The development and standardization of an ELISA for ovalbumin determination in influenza vaccines

A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of ovalbumin in influenza vaccines has been developed and standardized. Commercially available reagents were used. ELISA was compared to single radial immunodiffusion (SRD) and immunoelectro-osmophoresis (IEOP) techniques. The detection limit by ELISA was 0.5 ng/ml. This method was found to be at least 1000 times more sensitive than SRD and at least 200 times more sensitive than IEOP. It was concluded that ELISA is a specific, sensitive and reproducible method for the determination of the small amounts of ovalbumin found as an impurity in unconcentrated influenza vaccines.     

Development of a solid-phase immunoenzyme method for determining the antibody level in the blood sera of persons vaccinated with influenza vaccine

To control the effectiveness of vaccination against influenza, the optimum conditions for making the enzyme-linked immunosorbent assay (ELISA) with a view to determine the level of anti-influenza antibodies in human blood sera have been established. The kinetics of influenza virus adsorption in the wells of ELISA polystyrene plates and the kinetics of the interaction between the immobilized antigen and species-specific peroxidase-labeled antibodies have been studied. The method has been shown to be more sensitive than the hemagglutination inhibition test in the determination of seroconversion in persons immunized with influenza vaccine.     

The detection by serological methods of viruses infecting the rose

Homogenates of herbaceous test plants infected with arabis mosaic virus (AMV), prunus necrotic ringspot virus (PNRSV), or strawberry latent ringspot virus (SLRV), and purified virus preparations were used to assess the sensitivities of four serological methods (the enzyme-linked immunosorbent assay - ELISA, immunodiffusion in gels, the latex flocculation assay, and serologically specific electron microscopy -SSEM) for the detection of these viruses. The latex test was up to 250 times more sensitive than gel immunodiffusion, but SSEM and ELISA were respectively up to 1000 and 200 times more sensitive than the latex test. Gel immunodiffusion and latex tests failed to detect any of the viruses in infected roses. Although ELISA reliably detected PNRSV and SLRV when leaves from infected roses were homogenised in a leaf: buffer ratio of 1 g:10 ml, AMV was occasionally undetected. However, when a modified ELISA technique, which reduced non-specific reactions, was used some PNRSV-infected roses were also not detected. Detection by SSEM was c. twice as sensitive as ELISA for all three viruses in rose extracts. The relative advantages of ELISA and SSEM for the detection of plant viruses are discussed.     

Titer on Chip: New Analytical Tool for Influenza Vaccine Potency Determination

Titer on Chip (Flu-ToC) is a new technique for quantification of influenza hemagglutinin (HA) concentration. In order to evaluate the potential of this new technique, a comparison of Flu-ToC to more conventional methods was conducted using recombinant HA produced in a baculovirus expression system as a test case. Samples from current vaccine strains were collected from four different steps in the manufacturing process. A total of 19 samples were analysed by Flu-ToC (blinded), single radial immunodiffusion (SRID), an enzyme-linked immunosorbent assay (ELISA), and the purity adjusted bicinchoninic acid assay (paBCA). The results indicated reasonable linear correlation between Flu-ToC and SRID, ELISA, and paBCA, with regression slopes of log-log plots being 0.91, 1.03, and 0.91, respectively. The average ratio for HA content measured by Flu-ToC relative to SRID, ELISA, and paBCA was 83%, 147%, and 81%, respectively; indicating nearly equivalent potency determination for Flu-ToC relative to SRID and paBCA. These results, combined with demonstrated multiplexed analysis of all components within a quadrivalent formulation and robust response to HA strains over a wide time period, support the conclusion that Flu-ToC can be used as a reliable and time-saving alternative potency assay for influenza vaccines.     

