Single Radial Immunodiffusion Assay Research Articles

Establishment of Reference Reagents for Single-Radial-Immunodiffusion Assay on the 2022/23 Seasonal Influenza Vaccine in Japan and Their Quality Validation.

Potency tests for influenza vaccines are currently performed using a single-radial immunodiffusion (SRID) assay, which requires a reference antigen and anti-hemagglutinin (HA) serum as reference reagents. Reagents must be newly prepared each time a strain used for vaccine production is modified. Therefore, establishing reference reagents of consistent quality is crucial for conducting vaccine potency tests accurately and precisely. Here, we established reference reagents for the SRID assay to conduct lot release tests of quadrivalent influenza vaccines in Japan during the 2022/23 influenza season. The potency of reference antigens during storage was confirmed. Furthermore, we evaluated the cross-reactivity of each antiserum raised against the HA protein of the 2 lineages of influenza B virus toward different lineages of influenza B virus antigens to select a suitable procedure for the SRID assay for accurate measurement. Finally, the intralaboratory reproducibility of the SRID assay using the established reference reagents was validated, and the SRID reagents had sufficient consistent quality, comparable to that of the reagents used for testing vaccines during previous influenza seasons. Our study contributes to the quality control of influenza vaccines.

Assessing the stability-indicating properties of alternative potency assays for inactivated influenza vaccine

Determination of the potency of a vaccine is critical to ensuring that an appropriate dose is delivered, lot-to-lot consistency is maintained, and that the formulation is stable over the life of the vaccine. The potency of inactivated influenza vaccines is determined routinely by the Single Radial Immunodiffusion (SRID) assay. A number of alternative potency assays have been proposed and have been under evaluation in recent years. The aim of this study was to compare a surface plasmon resonance-based assay and two different enzyme linked immunoassays against the current potency assay, SRID, and against mouse immunogenicity when haemagglutinin antigen of the A(H1N1)pdm09 component of an inactivated influenza vaccine is stressed by elevated temperature, low pH and freezing. This analysis demonstrated that the alternative assays had good correspondence with SRID for samples from most stress conditions and that the immunogenicity in mice corresponded with potency in SRID for all stress samples. Subject to further analysis, the assays have been shown to have the potential to possibly replace, and at least complement, SRID.

Transfer of passive immunity and survival in Jersey heifer calves fed heat-treated pooled colostrum.

Acquisition of adequate transfer of passive immunity (ATPI) by calves depends on the absorption of sufficient mass of colostral immunoglobulin G (IgG). Several studies report conflicting evidence regarding the ability of feeding pooled colostrum to achieve ATPI. Pooling colostrum is practical and efficient for some dairies, and recommendations are required to prevent failure of transfer of passive immunity (FTPI) in calves following pooling. This study aimed to determine the effect of pooling colostrum on serum IgG concentrations, FTPI, and preweaning mortality in calves. A prospective study was performed on two conventional Jersey dairy farms where heat treatment of colostrum occurred in the same colostrum processing kitchen. Four to 10 cows contributed to colostrum pools. A sample of the colostrum pool fed to the calves and serum from calves at 24-72 h was collected for IgG concentration determination by single radial immunodiffusion assay. Multivariable and logistic regression analyses were performed to evaluate factors that predicated serum IgG concentrations and the probability of FTPI, respectively. A Cox proportional hazard model analysis was performed to determine risk factors for mortality over the preweaning period. A total of 164 calves fed 28 colostrum pools were enrolled. Birth weight, number of colostrum feedings and pool IgG concentrations were significant predictors of calf serum IgG concentrations at 24-72 h, whereas the number of colostrum feedings and age at bleeding to determine passive transfer status were not significant predictors of calf serum IgG concentrations at 24-72 h. The prevalence of FTPI was 4.9%. Birth weight, pool IgG concentrations, number of colostrum feedings, and age at bleeding to check for passive transfer status were not significant predictors of the probability of FTPI at 24-72 h. The incidence of mortality was 4.3%. Passive transfer status was not a predictor of mortality. Our study demonstrates the effect of pooling colostrum on serum IgG concentrations even in herds where colostrum with higher median colostrum IgG concentrations is fed to calves. The results emphasize the recommendations to assess pooled colostrum IgG concentrations before feeding calves.

