Naiyana Gujral

In-Vitro and In-Vivo Binding Activity of Chicken Egg Yolk Immunoglobulin Y (IgY) against Gliadin in Food Matrix

Chicken egg yolk immunoglobulin Y (IgY) is a promising alternative for the prevention of enteric gliadin absorption, the predisposing factor of celiac disease (CD). IgY antibody was produced from the egg yolk of Single Comb White Leghorn chickens during the immunization period for the development of an oral immunotherapeutic agent. Here, we report the potential use of spray dried IgY antibody formulation using sugar protectants (mannitol, sorbitol, or microcrystalline cellulose powder (MCCP)). The long-term stability of the spray dried egg yolk powder formulated with 37.5% mannitol (EYP-M) preserved IgY antibody activity at 99.9%, which was significantly higher than that with other protectants (p < 0.05). In a dissolution test, the EYP-M shows 82.4% IgY activity after 2 h in simulated gastric fluid (SGF). A competitive ELISA at 50% inhibition (IC(50)) shows that 1.6 mg/mL EYP-M bound to 7.6 mg/mL and 10.5 mg/mL gliadin in SGF without and with food matrix conditions, respectively, whereas in simulated intestinal fluid, the formulation bound to 10 mg/mL gliadin, regardless of a food matrix. In-vivo study: BALB/c mice fed with EYP-M and gliadin at a ratio of 1:5 (w/w) demonstrated that gliadin absorption in the gastrointestinal tract was minimal at <1%. Thus, EYP-M containing IgY antibody may be used in CD patients to eliminate the effects of ingested toxic gliadin.

Effect of anti-gliadin IgY antibody on epithelial intestinal integrity and inflammatory response induced by gliadin.

BackgroundPepsin-trypsin resistant gliadin (PT-gliadin) promotes intestinal tissue inflammation and increases paracellular permeability of immunogenic gliadin peptides into the lamina propria. This leads to the complications seen in the pathogenesis of celiac disease (CD). In this study, specific anti-gliadin IgY antibody was produced and evaluated for its efficacy on gliadin induced intestinal integrity impairment and proinflammatory effects on intestinal epithelial (Caco-2) cell culture model for CD.MethodsCaco-2 (passages 20-24) monolayers were subjected to 7 experimental conditions (n=3 each): phosphatebufferedsaline (PBS; control), pancreatic digested-casein (PD-casein; negative control), PT-gliadin (positive control), non-specific IgY with PT-gliadin, and anti-wheat gliadin IgY with PT-gliadin at a ratio of 1:6,000, 1:3,000 and 1:1,500. Caco-2 monolayers were then evaluated for effects of gliadin and/or anti-wheat gliadin IgY after 24 h exposure. Enzyme-linked immunosorbent assay (ELISA) was used to quantify anti-inflammatory markers (TNF-α and IL-1β) 5 days after cells were exposed to PT-gliadin and/or anti-wheat gliadin IgY.ResultsAmong other conditions, anti-wheat gliadin IgY at a ratio of 1:3,000 (anti-gliadin IgY: PT-gliadin) significantlyprevented gliadin toxicity on Caco-2 by maintaining intestinal integrity, inhibiting phenol red permeation, and inhibiting gliadin absorption and production of proinflammatory cytokines (TNF-α and IL-1β) as compared to PT-gliadin stimulated cultures (P < 0.05).ConclusionThe anti-wheat gliadin IgY antibody produced in this study has proved to inhibit absorption of gliadin and gliadin-induced inflammatory response in Caco2 cell culture model of CD. Anti-gliadin IgY, therefore has potential to be used as an oral passive antibody therapy to treat CD.

Growth Inhibition of Escherichia coli 987P by Neutralizing IgY Antibodies

Egg yolk antibodies, called IgY, were isolated from the egg yolks of chickens immunized with enterotoxigenic Escherichia coli (ETEC) 987P. Specific binding activity of IgY against ETEC 987P was found. Whole cell titers remained relatively high during the immunization period (up to 19 weeks). The IgY antibodies cross-reacted with other members of Enterobacteriaceae, Escherichia coli O157:H7, Salmonella enteritidis, and S. typhimurium by 28.3%, 21.1% and 19.7%, respectively. Specific IgY with a concentration higher than 0.54 mg/ml was found to inhibit the growth of ETEC 987P in a liquid medium. The difference in bacterial growth between the specific and the non-specific IgY was 6.1 log CFU/ml after 8 hr incubation. The specific binding activity of IgY to bacteria was further evaluated by immunofluorescence and immunoelectron microscopy, showing the binding of specific IgY to the bacterial surface. The immunoelectron microscopic observation revealed structural alterations on the bacterial surface bound by specific IgY. This proves that specific binding activity of IgY against ETEC 987P whole cells resulted in growth inhibition of effect ETEC 987P in vitro.