Evaluation of the single radial-immunodiffusion assay for measuring the glycoprotein content of rabies vaccines

The glycoprotein content of rabies vaccines containing the Pitman-Moore strain of rabies virus was measured by the single radial immunodiffusion assay and correlated with vaccine potency. The variability of this assay was 6.3% for a single vaccine lot tested over a one-year period. Using sera prepared against rabies virus glycoprotein from different strains of virus, the assay gave different values. These differences could be eliminated by using a homologous vaccine strain as an internal reference. Single radial-immunodiffusion values for Pitman-Moore vaccines correlated with the manufacturers' NIH potency assay, but required a mathematical transformation to convert values from one assay to the other. Single radial-immunodiffusion values for Street Alabama Dufferin and Flury-LEP vaccines did not correlate with NIH values. Modification of the single radial immunodiffusion technique and the feasibility of using this assay for the determination of rabies vaccine potency are discussed.     

The use of the Soviet detergents dezintegron-O and dezintegron-B for the solubilization of influenza virus glycoproteins]

Native detergents, desintegron-O-and desintegron-B, solubilize hemagglutinins of the influenza virus as intensively, as foreign drugs (mulgophen, zwittergent and sodium sarcozyl) do. This permits recommending desintegrons for quantitative determination of hemagglutinin of the influenza virus in virus-containing materials by means of the reaction of single radial immunodiffusion. Desintegron-O and desintegron-B permit extracting enzymatically active neuraminidase from influenza A viruses with 44.6-83.4% yield.     

Effectiveness of the combined use of an inactivated vaccine and a proteolysis inhibitor in the prevention of experimental influenza

The prophylactic effects produced by different types of antiviral preparations, used separately or in combination, in experimental lethal infection induced by influenza virus AO/32 (H0N1) in mice are compared. The use of inactivated vaccine and E-aminocaproic acid (E-ACA), a proteolysis-inhibiting agent, was studied. The qualitative characterization and quantitative evaluation of the anti-influenza effect were carried out by the method of multifactor analysis with the use of a computer after the optimum second-order plan based on the mathematical theory of experiment. This made it possible to determine the best combination of the preparations and their doses, to establish the time of the formation of reliable protection from influenza in mice. The prophylactic effect produced by the use of E-ACA alone and the capacity of this preparation for enhancing the protective action of inactivated influenza vaccine were established. Mathematical analysis revealed the optimum value of the four factors under study: the dose of the vaccine, the dose of E-ACA, the lapse of time between the injection of the preparations and the challenge of the animals, as well as the infective dose of the pathogenic virus. A special experiment made in the study of these data confirmed that the specific formation of a high level of anti-influenza protection in mice can be achieved by the combined use of the vaccine and the inhibitor of proteolysis.     

Application of deglycosylation to SDS PAGE analysis improves calibration of influenza antigen standards

Each year the production of seasonal influenza vaccines requires antigen standards to be available for the potency assessment of vaccine batches. These are calibrated and assigned a value for haemagglutinin (HA) content. The calibration of an antigen standard is carried out in a collaborative study amongst a small number of national regulatory laboratories which are designated by WHO as Essential Regulatory Laboratories (ERLs) for the purposes of influenza vaccine standardisation. The calibration involves two steps; first the determination of HA protein in a primary liquid standard by measurement of total protein in a purified influenza virus preparation followed by determination of the proportion of HA as determined by PAGE analysis of the sample; and second, the calibration of the freeze-dried reference antigen against the primary standard by single radial immunodiffusion (SRD) assay. Here we describe a collaborative study to assess the effect of adding a deglycosylation step prior to the SDS-PAGE analysis for the assessment of relative HA content. We found that while the final agreed HA value of the samples tested was not significantly different with or without deglycosylation, the deglycosylation step greatly improved between-laboratory agreement.     

Purification of a corpuscular influenza vaccine by means of immunoaffinity chromatography

Two immunoaffinity chromatographic methods for the purification of corpuscular influenza vaccine from the admixture of chick embryo components have been examined. The isolation of the virus on immobilized antiviral antibodies has proved to be unsuitable for preparative purposes. The method for the purification of the vaccine from ovalbumin with the use of immobilized anti-ovalbumin antibodies has proved to be highly effective. When introduced into guinea pigs in 3 injections, the vaccine purified by immunosorption has been found to produce no anaphylactic reactions.     

