Immunochromatographic assay using gold nanoparticles for measuring salivary secretory IgA in dogs as a stress marker

Abstract

The concentration of salivary secretory immunoglobulin A (sIgA) is a well-known stress marker for humans. The concentration of salivary sIgA in dogs has also been reported as a useful stress marker. In addition, salivary sIgA in dogs has been used to determine the adaptive ability of dogs for further training. There are conventional procedures based on enzyme-linked immunosorbent assay (ELISA) for measuring salivary sIgA in dogs. However, ELISA requires long assay time, complicated operations and is costly. In the present study, we developed an immunochromatographic assay for measuring salivary sIgA in dogs using a dilution buffer containing a non-ionic surfactant. We determined 2500-fold dilution as the optimum condition for dog saliva using a phosphate buffer (50 mM, pH 7.2) containing non-ionic surfactant (3 wt% Tween 20). The results obtained from the saliva samples of three dogs using immunochromatographic assay were compared with those obtained from ELISA. It was found that the immunochromatographic assay is applicable to judge the change in salivary sIgA in each dog. The immunochromatographic assay for salivary sIgA in dogs is a promising tool, which should soon become commercially available for predicting a dog's psychological condition and estimating adaptive ability for training as guide or police dogs.     

Purification of Immunoglobulin of Egg Yolk with Hydrophobic Charge Induction Chromatography: Comparison of Operation Modes with Packed Bed and Expanded Bed

Immunoglobulin of egg yolk (IgY) was separated from fresh hen eggs by three steps, water dilution, salt-out precipitation, and hydrophobic charge induction chromatography (HCIC). In the purification step, HCIC was used as a novel technology and two chromatographic operation modes, packed bed and expanded bed, were compared to improve the separation efficiency. For packed bed mode, IgY were separated with the purity of 92% and the recovery of 58.4% at the operation condition of pH 7.5 and sample (total protein concentration of 11.6 mg/mL, IgY purity of 31%) loading of 0.2 mL/mL adsorbent. For expanded bed mode, a similar purity of 90% and the recovery of 59.5% were found at the fluid velocity of 500 cm/h and same sample loading of 0.24 mL/ mL adsorbent at pH 7.5. The results demonstrated that HCIC in expanded bed mode is a potential platform technology for antibody separation both in high efficiency and low cost.     

Invited review: The importance of colostrum in the newborn dairy calf

It is critical that bovine maternal colostrum is fed to newborn calves during their first hours of life. Colostrum is the secretion a cow produces after mammary involution that is rich in various nutrients. In addition to the nutritive value for newborn calves, immunoglobulins are of interest due to their role in developing the naïve immune system of calves at birth. The process by which a calf acquires immunity via absorption of immunoglobulins is defined as passive immunity. When calves consume an adequate amount of immunoglobulins, they are classified as having successful passive immunity (SPI). In contrast, if they are deprived of adequate colostrum, they are considered to have had a failure of transfer of passive immunity (FPI). Transfer of passive immunity is assessed by measuring serum IgG concentrations at 24 to 48 h of age. The major factors that influence whether a calf has SPI or FPI are colostrum IgG concentration, quantity fed, and age of calf at colostrum feeding. Monitoring apparent efficiency of immunoglobulin absorption in calves is often recommended to evaluate overall colostrum management practices. Serum IgG analyses can be determined with direct (radial immunodiffusion) or indirect (refractometry) methods and used to assess SPI or FPI prevalence.     

Passive transfer of immunoglobulin G and preweaning health in Holstein calves fed a commercial colostrum replacer

