Radial Immunodiffusion Assay Research Articles

Rapid preparation of antiserum against influenza virus (H7N9) hemagglutinin for single radial immunodiffusion assay

Objective To establish a method for rapid preparation of antiserum against influenza virus (H7N9) hemagglutinin,and to study the possibility of using it in single radial immunodiffusion (SRID) assay for quantitative detection of antigen in H7N9 influenza vaccine.Methods Hemagglutinin proteins expressed in eukaryotic cells were used to immunize sheep.Serum samples were collected to detect antibody titers by ELISA and double immunodiffusion assay.Different concentrations of antiserum were used in SRID assay to get the optimized concentration.Results After 4 times of immunization,the antiserum titers achieved 1 ∶ 1 000 000 and 1 ∶ 32 as indicated by ELISA and double immunodiffusion assay,respectively.The antiserum could form a clear precipitation line in SRID assay.The detection of antigen in the range of 10 to 40 μg/ml showed good linearity in the standard curve.The antigen titers in six batches of H7N9 vaccine detected by this SRID assay were identical with those by SDS-PAGE assay.Conclusion The antiserum against H7N9 hemagglutinin for SRID assay was developed successfully,and could be used as a reagent for the quantitative detection of antigen in H7N9 influenza vaccine. Key words: A/H7N9 influenza virus; Antiserum; Single radial immunodiffusion

Use of plasma samples to assess passive transfer in calves using refractometry: comparison with serum and clinical cut-off point

Complete or partial failure of passive transfer (FPT) of immunoglobulins in the neonatal calf can result in an increased susceptibility to disease, a higher risk of mortality and decreased growth...

Use of Fourier‐Transform Infrared Spectroscopy to Quantify Immunoglobulin G Concentrations in Alpaca Serum

BackgroundRapid, economical, and quantitative assays for measurement of camelid serum immunoglobulin G (IgG) are limited. In camelids, failure of transfer of maternal immunoglobulins has a reported prevalence of up to 20.5%. An accurate method for quantifying serum IgG concentrations is required.ObjectiveTo develop an infrared spectroscopy‐based assay for measurement of alpaca serum IgG and compare its performance to the reference standard radial immunodiffusion (RID) assay.AnimalsOne hundred and seventy‐five privately owned, healthy alpacas.MethodsEighty‐two serum samples were collected as convenience samples during routine herd visits whereas 93 samples were recruited from a separate study. Serum IgG concentrations were determined by RID assays and midinfrared spectra were collected for each sample. Fifty samples were set aside as the test set and the remaining 125 training samples were employed to build a calibration model using partial least squares (PLS) regression with Monte Carlo cross validation to determine the optimum number of PLS factors. The predictive performance of the calibration model was evaluated by the test set.ResultsCorrelation coefficients for the IR‐based assay were 0.93 and 0.87, respectively, for the entire data set and test set. Sensitivity in the diagnosis of failure of transfer of passive immunity (FTPI) ([IgG] <1,000 mg/dL) was 71.4% and specificity was 100% for the IR‐based method (test set) as gauged relative to the RID reference method assay.Conclusions and Clinical ImportanceThis study indicated that infrared spectroscopy, in combination with chemometrics, is an effective method for measurement of IgG in alpaca serum.

Genetic Variability in Immunocompetence and Performance Status of Rhode Island Red Chicken Strains and its Crosses

The present investigation envisaged to assess genetic variability in immunocompetence and performance traits in three pure strains of Rhode Island Red (RIR) chicken, viz. RIRS, RIRC & RIRW, and its two crosses, viz. CARI-Sonali and CARI-Debendra. A total of 3232, 1263, 346, 1278 and 1258 eggs of RIRS, RIRC, RIRW, CARI-Sonali and CARI-Debendra, were set in three hatches for performance evaluation. Single hatched out 74, 74, 72, 82 and 81 chicks of corresponding genotypes were investigated for immunocompetence status assessed through haemagglutination (HA) test, Lysoplate Assay and Single Radial Immunodiffusion assay. The data on various immunocompetence and performance traits was recorded and analyzed by least squares analysis of variance. The investigation summarized that pure strains demonstrated better immunocompetence than crosses. RIRW demonstrated highest percent fertility followed by RIRC, RIRS, CARI-Sonali and CARI-Debendra. Highest percent hatchability on total egg set basis was observed in RIRC, whereas CARI-Debendra demonstrated highest percent hatchability on fertile egg set basis. CARI-Debendra demonstrated higher body weights than CARI-Sonali followed by RIRS, RIRW and RIRC strains. CARI-Sonali pullets demonstrated (p RIRW=CARI-Debendra>RIRC. There had been significant genotype×sex interaction effect on body weights at 2nd, 8th week and onwards. At 2nd week of age, CARI-Debendra-female showed highest body weight but subsequently CARI-Debendra-males were the heaviest. Effects of hatch, sex and chick weight-regression on growth traits as well as of hatch and housing weight-regression on layer production traits were significant. Crosses demonstrated least percent mortality than pure strains during brooding but reverse in laying stage.

