Radial Immunodiffusion Research Articles

Validation and method comparison of the use of densitometry to quantify monoclonal proteins in canine sera

Densitometric quantitation using serum protein electrophoresis (SPE) is used to monitor monoclonal proteins (M-proteins) in human patients but has not been validated in the dog. Serum globulin concentrations, species-specific radial immunodiffusion (RID), and ELISAs are currently used in veterinary medicine. We aimed to compare four methods that quantify M-proteins using densitometry and biuret protein (dM-protein) measurements. We also validated the best performing method and compared it with the RID and ELISA methods for measuring canine serum M-protein. Serum from six normal dogs and 83 serum samples from 46 dogs with confirmed monoclonal gammopathies were used. A spike and recovery experiment with purified monoclonal IgG and IgM, inter-run and intra-run variability, linearity under dilution, and lower limit of detection were performed. Results of commercial canine RID and ELISA kits for total class-specific immunoglobulin were compared with dM-proteins. The corrected perpendicular drop gating method had <20% error for IgG/γ-globulin and IgM/β-globulin M-protein quantifications. Linearity (r>.99), intra-run CV (1.1%-2.3%), and inter-run CVs (2.0%-3.5%) were acceptable. Correlation between the RID and densitometry results ranged from r=.25 to r=.88, depending on the class. The RID result was greater than that of the biuret total protein in 26/63 (41%) IgA cases. A panel of IgG, IgA, and IgM RIDs failed to correctly identify an IgM paraproteinemia in 6/6 (100%) cases. Densitometry was not comparable with any other tested method. Densitometric quantitation is a valid technique for measuring M-proteins in the β- and γ-globulin regions. Immunotyping via RID using the tested kit does not appear to detect IgM. Densitometry is recommended for measuring M-proteins in canine patients.

Development and validation of an ELISA for the quantification of bovine ITIH4 in serum and milk

Inter alpha trypsin inhibitor heavy chain 4 (ITIH4) is a serum protein belonging to the Inter alpha trypsin inhibitor (ITI) family, which was previously characterized by our group as a new APP in cattle. This protein was firstly described in pigs where is known to be a major acute phase protein, also denominated Pig-MAP. Increases of ITIH4 of up to 12 times the pre-infection values were previously reported in the serum of heifers with experimentally induced summer mastitis. ITIH4 was detected in the milk of cows with mastitis by western blot, but the method previously used to quantify this protein, radial immunodiffusion, was not sensitive enough to quantify it in milk samples. In this study we developed an ELISA method which allows the quantification of bovine ITIH4 in serum and milk samples. Previously developed antibodies were used to perform the assay, including anti bovine ITIH4 polyclonal antibodies and a monoclonal antibody against pig ITIH4 that also recognizes the bovine homologous protein. The ELISA developed showed an adequate precision, with inter and intra- assay coefficients of variation lower than 10% for serum and milk samples. The assay keeps linearity under dilution for both serum and milk samples. A good agreement was observed between the values measured by ELISA and radial immunodiffusion in serum samples.

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.

The influence of the closure format on the storage stability and moisture content of freeze-dried influenza antigen

Low moisture content is seen as crucial to achieving long term stability of freeze dried biologics and reference materials. Highly hygroscopic freeze-dried material are susceptible to moisture ingress over time which can lead to degradation and loss of biological potency. This study compared vials with unprocessed stoppers, vials with vacuum-oven dried stoppers and glass ampoules in order to determine the superior long term storage format in terms of moisture ingress and potency. B/Phuket influenza antigen was chosen as the model biological standard and the lyophilized antigen was stored at −20, 25 and 45 °C over a 1 year period. Ampoules had no significant moisture change across all storage temperatures as would be anticipated. Moisture content results at −20 °C showed no significant differences between ampoules, vials with vacuum-oven dried stoppers and vials with unprocessed stoppers over 12 months. Vials with vacuum-oven dried stoppers performed similarly to ampoules at −20 °C and 20 °C, but had a small increase in moisture content after 6 months at 45 °C. Vials with unprocessed stoppers preformed the worst and exhibited the largest moisture ingress after just 3 months at both 20 °C and 45 °C. Single radial immunodiffusion (SRD) potency assays showed at −20 °C and 20 °C there was no significant difference between all closure formats. At 45 °C there was a drop in potency for all closure formats, but ampoules and vials with vacuum-oven dried stoppers retained higher potency than vials with unprocessed stoppers. Thus, while ampoules are still considered to be the gold standard format for long term storage stability, using vials with vacuum-oven dried stoppers provides comparable stability and moisture integrity at −20 °C and 20 °C storage.