The objective of this study was to describe passive transfer of IgG and preweaning health in newborn calves fed a commercially available plasma-derived colostrum replacement (CR) product or maternal colostrum (MC). Twelve commercial Holstein dairy farms enrolled singleton newborn heifer calves to be fed fresh MC (n = 239 calves) or one dose of CR containing 125 g of Ig (n = 218 calves) as the first colostrum feeding. For 7 of these farms that routinely provided a second feeding of 1.9 L of MC to their calves 8 to 12 h after the first colostrum feeding, calves assigned to the CR treatment group were offered a second feeding consisting of 1.9 L of commercial milk replacer supplemented with one dose of a commercially available plasma-derived colostrum supplement, containing 45 g of Ig per dose, 8 to 12 h after the first colostrum feeding. A blood sample was collected from all calves between 1 to 8 d of age for serum IgG and total protein (TP) determination, and records of all treatment and mortality events were collected until weaning. Serum IgG and TP concentrations were significantly higher in calves fed MC (IgG = 14.8 ± 7.0 mg/mL; TP = 5.5 ± 0.7 g/dL) compared with calves fed CR (IgG = 5.8 ± 3.2 mg/mL; TP = 4.6 ± 0.5 g/dL). The proportion of calves with failure of passive transfer (serum IgG <10.0 mg/mL) was 28.0 and 93.1% in the MC and CR treatment groups, respectively. Though a trend was present, the proportion of calves treated for illness was not statistically different for calves fed MC (51.9%) vs. CR (59.6%). Total number of days treated per calf (MC = 1.7; CR = 2.0), treatment costs per calf (MC = $10.84; CR = $11.88), and proportion of calves dying (MC = 10.0%; CR = 12.4%) was not different between the 2 colostrum treatment groups. The mean serum total protein concentration predictive of successful passive transfer (serum IgG = 10 mg/mL) was 5.0 g/dL in calves fed MC or CR. Long-term follow-up of these calves (to maturity) is ongoing to describe the effects of feeding CR on longevity, productivity, risk for Johne's disease, and economics.     

Short communication: evaluation of a color method for testing immunoglobulin G concentration in goat colostrum

Colostrum samples (n = 1084) of first and second milking from Majorera goats were taken. The immunoglobulin (Ig) G concentrations estimated by measurement of the color of goat colostrum and by the radial immunodiffusion technique were compared. Least squares analysis of the relationship between the color measurement method and IgG concentration resulted in a significant linear relationship. Using 20 mg of IgG/ mL of colostrum as the cut-off point for colostrum selection, the sensitivity, specificity, and negative predictive value of the color method as a test of IgG concentration in goat colostrum were 93.03, 71.43, and 78.12%, respectively.     

Effects of lyophilized colostrum and different colostrum feeding regimens on passive transfer of immunoglobulin g in Majorera goat kids

Three experiments were conducted including 180 Majorera kids. In the first experiment, the effect of use of lyophilized colostrum vs. frozen colostrum on immunoglobulin G (IgG) blood serum concentration was evaluated. Kids (n = 40) received the same management and IgG mass [3368 mg/kg of body weight (BW)] during the colostrum feeding period. The IgG in blood serum of kids from the lyophilized colostrum group was greater than that for kids that received frozen colostrum. The second experiment evaluated the effect of total IgG ingested by kids (n = 60) on IgG in blood serum during the colostrum feeding period. Three groups of animals received 3368, 1684, and 842 mg of IgG/kg of BW in 4 feedings for 2 d [high IgG concentration (H-IgG), medium IgG concentration (M-IgG), and low-IgG concentration (L-IgG), respectively]. The IgG blood serum in the kids that received H-IgG was greater than in the other 2 treatment groups, and no statistical differences were found for IgG in blood serum of kids that received either M-IgG or L-IgG. The third experiment evaluated the effect of timing of lyophilized colostrum meals on IgG blood serum concentration. Four groups of kids (n = 80) were used. Two groups received 1684 mg of IgG/kg of BW (higher level-1 d and higher level-2 d) and the other 2 groups received 842 mg of IgG/kg of BW (lower level-1 d and lower level-2 d). Two groups received 2 feedings in 1 d, and the other 2 groups received 4 feedings over a 2-d period, as denoted. Higher level-1 d kids had greater IgG blood serum concentration than the higher level-2 d kids, and no statistical differences were found between lower level-1 d and lower level-2 d kids.     

