Establishment and evaluation of a fluorescent antibody assay against varicella-zoster virus membrane antigen based on Vero E6 cells / 中国输血杂志

【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.

Hydrogen reduces hyperoxic acute lung injury by inducing heme oxygenase 1 expression / 军事医学

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.

Isolation and purification of human haptoglobin by ion exchange chromatography / 军事医学

Objective To develop an effective process for isolating and purifying haptoglobin ( Hp) from Cohn fractionⅣby a new ion exchange chromatography and to preliminarily identify and analyze the product of each purification step . Methods The fraction was first diluted and impurities were adsorbed with Rivanol .Then, the supernatant was treated with 50%ammonium sulfate.Finally, the precipitate was redissolved , and Hp was purified further with Q Sepharose Fast Flow chromatography .Native-PAGE was used to measure the activity of the haptoglobin-bound hemoglobin , while SDS-PAGE analysis and immunoblot were used for identification of the target protein .Results After pretreatment , some of the impuri-ties were removed from the Cohn fraction Ⅳ, and the target protein was enriched .In our case, the target protein was Hp and Hp2-2 was the main phenotype in the human plasma fraction Ⅳ.Target protein band and high purity were identified by SDS-PAGE.Immunoblot analysis further proved that this method could successfully isolate the target protein Hp , and the activity of 2.8 U/ml was measured by Native-PAGE method.Conclusion Haptoglobin is successfully isolated from human Cohn fractionⅣwith this method.The purification process is simple and suitable for scale-up production with a good prospect.

Establishment and evaluation of a universal nucleic acid test method for detecting human parvovirus B19 / 军事医学

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 .

Establishment and preliminary application of a rapid microneutralization test for screening varicella-zoster virus special hyperimmune globulin in plasma donations / 军事医学

Objective To promote the progress in varicella-zoster virus (VZV) immunoglobulin preparation,a rapid microneutralization test ( RMNt) was set up for screening plasmapheresis donations with high titers of special neutralizing antibodies to VZV.Methods With reference to the VZV immunoglobulin (VZIG) preparation standard of FDA and VZIG international unit ( IU) , a screening standard was formulated; the amount of virus was analyzed to determine the optimal conditions for RMNt;screening technology was established and the IU was introduced as quality control ;twenty samples of apheresis plasma and fifteen samples of pooled plasma were diluted at 1∶2 to 1∶256 and tested by RMNt respectively;and the sensitivity of RMNt was also analyzed by the commercial ELISA kit .Results Plasma samples that were diluted at 1∶16 and had a titer more than 0.4 U/ml could be used in the production of VZIG .1500 PFU/ml titers of virus in RMNt pro-duced readable results in plasma screening .Eight apheresis plasma samples tested met the screening standard , but none of the pooled samples tested positive .RMNt had a good linear relationship with ELISA (r=0.895 24,P<0.0001).Conclu-sion The sensitivity, throughput and operability of the established RMNt can be used in the screening of plasma donations as key techniques for the production of VZIG .

Establishment and optimization ofα2-macroglobulin activity detection assay / 军事医学

Objective To establish an assay for detecting α2-macroglobulin activity in Cohn fraction Ⅳ.Methodsα2-M reacted with trypsin to form α2-M-trypsin complex.After the chromogenic substrate Na-benzoyl-DL-arginine 4-nitroanilide hydrochloride ( BAPNA) was added, absorption at 410 nm was detected with the microplate reader .α2-M activity in Cohn fractionⅣwas quantitatively detected according to the established standard curve of plasma α2-M activity. Result Several critical parameters in this assay were optimized .A standard curve of plasma α2-M activity was established . According to this standard curve ,α2-M activity in Cohn fraction Ⅳsample was detected to be 1.578 PU/ml.Conclusion Using normal human plasma as the reference material , theα2-M activity in Cohn fractionⅣcan be detected through chro-mogenic substrate assay.This study provides a simple method to detect α2-M activity during the purification process of α2-M from Cohn fraction Ⅳ.

