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【Objective】 To establish and evaluate a fluorescent antibody to membrane antigen (FAMA) method for detecting antibodies against varicella-zoster virus (VZV) based on Vero E6 cells. 【Methods】 Based on the adapted VZV-Oka-E6 strain that VZV-Oka live attenuated varicella vaccine strain grew in Vero E6 cells, Vero E6 cells were infected with VZV-Oka-E6 of three different doses (104.65, 104.95 and 105.25 TCID50), and the cytopathic effect was observed under a microscope to determine the optimal infection dose of VZV-Oka-E6 strain. Then the detectable sensitivity of the infected cell antigen slides prepared after fixing the infected cells with different fixatives was compared to determine the optimal fixative. As a result, the FAMA method based on Vero E6 cells for the determination of neutralizing anti-VZV has been developed. The established FAMA assay was used to detect the international standard for varicella zoster immunoglobulin with different titers to determine the sensitivity of the assay. The specificity of the assay was evaluated by detecting specific antibodies against human herpes simplex virus (HSV) type 1 and type 2. The neutralizing anti-VZV antibodies of the international standard for varicella zoster immunoglobulin were detected using VZV-infected cell antigen slides prepared in the same batch and four different batches, respectively, to determine the intra-assay repeatability and inter-assay repeatability. The international standard for varicella zoster immunoglobulin with three known titers was detected to evaluate the relative accuracy of this assay. The anti-VZV titers in 20 apheresis plasma samples were determined with the newly established FAMA test and ELISA test, respectively, and the detection results of the two methods were compared using Spearman’s correlation test. 【Results】 The optimal infection dose of the VZV-Oka-E6 strain in FAMA assay was determined to be 105.25 TCID50, and acetone precooled at -20℃ was used as the fixative. The FAMA test has a high sensitivity with a detecting limit of 31.25 mIU/mL for neutralizing anti-VZV titers. The negative result was observed when detecting herpes simplex virus (HSV) type 1 and 2 specific antibodies. The international standard for varicella zoster immunoglobulin was detected by VZV infected cell antigen slides prepared in the same batch and 4 different batches, with the coefficient of variation being 29.95% and 26.71%, respectively. The detection value of the international standard for varicella zoster immunoglobulin with three different titer levels was consistent with their theoretical value. The correlation coefficient of the detection results of 20 apheresis plasma samples by the FAMA test and ELISA test was 0.268. 【Conclusion】 The VZV FAMA assay has good sensitivity, specificity, repeatability, and relative accuracy in detecting neutralizing anti-VZV titers. It can be applied for detecting neutralizing anti-VZV titers in apheresis plasma samples as well as the varicella-zoster immunoglobulin (VZIG) preparations.
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Objective To investigate the mechanism by which hydrogen(H2) helps prevent acute lung injury induced by hyperoxia (HALI) in rats.Methods Thirty male Sprague-Dawley rats were randomly divided into three groups: control group, HALI group and H2 group, with 10 rats in each group.The control group was exposed to air at atmospheric pressure.Rats in HALI and H2 groups were exposed continuously to pure oxygen (100%O2) for 60 hours and during this period, 10 ml/kg of normal saline or H2-saturated normal saline was given every 12 hours by intraperitoneal injection to the HALI and H2 groups, respectively.After treatment, the arterial partial pressure of oxygen was examined and histopathological examination was conducted in each group.Then,RT-qPCR and Western blotting were performed to measure the transcriptional level and protein expression of heme oxygenase 1 (human heme oxygenase 1, HO-1) in rat lung tissue.Results Compared with the HALI group, H2 group showed significantly decreased severity of lung injury and a marked increase in the arterial oxygen saturation.Besides, H2 treatment induced up-regulation of HO-1 mRNA and protein levels.Conclusion The findings suggest that HO-1 may play an important role in the protection against HALI by H2.
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Objective To explore the correlation between aspirin resistance and COX-1, P2Y1, GPIa and GPIIIa gene polymorphism. Methods 35 case with aspirin resistant were selected and were given aspirin enteric coated tablets for oral treatment, 14 days.After the determination COX-1, P2Y1 and GPIa and GpIIIa gene polymorphism of each patient were analysis.Results The genotype AA of COX1 (A-842G) locus and CC of COX1 (C50T) locus was higher than other genotype (P<0.05);AG of P2Y1 (A1622G) locus genotype and CC of P2Y1 (C893T) locus genotype was higher than other genotype (P<0.05);CT of GPIa (C807T) locus genotype and GA of GPIa (G873A) locus genotype was higher than other genotype (P<0.05);PLA1/A1 of GPⅢa(T1565C)locus genotype was higher than other genotype (P<0.05).Conclusion P2Y1 (C893T), GPIa (C807T) allele is associated with aspirin resistance,With COX-1 (A-842G, C50T), GPIa (G873A), GP III A (T1565C), P2Y1 (A1622G) allele is more likely to induce the occurrence of aspirin resistance.
