Development and verification of an indirect ELISA method for determination of specific IgG antibody against SARS-CoV-2 variant strain in rhesus monkey serum / 中国生物制品学杂志

@#Objective To develop and verify an indirect ELISA method for determination of specific IgG antibody of rhesus monkey serum against SARS-CoV-2 variant strain. Methods An indirect ELISA method for the determination of specific IgG antibody was developed using inactivated SARS-CoV-2 Beta variant strain inactivated vaccine as coating antigen,and optimized for the coating antigen concentration(1,2 and 4 μg/mL),dilution of serum(1∶800～1∶12 800),blocking solution(PBST containing 1% BSA,2% BSA,1% skim milk,2% skim milk and 1% BSA + 1% skim milk),blocking time(30,60 and 90 min),dilution of secondary antibody(1∶5 000,1∶10 000,1∶15 000 and 1∶20 000),incubation time of serum and secondary antibody(30,60 and 90 min),and TMB reaction time(5,10,15,20,25 and 30 min). 60 negative serum samples of rhesus monkeys were detected by the developed method,and the negative and positive critical values were determined. The sensitivity and precision of the methodology were verified. In addition,the specific IgG antibody and neutralizing antibody against SARS-CoV-2 Beta variant strain in 44 serum samples of rhesus monkey were detected by the developed method and microneutralization method,and the correlation and consistency between the two methods were compared. Results The optimum detection conditions were determined:the coating antigen concentration was 1 μg/mL;the blocking solution was PBST containing 1% skim milk,and the blocking time was 30 min;the serum samples to be tested were diluted to 1∶1 600 and incubated for 90 min,and the secondary antibody was diluted to 1∶10 000 and incubated for 30 min;the color development time of substrate was 25 min. The positive critical value and negative critical value of the method was 0. 093 and 0. 084 respectively,and the detection values between them were judged as suspicious. The dilution of5 positive serum samples that showed positive results was 1∶51 200;the coefficients of variation(CVs)of precision were all less than 15%. There was a strong correlation between IgG antibody titer and neutralizing antibody level in the 44 rhesus monkey serum samples(r = 0. 858 0,P < 0. 000 1);the total coincidence rate of the two methods was 90. 9%,the positive coincidence rate was 93. 6%,and the negative coincidence rate was 84. 6%;the consistency test Kappa value was 0. 783 8(95% CI:0. 586 5～0. 981 0). Conclusion The developed indirect ELISA method for eletermination of specific IgG antibody against SARS-CoV-2 Beta variant strain in rhesus monkey serum has good sensitivity and precision,and has strong consistency with microneutralization method,which can be used for the determination of IgG antibody in rhesus monkey serum.

Caracterización de los anticuerpos anti-HLA en posibles receptores cubanos de trasplante cardiaco / Characterization of anti-HLA antibodies in Cuban possible receptors of heart transplant

Introducción: En la supervivencia del corazón trasplantado son de importancia el empleo de los anticuerpos contra el sistema principal de histocompatibilidad (anticuerpos anti-HLA). Hace seis años se introdujo en Cuba el porcentaje de anticuerpos anti-HLA frente a panel (PRA) por método de ensayo de inmunoabsorción ligado a enzima (ELISA) como parte de las pruebas de compatibilidad pretrasplante de los receptores de trasplante cardiaco. Objetivo: Caracterizar los anticuerpos anti-HLA en pacientes receptores cubanos de trasplante cardiaco. Métodos: Entre septiembre de 2013 y abril de 2017 se les realizó el PRA por ELISA a 38 muestras de pacientes recibidas en el laboratorio de histocompatibilidad del Instituto de Hematología e Inmunología. Se utilizó la comparación de proporciones para el análisis estadístico. Resultados: El 47,4 por ciento de los pacientes estudiados presentó anticuerpos anti-HLA, fueron los más frecuentes los de clase I. La proporción de pacientes con PRA del 0 por ciento fue mayor en PRA clase II que en I (p: 0,0027). Mientras que fue mayor la proporción de pacientes con PRA clase I entre el 20 y el 75 por ciento (p: 0,0046). El 77,8 por ciento de los pacientes tuvo un PRA clase I mayor al 10 por ciento y en el PRA clase II alcanzó el 80 por ciento. Conclusiones: El porcentaje de anticuerpos anti-HLA frente a panel por método de ensayo de inmunoabsorción ligado a enzima permitió una mejor caracterización de los anticuerpos anti-HLA, lo que contribuyó a mejorar la compatibilidad en este tipo de paciente(AU)