50 Years Ago in The Journal of Pediatrics: Poliomyelitis Antibodies in Human Colostrum and Milk

Human milk was always thought to have specific factors to protect infants from poliovirus, but it wasn’t until this landmark study that definitive antibodies to the virus were described both in the mother’s milk and serum.1 These investigators sampled the milk and sera of four women in the colostrum phase and four women on the thirteenth day postpartum, and identified anti-poliovirus antibody using neutralization assays and micro-immunodiffusion methods. Similar methods of radial immunodiffusion and enzyme-linked immunosorbent (ELISA) assays are still used today to identify antigen-specific antibodies in the milk after viral exposure. More specifically since the article 50 years ago, a functional relevance can be observed. Anti-influenza IgA in human milk can be identified by ELISA at a higher concentration with resultant fewer febrile illnesses in infants of women vaccinated with influenza vaccine during pregnancy compared to controls2; however, further study is needed to determine the exact mechanisms of human milk immunologic protection against these infectious diseases. New methods in the laboratory have recently been described by Lemay et al3 can determine pathways involved in the mammary gland by identifying the transcriptome in the milk fat globule. These remarkable findings will not just describe the milk properties but provide the opportunity to design interventional trials on the mother to influence milk production in beneficial ways for the infant.

Rapid Manufacture and Release of a GMP Batch of Avian Influenza A(H7N9) Virus-Like Particle Vaccine Made Using Recombinant Baculovirus-Sf9 Insect Cell Culture Technology

N ovavax reported that, in 28 days following public release of the avian influenza A/Anhui/1/2013 (H7N9) virus gene sequences, its recombinant DNA and baculovirus-Spodoptera frugiperda cell culture-based technology was used to produce a virus-like particle vaccine to avian influenza A(H7N9) virus, and murine animal challenge studies were initiated.[1] This report describes Novavax’s manufacturing process and the coordinated timing of critical activities necessary to produce and release a clinical batch of avian influenza A(H7N9) virus virus-like particle vaccine, under current good manufacturing practices, within three months from the time that the virus genomic sequences for this potential pandemic influenza virus were reported. The key enabling factors were: • A detailed, integrated project plan and daily coordination meetings • Advanced use of the baculovirus master virus seed to bypass production of the passage 3 virus stock • Successful functional testing of the master virus seed to establish process parameters • Drug substance quantitation with an alternate method prior to availability of single radial immunodiffusion assay reagents • Forward processing of intermediates prior to completion of quality control testing • A process that uses single-use manufacturing technology Introduction Since 1997, avian influenza A viruses of various subtypes, including H5N1, H9N2, H7N7, H5N2, and now H7N9 have infected humans. Unfortunately, there is no method to predict which existing, novel avian, or other influenza A subtypes—for which little or no pre-existing immunity in the human population exists—may emerge as human-tohuman transmissible and lead to the next pandemic. One of the best methods of protecting society from the health, social, and economic risks of an influenza pandemic is rapid immunization of the population with a potent vaccine prior to the major wave of infections that come with the onset of a pandemic. In the past decade, manufacturers have improved the response rate and timelines for influenza vaccine production through the use of cell culture and recombinant DNA technology— as compared to the traditional production method that uses embryonated chicken eggs with reassortant viruses adapted for high growth.[2] The industry is now preparing vaccines for avian influenza A(H7N9) virus through various novel manufacturing process technologies[3-7] in response to the recent human H7N9 infections resulting in severe respiratory symptoms, extended hospitalizations, and a high mortality rate, especially in the elderly. On March 31, 2013, the Chinese Health and Family Planning Commission noti f ied the World Health Organization (WHO) of the first confirmed human infections with avian influenza A(H7N9) virus.[8] Although the H7N9 virus is a low pathogen in avian species, three confirmed human avian influenza A(H7N9) virus-associated infections had occurred in Shanghai and Anhui.[9,10] During the first weeks of April, the incidence of confirmed cases rose rapidly, spreading to eight provinces of eastern China.[11] Patients were admitted to the hospital displaying severe Article Published Online: 29 July 2013 TRENDS & DEVELOPMENTS IN BIOPROCESS TE Article Published Online: 29 July 2013

Analytical technologies for influenza virus-like particle candidate vaccines: challenges and emerging approaches.