The production and influence of anti-Vibrio parahaemolytics IgY against experimental infection of V. parahaemolyticus in cultured Fenneropenaeus indicus

The increasing number of antibiotic-resistant bacteria emphasizes the need to find alternatives to complement antibiotics. Immunotherapy may also be used as a complementary treatment against pathogens that are difficult to treat with traditional antibiotics. Eggs are normal dietary components and there is practically no risk of toxic side effects of IgY given orally. In the present study, pathogenic Vibrio parahaemolyticus was isolated from infected shrimp and studied their virulence factors including LD50 (by challenging with Fenneropenaeus indicus), proteolytic and hemolytic activities. The edible antibody IgY was raised by injecting the antigen of Extra Cellular Products (ECP) of V. parahaemolyticus to Gallus gallus domesticus during layoff period with and without the herbal immunoadjuvants, Asparagus racemosus and Glycine max (V.p wo: V. parahaemolyticus ECP without adjuvant; V.p A: V. parahaemolyticus ECP with A. racemosus and V.p G: V. parahaemolyticus ECP with G. max). Eggs were collected after five weeks of immunization and anti- V. parahaemolyticus IgY was extracted and purified. Physicochemical properties of the immunized Chickens' serum and anti- V. parahaemolyticus IgY's cross reactivity, growth inhibition assay, single radial immunodiffusion assay and bacterial agglutination were studied. The results revealed that, the serum protein parameters were significantly (P ≤ 0.001) increased in experimental groups from control group. The antibody raised with immunoadjuvants had significantly (P ≤ 0.001) higher cross reactivity, growth inhibition, single radial immunoassay and bacterial agglutination when compared with and without immunoadjuvant and control groups. Further the control and experimental anti-V. parahaemolytics IgY coated artificial diets were fed to F. indicus for 60 days. After 30 and 60 dpv (days of post vaccination), shrimps from each groups were challenged with virulent V. parahaemolyticus and studied the survival, haematological and immunological parameters. The IgY coated diets (V. p A and V.p G) fed shrimps had decreased cumulative mortality, significantly (P ≤ 0.001) improved coagulase activity, total haemocyte count and oxyhaemocyanin. The immunological parameters such as prophenoloxidase, intracellular anion production, lysozyme production and phagocytosis also improved significantly (P ≤ 0.001) in IgY treated shrimps.

Robust humoral immune response against rabies virus in rabbits and guinea pigs immunized with plasmid DNA vectors encoding rabies virus glycoproteins – An approach to the production of polyclonal antibody reagents

DNA-based immunization has been previously shown to be an efficient approach to induce robust immunity against infectious diseases in animals and humans. The advantages of DNA vaccines are simplicity of their construction and production, low cost, high stability, and ability to elicit a full spectrum of immune responses to target antigens. The goals of this study were (i) to assess the antibody immune response to rabies virus glycoproteins (rGPs) in rabbits and guinea pigs after intramuscular immunization with pTargeT and pVAC2-mcs mammalian expression vectors encoding either the wild-type (WT) or codon-optimized (cOPT) rGP genes; and (ii) to prepare in-house rabbit anti-rGP polyclonal antibody reagents suitable for in Single Radial Immunodiffusion (SRID) and Indirect Fluorescent Antibody (IFA) assays. The maximum antibody responses against rabies virus in rabbits and guinea pigs were observed after immunization series with 500 μg/dose of pVAC2-mcs vector encoding either the WT or cOPT rGP genes adjuvanted with Emulsigen-D. No significant difference in the anti-rabies virus neutralizing antibody titers was observed in rabbits immunized with the WT and cOPT rGPs. The in-house rabbit anti-rGP polyclonal antibody reagents reacted comparable to the current reference reagents in SRID and IFA assays. The results of the study demonstrated that the DNA immunization of animals with the WT or cOPT rGPs is a promising approach to either induction of high anti-rabies virus neutralizing antibody titers in vivo or for production of polyclonal antibody reagents against rabies.

Towards integrated production of an influenza A vaccine candidate with MDCK suspension cells.

