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Stability of orally administered immunoglobulin in the gastrointestinal tract
Journal of Immunological Methods 384 (2012) 143–147
Contents lists available at SciVerse ScienceDirect
Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim
Technical note
Stability of orally administered immunoglobulin in the gastrointestinal tract Jeongmin Lee a, b, Hae-Eun Kang a, 1, Hee-Jong Woo a,⁎ a b
Laboratory of Immunology, College of Veterinary Medicine, Seoul National University, Seoul 151‐742, Republic of Korea Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151‐742, Republic of Korea
a r t i c l e
i n f o
Article history: Received 22 March 2012 Accepted 1 June 2012 Available online 9 June 2012 Keywords: Oral administration Encapsulation Immunoglobulin Passive immunization
a b s t r a c t Oral administration of immunoglobulin in the colostrum or egg yolk has been considered an effective tool for preventing enterobacterial infection via passive immunization. During this process, the transmission and residence of the active immunoglobulin are the most important conditions for successful protection. We investigated the stability of encapsulated colostrum and egg yolk immunoglobulin for the effective transmission of immunoglobulin in the gastrointestinal (GI) tract. First, we measured GI transit time. Contrast media passed through and reached the stomach within 10 min, the small intestine within 3.5 h, and the cecum within 5 h. Both the encapsulated colostrum containing anti-hepatitis A virus (HAV) antibody (IgG) and egg yolk with anti-rotavirus antibody (IgY) showed lower antibody activity than the nonencapsulated colostrum did in the stomach after administration; however, significantly higher antibody activities were observed in the encapsulated groups than in the non-encapsulated groups in the small intestine 3.5 h after the administration. In the large intestine, the antibody activities of the encapsulated groups were maintained or slightly increased in a timedependent manner; however, the titers of each non-capsulated control were as low as the negative controls. Therefore, this encapsulation is considered a useful tool for the delivery of active antibody through the GI tract. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Passive transfer of immunoglobulin is considered an important process that protects newborns against endobacterial infection. Colostrum, a milky fluid secreted on the first day or early period after parturition, can protect newborn mammals against environmental pathogens; in particular, bovine colostrum contains high concentrations of immunoglobulins, mainly immunoglobulin G (IgG), which has a pivotal role in passive immunization (Korhonen et al., 2000; Marnila et al., 2003). Bovine colostrum also has a large number of immuneregulating constituents—antimicrobial compounds, including
⁎ Corresponding author at: Laboratory of Immunology, College of Veterinary Medicine, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151‐742, Republic of Korea. Tel.: +82 2 880 1262; fax: +82 2 877 8284. E-mail address: [email protected] (H.-J. Woo). 1 Present address: Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado 80523-1619, USA. 0022-1759/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2012.06.001
active complement and lactoferrin, and other bioactive substances like growth factors and oligosaccharides; these bioactive molecules can be used for the protection of the gastrointestinal mucosa (Korhonen et al., 2000; Thapa, 2005). Immunoglobulin Y (IgY) is the major antibody in birds, reptiles, and lungfish blood. It is also found in chicken egg yolk, and has functional similarities with mammalian IgG, although their physical and chemical properties are different (Larsson et al., 1993). Because IgY is extracted from the yolks of laid eggs, the method of antibody production is non-invasive, and large quantitative production is possible without having to bleed the animal. Therefore, its application to specific foods or drugs for protection from enteric pathogens such as Salmonella, Helicobacter pylori, enterotoxigenic Escherichia coli (ETEC), and Campylobacter jejuni has been investigated (Horie et al., 2004; Chalghoumi et al., 2009). In addition, because the abuse of medical drugs, including antibiotics, has been considered a serious threat to public health as well as animal husbandry, practical uses and trials of IgY are becoming a more attractive
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and alternative approach for accomplishing protective immunity in newborn or immunologically weak individuals (Chalghoumi et al., 2009). Regarding the application of both colostrum and egg yolk for passive immunization, the critical limitations of oral administration are mainly due to the degradation and inactivation of antibodies by digestive enzymes and lytic molecules in the alimentary canal. Specific antibody properties are gradually decreased in the gastric juice and proteolytic enzymes in the intestine; therefore, for safe and effective transmission of antibodies in the colostrum or egg yolk, protecting antibodies from these unfavorable environments should be considered for establishing passive immunity via oral administration. In a previous study, anti-hepatitis A virus antibody and antirotavirus antibody were obtained from bovine colostrum and from hen egg yolk, respectively. Then the encapsulation process for these antibodies was developed and patented (Cho et al., 2005) (Korea patent numbers 1005431100000, 1005503740000, and 1007488420000). In this study, we investigated the stability of encapsulated immunoglobulin of the colostrum and yolk for effective transmission of immunoglobulin during digestion in the mouse alimentary canal. The encapsulation efficacy for transmission was calculated and compared with respect to time.