Selective Capture and Determination of Receptor-Binding Hemagglutinin in Influenza Vaccine Preparations Using a Coupled Receptor-Binding/RP-HPLC Assay.

Influenza vaccine potency is determined by the quantification of immunologically active hemagglutinin capable of eliciting neutralizing antibodies upon immunization. Currently, the single radial immunodiffusion (SRID) method is the standard in vitro potency assay used for lot release of seasonal inactivated influenza vaccines. Despite the proven usage of SRID, significant limitations such as the time-consuming preparation of reagents and limited dynamic range warrant the need for the development of alternative potency assays. Such alternative approaches need to discriminate and quantify relevant hemagglutinin material, provide strain identity, and be independent of strain-specific and seasonal reagents. Herein, we present a proof of concept method that combines the capture of conformationally well-folded hemagglutinin via a sialic acid binding step with the resolving power of reversed-phase high-performance liquid chromatography for strain identity and determination. Details of the protocol for the selective capture of receptor-binding hemagglutinin, its release from the receptor, and its relative determination are presented. This approach was found to provide flexibility for the reagents to be used and was adaptable to varying strain compositions of influenza vaccines. This proof of concept approach was developed as an antibody-independent methodology.

Short communication: Use of a digital refractometer in assessing immunoglobulin G concentrations in colostrum and the first 5 transition milkings in an Irish dairy herd

Transition milk is a source of immunoglobulin G (IgG) and could potentially be used to provide calves with passive immunity, when the IgG concentration is ≥50 g/L. Assessment of IgG concentrations in transition milk would be required before feeding and could be conducted using cow-side tests such as refractometers. Currently, limited information is available on the ability of refractometers to assess transition milk quality. We hypothesized that digital refractometry could be used to provide an accurate cow-side assessment of IgG concentrations in colostrum and transition milk, and IgG concentration in colostrum and one or more transition milking in an Irish herd is >50 g/L. The objectives of this study were to determine the IgG concentrations in colostrum and first, second, third, fourth, and fifth transition milk, and determine the utility of a digital refractometer in assessing quality of colostrum and transition milk produced by cows in a pasture-based dairy production system. A convenient sample of 75 dairy cows were enrolled. Colostrum and transition milk IgG concentrations were determined by radial immunodiffusion and refractometry. Sensitivity and specificity of the refractometer were determined and cut-off points that maximized sensitivity and specificity were determined using receiver operating characteristic curves. Median (range) IgG concentrations in colostrum and first, second, third, fourth, and fifth milking were 99.6, 43.5, 12.5, 5.3, 1.9, and 1.8 g/L, respectively. The sensitivity (0.8-1) of digital refractometry in identifying samples with low IgG concentrations in colostrum, first, second, and third transition milk was acceptable. In contrast, digital refractometry was not useful for assessing IgG concentrations in the fourth and fifth milking due to low IgG concentrations.

Opposite Profiles of Complement in Antiphospholipid Syndrome (APS) and Systemic Lupus Erythematosus (SLE) Among Patients With Antiphospholipid Antibodies (aPL).

APS is the association of antiphospholipid antibodies (aPL) with thromboses and/or recurrent pregnancy loss (RPL). Among patients with SLE, one-third have aPL and 10–15% have a manifestation of secondary APS. Animal studies suggested that complement activation plays an important role in the pathogenesis of thrombosis and pregnancy loss in APS. We performed a cross-sectional study on complement proteins and genes in 525 patients with aPL. Among them, 237 experienced thromboses and 293 had SLE; 111 had both SLE and thromboses, and 106 had neither SLE nor thrombosis. Complement protein levels were determined by radial immunodiffusion for C4, C3 and factor H; and by functional ELISA for mannan binding lectin (MBL). Total C4, C4A and C4B gene copy numbers (GCN) were measured by TaqMan-based realtime PCR. Two to six copies of C4 genes are frequently present in a diploid genome, and each copy may code for an acidic C4A or a basic C4B protein. We observed significantly (a) higher protein levels of total C4, C4A, C4B, C3, and anticardiolipin (ACLA) IgG, (b) increased frequencies of lupus anticoagulant and males, and (c) decreased levels of complement factor H, MBL and ACLA-IgM among patients with thrombosis than those without thrombosis (N = 288). We also observed significantly lower GCNs of total C4 and C4A among aPL-positive patients with both SLE and thrombosis than others. By contrast, aPL-positive subjects with SLE had significantly reduced protein levels of C3, total C4, C4A, C4B and ACLA-IgG, and higher frequency of females than those without SLE. Patients with thrombosis but without SLE (N = 126), and patients with SLE but without thrombosis (N = 182) had the greatest differences in mean protein levels of C3 (p = 2.6 × 10−6), C4 (p = 2.2 × 10−9) and ACLA-IgG (p = 1.2 × 10−5). RPL occurred in 23.7% of female patients and thrombotic SLE patients had the highest frequency of RPL (41.0%; p = 3.8 × 10−10). Compared with non-RPL females, RPL had significantly higher frequency of thrombosis and elevated C4 protein levels. Female patients with homozygous C4A deficiency all experienced RPL (p = 0.0001) but the opposite was true for patients with homozygous C4B deficiency (p = 0.017). These results provide new insights and biomarkers for diagnosis and management of APS and SLE.