QUANTITATIVE DOUBLE ANTIBODY SANDWICH ELISA FOR THE DETERMINATION OF GLIADIN

A sensitive double antibody sandwich ELISA (DAS-ELISA) based on chicken anti-gliadin IgY and biotinylated monoclonal antibody (mAb) was developed for the quantification of gliadin in foods. The anti-gliadin IgY and mAb specifically detected gliadin in wheat, barley, and rye by indirect ELISA and Western-blot assay. Using anti-gliadin IgY as capture antibody and biotinylated mAb as detecting antibody, the sensitivity of DAS-ELISA has a linear standard range of 4–40 ng/mL, showing that the limit of detection (LOD) corresponds to 4 ng/mL gliadin in assay buffer, equivalent to 0.8 ppm in foods. The intra-assay expressed as percentage of coefficients of variation (%CV) was 7.25% average of six food samples. The interassay precision was 9.51% in food samples. The combination of anti-gliadin IgY and biotinylated mAb in the DAS-ELISA provides a reliable, sensitive, and inexpensive tool for the detection of gliadin in gluten-free and gluten-containing food products.

Chondroitin sulphate extracted from antler cartilage using high hydrostatic pressure and enzymatic hydrolysis

Chondroitin sulphate (CS), a major glycosaminoglycan, is an essential component of the extracellular matrix in cartilaginous tissues. Wapiti velvet antlers are a rich source of these molecules. The purpose of the present study was to develop an effective isolation procedure of CS from fresh velvet antlers using a combination of high hydrostatic pressure (100 MPa) and enzymatic hydrolysis (papain). High CS extractability (95.1 ± 2.5%) of total uronic acid was obtained following incubation (4 h at 50 °C) with papain at pH 6.0 in 100 MPa compared to low extractability (19 ± 1.1%) in ambient pressure (0.1 MPa). Antler CS fractions were isolated by Sephacryl S-300 chromatography and identified by western blot using an anti-CS monoclonal antibody. The antler CS fraction did not aggregate with hyaluronic acid in CL-2B chromatography and possessed DPPH radical scavenging activity at 78.3 ± 1.5%. The results indicated that high hydrostatic pressure and enzymatic hydrolysis procedure may be a useful tool for the isolation of CS from antler cartilaginous tissues.

Competitive and double antibody sandwich ELISA for the quantification of lactoferrins by using monoclonal and chicken egg yolk IgY antibodies.

Two effective competitive and double antibody sandwich ELISA based on monoclonal (MAb) and chicken egg yolk IgY antibodies were developed to determine lactoferrin (LF) content in infant and milk formulas. Leghorn laying hens were immunized with purified bovine and human LFs to produce anti-bovine LF and anti-human LF IgY antibody in the egg yolk. After 5–8 weeks of the immunization, anti-LF IgY was extracted and analyzed by ELISA. Specific IgY antibodies against LFs cross reacted with human and bovine LFs, examined by ELISA and western-blot assay. Such cross-reactivity suggested the presence of common antigenic determinants between human and bovine LFs. An indirect competitive ELISA was preferred to quantify LF in milk and infant formulas, since the range of detection is 3.125–50 μg/mL, which is broader compared to the biotinylated ELISA system (5–50 ng/mL). As per the indirect competitive ELISA system, IgY at a concentration of the 150 μg/mL was incubated with various concentrations of bovine LF ranged from 0.003–100 μg/mL. The immunoassay was used to estimate the total bovine LF content in the milk samples and infant formulas, ranging from 49.28–80.96 μg/mL and 29.92–60.00 μg/mL, respectively.

Double antibody sandwich enzyme linked immunoassay and rapid Immunoswab assay for detection of gliadin in food

Abstract Celiac (gluten intolerant) sprue is a life-long threatening disease. The only effective treatment thus far is to maintain a gluten-free diet. Sensitive and reliable double antibody sandwich enzyme-linked immunosorbent assay (ELISA) and Immunoswab assay were developed by using chicken egg yolk anti-gliadin immunoglobulin Y as capturing antibody and monoclonal anti-gliadin IgG (HYB-314) antibody as detecting antibody for the detection of gliadin. The detection limits of the double antibody sandwich ELISA and Immunoswab assay were 5 ng/ml in phosphate buffered saline and 1.25 µg/ml in 60% ethanol, respectively. The Immunoswab was a more convenient and rapid detection system, but was less sensitive as compared to the double antibody sandwich ELISA developed.