Table 1 Experimental groups for investigation of antibody activity in the alimentary canal. Experimental Oral administration group
Number of mice
Group 1
15
Group Group Group Group
2 3 4 5
Bovine colostrum with IgG (nonencapsulated)a Encapsulated bovine colostrum with IgGa Egg yolk IgY (non-encapsulated)b Encapsulated egg yolk IgYb PBS (negative control)
15 15 15 15
The samples for oral administration were supplied by Korea Yakult Co., Ltd. a Anti-hepatitis A virus (HAV) antibodies (IgG) from bovine colostrum were raised from cows immunized with HAV antigen. b Anti-rotavirus antibodies (IgY) from egg yolk were raised from hens with rotavirus antigen.
(group 5) (Table 1). Three hundred microliters of each sample was orally administered to the animals. After oral administration, contents in the stomach, small intestine, and large intestine were recovered by adding 1 mL
2. Materials and methods 2.1. Sample preparation The samples for oral administration were obtained from Korea Yakult Co., Ltd. Non-encapsulated and encapsulated antihepatitis A virus (HAV) antibodies (IgG) in bovine colostrum were raised from cows immunized with HAV VP1 antigen; non-encapsulated and encapsulated anti-rotavirus antibodies (IgY) in egg yolk were raised from hens immunized with rotavirus VP5 and VP8 antigens. The procedures for immunization, antibody quantification, and encapsulation have been patented and described in previous studies partially (Cho et al., 2005). 2.2. Estimation of transit time in the mouse alimentary canal To estimate the transit time in the mouse digestive tract, mice were fed with 300 μL of 40% barium sulfate after a 24-h starvation, and were then examined by radiography every 10 min after oral administration. 2.3. Oral administration to mice Animal experiments were performed in a closed system under the control of the Animal Welfare Committee of Seoul National University Institutional Animal Care and Use Committee (SNUIACUC) in accordance with the laboratory's animal ethics guidelines. Seven-week-old female BALB/c mice (Charles River Laboratories, MA, USA) were randomly selected and divided into the following 5 groups, 15 animals each: non-encapsulated bovine colostrum with anti-HAV IgG (group 1), encapsulated bovine colostrum with anti-HAV IgG (group 2), non-encapsulated egg yolk with anti-rotavirus IgY (group 3), encapsulated egg yolk with anti-rotavirus IgY (group 4), and PBS for negative control
Fig. 1. Estimation of transit time in the alimentary canal by using contrast medium in mice. After administration of 40% barium sulfate, mice were examined by radiography after 10 min (A), 20 min (B), 30 min (C), 3.5 h (D), and 5 h (E).
J. Lee et al. / Journal of Immunological Methods 384 (2012) 143–147
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Fig. 2. Antibody activity after oral administration of non-encapsulated (□) and encapsulated (■) colostrum in the gastrointestinal contents (A), small intestinal contents (B), and large intestinal contents (C), respectively. Active antibody was measured by indirect ELISA for 10 min, 3.5 h, and 5 h, and then calculated using the following equation: [OD value of experiment] / [OD value of positive control]. Values are presented as a percentage. The statistically significant difference between encapsulation and non-encapsulation is indicated with an asterisk (p b 0.05).
of PBS from 5 animals per group, respectively, with intervals of 10 min, 3 h 30 min, and 5 h. These intestinal fluids were administered to measure antibody activity.
2.4. Indirect ELISA for antibody titration Active antibodies in intestinal fluid were titrated by indirect ELISA. One microgram of each protein (HAV VP1 antigen for the colostrum and rotavirus VP5 and VP8 antigens for yolk IgY) was used as antigens. The procedures were described previously (Lee and Woo, 2010). All samples were subsequently tested in duplicate. The antibody activity was calculated by the following equation: [OD value of experiment] / [OD value of positive control]. The value was presented as a percentage.