INDICES OF PECULIARITIES OF IMMUNE REGULATION DETECTED IN CHILDREN EXPOSED TO ENVIRONMENTAL CONTAMINATION WITH METALS

Introduction. The technogenic development of the habitat determines the need of study of the negative impact of environmental factors on public health. The aim of the work is to study the peculiarities of changes of indices of immune regulation, specific and non-specific sensitization in the children’s population, living in conditions of air pollution with metals. Material and methods. A survey of the children population, living in the exposure zone of the studied risk factors was conducted. Mass spectrometry was used to determine the concentrations of chemical elements in biological media. Phagocytic activity was determined using sheep erythrocytes, serum immunoglobulin concentrations - using radial immunodiffusion, total IgE and cytokine levels - using enzyme immunoassay, specific IgG and IgE antibodies to metals - using allergen sorbent testing method. Statistical analysis was performed using Statistica 6.0. Results. In the observation group №. 1 the level of blood contamination with aluminum and chromium compounds when compared to the indices of the comparison group was higher, as well as there was a gain in aluminum, manganese, nickel, chromium in the blood level, compared with the values of the observation group №. 2. In the observation group № 1, the relative phagocytosis and phagocytic number were lower in relation to the norm, to the comparison group and the observation group № 2. Specific antibodies to metals exceeded the reference levels in the observation groups № 1 and № 2. The production of interleukin-1beta in the observation groups № 1 and № 2 exceeded the comparison indices; high levels of interleukin-8 and interferon-gamma were noted. A violation of cellular immunity in the observation group № 1 was established. In observation group № 2, changes in immune reactivity were expressed in a less degree, which was characterized by reduced absolute values of metals sensitivity indices. Conclusion. Excessive blood contamination with aluminum and chromium compounds, a decrease in phagocytic activity, an increase in specific sensitization by the criterion for the content of specific antibodies to metals, as well as an imbalance of cytokine immune mediators, were shown.

THE EFFECTIVENESS OF IMMUNOVAC VP-4 FOR IMMUNOLOGICAL PARAMETERS IN FREQUENTLY AND LONG-TERM ILL CHILDREN

Aim. To study the level of specific antibodies of different isotypes to the antigens of Staphylococcus aureus and Klebsiella рneumoniae in the serum, saliva and nazal secret and the concentration of IgA,sIgA,IgG in saliva from frequently and long-term ill children in nasal-oral administration of Immunovac VP-4. Materials and methods. Specific antibodies to S. aureus and K.pneumoniae, contained in saliva, nasal, and serum were determined by the method of enzyme immunoassay. Concentrations of immunoglobulins of classes G, A and sА in saliva were determined by radial immunodiffusion using a commercial kit produced by the NPC «Medical immunology». Results. The high level of specific antibodies contained in the serum and nasal secretions, the level of antibodies in saliva is negligible. The serum is dominated by IgG-isotype antibodies, saliva and nasal secret — antibodies of IgA-isotope. After the introduction of Immunovac VP-4 there was a statistically significant increase in the level of specific antibacterial antibodies in serum, saliva and nasal secret, and increasing levels of IgG and sIgA in saliva. Conclusion. Obtained data demonstratet that the nasal-oral scheme of administration of Immunovac VP-4 frequently and long-term ill children allowed to increase the level of specific antibodies in serum, saliva and nasal secret to bacterial antigens that are part of the vaccine and to normalize the local synthesis of IgG and sIgA, which play a major role in the protection of the respiratory tract and mucous membranes of the upper respiratory tract.

