Detección de trazas de soja y de leche en productos libres de gluten: desarrollo de dos enzimoinmunoensayos competitivos / Detection of traces of soy and milk in gluten-free products: development of two competitive enzyme immunoassays / Detecção de vestígios de soja e leite em produtos livres de glúten: desenvolvimento de dois enzimoimunoensaios competitivos

Resumen El objetivo del presente trabajo fue el desarrollo de dos enzimoinmunoensayos competitivos (EIC) para la detección de trazas de soja y de leche en productos libres de gluten. Como anticuerpos primarios se utilizaron antisueros policlonales de conejo específicos contra proteínas de soja o de leche. Se determinaron las concentraciones óptimas de antígenos a inmovilizar en la placa y las concentraciones de anticuerpos primarios a utilizar en la competencia. Las curvas de calibración se ajustaron utilizando concentraciones crecientes de un extracto de producto de soja y de un extracto de leche descremada en polvo. El producto de soja y la leche descremada se extrajeron con buffer Tris-HCl 0,0625 M con dodecilsulfato de sodio al 3% y sulfito de sodio 0,1 M al 2%. Se evaluaron los parámetros de validación: linealidad, límites de detección y de cuantificación, recuperación y precisión en el día y entre días, los cuales resultaron adecuados. Se analizaron 9 productos libres de gluten con los EIC desarrollados y con kits de ELISA comerciales. Ambos EIC se comportaron de manera similar con respecto a los kits comerciales. Los EIC permitieron confirmar la presencia de leche en las muestras que la declaraban. En algunas muestras que no declaraban ni leche ni soja, ambos EIC detectaron su presencia (resultados confirmados con los kits comerciales). Los EIC desarrollados poseen menor costo que los kits y, por lo tanto, éstos podrían utilizarse como métodos de screening. Cuando esta metodología resulte negativa, debe confirmarse con un método más sensible (comercial) para garantizar la ausencia de proteínas de soja o de leche.

Abstract The aim of this study was to develop two competitive enzyme immunoassays (CEI) to detect the presence of traces of soy and milk in gluten-free products. Specific rabbit polyclonal antiserums against soy protein and other against elemilk protein were used as primary antibodies. Optimal antigen concentrations to be immobilized on the plate and primary antibody concentrations to be used in competition were determined. The calibration curves were fitted using increasing concentrations of an extract of soy product and of defatted milk powder. The soy product and the defatted milk were extracted with Tris-HCl buffer 0,0625 M with 3% sodium dodecyl sulfate and 2% sodium sulfite 0.1 M. The validation parameters were evaluated: linearity, limit of detection and quantification, recovery and precision on the day and in between days. They were appropriate. Nine commercial samples of gluten-free products were analyzed with these developed CEI and commercial ELISA kits. It was observed that both CEI behaved similarly with respect to the commercial kits. The enzyme immunoassays confirmed the presence of milk in samples that declared it. In some samples that did not declare the presence of milk or soy, both enzyme immunoassays detected their presence -these results were confirmed using commercial kits. The developed CEI have a lower cost than the commercial kits, so these could be used as screening methods. When this methodology is negative, it should be confirmed with a more sensitive (commercial) method to ensure the absence of soy or milk protein.

