Development of B cell epitopes-based enzyme linked immune sorbent assay for detection of bovine anti-Mullerian hormone.

The present study aimed to generate antibodies against predicted B cell epitopic peptides encoding bAMH for developing different ELISA models. Sandwich ELISA was determined to be an excellent technique for assessing bAMH in bovine plasma based on sensitivity tests. The assay's specificity, sensitivity, inter- and intra-assay CV, recovery %, Lower limit of quantification (LLOQ), and Upper limit of quantification (ULOQ) were determined. The test was selective since it did not bind to AMH-related growth and differentiation factors (LH and FSH) or non-related components (BSA, progesterone). The intra-assay CV was 5.67%, 3.12%, 4.94%, 3.61% and 4.27% for 72.44, 183.11, 368.24, 522.24 and 732.25 pg/ml AMH levels, respectively. At the same time, the inter-assay CV was 8.77%, 7.87%, 4.53%, 5.76% and 6.70% for 79.30, 161.27, 356.30, 569.33 and 798.19 pg/ml AMH levels, respectively. The average (Mean ± SEM) recovery percentages were 88-100%. LLOQ was 5 pg/ml and ULOQ at 50 µg/ml (CV < 20%). In conclusion, we developed a new highly sensitive ELISA against bAMH using epitope specific antibodies.

Bacteriocins: Classification, synthesis, mechanism of action and resistance development in food spoilage causing bacteria.

Huge demand of safe and natural preservatives has opened new area for intensive research on bacteriocins to unravel the novel range of antimicrobial compounds that could efficiently fight off the food-borne pathogens. Since food safety has become an increasingly important international concern, the application of bacteriocins from lactic acid bacteria that target food spoilage/pathogenic bacteria without major adverse effects has received great attention. Different modes of actions of these bacteriocins have been suggested and identified, like pore-forming, inhibition of cell-wall/nucleic acid/protein synthesis. However, development of resistance in the food spoilage and pathogenic bacteria against these bacteriocins is a rising concern. Emergence and spread of mutant strains resistant to bacteriocins is hampering food safety. It has spurred an interest to understand the bacteriocin resistance phenomenon displayed by the food pathogens, which will be helpful in mitigating the resistance problem. Therefore, present review is focused on the different resistance mechanisms adopted by food pathogens to overcome bacteriocin.

Estimation of steviol glycosides in food matrices by high performance liquid chromatography.

Steviol glycosides (SGs) are non-caloric, natural sweetener obtained from plant Stevia rebaudiana and are used as sugar substitute in foods. The level of SGs in foods should not exceed maximum permissible limit defined by regulatory agencies. Thus analytical methods are required for assay of stevioside (Stev) and rebaudioside A (Reb A), which are two major constituents of SGs, in foods. A method for extraction of Stev and Reb A from dairy viz., flavoured milk, flavoured yoghurt and non-dairy foods viz., carbonated water, jam, chewing gum and estimation of these by HPLC has been described. Extraction of SGs from dairy samples was achieved by treating samples with 20% acetonitrile in presence of Carrez solutions while these can be simply extracted with water from non-dairy samples. Separation and estimation of these two glycosides was achieved on C18 column (length: 4.6 × 250 mm, particle size: 5 µm) using isocratic mobile phase prepared by mixing of acetonitrile and 10 mM sodium phosphate buffer (pH 2.6) in ratio of 32:68 (v/v). Recovery of two SGs was quantitative. Separation and estimation of SGs by HPLC was robust. Limit of detection and limit of quantitation for Reb A in different food was in range from 1.057-1.834 to 3.525-6.114 mg kg-1 while that of Stev was from 1.679-2.912 to 5.596-9.707 mg kg-1, respectively. Neotame, an artificial sweetener can be used as internal standard for separation of SGs.

Construction of a lateral flow strip for detection of soymilk in milk.

A lateral flow based detection method for ascertaining the presence of soymilk in whole bovine milk has been described. The method uses commercially available rabbit anti-soy protein antibodies conjugated to gold nanoparticles (AuNPs) wherein soymilk protein in adulterated milk and soymilk protein at test line competes for limited antibodies. At control line, anti-rabbit immunoglobulin was immobilized for ensuring flow properties of antibody-conjugated AuNPs. Absence or diminished intensity of band at test line indicates presence of soymilk in milk. The soymilk detection limit was 1.75% (v/v) in whole bovine milk and results are available in 5 min. Constructed lateral flow device can be used for on-spot examination of soymilk in milk.