The use of the single radial haemolysis test for assessing antibody response and protective antibody levels in an influenza B vaccine study

Single radial haemolysis (SRH) and HAI tests were used to determine the levels of antibody in sera obtained from 118 volunteers taking part in a study of influenza B/Hong Kong/73 subunit vaccine. The two tests gave similar overall results, and showed good correlation, but SRH appeared to be more sensitive for the detection of low concentrations of antibody. A greater than or equal to 45% increase in SRH zone area was found to be equivalent to a significant increase in antibody titre and on the basis of this value, the HAI and SRH tests gave similar results for responses to immunization. The concentration of antibody equivalent to a 50% protective level against a challenge virus infection was a zone area of 45 mm2 for the SRH test; this was equivalent to an HAI titre of 1:20 which has previously been established as the 50% protective level.     

流感病毒裂解疫苗裂解剂去除工艺改进及稳定性研究

目的改进流感病毒裂解疫苗裂解剂去除工艺,降低残余卵清蛋白和裂解剂含量,提高疫苗质量,降低成本。方法分别将A1、A3和B型流感病毒纯化液用磷酸缓冲液（PB）沉淀法去除裂解剂,经超滤、除菌制备原液,配制6批半成品,其中3批不含硫柳汞,3批含硫柳汞,经全面检定,并观察放置37℃、25℃和2～8℃不同时间的稳定性。结果该疫苗各项指标均符合《中国药典》（2010年版）三部要求,其中卵清蛋白平均为3．83ng／mL,裂解剂平均为57μg／mL,比改进前分别降低97．9％和69％。37℃放置4周、25℃3个月及2-8℃12个月后检定全部合格。结论该工艺步骤简单,去除卵清蛋白和裂解剂效果明显,是进一步提高疫苗质量和降低成本的有效工艺。     

区带超速离心提纯流感病毒的初步探讨

在制备灭活流感疫苗中,采用两次蔗糖速率区带超离心从流感病毒感染的鸡胚尿液中提纯流感病毒是较简便、实用的方法,根据流感病毒分子特性设计了超离梯度并采用适当试验条件,获得了大量提纯流感病毒的初步结果。经血凝、电镜、蔗糖浓度、浮力密度等检验证明此方法是可行的。     

异型流感病毒感染小鼠肺细胞因子水平变化

为了制备能够抵御不同型别流感病毒感染的疫苗,揭示机体对异型流感病毒感染交叉免疫保护作用的主要机制,用流感病毒疫苗免疫小鼠后分别感染同型、异型流感病毒,另设使用免疫增强剂IL-2后感染异型流感病毒组,观察小鼠的一般状况和肺指数,并用ELISA方法测定肺匀浆中细胞因子IFN-γ、IL-2、IL-4及IL-10的含量。结果显示,异型免疫组和异型免疫加强组病毒感染后细胞因子IFN-γ含量明显高于感染前（P〈0.05）。研究表明,异型病毒感染后IFN-γ水平明显增高,此细胞因子可能在流感病毒异型间交叉保护免疫反应中起重要作用,其机制有待进一步研究确定。     

Determination of bacterial endotoxin admixtures in inactivated influenza vaccines

The materials substantiating the possibility of using the method for the determination of the lethal effect of endotoxin on dactinomycin-treated mice are presented. This determination is made with a view to detecting the admixtures of endotoxins in whole-virion and subvirion inactivated influenza vaccines at different stages of their manufacture, as well as in the final product. The proposed test is highly sensitive, rather simple in its practical realization and can be used for evaluating the degree of the purification of influenza vaccines from endotoxins.     

Free haemagglutinin in inactivated whole virus influenza vaccines

While studying the haemagglutinin content of whole virus inactivated influenza vaccines by the single radial diffusion test and quantitative electron microscopy, it was found that not all haemagglutinin measured by single radial diffusion was bound to virions, a part of it being in a free state. The influence of unbound haemagglutinin on the immunogenicity of whole virus inactivated influenza vaccine is discussed. In addition, the use of single radial diffusion for the assessment of unbound haemagglutinin is suggested.     

