Agarose gel electrophoretic fractionation of serum proteins in adult cattle. II. A study of cows with different diseases.

In 283 dairy cows suffering from internal disorders the serum proteins were studied by agarose gel electrophoresis supplemented by total protein and albumin determinations. The clinical diagnoses could be grouped according to protein pattern. Group 1 (abomasal displacement and traumatic muscle injury) did not appreciably affect the serum protein concentrations and represented primarily non-inflammatory diseases or diseases of non-infectious origin. Group 2 (leukosis) occupied an exceptional position, with heavy lowering of albumin unaccompanied by a corresponding rise of total globulin. The γ-globulin concentration was significantly lowered. Group 3 (acute traumatic peritonitis) represented an acute inflammatory process with increase chiefly of the α-globulins, while the γ-globulin concentration was normal. Group 4 (chronic traumatic peritonitis, summer mastitis, chronic subclinical mastitis, chronic laminitis and polyarthritis, urinary tract infection, abscess, and sub-acute-chronic pneumonia) comprised diseases chiefly of chronic inflammatory character and with infectious origin. Especially characteristic was the heavy rise of globulins in general and of γ-globulin in particular. In most cases there was a large increase also of the α- and p-globulin fractions.     

Studies of Transferrin Polymorphism in Swedish Cattle Using Agarose Gel Electrophoresis

The polymorphic transferrin picture in the sera from 894 Swedish cattle was investigated with an agarose gel electrophoresis technique. The serum transferrin bands in the electrophoresis pattern were first identified by labelling with ⁵⁹Fe. Six existing phenotypes based on the alleles Tf^A, Tf^D and Tf^E could be detected. The frequencies of transferrin types and transferrin alleles are presented, and it is concluded that there are great differences in the frequencies between the Swedish Red and White and the Swedish Friesian.     

Agarose gel electrophoretic fractionation of serum proteins in adult cattle. I. A study of clinically healthy cows.

Blood samples were taken from 25 clinically healthy Swedish red-and-white dairy cows on 11 occasions in the course of about one year. The blood serum proteins were studied, chiefly by agarose gel electrophoresis, in respect of relative and absolute concentrations. The following parameters were studied: Total protein, albumin, total globulin, A/G quotient, α₁-, α₂-, inter-α-β-, β₁-, β₂- and γ-globulin. The material was analysed statistically in respect of variation between and within cows, and of age, pregnancy and seasonal variation. The variation between cows was found to be significantly greater than that between different samplings of the same cow. Older cows exhibited significantly higher γ-globulin concentrations. The variation with season and stage of pregnancy was less pronounced for most serum protein fractions.     

Analysis of serum proteins in clinically healthy goats (Capra hircus) using agarose gel electrophoresis

Background: Electrophoretic patterns of serum proteins provide useful information on pathological conditions in ruminants. Their reference values, however, are dissimilar to those of other species. Reference values for goats using agarose gel as the supporting matrix have not been reported. Objective: The aim of this study was to evaluate serum concentrations of total protein and protein fractions (albumin and globulins) by means of agarose gel electrophoresis (AGE) in goats in order to establish electrophoretic reference intervals and to evaluate potential changes associated with aging. Methods: Blood was collected from 105 clinically healthy Girgentana goats by means of jugular venipuncture. Serum protein concentrations were assessed by AGE. Three age groups were compared: 1–1.5 years, 2–4 years, and 5–12 years. Results: Values (mean ± SD) were determined for concentrations of total protein (72.26 ± 6.40 g/L), albumin (31.80 ± 4.00 g/L), α‐globulins (6.40 ± 1.23 g/L), β₁‐globulins (10.50 ± 2.58 g/L), β₂‐globulins (5.18 ± 1.60 g/L), and γ‐globulins (18.65 ± 5.90 g/L) and for albumin/globulin (A/G) ratio (0.82 ± 0.20). One‐way ANOVA showed statistically significant age‐related differences for total protein and α‐globulin concentrations and A/G ratios. Age influenced protein concentrations with the 5–12‐year‐old group having higher total protein and α‐globulin concentrations and lower albumin concentration and A/G ratios than the 2–4‐year‐old group. Conclusions: This study provides reference values for total protein concentrations and protein fractions obtained by AGE in goats. Some values vary with age. Age‐specific reference intervals are reported in order to provide clinicians with an additional diagnostic aid.     