Rapid screening test for detection of oxytetracycline residues in milk using lateral flow assay.

A rapid, semi-quantitative lateral flow assay (LFA) was developed to screen the oxytetracycline (OTC) antibiotics residues in milk samples. In this study a competitive immuno-assay format was established. Colloidal gold nano-particles (GNP) were prepared and used as labelling material in LFA. Polyclonal antibodies were generated against OTC molecule (anti-OTC), purified and the quality was assessed by enzyme linked immuno sorbet assay. For the first time membrane components required for LFA in milk system was optimized. GNP and anti-OTC stable conjugate preparation method was standardized, and then these components were placed over the conjugate pad. OTC coupled with carrier protein was placed on test line; species specific secondary antibodies were placed on the control line of the membrane matrix. Assay was validated by spiking OTC to antibiotic free milk samples and results could be accomplished within 5min. without need of any equipment. The visual detection limit was 30ppb.

Expression of developmental genes in brown fat cells grown in vitro is linked with lipid accumulation.

En1, Nr2f1, Gpc4, Sfrp2, Shox2, Tbx15 and Thbd are among the genes involved in development process of an organism in a number of tissues, in particular adipose tissue. Considering the involvement of isobutyl-methyl-xanthine (IBMX), indomethacin, dexamethasone (DEX), triiodothyronine (T3), and insulin in adipocyte differentiation, we propose that these differentiation-inducing agents may regulate differentiation in brown adipose tissue through a developmental process. Stromavascular cells isolated from interscapular brown fat of mice were cultured in DMEM-LG medium. Proliferating brown preadipocytes were differentiated in the presence of IBMX, indomethacin, DEX, T3 and insulin. Pref1 (marker of proliferation stage) and uncoupling protein 1 (UCP1, marker for differentiation stage) were upregulated during proliferation and differentiation, respectively. Relative expression of Pref1, UCP1 and developmental genes was determined in different stages of adipogenesis. En1, Gpc4, Nr2f1, Sfrp2 and Shox2 were upregulated during differentiation. Differentiation of preadipocytes in the absence of IBMX, indomethacin, and DEX resulted in drastic reduction in fat accumulation in differentiated adipocytes with simultaneous decrease in En1, Gpc4, Nr2f1, Sfrp2, Shox2 and Tbx15 gene expression. T3 upregulated the expression of En1, Gpc4, Sfrp2 and Tbx15 genes during differentiation and downregulated Shox2 expression as compared to proliferated state. Insulin upregulated the expression of Shox2.

Synthesis and application of cephalexin imprinted polymer for solid phase extraction in milk.

Molecular imprinted polymer (MIP) against cephalexin was synthesized by co-polymerization of functional monomer, cross-linker, radical initiator, along with target molecule (cephalexin) in a porogenic material. Binding of cephalexin towards prepared MIP was studied in different solvents (water, methanol, 1M NaCl, acetone and acetonitrile) and best binding was observed in methanol. Partition coefficient and selectivity of prepared imprint and non-imprint was also studied. Cross reactivity in terms of binding efficiency was also assessed with other antibiotics. Chromatographic study of MIP was carried out by packing prepared imprint into glass column. MIP was used as matrix in solid phase extraction (SPE) for recovery of cephalexin from spiked milk samples for further estimation by high performance liquid chromatography. No interference was observed from milk components after elution of cephalexin from MIP, indicating selectivity and affinity of MIP. On the other hand, interference was observed in eluate obtained from C18 SPE column.

Selection of aptamers for aflatoxin M1 and their characterization.

In the present work, aptamers against aflatoxin M1 and aflatoxin B1 were generated and tested for creating proof of principle of recognition of aflatoxin M1 by generated aptamers. The aptamers were selected through the process referred as systematic evolution of ligands by exponential enrichment. A total of 41 different aptamer (36 aptamers for aflatoxin M1 and 5 for aflatoxin B1) sequences were obtained. The determination of dissociation constant (Kd ) values revealed that aptamers generated against aflatoxin M1 exhibited Kd values in the range of 35-1515 nM. Selected aptamers were grouped on the basis of the presence of common motifs or G-quadruplex. We find it interesting that one aptamer with no conserved motif or G-quadruplex had lowest Kd value (Kd = 35 nM). This structural motif is very distinct from motifs present in other aptamers. The Kd values of selected aptamers for aflatoxin B1 were in the range of 96-221 nM. One aptamer from each group was further tested for its ability to be used in aptasensor. The aptamer recognized aflatoxin M1 as indicated by color change (red to purple or blue) of aptamer-coated gold nanoparticles in the presence of 250-500 nM aflatoxin M1. The aptamers can be used in developing methods for detection/estimation/separation of aflatoxin or antidote for aflatoxin toxicity.