Seasonal influenza epidemics occur both in northern and southern hemispheres every year. Despite the differences in influenza virus surface antigens and virulence of seasonal subtypes, manufacturers are well-adapted to respond to this periodical vaccine demand. Due to decades of influenza virus research, the development of new influenza vaccines is relatively straight forward. In similarity with the ongoing coronavirus disease 2019 pandemic, vaccine manufacturing is a major bottleneck for a rapid supply of the billions of doses required worldwide. In particular, egg-based vaccine production would be difficult to schedule and shortages of other egg-based vaccines with high demands also have to be anticipated. Cell culture-based production systems enable the manufacturing of large amounts of vaccines within a short time frame and expand significantly our options to respond to pandemics and emerging viral diseases. In this study, we present an integrated process for the production of inactivated influenza A virus vaccines based on a Madin-Darby Canine Kidney (MDCK) suspension cell line cultivated in a chemically defined medium. Very high titers of 3.6 log10 (HAU/100 µl) were achieved using fast-growing MDCK cells at concentrations up to 9.5 × 106 cells/ml infected with influenza A/PR/8/34 H1N1 virus in 1 L stirred tank bioreactors. A combination of membrane-based steric-exclusion chromatography followed by pseudo-affinity chromatography with a sulfated cellulose membrane adsorber enabled full recovery for the virus capture step and up to 80% recovery for the virus polishing step. Purified virus particles showed a homogenous size distribution with a mean diameter of 80 nm. Based on a monovalent dose of 15 µg hemagglutinin (single-radial immunodiffusion assay), the level of total protein and host cell DNA was 58 µg and 10 ng, respectively. Furthermore, all process steps can be fully scaled up to industrial quantities for commercial manufacturing of either seasonal or pandemic influenza virus vaccines. Fast production of up to 300 vaccine doses per liter within 4-5 days makes this process competitive not only to other cell-based processes but to egg-based processes as well.

The Lucerne Protocol for the Production of Autologous Serum Eyedrops.

There are a variety of protocols for manufacturing autologous serum (AS) eye drops. The Lucerne protocol for the production of AS eye drops uses a slightly reduced gravitational (g)-force and time for the centrifugation process (2500 × g for 10 minutes), compared to previously published optimised protocols, to obtain high levels of epitheliotropic growth factors (3000 × g for 15 minutes). The goal of this study was to compare the concentrations of growth factors, albumin and lysozyme in autologous serum eye drops manufactured with these protocols. Blood from 5 healthy volunteers was placed in plastic tubes without an anticoagulant. Tubes from each donor were left in a vertical position for 2 hours at room temperature to facilitate coagulation, followed by centrifugation at either 2500 × g for 10 minutes or at 3000 × g for 15minutes at room temperature. The serum levels of beta nerve growth factor (β-NGF), transforming growth factor β1 (TGF-β1), epidermal growth factor (EGF), hepatocyte growth factor (HGF), platelet-derived growth factor BB (PDGF-BB) and vascular endothelial growth factor A (VEGF-A) were measured in triplicate with a multi-analyte Simple Plex platform. The Simple Plex cartridge allows each sample to be run in triplicate for each analyte and prevents any interaction between the antibody components for each biomarker. The serum level of albumin was measured by turbidimetric immunoassay Tina-quant and of lysozyme by single radial immunodiffusion assay. For all analytes, the reduced g-force and centrifugation time did not result in a significant difference in serum levels. The Lucerne protocol for the production of autologous serum eye drops with reduced g-force and a shorter centrifugation time does not affect the concentrations of the main epitheliotropic growth factors, albumin and lysozyme, in AS eye drops.

HA1 (Hemagglutinin) quantitation for influenza A H1N1 and H3N2 high yield reassortant vaccine candidate seed viruses by RP-UPLC

The only effective measure to decrease morbidity and mortality caused by the influenza virus in the human population is worldwide vaccination. Vaccination produces neutralizing antibodies that target the HA1 subunit of the HA (hemagglutinin) protein and are strain specific. The effectiveness of new influenza vaccines are linked to two factors, the correct prediction of the circulating strains in the population in a particular season and the concentration of the HA1 protein in the vaccine formulation. With the advent of the licensing of quadrivalent vaccines, pharmaceutical manufacturers are under considerable pressure due to time constraints and dedicated resources to deliver 194–198 million doses (2020–2021 U.S. market) of vaccine. Considering the valuable resources needed to produce the influenza vaccine in a timely manner, the efficient quantitation of the HA1 protein (the main component in the influenza vaccine) is required. Currently the only method approved by regulatory agencies for quantitation of the HA antigen in vaccines is the single radial immunodiffusion assay (SRID), an antibody dependent assay that is not time efficient. Time efficient methods that are antibody independent e.g. reverse phase-high performance liquid chromatography (RP-HPLC) or size exclusion-HPLC (SE-HPLC) are available. An improved method implementing reverse phase-ultra performance liquid chromatography (RP-UPLC) has been developed to quantitate the HA1 protein antigen present in the high yield reassortant vaccine seed viruses from influenza A H1N1 and H3N2 subtypes harvested from inoculated embryonated chicken eggs. This method differentiates between high yield and lower yielding reassortants in order to select the best vaccine candidate seed virus with the highest growth ‘in ovo’. This direct capability to monitor the HA1 concentration of potential reassortant seed viruses and to choose the best yielding HA influenza reassortant when faced with multiple viral seed candidates provides a major advantage on the industrial scale to the influenza vaccine process.