Influenza virus-like particle vaccines are one of the most promising ways to respond to the threat of future influenza pandemics. VLPs are composed of viral antigens but lack nucleic acids making them non-infectious which limit the risk of recombination with wild-type strains. By taking advantage of the advancements in cell culture technologies, the process from strain identification to manufacturing has the potential to be completed rapidly and easily at large scales. After closely reviewing the current research done on influenza VLPs, it is evident that the development of quantification methods has been consistently overlooked. VLP quantification at all stages of the production process has been left to rely on current influenza quantification methods (i.e. Hemagglutination assay (HA), Single Radial Immunodiffusion assay (SRID), NA enzymatic activity assays, Western blot, Electron Microscopy). These are analytical methods developed decades ago for influenza virions and final bulk influenza vaccines. Although these methods are time-consuming and cumbersome they have been sufficient for the characterization of final purified material. Nevertheless, these analytical methods are impractical for in-line process monitoring because VLP concentration in crude samples generally falls out of the range of detection for these methods. This consequently impedes the development of robust influenza-VLP production and purification processes. Thus, development of functional process analytical techniques, applicable at every stage during production, that are compatible with different production platforms is in great need to assess, optimize and exploit the full potential of novel manufacturing platforms.

Potency under pressure: the impact of hydrostatic pressure on antigenic properties of influenza virus hemagglutinin

Influenza vaccines are effective in protecting against illness and death caused by this seasonal pathogen. The potency of influenza vaccines is measured by single radial immunodiffusion (SRID) assay that quantifies antigenic forms of hemagglutinin (HA). Hydrostatic pressure results in loss of binding of influenza virus to red blood cells, but it is not known whether this infers loss of potency. Our goal was to determine the impact of pressure on HA antigenic structure. Viruses included in the 2010-2011 trivalent influenza vaccine were subjected to increasing number of cycles at 35,000 psi in a barocycler, and the impact of this treatment measured by determining hemagglutination units (HAU) and potency. Potency was assessed by SRID and immunogenicity in mice. After 25 cycles of pressure, the potency measured by SRID assay was below the limit of quantification for the H1N1 and B viruses used in our study, while the H3N2 component retained some potency that was lost after 50 pressure cycles. Pressure treatment also resulted in loss of HAU, but this did not strictly correlate with the potency value. Curiously, loss of potency was abrogated when influenza A, but not B, antigens were exposed to pressure in chicken egg allantoic fluid. Protection against pressure appeared to be mediated by specific interactions because addition of bovine serum albumin did not have the same effect. Our results show that pressure-induced loss of potency is strain dependent and suggests that pressure treatment may be useful for identifying vaccine formulations that improve HA stability.

A Novel Synthetic Receptor-Based Immunoassay for Influenza Vaccine Quantification

Vaccination is the most effective prophylactic method for preventing influenza. Quantification of influenza vaccine antigens is critically important before the vaccine is used for human immunization. Currently the vaccine antigen quantification relies on hemagglutinin content quantification, the key antigenic component, by single radial immunodiffusion (SRID) assay. Due to the inherent disadvantages associated with the traditional SRID; i.e. low sensitivity, low throughput and need for annual reagents, several approaches have been proposed and investigated as alternatives. Yet, most alternative methods cannot distinguish native hemagglutinin from denatured form, making them less relevant to antigenic analyses. Here, we developed a quantitative immunoassay based on the sialic acid binding property of influenza vaccine antigens. Specifically, we chemically synthesized human and avian influenza virus receptors analogues, N-acetylneuraminic acid-2,6-lactose and N-acetylneuraminic acid-2,3-lactose derivatives with an azidopropyl aglycon, using α-2,6- and α-2,3-sialyltransferases, respectively. The azido group of the two sialyllactose-derivatives was reduced and conjugated to mouse serum albumin through a squarate linkage. We showed that the synthetic α-2,6- and α-2,3-receptors selectively bound to human and avian-derived hemagglutinins, respectively, forming the basis of a new, and robust assay for hemagglutinin quantification. Hemagglutinin treated at high temperature or low pH was measured differentially to untreated samples suggesting native conformation is dependent for optimal binding. Importantly, this receptor-based immunoassay showed excellent specificity and reproducibility, high precision, less turnaround time and significantly higher sensitivity and throughput compared with SRID in analyzing multiple influenza vaccines.

