade de 72% e coeficiente Kappa de 0.55. A falta de concordância entre os métodos diagnósticos deveu-se, provavelmente, à elevada sensibilidade do ELISA, que possibilita detectar anticorpos mesmo em situações com baixos teores séricos. Apesar da IDGA se mostrar até o momento um teste eficiente, em etapas mais avançadas de um programa de controle e erradicação da LEB, com baixos índices de prevalência, o ELISA apresentará melhor desempenho, por possuir maior sensibilidade, evitando-se a permanência de animais disseminadores da doença nos rebanhos.​(AU)

Assessment of different methods to estimate bovine colostrum quality on farm.

AIMS: To evaluate two different hydrometers and an optical and a digital Brix refractometer for the assessment of bovine colostrum quality, in terms of accuracy and precision compared with the measurement of IgG concentrations using radial immunodiffusion (RID), and to evaluate the reliability and repeatability of the Brix refractometers. METHODS: To determine reliability and repeatability, 145 colostrum samples were tested by two independent observers twice, using the optical and digital Brix refractometers. A further 193 colostrum samples from Holstein cows were collected on one commercial dairy farm at first milking and tested with two hydrometers and an optical and digital Brix refractometer. An aliquot of each sample was frozen for RID measurement of IgG concentrations and samples were classified as poor (≤50 g IgG/L) or good (>50 g IgG/L) quality colostrum. Intraclass correlation coefficients (ICC) were used to determine inter- and intra-observer reliability and repeatability. Optimised cut-off values for the four devices were determined using receiver operating characteristics (ROC) analysis with the RID results as the reference. Using these cut-offs, sensitivities and specificities for determining good quality colostrum were calculated. RESULTS: The ICC for inter-observer reliability was 0.98 for the optical Brix refractometer, and for intra-observer repeatability was 0.97 and 0.98 for the optical and the digital Brix refractometers, respectively. For the 193 colostrum samples, 67 (34.7%) had concentrations of IgG ≤50 g/L determined by RID. Optimised cut-off values evaluated by ROC analysis were higher for all devices compared with manufacturer reference or previously published values. Using these values, the sensitivities for the two hydrometers, and the optical and the digital Brix refractometers were 0.73, 0.71, 0.56 and 0.79, respectively; specificities were 0.72, 0.61, 0.90 and 0.69, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: The Brix refractometers provided the most accurate assessment of colostrum quality of the devices evaluated, and demonstrated excellent precision in terms of repeatability. To provide optimal health for newborn calves, a sufficient intake of good quality colostrum is essential. The Brix refractometers provide rapid, convenient tools for classification of colostrum quality.

Assessment of four serological techniques in the immunological diagnosis of farmers' lung disease.

Farmers' lung disease (FLD) is a pulmonary disease that results from repeated inhalation of antigens from mouldy hay or straw. The objective of this prospective study was to assess the reliability of four serological techniques in FLD diagnosis. Sera from 15 consecutive patients with FLD, 15 healthy control farmers and 30 urban controls were analysed using four serological techniques [electrosyneresis (ES), Ouchterlony double diffusion (DD), ELISA and Western blot (WB)] with four antigens (Absidia corymbifera, Eurotium amstelodami, Wallemia sebi and Saccharopolyspora rectivirgula). In the authors' region, ES on cellulose acetate with A. corymbifera antigen was the most relevant diagnostic tool for discriminating FLD patients from healthy exposed farmers (sensitivity 87 %, specificity 100 %). DD tests were in accordance with ES, but their discriminatory power was lower. No threshold indicating both good sensitivity and specificity could be established with ELISA. WB analysis failed to identify specific bands for FLD. This study demonstrates the efficacy of determining precipitin levels with an appropriate technique, using a panel of antigens consistent with the specific exposure of a given area.