Specific human immunoglobulin in prevention and treatment of infectious diseases / 军事医学

The specific human immunoglobulin is a hyperimmune globulin against a particular pathogen or biotoxin .It′s an important variety in plasma derivatives .Specific human immunoglobulin is usually used to prevent and treat pathogen in -fections with high morbidity , severe outcomes and no efficient treatment available .Thus it has unique advantages in preven-tion and management of infectious diseases .A variety of specific human immunoglobulins have been licensed abroad , but the development of new specific human immunoglobulins is slow in China due to technical constraints , limited economic benefits or for other reasons .Here we reviewed some specific human immunoglobulins and their preventive and therapeutic effect on infectious diseases .

Contamination of human parvovirus B19 in source plasma and coagulation factor products / 军事医学

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 .

Advances in securing viral safety for plasma products / 军事医学

Plasma products are considered to be special medicinesderived from healthy human plasma .During 1980′s, events of transmission of human immunodeficiency virus through plasma products were frequently reported .Since then, ensuring the viral safety of plasma products has raised great concerns all over the world .So far, with decades of effort , most countries in the world have established rigorous systems with preventive measures to ensure the viral safety of plasma prod -ucts.These measures include control of source plasma , validated inactivation/removal of infectious agents , the adherence to current good manufacturing practices .Nevertheless , new infectious agents which may be threats to viral safety require continuous studies on appropriate countermeasures .

Screening of biological amniotic immunization safety / 国际药学研究杂志

Objective To evaluate the immunity efficacy of human amniotic membranes on rats. Methods One hundred and fifty Wistar rats were randomly divided into five groups:biological amnion group,immunosuppression group,immunostimulation group, sham-operated group and blank control group. According to the study period,each group of thirty rats would be randomly divided into five experimental operation subgroups:the 1st week,the 2nd week,the 4th week,the 8th week group and the 12th week groups. The rats were implanted subcutaneously,then intramuscular injection of gentamicin sulfate for 3 days to resist the infection ,and the immune organ coefficient,and the killing abilities of NK cell ,IL-1β,IL-6 and TNF-αserum levels were detected according to the study period.Results At 1st ,2nd ,4th ,8th and 12th week after amniotic membrane implantation in rats,compared with the sham-operated and blank control groups,the biological amnion group had nonsignificant differences (P>0.05). At 1st week after amniotic membrane implantation in rats, immunosuppression group showed different levels of the immunosuppressive effect,such as the analysis of immune organ coefficient , which had significant differences compared with other groups (P<0.01). At 1st week after amniotic membrane implantation in rats,the imunostimulation group showed a certain degree of the immunostimulant effect,such as the killing abilities of NK cell,which had marked differences compared with other groups (P<0.05). Conclusion The amniotic membranes have satisfactory immune safety with implantation in rats and do not cause significant adverse immune reactions.

Analysis of the differential expression of Stathmin in HEK293 cells infected with human-tropic porcine endogenous retrovirus / 中华微生物学和免疫学杂志

Objective To analyze the differential expression of Stathmin in human cells infected with human-tropic porcine endogenous retrovirus(PERV)and to explore the potential molecular effect of human-tropic PERV on human cells.Methods HEK293 cells were infected with the human-tropic PERV infectious molecular clone.PCR,real-time RT-PCR and immunofluorescence analysis were applied to confirm that HEK293 cells were infected.Then real-time RT-PCR and Western blot were carried out to analyze the differential expression of Stathmin at the mRNA level and protein level,respectively.Results HEK293 cells were infected by human-tropic PERV.Real-time RT-PCR and Western blot analysis showed that Stathmin was up-regulated in HEK293 cells infected with PERV compared with the control cells.Conclusion Stathmin was up-regulated in HEK293 cells infected with human-tropic PERV.These studies will be helpful for revealing the interaction of PERV and human cells,and for understanding the molecular effect of humantropic PERV on human cells.In addition,it suggested that PERV infection may infect cell growth and physiological functions,even be pathogenic.These will help to clarify the biologic characteristics of PERV and evaluate the safety of PERV in pig to human xenotransplantation.