Determination of the immunoglobulin G supply in the newborn calf

A sufficient supply of colostral antibodies within the first hours of life is crucial for the development and the health status in young calves. It is rational to examine the immunoglobulin uptake of single animals, but particularly on a herd basis, during herd controls and consultations. This enables economical calf rearing in accordance with animal welfare. Because of the costly, laboratory-dependent and in part time-consuming direct measurement of the absorbed immunoglobulins using radial immunodiffusion (RID) or ELISA, multiple studies attempted to develop indirect methods, which would be affordable and operational in the field. These aim to draw an inference for the absorbed quantity of colostral antibodies based on other correlated parameters. Multiple validations showed in part significant differences between various methods concerning specificity and sensitivity in comparison to the direct methods. In addition to RID and ELISA, this article presents the measurement of the γ-glutamyltransferase (GGT) activity, the determination of the total serum protein concentration using refractometry and the zinc sulphate turbidity test, and describes the advantages and disadvantages of their application. Refractory measurement and determination of the GGT activity represent a valuable alternative to a laboratory-dependent immunoglobulin G measurement. Nevertheless, there is no ideal rapid test method, such that several influencing factors have to be considered.

A cost-effective method for purification and characterization of human urinary albumin

We describe a simplified approach for the purification and characterization of urinary albumin, a key biomarker currently used for understanding the onset and prognosis of microalbuminuria. Urinary albumin was purified from human urine collected from diabetic kidney disease patients by using 2-stage tangential flow filtration process and set of column chromatography steps. The relative molecular mass of urinary albumin is 66,871 Da (SYNAPT G2 High Definition Mass Spectrometry System). Isolated urinary albumin was analyzed by SDS-PAGE, Western blotting, immunoelectrophoresis, Ouchterlony double-immunodiffusion, single radial immunodiffusion, size-exclusion HPLC and peptide mass fingerprint analysis. The size-exclusion HPLC elution profile of the purified urinary albumin was similar to that of a reference form of native albumin. Peptide mass fingerprint analysis of the purified urinary albumin yielded peptides that partially matched with known sequence of ALBU_HUMAN (P02768). This is the first report of purification and validation of immunochemically reactive form of urinary albumin from a large volume of urine of diabetic kidney disease patients. In this purification approach, the cost of the purified albumin is significantly lower.

Prevalence of Failure of Passive Transfer of Immunity in Dairy Calves in the Czech Republic

Prevalence of failure of passive transfer (FPT) of immunity remains relatively high worldwide. The aim of this study was to estimate the FPT prevalence in Czech dairy calves and to evaluate the selected factors – breed, herd size, sex of calves, single versus twin births and the influence of the season of birth. A total of 1,175 serum samples were taken from calves of Czech Fleckvieh and Holstein breed from 33 herds between October 2015 and October 2017. Serum IgG concentration was determined by reference method for IgG determination – radial immunodiffusion. Statistical evaluation was performed by Kruskal‑Wallis test. The concentration of IgG ranged from 1.5 to 46.6 g/L with average value 13.7 g/L and was significantly influenced by breed, size of the herd and season. Using the criterion IgG &lt; 10 g/L, it was found that 34.6 % of calves had FPT. The prevalence of FPT by breed was 42.9 % vs. 24.2 % (Czech Fleckvieh vs. Holstein), by size of the herd 45.0, 44.4, 25.5 and 22.0 % (&lt; 200, 200–399, 400–599 and ≥ 600 cows per herd, respectively) and by season 25.3, 34.6, 29.9 and 52.5 % (spring, summer, autumn and winter, respectively). The sex of calves was not found to be a statistically significant factor. The study in newborn calves showed that FPT is still an important problem in Czech dairy herds, especially in the Czech Fleckvieh breed. In smaller herds and especially in the winter, the prevalence of FPT was very high.