牛初乳加工预处理方式对免疫球蛋白活性的影响

以新鲜牛初乳为原料,考察不同的贮藏方法(直接冷冻保存,脱脂牛初乳冷冻保存,乳清冷冻保存)及解冻方法(60℃热水水浴解冻,室温解冻,微波解冻),对牛初乳中免疫球蛋白的提取率和活性的影响。实验结果表明:工业化提取牛初乳中免疫球蛋白时,将生鲜牛初乳从牧场取回后直接放入-20℃冷冻贮藏一个月,解冻时采用60℃水浴加热解冻。     

Detection of alkaline phosphatase by competitive indirect ELISA using immunoglobulin in yolk (IgY) specific against bovine milk alkaline phosphatase

Immunoglobulin in yolk (IgY) (with a titer of 1.3 × 10⁶) specific against bovine milk (BM) alkaline phosphatase (ALP) was obtained by intramuscularly immunizing hens on the thigh and was used as the primary antibody to conduct competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) to determine BMALP in ALPs from BM and Escherichia coli sources. A relationship between the ELISA value and the BMALP level (0.01–10 μg/mL) in whole milk (R² = 0.9019) or in skimmed milk (R² = 0.9402) was observed. The maximal inhibition (%) of BMALP on the microtiter plate by free BMALP at 10 μg/mL whole milk (3.89 mU/μg BMALP) was about 50%, while no inhibition (%) of BMALP by free E. coli ALP at concentrations between 0.01 to 10 μg/mL (60 mU/μg E. coli ALP) was determined. At BMALP levels higher than 0.1 μg/mL, CI-ELISA was proved to be effective in differentiating between BMALP and E. coli ALP and quantifying BMALP in whole milk or skimmed milk in the presence of E. coli ALP with an activity of 0.6 U/mL. Higher inhibition (about 70%) of BMALP on the microtiter plate by free BMALP in diluted (10¹–10⁴ fold) milk samples was observed. The optimal conditions for CI-ELISA in determining BMALP (0.1–10 μg/mL) from ALPs in milk samples were using 10³-fold diluted crude IgY specific against BMALP as primary antibody and 10³-fold diluted goat anti-chicken IgG–ALP conjugate as the secondary antibody.     

AOT-异辛烷反胶束萃取技术分离牛初乳IgG的研究

在一个琥珀酸二(2-乙基己基)酯磺酸钠(AOT)-异辛烷反胶束体系中，研究了水相pH值、离子强度和有机相表面活性剂AOT浓度等因素对牛初乳乳清蛋白中IgG分离效果的影响。结果表明，在以下两组优化条件：①pH值为7．668，[Na^ ]浓度为0．270mol／L，[AOT]浓度为0．088mol／L；②pH值为6524，[Na^ ]浓度为0．205mol／L，[AOT]浓度为0．175mol／L，水相IgG的残留率或纯度分别达到最大值，分别为90．23％和98．35％．RID分析表明，反胶束萃取过程对原牛初乳乳清IgG的免疫反应活性基本无影响。     

Short communication: serum immunoglobulin G and total protein concentrations in dairy calves fed a colostrum-replacement product

Neonatal calf health is largely dependent on the ingestion and absorption of maternally derived antibodies via colostrum administration. The objective of this study was to evaluate the efficacy of a commercially available plasma-derived colostrum-replacement (CR) product as compared with bovine colostrum. Holstein calves were removed from the dam immediately after birth and randomly allocated to 1 of 3 groups. Group 1 calves (n = 22) were fed 1 package of the CR product; group 2 calves (n = 22) were fed 2 packages of the CR product; and group 3 calves (n = 22) were fed 3 L of bovine colostrum. Blood samples were collected from all calves 24 h after colostrum or CR feeding and analyzed for serum IgG and total protein concentrations. Calves fed bovine colostrum had significantly higher serum IgG and total protein concentration than calves in either group fed the CR product. Group 1 calves (1 package of CR product) had a significantly higher incidence of failure of transfer of passive immunity than calves in groups 2 or 3. The results of this study indicated that 2 packages of this CR product achieved adequate IgG concentrations in calves. However, calves fed 1 package of this CR product consistently had failure of transfer of passive immunity.