Introduction: In survival after heart transplantation, the use of antibodies against the main histocompatibility system (anti-HLA antibodies) is important. Six years ago, the percentage of anti-HLA antibodies against panel (PRA) by enzyme-linked immunosorbent assay (ELISA) method was introduced in Cuba as part of the pre-transplant compatibility tests of heart transplant recipients. Objective: To characterize anti-HLA antibodies in Cuban heart transplant recipients. Methods: Between September 2013 and April 2017, PRA by ELISA was performed on 38 patient samples received in the histocompatibility laboratory of the Institute of Hematology and Immunology. Comparison of proportions was used for statistical analysis. Results: 47.4 percent of the study patients presented anti-HLA antibodies; those in class were the most frequent. The proportion of patients with PRA of 0 percent was higher in PRA class II than in class I (p=0.0027). The proportion of patients with PRA class I was greater, accounting for 20-75 percent (p=0.0046). 77.8 percent of the patients had a class I PRA greater than 10 percent, while in class II PRA it reached 80 percent. Conclusions: The percentage of anti-HLA antibodies versus a panel of enzyme linked immunosorbent assay method allowed better characterization of anti-HLA antibodies, which contributed to improving compatibility in this type of patient(AU)

Establishment of ELISA method for testing tobacco mosaic virus (TMV) infecting Zingiber officinale Rosc / 中国免疫学杂志

Objective:In order to establish ELISA method for testing tobacco mosaic virus (TMV) infecting Zingiber officinale Rosc.Methods: Tobacco mosaic virus(TMV) infecting Zingiber officinale Rosc was tested by RT-PCR.Zingiber officinale Rosc leaves which only contained TMV were choiced.TMV particle was purified by centrifugation method.TMV CP was purified through preparation electrophoresis including 12％ SDS-PAGE first and then 5％-20％ gradient SDS-PAGE.Polyacrylamide gel contained TMV CP was ground into suspension.Mice were immuned with the suspension and antiserum was obtained.Antiserum quality was tested by Western blot and ELISA test.IgG was purified through affinity chromatography method.IgG solution was concentrated and dialyzed to a suitable concentration.The IgG then mixed with glycerol.Results: IgG in antiserum only combined with TMV CP protein and it could combine with nature TMV particle CP protein.Its quality was up to standard.Conclusion: Establishment of ELISA method for testing TMV infecting Zingiber offcinale Rosc is successfull by using this IgG.

Determination of Specific Antibodies in Allergic Rabbits Induced by 1,3-Di-caffeoylquinic Acid in Shuanghuanglian Injection / 中国药师

Objective: To determine the antibody titer in the serum of allergic rabbits after the injection of 1, 3-di-caffeoylquinic acid contained in Shuanghuanglian injection.Methods: The complete antigen was prepared by incubating the suspected small molecular hapten 1, 3-di-caffeoylquinic acid contained in Shuanghuanglian injection with the serum from the normal rabbits.The specific antibody was obtained in the immunized rabbits.The antibody titers of antiserum were measured by ELISA kits.Indirect competitive ELISA was used to determine serological specificity, and the obtained data was used to plot the inhibition curves.The content of IgE antibody in the antiserum of rabbits was detected by rabbit immunoglobulin E (IgE) ELISA kits.Results: The antibody titer (A) of 1,3-di-caffeoylquinic acid was 2 times higher than that of the negative control, which indicated its potential allergenicity.The regression equation was I=0.170 6 lg C + 0.317 5 , which was with the correlation coefficient of r=0.985 4 , the detection limit of IC 10 =57.40 μg·ml-1 and the half inhibitory concentration of IC 50 =8.732 0 mg·ml-1.Furthermore, the exogenous IgE antibody was produced in the rabbits.Conclusion: The results indicated that the hapten substance 1,3-di-caffeoylquinic acid in Shuanghuanglian injection was allergenic under the present experimental conditions.