Distribution and determinants of circulating complement factor H concentration determined by a high-throughput immunonephelometric assay

BackgroundResearch on complement factor H (fH) in human disease is hampered by lack of an assay suitable for use in large-scale epidemiological studies. We describe the development and validation of a high throughput nephelometric assay for fH. MethodsReagents from a commercial radial immunodiffusion (RID) assay (The Binding Site) were adapted for use on the Siemens BNII high throughput nephelometric instrument. The assay was calibrated with a highly purified human fH preparation with rigorously determined concentration, and assay performance was comprehensively evaluated using samples from healthy human volunteers, with the commercial RID assay as a comparator. The distribution and determinants of circulating fH concentration in humans were then investigated in a large representative population sample. ResultsThe nephelometric assay had recovery close to 100%, was reproducible with intra- and inter-assay CV's of 11% and 5–15% respectively, and had a wider operating range than the RID assay. fH values were unaffected after multiple freeze-thaw cycles demonstrating that it is evidently a stable analyte for immunoassay. fH concentration was unaltered by an acute inflammatory stimulus. The population study showed that plasma fH concentration is associated with circulating lipids and indices of body fat. ConclusionWe present the first high throughput assay for circulating fH; the assay is accurate and reliable with reproducible measures from stored samples. It has established the distribution of fH values at a population level and demonstrated important associations with circulating lipids and indices of body fat, thus providing an important reference for future clinical and epidemiological investigations.

Comparison of three immunoglobulin G assays for the diagnosis of failure of passive transfer of immunity in neonatal alpacas

Measurement of serum immunoglobulin G (IgG) is used for the assessment of passive transfer of immunity in neonatal crias, with an IgG concentration <10 g/l being suggestive of failure of passive transfer (FPT). The purpose of the current study was to determine whether 3 commercially available immunologic assays yielded comparable results for IgG in alpacas. Serum samples from 91 alpacas were used and were stored frozen until batch analysis on the same day with the 3 assays. Immunoglobulin G was measured by radial immunodiffusion (RID) and 2 immunoturbidimetric (IT) assays (IT1, configured for automated chemistry analyzers; IT2, a point-of-care test). Median IgG concentrations were significantly different between the 3 assays, with the RID (median: 15 g/l) and IT1 (median: 16 g/l) assays, which used the same standard, yielding significantly higher IgG values than IT2 (median: 11 g/l). Results indicated a diagnostic discordance in 1-17% of samples at an IgG threshold of 10 g/l. Protein electrophoresis revealed that the RID and IT1 standard contained mostly albumin (>60%), whereas the IT2 standard consisted of beta and gamma globulins. The discrepant results between assays IT1 and IT2 were eliminated when the same standard was used (IT1: median 11 g/l; IT2: 10 g/l; n = 19 and 17, respectively). The IT1 assay had the highest precision, while the RID assay had the lowest. The results indicate that camelid IgG measurement is highly dependent on the assay standard and is not directly comparable between assays, potentially resulting in underdiagnosis of FPT in some crias.

Characterization of Alpha-fetoprotein Levels in Three Dolphin Species: Development of Sensitive Immunoassays for Analysis of the Pregnancy-associated Variations

A single radial immunodiffusion (SRID) assay and a chemiluminescent immunoassay (CLIA) were initially developed for alpha-fetoprotein (AFP) of the striped dolphin. Utilizing these developed assays, we investigated pregnancy-associated changes in the levels of AFP in the sera of fetuses and pregnant females of three dolphin species; samples were either collected from captive individuals or obtained as fishery by-products. The concentrations of AFP in the fetal serum ranged from 419.0 to 2026.3 μg/ml in the striped dolphin, 12.6 to 1218.7 μg/ml (for an AFP equivalent; eqAFP) in the common bottlenose dolphin and 770.6 to 3129.1 μg eqAFP/ml in the Risso's dolphin. AFP levels decreased with increased fetal size in fetuses over 20 cm in length. The concentrations of AFP in sera of pregnant females ranged from 7.18 to 8068.7 ng/ml in the striped dolphin, 6.6 to 1241.1 ng eqAFP/ml in the common bottlenose dolphin and 3.4 to 2868.7 ng eqAFP/ml in the Risso's dolphin. The levels in most pregnant females were equal to or lower than those found in males and nonpregnant individuals, although a few pregnant females exhibited extremely high levels (in the range of hundreds to thousands of nanograms per milliliter). Such high levels of AFP were not observed during pseudopregnancy. To our knowledge, this is the first report on basal profiles for serum AFP levels in small odontocetes. The profiles indicated that AFP may play a significant role during embryonic development, although maternal levels do not appear to be a diagnostic biomarker for monitoring pregnancy.