Evaluación del desempeño diagnóstico del medio de difusión en agar Etest para el estudio de sensibilidad a los antifúngicos / Assessment of diagnostic performance of Agar Etest for antifungal sensitivity testing

El aumento de la incidencia de las micosis profundas ha generado la necesidad de desarrollar técnicas para el estudio de la susceptibilidad in vitro a los antifúngicos. El método de microdilución en caldo es el de referencia, sin embargo métodos alternativos surgen ante la necesidad de diagnóstico en el laboratorio clínico, siendo el Etest el método de difusión en agar m s utilizado en nuestro medio. En este trabajo se evaluó el desempeño diagnóstico del Etest en comparación con la técnica de referencia y se correlacionaron las concentraciones inhibitorias mínimas (CIMs) por ambos métodos. Se estudiaron 80 cepas de Candida spp., proces ndose por Etest y por microdilución en caldo (protocolo M27-A2 del National Committee for Clinical Laboratory Standards [NCCLS]). Se probaron: anfotericina B (AB), itraconazol (ITZ) y fluconazol (FLZ). Se evaluó el desempeño diagnóstico del Etest con respecto a la metodología de referencia (EPIDAT, versión 2.0 para Windows) y se calculó la correlación mediante el coeficiente de Pearson. El Etest presentó para el ITZ una especificidad y sensibilidad diagnóstica de 44 por ciento y 92 por ciento respectivamente, valor predictivo positivo (VPP) de 60por ciento y valor predictivo negativo (VPN) de 85 por ciento. Para el FLZ la especificidad y sensibilidad fue de 85 por ciento y 12,5 por ciento, respectivamente, VPP de 45 por ciento y VPN de 49 por ciento. Para AB el Etest no captó ninguna de las cepas resistentes. Los valores predictivos fueron calculados para detectar resistencia empleando los puntos de corte del National Committee for Clinical Laboratory Standards (NCCLS). Los coeficientes de correlación fueron los siguientes: r = - 0,02 (AB), r = - 0,03 (FLZ) y r = 0,4 (ITZ). No se validó el método desde el punto de vista analítico por no existir correlación entre las CIMs obtenidas por el Etest y la técnica de referencia. De acuerdo con los resultados obtenidos, el desempeño diagnóstico de la técnica es poco confiable. Analizando el desempeño global del Etest a la luz de los conocimientos actuales no parece un método confiable para su uso en los laboratorios de an lisis clínicos.

Evaluation of a Pourquier ELISA kit in relation to agar gel immunodiffusion (AGID) test for assessment of the humoral immune response in sheep and goats with and without Mycobacterium paratuberculosis infection.

The present study was designed to evaluate a commercial ELISA kit (Institut Pourquier) for the diagnosis of ovine and caprine paratuberculosis under Australian conditions and to compare its accuracy with the existing AGID test. The sensitivity of the ELISA in sheep and goats was 34.9% and 56.4%, with a specificity of 98.8% and 100.0%, respectively. Sensitivity of AGID was 13.8% for sheep and 39.5% for goats, with specificity of 100.0% for both species. The sensitivity of the ELISA in sheep depended on the category of histological lesions. AGID and ELISA were conditionally independent, and appeared to detect overlapping but distinct subgroups of infected animals. The ELISA was significantly more sensitive than the AGID. The ELISA was simple to perform, robust and repeatable. Coefficients of variation of <12.0% were observed for positive and negative controls included on 193 plates over a 10-month period and there was a high level of intraassay repeatability with 12.0% of the duplicate samples having CV of >15.0%.

[Assessment methods of autoantibodies in autoimmune diseases]. / Nowoczesne metody wykrywania autoprzeciwcial w chorobach z autoagresja.

In most of the autoimmune diseases, a humoral and cellular immune response is characteristically seen, with autoantibodies and cells directed to distinct intracellular antigens. This phenomenon can be shown in systemic diseases like sclerosis, systemic lupus erythematosus, Sjogren's syndrome, mixed connective tissue disease, polymyositis and rheumatoid arthritis. It is also evident that autoantibodies are present in many the autoimmune diseases (called organ-specific) like for example Hashimoto, Graves-Basedow or Addison disease. The presence of autoantibodies is important items to be considered in establishing a diagnosis and because of that autoantibodies are included in the diagnostic criteria of many autoimmune diseases. They are useful prognostic markers in some situations and facilitate clinical and treatment follow-up. Since year 1950 (discovering of the first autoantibody--classical rheumatoid factor IgM class) many new methods like: immunoelectrophoresis, counter-electrophoresis, double and radial immunodiffusion in gels, immunoagglutination and haemolytic methods have been used for autoantibodies assessment. It seems to us the indirect immunofluorescence method (IIF) was a most powerful, sensitive and comprehensive test for screening of autoantibodies, until an immunoenzymatic (EIA) methods (ELISA, Western-blotting) in late 60-s was worked out. The immunoenzymatic tests are very useful because of their simplicity and reliability. But there is one more excellent test hybrid named "Colorzyme" (presented by Immuno-Concept Corporation from USA) worked out by combining of the EIA and IIF tests. Instead of FITC-conjugates (like in IIF) a HRP-conjugates for developing of typical ANA-test based on glass fixed Hep-2 cells have been used. The nuclear type of pattern we get using "Colorzyme" test are very strong, nit and rich in details. The prevalence of the "Colorzyme" test relies on that it can be properly done and interpreted by unexperienced technician. More and more new-founded resources of marker autoantibodies and methods force to introduction into standardization both methods and specimens on which they are marked.