Resumo O objetivo do presente trabalho foi o desenvolvimento de dois enzimoimunoensaios competitivos (EIC), para a detecção de vestígios de soja e leite em produtos livres de glúten. Antissoros policlonais de coelho específicos contra proteínas de soja ou de leite foram utilizados como anticorpos primários. Foram determinadas as concentrações ótimas de antígenos a serem imobilizados na placa e as concentrações de anticorpos primários a serem utilizadas na competição. As curvas de calibração foram ajustadas usando concentrações crescentes de um extrato de produto de soja e de um extrato de leite em pó desnatado. O produto de soja e o leite desnatado foram extraídos com tampão Tris-HCl 0,0625 M com dodecil sulfato de sódio a 3% e sulfito de sódio 0,1 M a 2%. Os parâmetros de validação foram avaliados: linearidade, limite de detecção e quantificação, recuperação e precisão no dia e entre os dias, os quais resultaram adequados. Nove produtos livres de glúten foram analisados com os EIC desenvolvidos e com kits de ELISA comerciais. Os dois EICs se comportaram de maneira semelhante em relação aos kits comerciais. Os EIC permitiram confirmar a presença de leite nas amostras que o declararam. Em algumas amostras que declaravam nem leite nem soja, ambos os EIC detectaram sua presença (resultados confirmados usando kits comerciais). Os EIC desenvolvidos têm um custo menor que os kits, portanto, eles poderiam ser utilizados como métodos de triagem. Quando esta metodologia é negativa, deve ser confirmada com um método mais sensível (comercial) para garantir a ausência de proteínasda soja ou do leite.

Saúde nas fronteiras: análise quantitativa e qualitativa da clientela do Centro Materno Infantil de Foz do Iguaçu, Brasil / Health at the border: quantitative and qualitative analysis of patients treated at the Maternal and Child Care Center in Foz do Iguaçu, Brazil

Resumo Foz do Iguaçu participa do SIS-Fronteiras e instalou o Centro Materno Infantil (CMI), ofertando atendimento ao pré-natal das gestantes brasileiras moradoras no Paraguai (brasiguaias). Para analisar as características do CMI e comparar o perfil de brasiguaias com gestantes brasileiras residentes no Brasil, conciliou-se abordagem quanti-qualitativa na metodologia. Verificou-se que gestantes brasiguaias atendidas no CMI procuram o local devido à precariedade do sistema de saúde paraguaio. Elas são mais jovens, apresentam maior paridade, menor escolaridade e não têm companheiro, quando comparadas às moradoras no Brasil. Elas omitem onde moram, tentando minimizar a possiblidade de terem atendimento inferior ao das brasileiras do local, ou terem negado seu direito à consulta; e buscam o serviço de obstetrícia tardiamente para evitar a negativa do atendimento. Elas geram custo alto para o município, sobretudo pela desinformação sobre a sua história reprodutiva e gestacional, o que aumenta as chances de serem submetidas a parto cesáreo e de internação da mãe e/ou do bebê, por complicações. Ações efetivas em relação à saúde materno-infantil nas zonas de fronteira precisam ser priorizadas.

Abstract Foz do Iguaçu participates in the SIS-Fronteiras program and installed the Maternal and Child Care Center (CMI) to offer prenatal care service to pregnant Brazilian women resident in Paraguay (Brasiguaias). To analyze the characteristics of the CMI and compare the profile of Brasiguaias with pregnant Brazilian women resident in Brazil, a quantitative and qualitative approach in methodology was applied. It was found that Brasiguaias go to the CMI because of the precariousness of services of the Paraguayan Health System. They tend to be younger, bear more children, have lower education and are unmarried compared with pregnant Brazilian woman resident in Brazil. They omit where they live to avoid being denied the right or receiving inferior treatment than local pregnant Brazilian women and seek obstetric treatment later to avoid being denied attendance. Pregnant Brazilian women resident in Paraguay are onerous to the municipality, especially due to misinformation about their reproductive and pregnancy history, which increases the chances of undergoing cesarean delivery and hospitalization of the mother and/or infant due to complications. Effective actions in relation to maternal and child health in the border areas need to be prioritized.

Functional estimation of mannose binding lectin associated serine protease (MBL-MASPs) in human serum.