PRUEBAS DE CAMPO PARA EVALUAR CALIDAD CALOSTRAL EN LA ALPACA.

In the majority of livestock species, the concentration of maternal immunoglobulin (Ig) in colostrum determines the Ig level in their offspring, and the failure of passive transfer of maternal immunoglobulins results in their susceptibility to disease. This study evaluates two methods (visual assessment and Brix refratometry) for determining alpaca colostrum quality in the field. Evaluations of 26 fresh colostrum samples from 77 alpaca mothers were compared with the results obtained by radial immunodiffusion assay for 77 blood serum samples from 36-48 hours old offspring, and 26 colostrum samples. The colostrum was collected from the dams immediately postpartum and pre-suckling and blood was taken from the crias by jugular venipuncture. Grades of calostral viscosity were assessed visually, with 60% at 2-5, and total calostral solids measured by Brix sugar refractometry averaged 37.3%. The radial immunodiffusion test yielded newborn IgG serum levels of 2679 ± 603.4 mg/dL and colostral IgG levels of 28337± 5593 mg/dL, and only one animal registered failure of immune transfer with serum levels of 750 mg/dL. Results obtained by visual assessment of the colostrum coincide with those obtained by refractometry (p=0.0007), but differ from the serum IgG concentrations in the newborn animals (p=0.15), as do the Brix refractometry readings (p=0.338). Due to the lack of low IgG levels in the colostrum samples, it was impossible to determine if a relationship exists between observed viscosity and colostral IgG concentration. Nonetheless, the positive correlation (p&lt;0.001) between viscosity and Brix refractometry readings point to the need for further research.

Short communication: Evaluation of serum immunoglobulin G concentrations using an automated turbidimetric immunoassay in dairy calves

The absorption of maternal antibodies associated with colostrum feeding is critical to the health of calves. Multiple assays have been described to assess serum immunoglobulin G (IgG) concentrations in calves. However, none are ideal for routine use on farms. The purpose of this study was to evaluate the reliability of a new commercially available immunoassay and portable analyzer for measuring serum IgG concentrations in dairy calves. Serum from 100 Holstein calves that had received colostrum was collected for this study. Immunoglobulin G concentrations were run on each calf using both the rapid immunoassay method and radial immunodiffusion assay. Serum IgG concentrations in calves from this study ranged from 460 to 3,640mg/dL (mean ± SD: 1,515±71) as measured by radial immunodiffusion and 402 to 3,586mg/dL (mean 1,473±70) as measured by the immunoassay. Based on regression analysis, the automated results closely paralleled those obtained by radial immunodiffusion with a coefficient of determination value of 0.98. Based on the results of this study, the immunoassay technique using the portable analyzer represents a reliable method that can be run within 15min and provide an accurate serum IgG level. Although the cost is not insignificant, this assay could be easily implemented on a dairy farm to help monitor transfer of passive immunity.

A one step separation of immunoglobulin g from bovine serum by pseudobioaffinity chromatography on histidine grafted to epoxy activated sepharose

The selective adsorption of proteins and macromolecules on activated matrix grafted with histidine has been shown to be dependent on certain separation parameters like, pH and buffer type. In the present study, the significant potential of the histidine ligand to separate bovine IgG from other bovine serum proteins has been demonstrated. The successful separation was carried out by pseudobioaffinty chromatography on histidine grafted-epoxy activated sepharose. The method was applied to sterile bovine serum. Bovine IgG was completely separated in the form of a single peak with 25 mM MES buffer containing 0.2 M NaCl at pH 5. The purity of the separated bovine IgG was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Ouchterlony radial immunodiffusion (ODD) assay showed that bovine IgG was the main component present in the elution fraction.

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.