Measurement of apolipoproteins B and A by radial immunodiffusion: methodological assessment and clinical applications.

The clinical evaluation of apolipoproteins is of interest in order to characterize the risk profile for ischemic heart disease both in normolipidemic and hyperlipidemic subjects. In the non-specialized and/or small practice clinical laboratory, the measurement of some apolipoproteins can be undertaken by simple methods of immunological analysis, among which radial immunodiffusion can be of interest due to its simplicity of use and because it does not require specific equipment. In this work several methodological questions concerning the measurement of plasma apolipoproteins B and A by radial immunodiffusion have been addressed; the results show that this method is particularly reliable for the apo B assay. Regression analysis between values obtained with radial immunodiffusion and radioimmunoassay was r = 0.972 for apo B and r = 0.782 for apo A. The recovery rate was above 90% for both apolipoproteins (93.8% for apo B and 99.5% for apo A). The inter and intraassay coefficients of variation were below 5%, and the detection limits were estimated as 9.6 mg/dl for apo A and 6.9 mg/dl for apo B. Neither the ingestion of a standard breakfast (500 Cal, 17 g fat, 120 mg cholesterol) 2 h prior to testing nor freezing the sample significantly affected the measurement of apolipoproteins B and A. Mean plasma concentrations of both apolipoproteins measured by radial immunodiffusion in normo and hyperlipidemic subjects are also presented.

Apoprotein assays: radial immunodiffusion versus an immunoturbidometric technique.

The measurement of apolipoprotein using radial immunodiffusion (RID) and Cobas-Bio immunoturbidometric assays is compared. Samples were obtained from 100 patients in a fiber-diet study. RID assays were performed using plates, standards, and diluent from Tago Inc. The immunonephelometric assays were performed on a Cobas-Bio with DENS program centrifugal analyzer. The correlation coefficient for the apolipoprotein Al was 0.90 and for apolipoprotein B was 0.91. The bias of the RID was to report higher values. The Cobas-Bio system affords the clinical laboratory with a reliable and cost-effective means for assessing these apolipoproteins.

Quantitation of serum immunoglobulins.

This article reviews critically the current "state of the art" in the quantitation of immunoglobulins in serum and other body fluids. The methodologies reviewed include (a) those occurring in solution, for example, automated immunoprecipitin and laser nephelometric techniques, rate analysis techniques and radioimmunoassays; (b) techniques involving radial diffusion in agarose gels, with and without secondary development steps; and (c) those techniques involving solid supports, such as fluoroimmunometric assays and solid phase radioimmunoassays. The theory of each technique is presented with an analysis of the strengths and weaknesses, particularly sources of error, and the techniques are compared in terms of accuracy, precision, sensitivity, test cost, equipment cost, feasibility for use in different laboratory settings, and ease of handling. Problems associated with the antisera used, with standards, and with quality controls are discussed and solutions suggested.

[An economical modification of radial immunodiffusion for the determination of immunoglobulins in the cerebrospinal fluid]. / Eine ökonomische Modifikation der radialen Immundiffusion zur Bestimmung von Immunglobulinen im Liquor cerebrospinalis.

In a total of fifty cerebrospinal fluids, such immunoglobulins as IgG, IgA, and IgM were quantitatively determined in a parallel manner on LC partigen plates and on slides (SEVAC set of means of testing). The comparability of the two methods of test proved excellent with good precision. The sensitivity of IgA determination on slides was made higher, the measurability of all precipitates was improved, and the economic advantage increased by cutting the cost of one determination fifteen times.