Evaluation of colostrum quality in the Czech Republic using radial immunodiffusion and different types of refractometers

The objectives of this study were to determine the immunoglobulin G concentration of colostrum in Czech dairy cows, to compare refractometer results with results achieved using the radial immunodiffusion method and to evaluate the reliability of three types of refractometers and recommend the best solution for the evaluation of colostrum quality. Colostrum samples (n = 1522) were collected from 38 herds between 2015 and 2017. The immunological quality of colostrum was estimated using Brix refractometers (optical, simple digital, digital Misco) and compared with the immunoglobulin G concentration assessed using radial immunodiffusion. We found high variability in the quality of colostrum. The minimum, maximum and median of individual measurements were the following: radial immunodiffusion immunoglobulin G – 5.2, 199.1, 76.9 g/l; optical refractometer – 9.5, 32.0, 23.1% Brix; simple digital refractometer – 5.4, 35.0, 19.1% Brix; digital refractometer Misco – 9.8, 37.4, 23.2% Brix. On the basis of immunoglobulin G concentration assessed using radial immunodiffusion, 20.9% of colostrum samples were of low quality (immunoglobulin G &amp;lt; 50 g/l). The Spearman correlation coefficients between radial immunodiffusion and the Brix refractometer readings were 0.62–0.67 (P &amp;lt; 0.001) according to the type of refractometer. The cut-off evaluation of the readings from optical and Misco digital refractometers both showed 20% Brix, with sensitivities of 89.4% and 88.2%, specificities of 73.2% and 74.5% and accuracies of 86.0% and 85.4%, respectively. The cut-off level for the simple digital refractometer showed 17% Brix with a sensitivity of 77.5%, specificity of 80.4% and an insufficient accuracy of 78.1%. For optical and Misco refractometers we recommend the use of two cut-off levels for the evaluation of colostrum: 23% Brix for the selection of good quality colostrum suitable for freezing and 19% Brix to discard poor quality colostrum. The different cut-off levels obtained by measuring with different types of refractometers indicate the need to check the quality of the instruments prior to their use in practice and, where appropriate, to determine their cut-off levels by comparison with results obtained using the reference method.

SPRi-based hemagglutinin quantitative assay for influenza vaccine production monitoring

Influenza vaccine manufacturers lack tools, whatever the involved production bioprocess (egg or cell-based), to precisely and accurately evaluate vaccine antigen content from samples. Indeed, the gold standard single-radial immunodiffusion (SRID) assay, which remains the only validated assay for the evaluation of influenza vaccine potency, is criticized by the scientific community and regulatory agencies since a decade for its high variability, lack of flexibility and low sensitivity. We hereby report an imaging surface plasmon resonance (SPRi) assay for the quantification of both inactivated vaccine influenza antigens and viral particles derived from egg- and cell-based production samples, respectively. The assay, based on fetuin-hemagglutinin interactions, presents higher reproducibility (<3%) and a greater analytical range (0.03–20 µg/mL) than SRID for bulk monovalent and trivalent vaccine and its limit of detection was evaluated to be 100 times lower than the SRID’s one. Finally, viral particles production through cell culture-based bioprocess was also successfully monitored using our SPRi-based assay and a clear correlation was found between the biosensor response and total virus particle content.

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.

Diagnostic performance of direct and indirect methods for assessing failure of transfer of passive immunity in dairy calves using latent class analysis

Accurate diagnosis of failure of transfer of passive immunity (FTPI) in newborn calves is an essential component of dairy farm management plan. Several methods (direct and indirect) are available for diagnosis of FTPI in dairy calves. However, the indirect methods offer an advantage over the direct methods in not requiring an experienced veterinarian, rapid, cost efficient and can be performed under field-setting. The objective of this study was to estimate the diagnostic performance of radial immunodiffusion (RID) assay, transmission infrared (TIR) spectroscopy and digital Brix refractometer for diagnosis of FTPI in dairy calves using latent class models at four cut-off values of digital Brix refractometer. Holstein calves (n = 691) from 40 commercial dairy farms in the four Atlantic Canada provinces were blood-sampled and tested for detection of FTPI. Results showed that the number of calves with FTPI was 253 (36.6%) by RID, 194 (28.1%) by TIR and 204 (29.5%) by Brix refractometer at cut-off value of 8.2%. Estimates of SeRID was higher than SeTIR and SeBrix, at all Brix refractometer cut-offs, but with increase of Brix refractometer cut-off from 8.2 to 8.5%, SeRID and SeTIR were decreased from 96.0% (95% PCI: 88.0–99.0) and 79.0% (95% PCI: 70.0–85.0), to 92.0% (95% PCI: 77.0–99.0) and 74.0% (95% PCI: 61.0–82.0), respectively. SpRID and SpTIR were always higher than SpBrix at all tested cut-offs and were above 92.0%, and 96.0%, respectively. With increasing the cut-off of Brix refractometer from 8.2 to 8.5%, SeBrix estimate has remarkably increased from 79.0% (95% PCI: 70.0–96.0) to 95.0% (95% PCI: 87.0–100.0), respectively. Whilst, SpBrix was decreased from 95.0% (95% PCI: 91.0–98.0) at cut-off 8.2% to 84.0% (95% PCI: 78.0–94.0) at cut-off 8.5%. In conclusion, RID has a higher Se than TIR and Brix, if the latter is used with cut-offs of 8.2% or 8.3%. However, the higher the cut-off, the more comparable sensitivities of RID and digital Brix refractometer. The median estimate of SpTIR was always higher than SpRID and SpBrix at all tested cut-offs. However, the 95% confidence interval estimates of the three tests were overlapping across the tested cut-offs of digital Brix refractometer reflecting the inability to prefer a test over the other based on the Sp estimate.