流感疫苗单向免疫扩散试验中抗血清制备新方法的研究

流感疫苗血凝素含量检测方法为单向免疫扩散试验,其抗血清通常由WHO参比实验室提供,通过纯化病毒、蛋白酶切获得血凝素主要抗原片段,然后再免疫动物获得抗血清。该方法制备时间较长,是制约流感疫苗研发及检测的主要因素。以RT-PCR方法获取甲型H1N1流感病毒血凝素中主要抗原片段基因,构建重组质粒并在大肠杆菌中表达,表达产物经纯化、复性后,以蛋白电泳及免疫印迹方法进行了鉴定。以纯化蛋白免疫家兔,制备了相应的抗体,初步证明可用于常规的单向免疫扩散试验。以该法可以快速获得抗血清,尤其是在大流行流感疫苗的研发中,加快疫苗研发进程。     

Synthesis and properties of an affinity adsorbent for purifying neuraminidases of varying origins

Neuraminidases (NA) from Clostridium perfringens, noncholera vibrios and influenza virus were purified by affinity chromatography on Sepharose coupled to para-aminophenyl oxamic acid. Adsorption was carried out at pH 5.5. The effect of elution conditions on purification of NA was studied. The use of the pH gradient enhances 10-fold the purification degree as compared with the pH shift-elution process, 90% of the activity being eluted from the column within a pH range from 6.0 to 6.6. According to the described procedure, electrophoretically homogeneous preparations of NA were obtained from noncholera vibrios and influenza virus.     

应用单向放射免疫扩散法测定流感疫苗血凝素含量的研究

分析了单向放射免疫扩散法快速、准确测定流感疫苗血凝素含量的可行性。利用在打孔器孔径一定的条件下抗原浓度只与扩散圈直径的平方成线性关系,而与打孔器无关,测定流感疫苗血凝素含量。结果可见,流感疫苗3个型别血凝素扩散圈直径的平方均与其浓度成线性,r均大于0.99,变异系数均小于1%,平均回收率99.7%。单向放射免疫扩散法方法简便、快速、准确,适用于流感疫苗血凝素含量的测定。     

The effect of strain differences on the assay of rabies virus glycoprotein by single radial immunodiffusion

Antigenic differences between rabies virus strains used for vaccine manufacture can be demonstrated using monoclonal antibodies. We have shown that these differences are sufficiently large to affect the potency values of vaccines measured in single radial immunodiffusion (SRD) assays if the reference and test vaccines are antigenically heterologous. The production of reagents for use in SRD assays for each strain of rabies virus should be considered.     

Genetic fusion protein 3×STa‐ovalbumin is an effective coating antigen in ELISA to titrate anti‐STa antibodies

Heat‐stable toxin type I (STa)‐ovalbumin chemical conjugates are currently used as the only coating antigen in ELISA to titrate anti‐STa antibodies for ETEC vaccine candidates. STa‐ovalbumin chemical conjugation requires STa toxin purification, a process that can be carried out by only a couple of laboratories and often with a low yield. Alternative ELISA coating antigens are needed for anti‐STa antibody titration for ETEC vaccine development. In the present study, we genetically fused STa toxin gene (three copies) to a modified chicken ovalbumin gene for genetic fusion 3×STa‐ovalbumin, and examined application of this fusion protein as an alternative coating antigen of anti‐STa antibody titration ELISA. Data showed fusion protein 3×STa‐ovalbumin was effectively expressed and extracted, and anti‐STa antibody titration ELISA using this recombinant protein (25 ng per well) or STa‐ovalbumin chemical conjugates (10 ng/well) showed the same levels of sensitivity and specificity. Furthermore, mice immunized with this fusion protein developed anti‐STa antibodies; induced antibodies showed in vitro neutralization activity against STa toxin. These results indicate that recombinant fusion protein 3×STa‐ovalbumin is an effective ELISA coating antigen for anti‐STa antibody titration, enabling a reliable reagent supply to make standardization of STa antibody titration assay feasible and to accelerate ETEC vaccine development.