Determination of the potency of a cell-based seasonal quadrivalent influenza vaccine using a purified primary liquid standard

In Japan, the practical application of completely cell-based seasonal influenza vaccines is under consideration. Considering the good correlation between the immunogenicity of egg-based influenza vaccines and the hemagglutinin (HA) content determined by the single radial immunodiffusion (SRD) assay, we determined the potency of the first cell-based quadrivalent vaccine experimentally generated in Japan using the SRD assay in this study. A primary liquid standard (PLS) and reference antigen were generated from the purified vaccine virus, and a sheep antiserum was produced against the HA of the vaccine virus. Since the purity of the PLS affects the reliability of vaccine potency testing, the purification steps are significant. We successfully prepared a purified PLS nearly free of cell debris. The HA content in the PLS was first estimated from the total amount of viral protein and the percentage of HA content determined by SDS-PAGE analysis. The HA content in the reference antigen was calibrated to that in the PLS via the SRD assay. The vaccine potency, that is, the HA content in each vaccine, was finally measured using the corresponding reference antigen. Ultimately, the measured vaccine potency of the monovalent vaccine was similar to that of the quadrivalent vaccine.

Development and characterization of standard reagents for cell-based prepandemic influenza vaccine products

ABSTRACT Outbreaks of infection by novel avian influenza virus strains in humans cause public health issues worldwide, and the development of vaccines against such novel strains is the most effective method for the prevention of these virus outbreaks. All types of vaccines must be tested for potency before use; thus, quantitative potency assays are needed for influenza vaccines. The single radial immunodiffusion (SRID) assay is considered the gold standard for quantification of influenza virus antigens, and the SRID reference reagents are essential for the determination of vaccine potency. However, it remains debatable whether reference reagents derived from egg-based vaccine platforms can be used to precisely quantify non-egg-derived vaccines; thus, influenza vaccine production using cell-based platforms has attracted increasing attention. To evaluate the utility of reference reagents derived from a cell-based influenza vaccine platform, we prepared cell-based reference reagents from MDCK cell-grown viruses and compared them with egg-derived reference reagents. A primary liquid standard (PLS) was purified from cell-derived candidate influenza vaccine viruses, and hemagglutinin (HA) antigen content was determined by a densitometric method. The produced PLS could be stored at 4°C for more than 10 months. We also established a simple HA protein purification method for goat antiserum preparation, and the performance of the resulting antiserum was compared to that of standard reagents obtained using different production platforms. The results of this study indicate that these reference reagents can be used for both cell-based and egg-based production platforms and that the differences between these two types of platforms are negligible.

The Effect of Bacterial Infections on the Immune States in Eczematic Patients

Background :Eczema is one of most important global diseases that affects both genders at all age groups, the relationship between bacteria and allergy in Eczematic patients is still as a great need of studies to show more of the microbial role in eczema. Objective:This study has been investigating the main commonbacterial secondary infections associated with Eczema lesionand estimation the serum levels of immunoglobulin types and cytokines in both patients and control groups. Material and Method: Swabs samples were taken from infected eczema lesions in patients consulting in Dermatology Consultation Unit in Medical Marjan City in Al-Hilla Province. Microbiology investigation for swabs were done with antibiotic sensitivity test. Blood was collected for measuring the concentrations of serum IgG, IgA, IgM by single radial immunodiffusion assay, IgE, IL-1β, IL-4, and IL-10 were measured using ELISA kits. Results: The noted profile of bacteria associated with cases of pyogenic eczema were Staphylococcus aureus (81.08%), Streptococcus pyogenes (5.1%) and Pseudomonas aeruginosa (8.1%). Several skin areas were affected, such as Hands (35.13%), Thighs (18.9%) and Feet (18.9%). The relation between pyogenic Eczema lesions and Occupation, Season and Geography were studied.At the immunological level, we found a significant increased (p≤0.05) in the concentrations of IgM, IgG, IgA and IgE compared with control groups with a significantly increased (p≤0.05) in the concentrations of cytokines (IL-1β, Il-4, and IL-10) that stimulated the Th2 antibody production. Conclusion: We concluded that the bacterial infections played an important role in systemic humoral and cellular immunity in eczematic patients.