Development and qualification of the parallel line model for the estimation of human influenza haemagglutinin content using the single radial immunodiffusion assay

Infection with human influenza virus leads to serious respiratory disease. Vaccination is the most common and effective prophylactic measure to prevent influenza. Influenza vaccine manufacturing and release is controlled by the correct determination of the potency-defining haemagglutinin (HA) content. This determination is historically done by single radial immunodiffusion (SRID), which utilizes a statistical slope-ratio model to estimate the actual HA content. In this paper we describe the development and qualification of a parallel line model for analysis of HA quantification by SRID in cell culture-derived whole virus final monovalent and trivalent influenza vaccines. We evaluated plate layout, sample randomization, and validity of data and statistical model. The parallel line model was shown to be robust and reproducible. The precision studies for HA content demonstrated 3.8-5.0% repeatability and 3.8%-7.9% intermediate precision. Furthermore, system suitability criteria were developed to guarantee long-term stability of this assay in a regulated production environment. SRID is fraught with methodological and logistical difficulties and the determination of the HA content requires the acceptance of new and modern release assays, but until that moment, the described parallel line model represents a significant and robust update for the current global influenza vaccine release assay.

Quantification of Immunoreactive Viral Influenza Proteins by Immunoaffinity Capture and Isotope-Dilution Liquid Chromatography–Tandem Mass Spectrometry

An immunocapture isotope dilution mass spectrometry (IC-IDMS) method was developed to quantify antibody-bound influenza hemagglutinins (HA) in trivalent influenza vaccines (TIV). Currently, regulatory potency requirements for TIV require HA quantification based on the single radial immunodiffusion (SRID) assay, which is time-consuming, laborious, and requires production of large quantities of reagents globally. In IC-IDMS, antiserum to the HA of interest captured viral proteins that were in the correct conformation to be recognized by the antibodies. The captured proteins were digested, and evolutionarily conserved tryptic peptides were quantified using isotope-dilution liquid chromatography-tandem mass spectrometry. IC-IDMS relies on antibody-antigen binding similar to SRID but incorporates the accuracy and precision of IDMS. Polyclonal antibodies (pAb-H3) prepared by injection of sheep with purified H3 HA captured 82.9% (55.26 fmol/μL) of the total H3 HA (66.69 fmol/μL) from the commercial TIV and 93.6% (57.23 fmol/μL) of the total H3 HA (61.14 fmol/μL) in purified virus. While other HA (H1, B), neuraminidase (N1, N2, NB), viral matrix proteins, and nucleoproteins were also captured by this antiserum, our results were not affected due to the specificity of the mass spectrometer. IC-IDMS is an accurate, precise, sensitive, and selective method to measure antibody-bound HA in purified virus and commercial vaccines.

PCPP-Formulated H5N1 Influenza Vaccine Displays Improved Stability and Dose-Sparing Effect in Lethal Challenge Studies

The potential impact of an influenza pandemic can be mitigated through the realization of a successful vaccination program. The implementation of antigen stabilization and dose-sparing technologies is an important step in improving availability of vaccines at the time of a pandemic outbreak. We investigated poly[di(carboxylatophenoxy)phosphazene] (PCPP) as a potential stabilizing and immunostimulating agent for H5N1 influenza vaccine. Physicochemical characterization of PCPP-formulated H5N1 influenza vaccine revealed macromolecular complexation in the system, whereas single radial immunodiffusion assay verified antigenicity of the formulation in vitro. PCPP-enhanced formulation displayed a fourfold increase in the half-life at 40°C compared with a nonadjuvanted vaccine. Lethal challenge studies in ferrets demonstrated 100% protection for low-antigen dose PCPP-adjuvanted formulations (1 μg of hemagglutinin) and at least a 10-fold antigen-sparing effect. Therefore, PCPP demonstrated an ability to improve thermal stability of H5N1 influenza vaccine in solutions and provide for a substantial dose-sparing effect in vivo.

An evaluation of Brix refractometry instruments for measurement of colostrum quality in dairy cattle

Acquisition of high quality colostrum is an important factor influencing neonatal calf health. Many methods have been used to assess the Ig concentration of colostrum; however, improved, validated evaluation tools are needed. The aims of this study were to evaluate both optical and digital Brix refractometer instruments for the measurement of Ig concentration of colostrum as compared with the gold standard radial immunodiffusion assay laboratory assessment and to determine the correlation between Ig measurements taken from fresh and frozen colostrum samples for both Brix refractometer instruments. This research was completed using 288 colostrum samples from 3 different farms. It was concluded that the optical and digital Brix refractometers were highly correlated for both fresh and frozen samples (r=0.98 and r=0.97, respectively). Correlation between both refractometer instruments for fresh and frozen samples and the gold standard radial immunodiffusion assay were determined to be very similar, with a correlation coefficient between 0.71 and 0.74. Both instruments exhibited excellent test characteristics, indicating an appropriate cut-off point of 22% Brix score for the identification of good quality colostrum.