Analysis of mouse, rat, dog, marmoset, and human serum proteins by capillary electrophoresis: comparison with agarose gel electrophoresis

BACKGROUND: Serum protein analysis in both humans and experimental animal species has so far been carried out by labor-intensive techniques, such as agarose gel electrophoresis (AGE). OBJECTIVE: The objective of this study was to evaluate capillary electrophoresis (CE) as an alternative technique to AGE for the analysis of serum proteins from healthy animals. METHODS: Blood samples were collected into tubes without anticoagulant from 6 fasted healthy male mice, rats, dogs, marmosets, and humans. Serum proteins were separated by CE using a technique standardized for the analysis of human proteins, and the results (efficiency, resolution, and precision) were compared with those obtained through AGE. RESULTS: Compared with AGE, CE resulted in narrower peaks and more peaks. The efficiency of protein separation by CE was significantly higher for all species, and resolution (R) was significantly higher in samples from dogs. Using rat serum, intraday reproducibility was lower for all protein fractions, and interday reproducibility was lower for most peaks, compared with AGE. CONCLUSIONS: We conclude that CE is a viable alternative to AGE for the determination of protein electrophoresis in a routine veterinary clinical pathology laboratory. The minimal sample requirement (2 microL), complete automation, and quantitative results make CE an especially valuable technique for protein analysis in experimental animal models.     

Agarose gel electrophoresis of cerebrospinal fluid proteins of dogs after sample concentration using a membrane microconcentrator technique

BACKGROUND: Cerebrospinal fluid (CSF) is produced in the cerebral ventricles through ultrafiltration of plasma and active transport mechanisms. Evaluation of proteins in CSF may provide important information about the production of immunoglobulins within the central nervous system as well as possible disturbances in the blood-brain barrier. OBJECTIVE: The objective of this study was to measure the concentration and fractions of protein in CSF samples using a membrane microconcentrator technique followed by electrophoresis, and to compare the protein fractions obtained with those in serum. METHODS: CSF samples from 3 healthy dogs and 3 dogs with canine distemper virus infection were concentrated using a membrane microconcentrator having a 0.5 to 30,000 d nominal molecular weight limit (Ultrafree, Millipore, Billerica, MA, USA). Protein concentration was determined before and after concentration. Agarose gel electrophoresis was done on concentrated CSF samples, serum, and serial dilutions of one of the CSF samples. RESULTS: Electrophoretic bands were clearly identified in densitometer tracings in CSF samples with protein concentrations as low as 1.3 g/dL. The higher CSF protein concentration in dogs with distemper was mainly the result of increased albumin concentration. CONCLUSION: The microconcentrating method used in this study enables characterization of the main protein fractions in CSF by routine electrophoresis and may be useful for interpreting the underlying cause of changes in CSF protein concentrations.     

Multiple forms of alkaline phosphatase in horse bone,liver, kidney,duodenum, caecum and serum separated by agarose gel electrophoresis with and without neuraminidase pretreatment

Horse bone, liver, duodenum, caecum and kidney alkaline phosphatases were separated by a commercial agarose gel electrophoresis method with and without neuraminidase pretreatment, following the manufacturer's directions. Tissue extracts were obtained in saline solution and ALP extracted from cell membranes by the butanol method. Electrophoresis was performed using a TRIS/barbital/sodium barbital buffer with detergent, pH 8.6 to 9.0, at 250 V for 30 minutes. Bone, liver and kidney untreated extracts showed two ALP bands each, but with different relative migration (compared to albumin migration). When they were preincubated with neuraminidase, the two bone bands showed a marked decrease in their migration, followed by the kidney ALP bands and the most anodic band of liver Both intestinal untreated extracts showed three bands but with different mobilities. After preincubation with neuraminidase, the three bands of caecum mucosa decreased in their migration, and the most anodic duodenum band disappeared, overlapping the second one. When tissue extracts were incubated with wheat germ-lectin (WGL), 74.5% of bone extract ALP and 67.2% of caecum extract ALP precipitated, which demonstrated that the ALP band of both tissues have similar groups in the carbohydrate side chains. Horse serum showed two electrophoretic bands, which increased to three bands when treated with neuraminidase. ALP from hepatocytes was the dominant isoform, followed by a caecum band. Because the electrophoretic mobilities of some of the tissue bands studied were identical, the neuraminidase agarose electrophoretic method appeared to be a satisfactory alternative to separate them.     