High Hydrostatic Pressure-Assisted Enzymatic Treatment Improves Antioxidant and Anti-inflammatory Properties of Phosvitin

BACKGROUND Phosvitin (PV) is a highly-phosphorylated metal-binding protein in egg yolk. Phosphoserine clusters make PV resistant to enzymatic digestion, which might be nutritionally undesirable. OBJECTIVE This study was designed to determine the effects of high hydrostatic pressure and enzymatic hydrolysis (HHP-EH) on the antioxidant and anti-inflammatory properties of PV hydrolysates (PVHs). METHODS PV was hydrolyzed by alcalase, elastase, savinase, thermolysin, and trypsin at 0.1, 50, and 100 MPa pressure levels. PVHs were evaluated for degree of hydrolysis, molecular weight distribution patterns, antioxidant and anti-inflammatory properties in chemical and cellular models. The effect of PVH on gene expression of pro-inflammatory cytokines (TNF-α and IL-1β) was also evaluated using real time-PCR. The hydrolysate with most potent antioxidant and anti-inflammatory properties was subjected to LC-MS/MS analysis to identify the peptide sequence. RESULTS Hydrolysates produced at 100 MPa exhibited higher degree of hydrolysis and greater reducing power and free radical scavenging activity compared to those obtained at atmospheric pressure. After adjusting the phosphate content, alcalase- and trypsin-digested PVHs showed superior iron chelation capacity (69-73%), regardless of pressure. Both alcalase- and trypsin-digested PVHs significantly inhibited nitric oxide production by RAW264.7 macrophage cells. LPS-stimulated up-regulation of proinflammatory cytokines was also suppressed by alcalase-digested PVH. CONCLUSION The HHP-EH method could play a promising role in the production of bioactive peptides from hydrolysis-resistant proteins. HHP-assisted PVH may be useful in preparing a potential pharmaceutical with antioxidant and anti-inflammatory properties.

Sensitive double antibody sandwich ELISA for the quantification of phosvitin

ABSTRACT An effective double antibody sandwich ELISA (DAS-ELISA) method based on monoclonal (mAb) and chicken egg yolk IgY antibodies was developed to determine phosvitin (PV) content in therapeutic and functional products. Leghorn laying hens were immunized with purified PV to produce anti-PV IgY antibody in the egg yolk. High anti-PV IgY titer obtained from the egg yolks collected during 4–10 weeks of the immunization period contained approximately 6.2% of specific anti-PV IgY in total IgY. The PV detection range of the DAS-ELISA and biotinylated DAS-ELISA was 16.8–90 and 7.5–40 ng/mL, respectively. However, biotinylated DAS-ELISA was the better method for PV quantification in terms of accuracy and sensitivity. This highly efficient PV detection method may recuperate the performance of the existing protein assay methods as well as facilitate future research on PV bioactivities and applications.

A Combination of Egg Yolk IgY and Phosvitin Inhibits the Growth of Enterotoxigenic Escherichia coli K88 and K99

BACKGROUND Enterotoxigenic Escherichia coli (ETEC) is the main cause of fatal diarrhea in piglets during the first week of life and over the time of weaning. Pathogenesis of ETEC-causing diarrhea involves intestinal colonization mediated by fimbriae. Although, both IgY and egg yolk phosvitin (PV) possess antimicrobial activity, their combined activity has not been explored. A combination of IgY specific for ETEC and metal-chelating PV may show synergistic effect in reducing the growth of ETEC by inhibiting bacterial proliferation and stipulating protection against ETEC infection. OBJECTIVE The goal of this study was to determine the effects of anti-ETEC IgY and PV on in vitro growth inhibition of ETEC strains possessing K88 and K99 fimbriae prevalent in the porcine population. METHODS Anti-K88 and -K99 IgY antibodies were obtained from egg yolks of 23-week-old Single- Comb White Leghorn hens immunized with K88 and K99 fimbriae of ETEC, respectively, with high titres sustained over 6 to 8 weeks of the immunization period. Specific IgY, PV, and PV-hydrolysate from alcalase-hydrolysis under high hydrostatic pressure (PVH-Alc-HHP) alone or in combination, were used to treat ETEC K88 and K99 cultures at optimal concentrations of 100 μg/mL, 1 mg/mL, and 1 mg/mL, respectively, for 24 h. RESULTS PVH-Alc-HHP demonstrated the highest degree of hydrolysis, 38.9%. Combined use of IgY and PVH-Alc-HHP showed the highest bactericidal effect resulting in ETEC K88 and K99 growth inhibition of 2.8 and 2.67 log CFU/mL, respectively. CONCLUSION Combined IgY-PVH effectively control ETEC, therefore holds a great potential for microbial control in veterinary pharmaceutical industry.