Background & objectives: Mannose binding lectin (MBL), a C-type or Ca2+ dependent lectin, plays a major role in lectin pathway of complement activation. MBL deficiency/insufficiency is associated with susceptibility to many infections. It is important to know the association of functional lectin levels with disease condition. Therefore, we carried out this study to develop a simple assay to estimate the functional MBL-associated serine proteases (MBL-MASPs) levels in human serum samples. Methods: A novel method was developed based on direct haemolysis of mannan coated human erythrocytes in autologous human serum for functional estimation of MBL and associated serine proteases (MBLMASPs complex). Functional MBL-MASPs serum levels in 75 healthy individuals was estimated. Results were compared with those obtained by ELISA based assay. Results: Lysis of mannan coated human RBC in autologous serum was highly specific and mediated by MBL-MASPs lectin complement pathway. Concentration of MBL-MASPs in serum of normal healthy individuals (n=75) was found to be 1.579 μg/ml (median= 1.149 μg/ml) by the haemolytic assay which was comparable to the values obtained by ELISA method. Interpretation & conclusions: Our findings showed that the method developed for the estimation of functional MBL-MASPs levels in human serum is simple, cost-effective and comparable with existing ELISA method.

Comparación de tres estrategias de tamizaje para la prevención de la infección perinatal por VIH en Colombia: análisis de decisiones / A comparison of three screening strategies for prevention of perinatal HIV infection in Colombia: a decision analysis model

OBJETIVO: Comparar mediante un modelo de análisis de decisiones tres estrategias de tamizaje de la infección por el VIH en mujeres embarazadas según su relación costo-efectividad y proponer la más apropiada para el sistema de salud colombiano. MÉTODOS: Estudio económico basado en el análisis mediante árboles de decisión según tres estrategias de tamizaje de la infección por el VIH en mujeres embarazadas: la voluntaria, la universal y la opcional. Se consideró a todas las mujeres colombianas embarazadas sin diagnóstico de infección por el VIH que se presentaban para el parto. Se emplearon los costos médicos directos desde la realización de la prueba hasta un año después del parto, según el Sistema General de Seguridad Social en Salud. Se compararon las razones costo-efectividad y el ahorro de cada estrategia analizada. RESULTADOS: Por cada 10 000 mujeres, la estrategia universal permitió detectar 5 casos más que la estrategia voluntaria y 7 casos más que la opcional. La estrategia universal generó costos aproximados de US$ 17,00 por cada recién nacido positivo, es decir, menos de la mitad que lo calculado para la estrategia voluntaria (US$ 38,00) y menor que para la opcional (US$ 24,00). Según el análisis bifactorial, la estrategia de tamizaje universal fue menos costosa que la voluntaria y más efectiva que las otras dos estrategias, independientemente de la prevalencia, la tasa de positivos falsos del sistema de diagnóstico empleado y la tasa de aceptación materna para realizarse la prueba de tamizaje. CONCLUSIONES: La estrategia de tamizaje voluntaria, que se utiliza actualmente en Colombia, es más costosa que la universal a mediano y largo plazos y tiene menor efectividad y capacidad de prevención. Se recomienda a las autoridades nacionales de salud realizar el tamizaje de la infección por el VIH a todas las embarazadas colombianas con pruebas de tercera generación.

OBJECTIVES: To apply decision analysis to compare the cost-effectiveness of three strategies for HIV screening of pregnant women and to recommend the one most appropriate for the health care system of Colombia. METHODS: An economic study applying decision analysis to three types of HIV screening of expectant women: voluntary, universal, and optional. All the women in Colombia with unknown HIV status who were admitted for child birth were included. The study included all the direct medical costs incurred from the time of testing through the first year following delivery, according to the General System for Healthcare Social Security. Cost-effectiveness ratio and the savings of each of the strategies were compared. RESULTS: For every 10 000 women, the universal strategy detected five cases more than the voluntary strategy and seven cases more than the optional. The universal strategy carried a cost of approximately US$ 17 for each HIV-positive newborn; that is, less than half of that of the voluntary strategy (US$ 38) and less than the optional (US$ 24). According to the bifactorial analysis, the universal screening strategy was less costly than the voluntary and more effective than both of the others, regardless of prevalence, the false-positive rate of each method, and the rate of maternal compliance with screening. CONCLUSIONS: The screening strategy currently in use in Colombia is more costly (in both the medium- and long-term), less effective, and less capable of prevention, than the universal screening strategy. The recommendation to the national health authorities of Colombia is to begin screening all pregnant women for HIV infection using third-generation testing.