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Objective To establish and evaluate a universal real-time fluorescent quantitative PCR(qPCR)method for identifying and quantifying three human parvovirus B 19 ( B19V) genotypes.Methods Firstly, following a bioinformatic analysis of a subset of B19V genomic sequences available in the NCBI nucleotide database ,representative of genotypes 1 to 3,a set of suitable universal primers and TaqMan probes was designed from the NS 1 gene of B19V.Aplasmid was used as a quantitative standard that contained the identical sequence of the B 19 target sequence .An internal control ( IC ) was included to prevent false negative results .Then,serial 1-log dilutions of quantitative standards were prepared and used in the qPCR assays for generation of a standard curve .Finally,the specificity,sensitivity and reproducibility of the assay were assessed.Results A linear relationship of the real-time PCR method for detecting B19V from 1 ×109copies/μl to 1 ×103 copies/μl was observed .The developed qPCR protocols allowed for the detection of genotypes 1 to 3 with a limit of detection ( LOD) of 10 copies/μl.Furthermore, the assay did not amplify other blood-borne viruses.The inter-and intra-assay variability analyses showed good reproducibility of the assay .Conclusion A universal real-time qPCR method for the detection of B19V DNA is established,which will facilitate the diagnosis of B19V infections and the screening of blood and plasma-derived products , thereby improving the viral safety of transfusion and plasma-derived products .
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Objective To detect human parvovirus B19(B19V)DNA in source plasma pools and coagulation factor products and determine its prevalence and the level of contamination .Methods A pair of primers and a probe selected from the highly conserved sequences encoding the non-structural protein(NS1)of B19 were designed and synthesized.With the primer-probe combination ,source plasma pools and four types of coagulation factor products were determined for B 19V DNA by TaqMan real-time quantitative PCR.Results One-hundred and sixteen from 195 (59.49%) source plasma pools contained B19 DNA and concentrations up to 1.35 ×1010 copies/ml were measured.High frequencies of contamination were detected in factor Ⅷ (29 of 31; 93.55%), thrombin (10 of 10; 100%), fibrinogen (6 of 7; 85.71%) and prothrombin complex (8 of 9;88.89%).Conclusion These data show that B19V is a common contaminator in Chinese source plasma pools and coagulation factor products .Thus,B19V screening in Chinese source plasma seems desirable and significant for the safety of plasma derivatives in China .
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Objective To determine the neuroprotective effect of clonidine on primary cultured cortical neurons in rats exposed to oxygen-glucose deprivation ( OGD) injury. Methods Cortical neurons cultured for 8 days were randomly assigned to the three groups: normal control group, model control group, and clonidine pretreatment group. OGD injury model was established by chemical hypoxia and glucose deprivation in incubation liquid for 4 h. Clonidine (1. 0, 3. 0, 10 μmol·L-1 ) was added 24 h before OGD injury. Neuronal injury was evaluated by MTT staining and the release of lactate dehydrogenase ( LDH) . Results Under the microscope, primary cultured cortical neurons in normal control group presented great density, round size, smooth edge, and high diopter,The suvival rate of neurons and the percentage of LDH releasing were (100. 00±32. 12)% and (100. 00 ± 37. 51 )%, respectively. After exposure to OGD injury, cortical neurons showed karyopyknosis, incomplete cell membranes, low diopters and a significant reduction in optical density of MTT staining. In addition, the suvival rate of neurons and the percentage of LDH releasing were (53. 61±7. 62)% and (166. 07±9. 65)% separately compared with normal control group. In the group with pretreatment of different concentrations of clonidine (1. 0, 3. 0, 10μmol·L-1), morphological changes induced by OGD injury were significantly reversed and optical density of MTT staining was dose-dependently raised. The percentages of survival neurons much higher than that of model control group were [(67. 53±10. 54)%, (71. 50±9. 79)% and (87. 48±5. 29)%, separately] and the obvious reductions of LDH releasing were [(136. 45±25. 72)%, (130. 92±24. 94)%and (121. 63±32. 68)%, respectively]. Conclusion Clonidine can exert neuroprotection against OGD-induced injury in primary cultured cortical neurons in rats.