Drug metabolism distribution of FA in uterine fibroids and its effect on PGE2 / 中国生化药物杂志

Objective To investigate drug metabolism distribution of flurbiprofen axetil(FA) in uterine fibroids and its effect on prostaglandin E2 (PGE2).Methods A total of 86 patients with uterine fibroids from January 2014 to February 2016 in The NO.2 Hospital of Ningbo were randomly divided into control group of 44 cases and observation group of 42 cases.The control group and the observation group were given 0.9% sodium chloride solution and 1 mg/kg FA at 15 min before operation,detection of the active metabolite of FA flurbiprofen (FP),and the concentration of PGE2 in tissue homogenate was detected by ELISA.Results The observation group FP concentration of normal and tumor tissue were (0.70 ±0.13)μg/mL and (1.72 ± 0.13)μg/mL,significantly higher than the control group(0.00 ±0.00)μg/mL and (0.00 ±0.00)μg/mL, the difference was statistically significant(P<0.05), the FP concentration of tumor tissue in the observation group was significantly higher than that in the normal group, the difference was statistically significant(P<0.05), the normal tissue and tumor tissue PGE2 concentration in the observation group were (189.29 ±26.38) pg/mL and (260.01 ±46.63) pg/mL,which were significantly lower than those in the control group(210.03 ±35.22)pg/mLand(390.20 ±92.10)pg/mL, the difference was statistically significant(P<0.05), the observation group plasma FP was (5.50 ±0.72)μg/mL,significantly higher than the control group (0.00 ±0.00)μg/mL, the difference was statistically significant(P <0.05),and PGE2 was (602.38 ±84.09) pg/mL,significantly lower than the control group(920.13 ±89.05)pg/mL, the difference was statistically significant(P <0.05).Conclusion FA in the uterine fibroids have a certain distribution of targeted,can reduce the concentration of PGE2 ,so as to alleviate the pain of patients.

Clinical application of three methods for the detection of brucellosis / 中国基层医药

Objective To detect serum brucellosis in high risk population,three methods were used and their advantages and disadvantages was compared.Methods In accordance with the surveillance standard for brucello-sis(GB 16885 -1997)and brucellosis diagnostic criteria(WS269 -2007)in the prescribed method,rose -bengal plate agglutination test (RBPT),standard -tube agglutination test (SAT)and enzyme linked immunoassay assay (ELISA)were used to detect brucellosis and analysis of its diagnostic significance in the high risk population of sheep farm of Guoyang county in Anhui province.Results The positive rates of RBPT,SAT and ELISA were 19.1%,12.1% and 16.3% in 257 blood samples,respectively.Compared to SAT,the sensitivity,specificity,accura-cy and the area under the ROC curve of RBPT were 88.5%,91.8%,91.4%,0.81,respectively,which of ELISA were 93.9%,95.1%,94.9%,0.88.Conclusion The sensitivity,specificity,accuracy and the area under the ROC curve of ELISA were higher than those of other methods.Proper method,early surveillance and effective technology can help to control the occurrence and epidemic of brucellosis in the actual test work promotion.