Synergistic effects of estradiol and 11-ketotestosterone on vitellogenin physiology in the shortfinned eel (Anguilla australis).

Estradiol-17β (E2) and 11-ketotestosterone (11KT) have been implicated in vitellogenesis and in regulating expression of the follicle-stimulating hormone receptor (fshr), respectively. To override the captivity-induced reproductive block in shortfinned eel, Anguilla australis, we hypothesized that in combination, 11KT and E2 would stimulate ovarian uptake of vitellogenin (Vtg). Early pubertal eels received hormone implants containing varying concentrations of E2 (0, 0.2, 2, 5 mg) with or without 11KT (1 mg). Vtg levels were determined in plasma, liver, and ovarian tissues by histological examination, qPCR, immunoblotting, or single radial immunodiffusion. The expression of gonadotropin-beta subunits and gonadotropin receptors in the pituitary and ovary, respectively, were analyzed to determine mechanisms by which steroid effects may be exerted. When administered alone, E2 increased hepatic production and plasma levels of Vtg. In contrast, 11KT decreased plasma levels of Vtg, seemingly reducing its production. Neither 11KT nor E2 could induce uptake of Vtg into oocytes, although E2 treatment appeared necessary for uptake to occur. This was the case despite 11KT dramatically increasing both oocyte size and fshr mRNA levels. Astonishingly, the uptake of Vtg was successfully induced by co-treatment with 11KT and E2, suggesting that 11KT might facilitate the incorporation of Vtg into the developing oocyte. These results highlight the potential of sex steroid co-treatment, an approach aimed at mimicking oogenesis in wild eels, to induce vitellogenesis, specifically ovarian yolk deposition, even in the absence of exogenous gonadotropin treatment.

N-Glycosylation of Seasonal Influenza Vaccine Hemagglutinins: Implication for Potency Testing and Immune Processing.

Prior to each annual flu season, health authorities recommend three or four virus strains for inclusion in the annual influenza vaccine: a type A:H1N1 virus, a type A:H3N2 virus, and one or two type B viruses. Antigenic differences between strains are found in the glycosylation patterns of the major influenza virus antigen, hemagglutinin (HA). Here we examine the glycosylation patterns of seven reference antigens containing HA used in influenza vaccine potency testing. These reagents are supplied by the Center for Biologics Evaluation and Research (CBER) or the National Institute for Biological Standards and Control (NIBSC) for use in vaccine testing. Those produced in hen egg, Madin-Darby canine kidney (MDCK), and insect (Sf9) expression systems were examined. They are closely related or identical to antigens used in commercial vaccines. The reference antigens studied were used in the 2014-2015 influenza season and included A/California/07/2009 H1N1, A/Texas/50/2012 H3N2, and B/Massachusetts/02/2012. Released glycan and HA-specific glycopeptide glycosylation patterns were examined. We also examined the sensitivity of the single radial immunodiffusion (SRID) potency test to differences in HA antigen glycosylation. Based on deglycosylation studies applied using standard assay procedures, the SRID assay was not sensitive to any HA antigen glycosylation status from any cell system. Mapping of glycosites with their occupying glycan to functional regions, including antigenic sites, lectin interaction regions, and fusion domains, was performed and has implications for immune processing, immune responses, and antigenic shielding. Differences in glycosylation patterns, as dictated by the cell system used for expression, may impact these functions.IMPORTANCE In the present study, the glycosylation patterns of the 2014-2015 influenza vaccine season standard antigens A/California/07/2009 H1N1, A/Texas/50/2012 H3N2, and B/Massachusetts/02/2012 were revealed, and the sensitivity of the single radial immunodiffusion (SRID) potency test to glycosylation was tested. Differences in hemagglutinin glycosylation site composition and heterogeneity seen in antigens produced in different cell substrates suggest differences in processing and downstream immune responses. The SRID potency test used for vaccine release is not sensitive to differences in glycosylation under standard use conditions. This work reveals important differences in vaccine antigens and may point out areas where improvements may be made concerning vaccine antigen preparation, immune processing, and testing.