Advantage of using a home-made ELISA kit for detection of Helicobacter pylori infection over commercially imported kits.

PURPOSE: To evaluate a home-made ELISA kit for detection of Helicobacter pylori (Hp) infection and comparison of its immunologic criteria with those of foreign commercial kits. METHODS: A home-made IgG ELISA kit was developed using soluble antigenic fractions of Hp proteins. Confirmed sera were tested and serological criteria were evaluated through assessment of 199 serum samples. RESULTS: The accuracy, sensitivity and specificity values of home-made kit were 92, 92 and 90.4%, respectively. These immunologic criteria for Trinity kit were 95.2, 95.2 and 95% in comparison with IBL kit (91.3, 92.2 and 88.5%), BIOHIT kit (72.4, 41.6 and 94.1%) and HelicoBlot2.1 (94.2, 93.4 and 100%). Kappa agreement assessment demonstrated that two of the imported ELISA kits had fair to moderate agreement with the home-made kit while the other one had a poor agreement value. CONCLUSIONS: Apart from comparable values between the home-made kit and the most efficient imported kit (Trinity) there was significant cost benefit. Therefore, we recommend the home-made kit as a suitable substitution for detection of Hp infection in the Iranian population.

Cost-effective analysis of different algorithms for the diagnosis of hepatitis C virus infection

We compared the cost-benefit of two algorithms, recently proposed by the Centers for Disease Control and Prevention, USA, with the conventional one, the most appropriate for the diagnosis of hepatitis C virus (HCV) infection in the Brazilian population. Serum samples were obtained from 517 ELISA-positive or -inconclusive blood donors who had returned to Fundação Pró-Sangue/Hemocentro de São Paulo to confirm previous results. Algorithm A was based on signal-to-cut-off (s/co) ratio of ELISA anti-HCV samples that show s/co ratio ≥95 percent concordance with immunoblot (IB) positivity. For algorithm B, reflex nucleic acid amplification testing by PCR was required for ELISA-positive or -inconclusive samples and IB for PCR-negative samples. For algorithm C, all positive or inconclusive ELISA samples were submitted to IB. We observed a similar rate of positive results with the three algorithms: 287, 287, and 285 for A, B, and C, respectively, and 283 were concordant with one another. Indeterminate results from algorithms A and C were elucidated by PCR (expanded algorithm) which detected two more positive samples. The estimated cost of algorithms A and B was US$21,299.39 and US$32,397.40, respectively, which were 43.5 and 14.0 percent more economic than C (US$37,673.79). The cost can vary according to the technique used. We conclude that both algorithms A and B are suitable for diagnosing HCV infection in the Brazilian population. Furthermore, algorithm A is the more practical and economical one since it requires supplemental tests for only 54 percent of the samples. Algorithm B provides early information about the presence of viremia.

Enzyme-linked immunosorbent assay (ELISA) immunoassaying versus microscopy: advantages and drawbacks for diagnosing giardiasis

CONTEXTO E OBJETIVO: A giardíase é comum no Brasil. Para o diagnóstico laboratorial, o método mais empregado é o exame microscópico de amostras fecais. O método imunoenzimático (ELISA) também é utilizado. O objetivo deste trabalho é verificar as vantagens e desvantagens do método microscópico quando comparado ao imunoenzimático para o diagnóstico de Giardia lamblia em uma única amostra fecal. TIPO DE ESTUDO E LOCAL: Estudo prospectivo, duplo cego, no Laboratório de Parasitologia, Faculdade de Medicina da Fundação ABC. MÉTODOS: As amostras foram preparadas para exame de acordo com os tradicionais métodos de sedimentação (Hoffman, Pons e Janer) e Faust. Um resultado positivo significa o encontro de Giardia lamblia por um dos métodos ou ambos. O kit Prospect ELISA foi utilizado para detecção do antígeno específico de Giardia lamblia, de acordo com as instruções do fabricante. Os resultados foram expressos em escala visual como negativos ou positivos (+, ++, +++ ou ++++). RESULTADOS: O teste ELISA é positivo mesmo quando uma significante proporção das correspondentes amostras examinadas por microscopia ainda é negativa, sendo esta tendência estatisticamente significante (p < 0,001). A concordância de resultados entre os dois métodos é apenas moderada (0,5 pelo teste kappa). CONCLUSÃO: O teste ELISA é recomendável quando se busca uma elevada sensibilidade para a detecção de antígenos específicos de Giardia lamblia, como em estudos de prevalência. Para a prática diária, recomendamos o método microscópico, que tem custo muito menor e pode detectar outros parasitas na mesma amostra. A baixa taxa de positividade do método no exame de uma única amostra pode ser contornada pelo exame de três amostras, como recomendado pela maioria dos autores.

