Hoon H. Sunwoo

A rapid point of care immunoswab assay for SARS-CoV detection

Abstract The emergence of severe acute respiratory syndrome (SARS) resulted in several outbreaks worldwide. Early tests for diagnosis were not always conclusive in identifying a SARS suspected patient. Nucleocapsid protein (NP) is the most predominant virus derived structural protein which is shed in high amounts in serum and nasopharyngeal aspirate during the first week of infection. As part of such efforts, a simple, easy to use immunoswab method was developed by generating a panel of monoclonal antibodies (MAbs), Bispecific MAbs and chicken polyclonal IgY antibody against the SARS-CoV nucleocapsid protein (NP). Employing the MAb-based immunoswab, an NP concentration of 200pg/mL in saline and pig nasopharyngeal aspirate, and 500pg/mL in rabbit serum were detected. BsMAb-based immunoswabs detected an NP concentration of 20pg/mL in saline, 500pg/mL in rabbit serum and 20–200pg/mL in pig nasopharyngeal aspirate. Polyclonal IgY-based immunoswabs detected an NP concentration of 10pg/mL in pig nasopharyngeal aspirate providing the most sensitive SARS point of care assay. Results show that the robust immunoswab method of detecting SARS-CoV NP antigen can be developed into an easy and effective way of identifying SARS suspected individuals during a future SARS epidemic, thereby reducing and containing the transmission. The key feature of this simple immunoswab diagnostic assay is its ability to detect the presence of the SARS-CoV antigen within 45–60min with the availability of the body fluid samples.

Isolation and characterization of proteoglycans from growing antlers of wapiti (Cervus elaphus)

Proteoglycans were extracted with 4 M guanidine-HCl from the zone of maturing chondrocytes, the site of endochondral ossification of growing antlers of wapiti (Cervus elaphus). Proteoglycans were isolated by DEAE-Sephacel chromatography and separated by Sepharose CL-4B chromatography into three fractions. Fraction I contained a high molecular mass (> 1000 kDa) chondroitin sulfate proteoglycan capable of interacting with hyaluronic acid. Its amino acid composition resembled that of the cartilage proteoglycan, aggrecan. Fraction II contained proteoglycans with intermediate molecular weight which were recognized by monoclonal antibodies specific to chondroitin sulfate and keratan sulfate. Fraction III contained a low molecular mass (< 160 kDa) proteoglycan, decorin, with a glucuronate-rich glycosaminoglycan chain.

Maternal dietary FA modulate the long-chain n-3 PUFA status of chick cardiac tissue.

The effect that egg yolk or maternal n−3 FA have on the cardiac tissue long-chain n−3 FA status of chicks during growth was investigated. Fggs with low, medium, and high levels of n−3 PUFA were obtained by feeding breeder hens a wheat/soybean meal-based diet containing 5% sunflower oil (Low n−3), 2.5% sunflower oil plus 2.5% fish oil (Medium n−3), or 5% fish oil (High n−3). The chicks hatched from Low, Medium, and High n−3 eggs were fed a diet containing 18∶3n−3, but devoid of long-chain n−3 FA. The FA composition of cardiac tissue was determined on days 0, 14, 28, and 42. At day 0, the cardiac FA reflected maternal diet. With time, the level of all the long-chain n−3 FA decreased compared with day 0, and this was true especially by day 14. These data show that dietary 18∶3n−3 fed to the chicks did not sustain high levels of EPA and DHA in cardiac tissue, despite the high content of long-chain n−3 FA in the maternal diet. At days 0 and 14, the chicks hatched from High and Medium n−3 eggs had higher 20∶5n−3, 22∶5n−3, and 22∶6n−3 contents with a concomitant reduction in 20∶4n−6 in the cardiac tissue compared with the Low n−3 egg group. Cardiac tissue of birds hatched from Medium n−3 eggs retained higher levels of 20∶5n−3 up to day 42 of growth when compared with other treatments (P<0.05). None of the treatments was effective in maintaining DHA levels after day 14 of growth.

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.

Overview of Plant-Derived Vaccine Antigens: Dengue Virus

This review highlights the advantages and current status of plant-derived vaccine development with special reference to the dengue virus. There are numerous problems involved in dengue vaccine development, and there is no vaccine against all four dengue serotypes. Dengue vaccine development using traditional approaches has not been satisfactory in terms of inducing neutralizing antibodies. Recently, these issues were addressed by showing a very good response to inducing neutralizing antibodies by plant-derived dengue vaccine antigens. This indicates the feasibility of using plant-derived vaccine antigens as a low-cost method to combat dengue and other infectious diseases. The application of new methods and strategies such as dendritic cell targeting in cancer therapy, severe acute respiratory syndrome, tuberculosis, human immune deficiency virus, and malaria might play an important role. These new methods are more efficient than traditional protocols. It is expected that in the near future, plant-derived vaccine antigens or antibodies will play an important role in the control of human infectious diseases.

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.

Diagnostics of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) nucleocapsid antigen using chicken immunoglobulin Y

Abstract The goal of this study was to develop a quantitative detection system for severe acute respiratory syndrome-associated coronavirus (SARS-CoV), targeting the nucleocapsid protein (NP), to determine the presence and degree of infection in suspected individuals. Because the NP is the viral protein shed during infection and its template mRNA is the most abundant subgenomic RNA, it is a suitable candidate for developing antibodies for diagnostic applications. In this study, we have prepared full-length SARS-CoV NP expressed in Escherichia coli and purified. Full-length NP was used for the preparation of mouse monoclonal antibody and chicken polyclonal IgY antibodies for the development of heterosandwich ELISA for early diagnostics of SARS-suspected individuals. The sensitivity of the developed heterosandwich ELISA can detect the viral antigen at 18.5 pg/mL of recombinant NP. This study describes ultrasensitive ELISA using 19B6 monoclonal antibody as the capture antibody and IgY as the detecting antibody against the most abundant SARS-CoV NP antigens. One of the most important findings was the use of inexpensive polyclonal IgY antibody to increase the sensitivity of the detection system for SARS-CoV at the picogram level. Furthermore, the immunoassay of SARS-CoV NP antigen developed could be an effective and sensitive method of diagnosing SARS-suspected individuals during a future SARS-CoV outbreak.

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.