Development and fit-for-purpose verification of an LC-MS method for quantitation of hemagglutinin and neuraminidase proteins in influenza virus-like particle vaccine candidates

Recombinant influenza Virus-Like Particle (VLP) vaccines are promising vaccine candidates to prevent influenza, contain two major viral antigenic glycoproteins, Hemagglutinin (HA) and Neuraminidase (NA), on the surface of recombinant VLPs. Accurate quantitation of the mass of these antigenic proteins is important to ensure the product quality and proper dosing. Currently, Single Radial Immunodiffusion (SRID) is a recognized assay for determination of the HA immuno-reactive concentration (potency) in vaccine products, based on immuno-reactivity of HA with strain-specific antisera. The SRID assay, however, requires availability of strain-specific and properly calibrated reagents, which can be time-consuming to generate and calibrate. In addition, the assay is not suitable for quantitation of low abundant proteins, such as NA. In order to accelerate the overall production cycle, we have developed and optimized a high-resolution (HR) LC-MS method for absolute quantitation of both HA and NA protein concentrations in influenza VLP vaccine candidates. In this work, we present the method development, optimization and verification of its suitability for the intended purpose, as a prerequisite for its potential application in Quality Control, by assessing specificity, precision and accuracy, detection characteristics, and dynamic linear range. The method can be also used for other HA/NA containing preparations including in-process samples, purified proteins, whole virus preparations, nano-particle and egg-based vaccine preparations, or for calibration of SRID reference antigens.

COMPARISON OF RADIAL IMMUNODIFFUSION AND SANDWICH-ELISA FOR THE MEASUREMENT IGG IN SERUM OF CALVES

This investigation compared single radial immunodiffusion (sRID) and sandwich enzyme-linked immunosorbent assay (sandwich-ELISA) for the detection concentration of IgG in serum of calves. The results from each methods was shown positively correlated and that the ELISA test procedure would give more precise estimates of IgG concentration in serum for express diagnosis of failure of transfer of passive immunity of calves

Purification method for cell-cultured influenza virus H5N1

Objective To reduce the residual proteins and DNA of host cells in the preparation of H5N1 influenza A virus. Methods Core 700 was firstly used to remove residual host cell proteins, and then Capto Q was used to remove host cell DNA. Several batches of H5N1 influenza A virus cultured in Madin-Darby canine kidney (MDCK) cells were purified using this method. The efficiency of purification was evaluated using many methods including quantitative real-time PCR, hemagglutination (HA) test and single radial immunodiffusion assay. Moreover, Benzonase nuclease was used for comparison. Results Without the use of Benzonase nuclease, the overall removal rates of host cell DNA and residual proteins were 99.62% and 98.1%, and the HA antigen recovery rate was 66.96%. Conclusions This study established a purification strategy with good effect for cell-based influenza vaccines. It can efficiently remove host cell DNA and proteins and achieve a high HA recovery rate. The purification result is no worse than that of adding Benzonase nuclease, suggesting the potential of its application in actual vaccine production. Key words: Influenza virus; MDCK cells; Host cell protein; Host cell DNA; Purification