Quantification of 20-hydroxyeicosatetraenoic acid by colorimetric competitive enzyme linked immunosorbent assay.

Analysis of 20-hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictor produced by the cytochrome P450 pathway, presently requires high-performance liquid chromatography (HPLC) and gas chromatography/ mass spectrometry (GC/MS). To simplify 20-HETE analysis, competitive ELISAs were developed using polyclonal anti-20-HETE coated ELISA plates to which free 20-HETE and 20-HETE conjugated to horseradish peroxidase (HRP) or alkaline phosphatase (AP) were added. Assays were developed with and without a pro prietary enhancer solution which allows for the extraction-free measurement of 20-HETE in urine samples. The bound 20-HETE-HRP or 20-HETE-AP was detected using 3,3 ,5,5, -tetramethylbenzidine and p-nitrophenyl phosphate, respectively. Sensitivities expressed as 80% B/B0, were 0.1 ng/ml for the HRP assay, and 0 5 ng/ml for the AP assay, with r2 = 0 99 for both formats. Of the 17 lipids tested for cross-reactivity, arachidonic acid showed the highest (0.32%) followed by racemic 5-HETE (0.07%) and 8,9-dihydroxyeicosatrienoic acid (DHET) (0.04%). Preliminary validation experiments examining serum and urine concentrations of 20-HETE yield values that fall within the ranges established by GC/MS in the literature. These ELISAs provide simple and inexpensive methods for the analysis of 20-HETE in biological samples.

The use of filter paper plasticized with polyvinyl alcohol-glutaraldehyde in ELISA

F1-antigen purified from Yersinia pestis was covalently linked to 5-mm diameter filter paper discs plasticized with polyvinyl alcohol-glutaraldehyde. These discs were used both for ELISA and dot-ELISA for the detection of anti-F1 IgG in rabbits. The best conditions were achieved using 1.25 µg of F1 antigen/disc, 3 percent w/v skim milk in PBS as blocking agent, anti-IgG peroxidase conjugate diluted 12,000 times, and serum from rabbits immunized or not against Y. pestis, diluted 6,400 times. The absorbance values obtained from the comparative study between this procedure and conventional ELISA were not significantly different but the low cost of the reagents employed in ELISA using the filter paper discs plasticized with polyvinyl alcohol-glutaraldehyde makes this method economically attractive.

Effect of serum dilution in diagnosis of malaria in community.

Recently, it has been shown that immunological methods can be used for the diagnosis of malaria other than sero-epidemiology. A study has been done to investigate optimum binding capacity of antigen-antibody (Ag-Ab) at different serum dilutions. For validating antigen-antibody (Ag-Ab) reaction at 1:100, 1:1000 and 1:1000 serum dilutions, have been tested in two different laboratories to establish validation of the ELISA method. Inter laboratory test on synthetic peptide (RI) ELISA was found comparable and meaningful for assessing malaria transmission in defined locality at 1:100 dilution. Results also showed that 1:1000 serum dilution can be useful for diagnostic purpose.