Niveles de Ocratoxina A y Aflatoxinas totales en cafés de exportación de Panamá por un método de ELISA

Se realizó un estudio de las condiciones del procesamiento del café de exportación en 15 beneficios, ubicados en Chiriquí, región occidental de Panamá. Además se analizaron 21 muestras de café procesado (grano verde), provenientes de los beneficios. Las muestras fueron analizadas microbiológicamente y se cuantificaron las Aflatoxinas totales (B1, B2, G1 y G2) y Ocratoxina A (OTA), mediante el método de inmunoafinidad ELISA. Se determinó un límite de detección de 0,017 ng/mL, para la Ocratoxina A, lo que equivale a una concentración de 0,829 μg/kg en la muestra, y un límite de detección de 0,027 ng/mL, para las Aflatoxinas totales, lo que equivale a una concentración de 1,350 μg/kg de Aflatoxinas totales. En la muestra, se encontró que cuatro de las 21 (19%) resultaron positivas a la presencia de Ocratoxina A y tres, a la presencia de Aflatoxinas totales (14%). Las muestras presentaron niveles de Ocratoxina A en el rango de 4,90-37,73 μg/kg; sólo tres de ellas superaron el límite máximo permitido por la Unión Europea, para la concentración de Ocratoxina, que es de 5,0 μg/kg. Las Aflatoxinas totales se encontraron en el rango de 1,51- 1,93 μg/kg, por debajo de los 10 μg/kg, que es el límite máximo permitido en el café por la Unión Europea. Los resultados nos indican que el procesamiento de café producido en Panamá cumple satisfactoriamente con los estándares internacionales de manejo poscosecha, lo que conduce a una baja incidencia de hongos productores de micotoxinas y niveles muy bajos de micotoxinas.

Levels of Ochratoxin A and total Aflatoxins in Panamanian exportation coffee by an ELISA Method. A study about processing conditions of exportation coffee in 15 benefits located in Chiriquí, western region of Panama, was conducted. In addition, 21 samples of processed coffee (green beans), from the benefits, were analyzed. The samples were microbiologically tested in order to quantify total aflatoxins (B1, B2, G1 and G2) and Ochratoxin A (OTA), using the immunoaffinity ELISA method. A detection limit of 0.017 ng/mL, was determined for Ochratoxin A, which is equivalent to a concentration of 0.829 μg/kg, and a detection limit of 0.027 ng/mL, for total aflatoxins, which is equivalent to a concentration of 1.350 μg/kg. It was found that four (19%) out of the 21 samples were positive to the presence of Ochratoxin A and three (14%) to the presence of total aflatoxins. Samples showed levels of Ochratoxin A in the range 4.90 - 37.73 μg/kg; only three of them exceeded the maximum limit allowed by the European Union, for the concentration of Ochratoxin, which is of 5.0 μg/kg. Total aflatoxins were found in the range 1.51 - 1.93 μg/kg, below 10μg/kg which is the maximum limit allowed for coffee by the European Union. The results indicate that the processing of coffee produced in Panama successfully meets international standards for postharvest handling, which leads to a low incidence of mycotoxins and very low levels of mycotoxin- producing fungi.

A Study on Serum Leptin Values by Elisa Method in Children / 대한소아소화기영양학회지

PURPOSE: Leptin is an adipocyte-derived blood-borne satiety factor that acts on its cognate leptin receptor in the hypothalamus, thereby regulating food intake and energy expenditure. We measured the leptin concentrations in serum of normal and obese children with human leptin ELISA kit, unlike previous study with leptin RIA kit and investigated the relationship between leptin concentrations and body mass index, gender, and age. METHODS: We measured serum concentrations of leptin in 67 children who were visited to the Department of Pediatrics, Chungnam National University Hospital during the period of 5 months from February, 1999 to June, 1999. Height, weight, obesity index, and body mass index were measured in 67 subjects. Leptin values in serum were measured by sandwich ELISA method. Data analysis was done according to the obesity, body mass index, gender and age. RESULTS: The mean concentration of leptin was 7.69±8.83 ng/ml in normal children group and 36.34±18.57 ng/ml in obese group. Serum leptin concentrations were significant correlation with the body mass index (p < 0.01). Serum leptin concentration was significant higher in the group of over 10 years of age (p < 0.01). Leptin levels showed no significant difference by gender. CONCLUSION: Serum leptin levels were significantly higher in obesity group than in control one, and they were correlated with body mass index and age. Measurements of leptin value by sandwich ELISA method are very useful and easily applicable to determine obesity.