Agarose gel electrophoresis of alkaline phosphatase isoenzymes in the serum of hyperthyroid cats

Cats with hyperthyroidism [(increased serum thyroxine (T(4))] commonly have increased serum alkaline phosphatase (ALP) activity in addition to other serum biochemical abnormalities. Serum biochemical profiles were obtained from 10 hyperthyroid cats which had increased serum ALP. Agarose gel electrophoresis of serum from these cats was performed and stained for alkaline phosphatase activity. Alkaline phosphatase activity was calculated for each of the separate bands obtained, and the results were compared to those of tissue extracts, serum from normal cats, and serum from normothyroid cats with increased serum ALP activity. The hyperthyroid cats had increased ALP activity in bands corresponding to isoenzymes originating in the liver, bone, and an unidentified tissue source.     

从鸡血中快速提取高质量基因组DNA方法的研究

介绍了一种经改进建立的从鸡血中快速提取高纯度基因组DNA的方法。该方法提取DNA耗时较短,只需2.5~3 h即可拿到DNA样品。提取的DNA样品经紫外分光光度计检测,DNA样品的A260/A280达1.782 2±0.009 3;经琼脂糖凝胶电泳检测,DNA样品的带型清晰、整齐,无拖尾现象;微卫星PCR扩增效果理想。结果表明,该方法所提DNA纯度较高,质量好,分子片段完整,完全能满足分子生物学实验的要求。     

Consecutive changes in serum alkaline phosphatase isoenzyme 3 activities in Holstein heifers during the first 18 months of life

This study investigated consecutive fluctuations in serum activities of bone-specific alkaline phosphatase (ALP) isoenzyme 3 (ALP3) in 11 clinically healthy Holstein heifers during the first 18 months of life. ALP3 activities at the first sampling time point after weaning (3 months) were significantly lower than those at multiple time points during the pre-weaning period. Those activities increased from a minimum at 3 months to a peak at 6 months during the post-weaning period. In the anthropometric data, daily body weight and wither height gains appeared to be below the public data at 4 months and 4–5 months, respectively. The data suggested that serum ALP3 activity can be used to monitor skeletal growth of heifers at weaning.     

Evaluation of serum iso-amylase in the normal dog using an improved electrophoretic technique

Six iso-amylase fractions are known to exist in human serum, three originating from the salivary glands and three from the pancreas. Although it is known that a different number and source of iso-amylase fractions occur in the dog, the routine detection of all the canine iso-amylase fractions has not been previously established. Earlier methods detected either two iso-amylase fractions or, in a proportion of cases, four fractions. A method is described which detects four iso-amylase fractions in 95% of normal canine serum samples. Trials have shown the method is reproducible and that freezing at -40°C has no effect on iso-amylase activity. The normal values for iso-amylase are recorded in 18 normal dogs.     

柴达木黄牛血清运铁蛋白的多态性

采用聚丙烯酰胺凝胶垂直平板电泳法对 2 33头柴达木黄牛血清运铁蛋白的多态性特征进行研究。结果发现 :柴达木黄牛血清运铁蛋白的多态性受TFA1 ,TFA,TFB,TFD,TFF和TFE6个等位基因的控制 ,共有 1 4个基因型 ,其中TFD 为优势等位基因 (0 643 8)。柴达木黄牛TF基因座的群体间基因分化程度 (Gst为 0 74% )和每一个等位基因的分化程度 (最大的Fst为 0 0 66 5)很小。     

Liver abscesses in intensively fed cattle. Serial investigations of serum proteins

An attempt was made to determine the time of onset of liver abscesses in intensively fed calves. Determinations of seram proteins (total protein, albumin, globulin, albumin/ globulin ratio, agarose-gel electrophoretic separation and formol-gel test) and, in a part of the material, of serum-GOT and -GPT were made in serial samples taken once a month from 125 calves. Liver abscesses were found in 48 (38.4 %) when slaughtered at 6–7 months of age.Total protein and globulin increased, the albumin-globulin ratio decreased with increasing age. Calves with liver abscesses had a significantly (0.05 > P > 0.01) higher globulin concentration at the last sampling. At this sampling the material was of a limited size and the difference has to be interpreted with caution. No other significant differences were found. Nor were any rises observed in S-GOT and S-GPT referable to the formation of liver abscesses.Bacteriological studies were not made but, in analogy with observations on a similar animal material in another part of Sweden, the infectious agent in the liver abscesses was assumed to be Sph. necrophorus. The explanation of the almost absent changes in the serum proteins in calves with liver abscesses may be the low antigenicity of this bacterium.In conclusion it is stated that the present serial investigations of the serum protein pattern in young cattle provide no definite guidance for establishing the time of onset of liver abscesses.     