Pesquisa de anticorpos contra o vírus da hepatite C em pool de 5 soros: sua utilizaçäo em inquéritos soroepidemiológicos / Detection of antibodies against hepatitis C virus in a 5 sera pool testing: its use in seroepidemiological survey

Para avaliar reduçäo de custo da pesquisa de anticorpos contra hepatite C, por ELISA, em pool de cinco soros, realizou-se o teste em grupos de baixo e alto risco. Dois terços dos conjuntos de alto risco tiveram que ser repetidos. A reduçäo do gasto de reagente foi de 80 por cento na populaçäo de baixo risco e de 13 por cento na amostra de alto risco. Estes dados sugerem que a pesquisa do anti-VHC em pool reduz o custo do procedimento em populaçöes de baixo risco

Development and assessment of ELISA for serodiagnosis of HIV-infection.

A home-made ELISA for serodiagnosis of HIV-infection was developed. It made use of the HIV viral lysate to coat on ELISA microplates. The purpose was to establish an ELISA for serodiagnosis of HIV-infection. The newly-developed ELISA, "H-ELISA", was applied to test 792 samples of HIV-positive serum as confirmed by Western blot. All 792 samples were positive by H-ELISA. It was also applied to test 540 samples of normal sera obtained from different laboratories in Srinagarind Hospital. A number 530 normal sera was negative, 8 samples were positive and confirmed by Western blot and 2 samples were false positive. It was concluded that the H-ELISA possessed 100% sensitivity with a false positive rate of 2/532(0.38%). The H-ELISA, the cost/test was less than 5 bahts, appears to be promising for substitution of imported commercial kits.

Comparative evaluation of bioassay and ELISA for detection of Japanese encephalitis virus in field collected mosquitos.

Comparative evaluation of enzyme-linked immunosorbent assay (ELISA) and bioassay (virus isolation in Toxorhynchites splendens larvae and identification by immunofluorescence test using virus specific monoclonal antibody) was carried out in order to define a suitable strategy for monitoring Japanese encephalitis virus infection in field mosquitos. A total of 8,850 adult female mosquitos in 177 pools (Culex tritaeniorhynchus 91, Cx. vishnui 59 and Cx. fuscocephala 27) collected from an endemic area of Tamil Nadu were examined by both the techniques. In ELISA, 9 pools which had optical densities (OD) equal to the mean of normal infected pools plus > or = 4 standard deviations (SD) mean considered positive and all of them were virus positive by the bioassay also. Sixty-five pools had OD = Mean + 2-3 SD and 103 pools had OD = Mean + < 2 SD of normal pools. From these groups, 12 (18.5%) and 8 (7.8%) pools respectively were found to be virus positive by the bioassay. In total 29 (16%) pools were positive by the bioassay as against 9 (5%) by ELISA. This study demonstrated that the bioassay is sensitive for estimation of true positives and ELISA is a rapid screening system. A protocol has now been developed for surveillance in which field pools are first screened by ELISA and only those with OD = Mean + > or 2 SD are assayed in Toxorhynchites. By excluding a large majority of pools with low OD (Mean + < 2 SD), which are likely to yield to only a small percentage of true positives, the cost, time and labor involved are greatly reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of the cost of testing for antibody to human immunodeficiency virus, without losing sensitivity, by pooling sera.

The sensitivity of testing pooled sera instead of individual sera for antibody against human immunodeficiency virus (HIV) was evaluated using a non-competitive enzyme-linked immunosorbent assay (ELISA). For this purpose, 42 HIV antibody positive sera were titrated and introduced into 42 sets of pools of 2, 4, 8, 16, 32 or 64 sera in such a manner that each pool had one positive sample and the rest, HIV antibody negative sera. When the pools were tested in ELISA, all pools with high titred antibody positive sera were reactive irrespective of pool size, while some of the pools containing medium or low titred sera were non-reactive when pool size exceeded 16. Subsequently the pool size was limited to 16. When 208 previously unscreened samples were tested in 52 pools of 4, 26 pools of 8 or 13 pools of 16 sera, or individually, 6 antibody positive sera were correctly identified. Thus, it was found that the pooling method did not reduce the sensitivity of ELISA test, whereas the cost was reduced to less than half of that of individual testing.