Rapid methods for assessing efficiency of heat treatment of milk.

Two methods based on the activity of γ-glutamyl transpeptidase and lactoperoxidase are developed for assessing the efficiency of heat treatment of milk at 80 °C for 15 s. Heat treatment of milk at 80 °C (15 s) completely inactivates both milk enzymes. Under the selected assay conditions, enzyme activities are related with intensity of pink colour of product. In contrast to raw (unheated) milk which gives pink colour under the test conditions, heated milk (80 °C, 15 s) remains either white or gives pink colour with significantly reduced intensity. It is recommended to always use raw milk as positive control for the enzyme assay. Principles of colour formation in enzymatic reactions are discussed.

Feed intake, milk production and composition of crossbred cows fed with insect-protected Bollgard II® cottonseed containing Cry1Ac and Cry2Ab proteins.

Twenty crossbred lactating multiparous cows were used in a 28-day study to compare dry matter intake (DMI), milk yield, milk composition and Bacillus thuringiensis (Bt) protein concentrations in plasma when fed diets containing Bollgard II(®) cottonseed (BGII) or a control non-genetically modified isogenic cottonseed (CON). Bollgard II cottonseed contains the Cry1Ac and Cry2Ab insecticidal proteins that protect cotton plants from feeding damage caused by certain lepidopteran insects. Cows were assigned randomly to the BGII or CON treatments after a 2-week adjustment period. Cows consumed a concentrate containing 40% crushed cottonseed according to milk yield and green maize forage ad libitum. All cows received the same diet but with different crushed cottonseed sources. Cottonseed was included to provide approximately 2.9 kg per cow daily (dry matter basis). The ingredient composition of the concentrate was 40% crushed cottonseed, 15% groundnut cake, 20% corn, 22% wheat bran, 1% salt and 2% mineral mixture. Milk and blood plasma were analyzed for Cry1Ac and Cry2Ab proteins. DMI, BW, milk yield and milk components did not differ between cows on the BGII and CON treatments. Although milk yield and milk fat percentage were not affected by treatment, 4% fat-corrected milk (FCM) production and FCM/kg DMI for cows on the BGII treatment (14.0 kg/cow per day, 1.12 kg/kg) were significantly improved compared with cows on the CON treatment (12.1 kg/cow per day, 0.97 kg/kg). Gossypol contents in BGII cottonseed and conventional cottonseed were similar. Cry1Ac and Cry2Ab2 proteins in Bollgard II cottonseed were 5.53 and 150.8 µg/g, respectively, and were not detected in the milk or plasma samples. The findings suggested that Bollgard II cottonseed can replace conventional cottonseed in dairy cattle diets with no adverse effects on performance and milk composition.

Nutritional evaluation of transgenic cottonseed in the ration of lactating dairy cows.

The effects of feeding transgenic (Bt) whole cottonseed (WCS) were studied in lactating cows. Twenty multiparous crossbred cows (Karan Swiss x Karan Fries) in early lactation were given a concentrate mixture containing 40% crushed delinted non-transgenic (non-Bt) WCS, 2 kg wheat straw and green fodder ad lib for a 15-day adaptation period. Thereafter, the cows were divided in two similar groups of 10 each on the basis of milk yield, body weight (BW) and date of calving. The non-Bt control group continued on same ration, while for the Bt group the non-Bt WCS was replaced by transgenic WCS, in a feeding trial of four weeks. The diets provided a minimum of 2 kg cottonseed/cow/d. Mean DMI/100 kg BW and milk yield of non-Bt and Bt groups was 3.48 and 3.45 kg and 11.4 and 12.0 kg/d, respectively. Intake of nutrients, digestibility, milk production and body condition score (BCS) did not differ between the groups (P > 0.05), but BW gain was higher (P < 0.05) in the Bt group than the non-Bt group, probably as a result of hoof problem in two cows of non-Bt group, which when compared excluding two animals from each group did not differ significantly (P > 0.05). Transgenic protein (Cry1C) was not detected in the weekly milk samples or in blood plasma at the end of the experiment, showing that delinted WCS containing Cry1C protein can safely be fed to lactating cows.