Detection of Alpha-1 antitrypsin and Interleukin-8 in meningitis patients

Meningitis is a meninges inflammation and the subarachnoid cerebrum spinal fluid that surrounds the spinal cord and brain. Infantile meningitisis a major cause of morbidity & mortality in various parts of the world; it is earnest disorder owing to the nearness of the location to the brain andspinal cord. It can be caused by virtually any organism; in general viral infections are more common than bacterial, fungal and parasitic infectionsrespectively. Objective: investigation of the Interleukin-8 (IL-8) and Alaph-1 Antitrypsin in the cerebrum spinal fluid of meningitis patients.Material and Method: A study was done at the Medical city and Al-kadimia hospitals included 47 and 20 beings of Patients and Control groupswith an overall age range between 11 days to 6 years old. According to the causative agents of the disease, they were divided into three groups:bacterial, viral and control groups. All subjects were monitored for the IL-8 by Enzyme Linked Immunosorbent Assay (ELISA) and Alaph-1Anti-trypsin by Single Radial Immunodiffusion assay (SRIA) in the cerebrospinal fluid the data was analyzed via using SPSS (chi-squire test)program (version 22). Results and discussion: The results revealed the presence of an elevation concentration of IL-8 among meningitis diseasepatients (484.0695 pg/ml ), high concentration of IL-8 seemed in bacterial meningitis (825.3250pg/ml) when compared with its level in viralmeningitis (142.8143pg ml) ( p= 0.002) and control group ( 56.3409 pg/ml) (p=0.00) but no significant difference was found between viral andcontrol groups (p= 0.679). Alpha 1 antitrypsin results appeared high concentration of (21.6462 mg/ dl) and (16.4000 mg/ dl) in bacterial and viralmeningitis groups in sequentially, and that concentrations showed differences arrived significantly. The statistical analysis of this research showeda correlation between IL-8 levels and both of neutrophil and lymphocyte in meningitis patients. IL-8 had significant correlation with lymphocyte(p=0.02) in viral group while no significant correlation was appeared between IL-8 and neutrophil. In bacterial group, lymphocytes with IL-8 hadweak correlation, in contrary the correlation between neutrophil and IL-8 revealed the significantly correlation (p=0.01). No correlation was establish concerning the Alpha-1 antitrypsin and the cell’s numbers and types. Conclusion: A study indicated that Alpha 1 antitrypsin and IL-8 playedan important role in meningitis disease.

The differential effect of sub-micron level HA aggregates on influenza potency assays

Influenza vaccines remain the most effective public health measure for the prevention of influenza-related illnesses. The primary immunogen in inactivated influenza vaccines is hemagglutinin (HA), the receptor binding protein of influenza. The concentration of HA during vaccine production and testing is standardized according to the level of antigen as measured by Single Radial Immunodiffusion Assay (SRID). This allows vaccine potency to be controlled such that individuals receive a dose known to provoke a clinically protective immune response. As compared to alternatives, SRID has the advantage of quantifying immunologically relevant forms of HA, but it depends on timely generation of novel reagents for each new vaccine strain. In recent years, a number of alternative assays have been suggested based on either epitope recognition, receptor binding or protection from proteolysis but it is unclear how they relate to vaccine potency in clinical trials. In this report we describe the development of a lectin-based, ELISA-type assay for HA potency and find it provides similar potency estimates to SRID except in the case of a vaccine with aggregated HA and other viral proteins. In that case, SRID predicted the immunologically active HA present and ELISA techniques did not. This difference was due to tested antibodies failing to pull down or bind to the HA present unless particle aggregates were first dissociated. Furthermore, detergent treatment alone was insufficient to complete this dissociation. While others have previously demonstrated that immunocapture-based techniques can misestimate the potency of influenza vaccines depending on the individual antibodies used we demonstrate that in this case the failure was due to an inability of all antibodies to capture HA contained in the aggregated influenza vaccine.

Universal monoclonal antibody-based influenza hemagglutinin quantitative enzyme-linked immunosorbent assay

Seasonal and pandemic influenza infections remain a serious public health concern. Many health authorities recommend annual vaccination as the most effective way to control influenza infection. Accordingly, regulatory guidelines ask vaccine manufacturers to determine vaccine potency at the time of release and throughout shelf-life to ensure vaccine quality. The potency of inactivated influenza vaccine is related to the quantity of hemagglutinin (HA). Since 1970s, single radial immunodiffusion (SRID) assay has been standardly used for the quantitation of HA in influenza vaccine. However, SRID is labor-intensive, inaccurate, and requires standard reference reagents that should be updated annually. Therefore, there have been extensive efforts to develop alternative potency assays. In this study, we developed and tested a new HA quantitative enzyme-linked immunosorbent assay (ELISA) using a universal monoclonal antibody that can bind to HAs from various subtypes in group 1 influenza A virus (IAV). We analyzed the conserved stalk domain of HA via a library approach to design a consensus HA antigen for group 1 IAV. The antigens were expressed as a soluble form in E. coli and were purified by Ni-affinity chromatography. When tested with variety of HAs from IAVs or influenza B viruses (IBVs), the mAbs exhibited specific binding to group 1 HAs, with potential exception to H9 subtype. Among various conditions of pH, urea, and reducing agents, pretreatment of HA at low pH exposing the conserved stalk domain was crucially important for optimal ELISA performance. Calibration curves for various HAs were generated to determine accuracy, specificity, sensitivity, and linear dynamic range. The ELISA method shows high sensitivity and accuracy compared with the SRID assay. The HA group specific universal mAbs against the consensus stalk domain of HA are conducive to establishing an ELISA-based standard procedure for the quantitation of HA antigens for annual vaccination against influenza infection.