Serum protein concentrations from clinically healthy horses determined by agarose gel electrophoresis

Background: Serum protein electrophoresis is a useful screening test in equine laboratory medicine. The method can provide valuable information about changes in the concentrations of albumin and α‐, β‐, and γ‐globulins and thereby help characterize dysproteinemias in equine patients. Reference values for horses using agarose gel as a support medium have not been reported. Objectives: The purpose of this study was to establish reference intervals for serum protein concentrations in adult horses using agarose gel electrophoresis and to assess differences between warm‐blooded and heavy draught horses. In addition, the precision of electrophoresis for determining fraction percentages and the detection limit were determined. Methods: Blood samples were obtained from 126 clinically healthy horses, including 105 Thoroughbreds and 21 heavy draught horses of both sexes and ranging from 2 to 20 years of age. The total protein concentration was determined by an automated biuret method. Serum protein electrophoresis was performed using a semi‐automated agarose gel electrophoresis system. Coefficients of variation (CVs) were calculated for within‐run and within‐assay precision. Data from warm‐blooded and draught horses were compared using the Mann–Whitney U test. Results: Within‐run and within‐assay CVs were <5% for all protein fractions. No significant difference was found between warm‐blooded and heavy draught horses and so combined reference intervals (2.5–97.5%) were calculated for total protein (51.0–72.0 g/L), albumin (29.6–38.5 g/L), α₁‐globulin (1.9–3.1 g/L), α₂‐globulin (5.3–8.7 g/L), β₁‐globulin (2.8–7.3g/L), β₂‐globulin (2.2–6.0 g/L), and γ‐globulin (5.8–12.7 g/L) concentrations, and albumin/globulin ratio (0.93–1.65). Conclusion: Using agarose gel as the supporting matrix for serum protein electrophoresis in horses resulted in excellent resolution and accurate results that facilitated standardization into 6 protein fractions.     

Analysis of differential strategies to enhance detection of low‐abundance proteins in the bovine serum proteome

Serum‐based biomarkers hold propitious applications for addressing livestock health, and management. However, discovery of protein biomarkers in complex biological fluids like serum is wholly intractable due to the large dynamic range of protein concentrations; that is, ?10–12 high abundance proteins constitute >90% of the total protein content and effectively mask proteomic detection of low‐abundance biomarkers. Toward addressing this limitation, we test a continuous elution size‐based fractionation method, and two approaches that use affinity interaction‐based separation of proteins in preparing bovine serum, and compare liquid chromatography tandem mass spectrometry protein identification to neat serum. Our results identify the high‐abundance proteins in bovine serum, and demonstrate dynamic range compression and improved protein identification with the different enrichment methods. Although these findings indicate the highest protein number identified in bovine serum (445 proteins, all methods combined), and by any single sample processing method (312 proteins) to date, they still remain lower than levels deemed necessary for biomarker discovery. As such, this investigation revealed limitations to resolving the bovine serum proteome, and the need for species‐specific tools for immunodepleting high‐abundance proteins. In concert, this study represents a step toward advancing sample preparation methods for bovine serum biomarker identification.     

间接ELISA检测奶牛乳房炎多联苗抗体方法的研究

利用超声波破碎法制备无乳链球菌、停乳链球菌和金黄色葡萄球菌可溶性抗原。对3种破碎抗原最佳包被浓度及包被条件、血清样品工作浓度、封闭液、酶标二抗的最佳工作浓度及作用时间等反应体系进行筛选和优化,初步建立了间接ELISA法检测奶牛乳房炎多联苗3种菌血清抗体的方法。对24头泌乳牛进行抗体检测,结果表明该3种抗原反应体系一致,3种抗原最适包被浓度为4—6μg／mL．血清样品最佳稀释度为1：200,该方法具有快速、简单、特异性和重复性好等优点。