Monoclonal antibodies against bovine growth hormone.

A number of mouse x mouse hybridomas producing monoclonal antibodies (MAbs) against bovine growth hormone (bGH) were prepared by fusion of spleen cells from bGH-primed mice (Balb/c) with non-secretory mouse myeloma cells (PAIOP3) and characterized. MAbs obtained from three fusion experiments belonged to IgM, IgG1 and IgG2b class/subclass of antibodies. Cross-reaction studies indicated that generated antibodies were against three different epitopes of bGH. VIA6E8 (IgG1) and VIIB2E11C9 (IgM) did not cross-react with ovine prolactin (oPRL), ovine leutinizing hormone (oLH) and porcine follicle stimulating hormone. Antibody VIB3C9E8 (IgM) exhibited cross-reaction with oPRL and oLH. Antibody VIC1F9 (IgG2b) cross reacted with oPRL. All MAbs were against conformational epitopes of bGH.

Immunosuppressive activity in buffalo placenta.

Immunosuppressive activity in buffalo placenta was evaluated by measuring proliferation of lymphocytes in presence of phytohaemagglutinin (PHA) alone or PHA plus placental proteins. The immunosuppressive activity was dose-dependent over the protein concentration range of 10-50 micrograms/ml. Proteins from both cotyledon and non-cotyledon portions of placenta exhibited immunosuppressive activity. Fractions obtained with 0-40, 40-60 and 60-80% saturated ammonium sulphate exhibited 70, 73 and 75% suppression, respectively. PBS-soluble placental proteins were resolved on G-100 column into three peaks that exhibited 69 (peak 1), 55 (peak 2) and 73% (peak 3) suppression. Exogenously added interleukin-1 (IL-1) failed to reverse the suppression caused by buffalo placental proteins.

Purification and characterization of an acid phosphatase from Arachis hypogaea.

An acid phosphatase from Arachis hypogaea (peanuts) has been purified. The electrophoretically homogeneous enzyme preparation is free of any phophodiesterase activity. The enzyme has a molecular weight of 120,000. Among the various phosphomonoesters tested, p-nitrophenylphosphate was found to be its most effective substrate. The Km for p-nitrophenylphosphate was 1.21 mM at pH 5.0 and 25 degrees C. The enzyme was thermostable and did not loose activity after 1 hr at 50 degrees C.

Preparation of heteroenzyme conjugates: trypsin-chymotrypsin and trypsin-alkaline phosphatase.

Hybrid enzymes which have two different enzyme activities linked together covalently may be useful reagents for various applications, such as the determination of complex biological structures. The present paper describes the preparation and purification of two such enzyme-enzyme conjugates, namely, trypsin-chymotrypsin and trypsin-alkaline phosphatase. Whereas the former has been prepared by using the well-known bifunctional reagent glutaraldehyde, the latter exploited the Schiff base formation between the oxidized carbohydrate moiety of alkaline phosphatase and the free amino groups of trypsin.

Coaggregate of trypsin and chymotrypsin.

The method of chemical aggregation of enzymes has the advantage of yielding an immobilized enzyme preparation wherein reactor volume can be significantly reduced because of the absence of an inert carrier. A coaggregate of trypsin and chymotrypsin formed by extensive cross-linking with glutaraldehyde is described. A significant property of this aggregate is the reduced autolysis of the trypsin component of the coaggregate.

A conjugate of trypsin and chymotrypsin.

A heteroenzyme conjugate retaining activities of two component enzymes from trypsin and chymotrypsin was prepared using N-succinimidyl pyridyl dithiopropionate as crosslinking reagent. The conjugate bound to both trypsin and chymotrypsin affinity columns. Trypsin and chymotrypsin were linked in the ratio of 1:1 on mol basis. The conjugate, when treated with dimethyladipimidate, showed decreased autolysis of its trypsin component.

Distribution of acid and alkaline phosphatase in buffalo spermatozoa.

The distribution of acid phosphatase (EC 3.1.3.1) and alkaline phosphatase (EC 3.1.3.2) was studied in the head, midpiece and tail fractions of buffalo spermatozoa. Fractionation was achieved by the basic principles of ultrasonication and density gradient centrifugation. The activity of acid phosphatase in the head, midpiece and tail fractions was 7.30, 11.55 and 47.10 and for alkaline phosphatase 1.74, 6.95 and 26.71 respectively; the unit being microgram of inorganic phosphorus liberated/mg of protein/h at 37 degrees C.