Estimation levels of Immunoglobulin and Complement in Amoebiasis patients (Amebic Dysentery)

Background: Amebic dysentery is an infections causing by parasitic protozoan as Entamoeba histolytica which caused light to severe intestinal damage and in sometimes its spreading to the lungs, liver, brain and others organs, so it can be causing by closely related species of Entamoeba hartmanni and Entamoeba coli are commensals. Patients and methods: The samples was collected from patient reviewers to teaching Hospital/Baghdad city and ask them about information as age, sex, educational level, Residency and Income level, During (February–May) 2018, and Stool specimens and blood (5ml) from patients suffering from diarrhea then examined all specimens within thirty min. by General stool examination (Macroscopic and Microscopic examination and determined the concentration of immunoglobulin and complement (C3 & C4) by using Single Radial Immunodiffusion (SRID). Results of current study were found higher percentage of patients in the age group (1) as 45% , whilst decreased in age groups (2) to 23% , also results showed highly percentage in male (53.4) than female (46.6)%, also percentage of infection with Entamoeba histolytica was highest in Secondary as (36.6)% and followed by Non – study (illiterate people) as ( 34.4)%, so in urban ( 74.8) % more than rural (25.2)% as well as low Income level ( 53.5%) more than both good and high income level, also this study explain all concentrations of immunoglobulin and Complement were found high significant (p<0.001) in patients group than control group, Conclusion The infection with E. histolytica were highest in young old with age range from (1-15) years who live in rural areas, and high rate concentration of IgM ; IgG and IgE. Conclusions current study, infection with E. histolytica were highest in young old with age range from (1-15) years who live in rural areas, also high rate concentration of IgG, IgM and IgE in sera of amoebiasis patients than control group as well as the complement levels (C3&C4).

Salivary IgG levels in neonatal calves and its association to serum IgG: an observational pilot study.

The diagnosis of inadequate transfer of colostrum immunoglobulin G (IgG) to calf serum, often known as failure of passive transfer (<10 g/L IgG1 at 24 to 48 h), necessitates blood sampling from the calf and in some instances the presence of a veterinarian. Sampling saliva is both less invasive and easy for the producer. Previous research has shown that quantification of saliva IgG is possible in juvenile and adult cattle. The objectives of this observational pilot study were to investigate whether IgG can be quantified in neonatal calf saliva, if it is correlated to serum IgG concentrations, and if the indirect quantification of saliva IgG is achievable by use of a digital refractometer. Paired blood and saliva samples were collected from 20 healthy dairy calves aged 1 to 3 d. In these samples, IgG was quantified directly with single radial immunodiffusion and indirectly by use of a digital refractometer indicating Brix % (a subsample of n = 12 saliva samples). A strong positive correlation (r = 0.7, P < 0.001) between saliva IgG (mean ± SD; 0.2 ± 0.11 g/L) and serum IgG (32.1 ± 11.94 g/L) was found. Saliva IgG ranged from the lowest detectable value, 0.1 g/L (n = 6 samples) to 0.6 g/L. Saliva Brix (1.2 ± 0.69%) was not significantly correlated to serum IgG (n = 12, r = 0.43, P = 0.155); however, it was significantly correlated to saliva IgG (n = 12, r = 0.7, P = 0.018) and Brix in serum (n = 12, r = 0.7, P = 0.013). We conclude that IgG was quantifiable in most of the saliva samples. For saliva IgG to be of any value with regards to detecting failure of passive transfer, future studies should investigate methods that can detect IgG <0.1 g/L. The results indicate that saliva IgG can be used to predict serum IgG at levels above 10 g/L, which may warrant further exploration of the use of saliva in the surveillance of failure of passive transfer. The results of the current pilot study did not support the potential usage of a Brix % refractometer to quantify saliva IgG.