Influence of Immunocompetence Levels on Layer Economic Traits in Rhode Island Red Chicken

This investigation was carried out to study the influence of immunocompetence levels of layers on its economic performance traits after long term selection in Rhode Island Red chicken (selected strain) maintained at experimental layer farm of this institute. Five weeks aged 100 chicks were immunized with 1% (v/v) sterile sheep erythrocytes suspension (SRBC). In vivo antibody response to SRBC, serum lysozyme and serum IgG concentrations were estimated through haemagglutination (HA) test, lysoplate and single radial immunodiffusion assays, respectively and were classified into high, medium and low levels of immunocompetence based on their means and standard deviations. Layer performance data under various immunocompetence levels were analyzed by least squares analysis of variance. The least squares means of HA titre, serum lysozyme and serum IgG concentrations were 9.33±0.34, 4.42±0.18 μg/ml and 8.72±0.41 μg/μl, respectively. Pullets with high serum lysozyme concentration laid highest (P

Comparison of single radial immunodiffusion, SDS-PAGE and HPLC potency assays for inactivated influenza vaccines shows differences in ability to predict immunogenicity of haemagglutinin antigen

The current gold-standard potency test for inactivated influenza vaccines is the single radial immunodiffusion (SRD) assay. A number of alternative potency tests for inactivated influenza vaccines have been proposed in recent years. Evaluation of these new potency tests commonly involves comparison with SRD, in order to ascertain that the new method obtains values that correlate with those measured by the standard potency test. Here, we extended comparison of two methods, reverse-phase HPLC and SDS-PAGE, with SRD by assessing the methods’ capacity to detect loss of potency induced by various deliberate treatments of vaccine samples. We demonstrate that neither of these methods detected the loss of potency observed by SRD; importantly, neither SDS-PAGE nor reverse-phase HPLC reflected results from mouse experiments that showed decreased immunogenicity and protection in vivo. These results emphasise the importance of assessing the stability-indicating nature, ie the ability to measure loss of vaccine potency, of any potential new potency assay.

VaxArray for hemagglutinin and neuraminidase potency testing of influenza vaccines

Practical methods to measure the potency of influenza vaccines are needed as alternatives for the standard single radial immunodiffusion (SRID) assay. VaxArray assays for influenza hemagglutinin (HA) and neuraminidase (NA) have been developed to address this need. In this report, we evaluate the use of these assays to assess the potency of HA and NA of an A/H3N2 subunit vaccine by determining the correlation between the amounts measured by VaxArray and the immunogenicity in mice. The antibody response after one and two doses of five formulations of the vaccine ranging from 5 µg/mL to 80 µg/mL of HA, was measured by hemagglutination inhibition (HAI) and neuraminidase inhibition (NAI) assays. For hemagglutinin, vaccine potency determined by VaxArray was equivalent to potency measured SRID and these amounts were predictive of immunogenicity, with excellent correlation between potency measured by VaxArray and the HAI geometric mean titers (GMT). Likewise, the amount of NA measured by VaxArray was predictive of the NAI GMT. The VaxArray NA assay reported non-detectable levels of intact NA for a sample that had been heat degraded at 56 °C for 20 h, demonstrating that the assay measures the native, active form of NA. Similarly, the HA potency measured by VaxArray in this heat-treated sample was very low when a monoclonal antibody was used to detect the amount of antigen bound. Importantly, the force degraded sample induced low HAI titers and the NAI titers were not measurable, supporting the conclusion that the VaxArray HA and NA assays measure the immunogenic forms of these A/H3N2 antigens. This study indicates that VaxArray assays can be used to assess the potency of HA and NA components in influenza vaccines as a proxy for immunogenicity.