3. Results and discussion Bovine milk and colostrum contain abundant bioactive components, including growth factors, immunoglobulin, lactoperoxidase, lysozyme, lactoferrin, cytokines, nucleosides, vitamins, peptides, and oligosaccharides, which are of increasing relevance to animal and human health (Gapper et al., 2007). Moreover, oral administration of bovine colostrum has been proved an effective method in the treatment of intestinal pathogen infections; the biological activity of bacteria and endotoxins was reduced by immunoglobulin in the colostrum (Dohler and Nebermann, 2002). In hens, IgY is passively transferred from their serum to the egg yolk, which protects the chicks from various infectious diseases (Sarker et al., 2001). Moreover, the amount of IgY
Fig. 3. Antibody activity after oral administration of non-encapsulated (□) and encapsulated (■) egg yolk in gastrointestinal contents (A), small intestinal contents (B), and large intestinal contents (C), respectively. Active antibody was measured by indirect ELISA for 10 min, 3.5 h, and 5 h, and then calculated and presented by the equation in the legend of Fig. 2. The statistically significant difference between encapsulation and non-encapsulation is indicated with an asterisk (p b 0.05).
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obtained from immunized hen eggs is about 18 times higher than that which can be obtained from immunized rabbit serum via conventional methods. It is, thus, much easier to produce desired antibodies in a large scale within a limited time because of high percentages of IgY in egg yolk (~1%). It is also more hygienic to produce IgY from egg yolk than from animal serum or colostrum. Several animal studies have shown effective protection against rotavirus by oral IgY in mice, calves, and cats (Hiraga et al., 1990; Kuroki et al., 1993, 1994). During oral administration of immunoglobulins, the most critical and considerable factor that influences induction of successful passive immunity is the delivery of large amounts of naïve and active antibodies to overcome the unfavorable digestive environment in the alimentary canal. Therefore, in this study, we investigated the stability of encapsulated immunoglobulins in the colostrum and egg yolk for effective transmission in the alimentary canal. First we estimated the transit time in the alimentary canal in mice (Fig. 1). The contrast medium was found in a connecting area between the stomach and the small intestine 10 min after administration, and then, in the small intestine 20 min, 30 min, and 3 h after administration, and finally, in the cecum 5 h after administration. During oral administration of protein-based compounds, GI residence and transit times have been recognized as crucial factors, because precise information regarding the location of compound release in situ allows the optimization of the pharmaceutical potential of oral administration (Schwarz et al., 2002). For evaluating the GI transit time, motility, and compound release, a large number of methods have been applied. Most prominent are the X-ray and scintigraphic techniques, which have been used to monitor orally ingested capsules containing radio opaque material or gamma-emitters. Noninvasive techniques, including ultrasound, metal detectors, magnetic field detectors, and dyes, have been used to avoid the adverse effects of ionizing radiation (Schwarz et al., 2002). In this study, we used radiography involving 40% barium sulfate for measuring GI transit time and it has been proven that this is an easy and non-laborious way of time-dependent tracking in small animals. The acidic environment in the stomach is a harmful condition that decreases antibody activity. For quantitative measurements of active colostrum antibody or egg yolk antibody in the alimentary canal, encapsulated colostrum with anti-HAV IgG or encapsulated egg yolk with anti-rotavirus IgY was administered to mice, and then chased along the digestive tract, respectively. The colostrum or egg yolk sample reached the stomach, the first organ in the GI tract, within 10 min after oral administration, and the antibody activity of the non-encapsulated samples was higher than that of the encapsulated ones; however, the activity levels were not maintained, and sharply decreased to levels comparable to those of the negative controls within 3.5 h and 5 h after administration (Figs. 2A and 3A). However, in the small intestine, encapsulated samples showed higher and more constant levels of antibody activity than the non-encapsulated ones (Figs. 2B and 3B). These results are in accordance with other studies. It has been reported that only 3% of antibodies were active after a 30-min incubation when the specific antibody was tested in a simulated gastric juice containing HCl with pepsin (1100 units/mL) at pH 2.0. At pH 3.0, 22–28% of specific antibodies were intact, and at pH 4.0, 25–39% were still active (Marnila et al., 2003). In the stomach, pepsin can digest
IgG into an F(ab′) fragment; however, colostrum IgG1, which is the most abundant immunoglobulin in colostrums, is resistant to this proteolytic cleavage, and can remain active in the intestine (Korhonen et al., 2000). Relatively high activity of non-encapsulated colostrum in the small intestinal tract can be explained on the basis of these previous reports. The active antibodies surviving in the stomach are usually degraded proteolytically in the small intestine by trypsin and in the large intestine by bacterial flora. Thus, the remnant activity of the antibody is undetectable in stool samples (Hilpert et al., 1987). In the case of IgY, the level of IgY activity is dependent on the amount administered, pH, enzyme activity, and passage time in the GI tract. Although IgY is proteolytically digested to Fab, Fab′2, and Fc fragments by pepsin and trypsin in the GI tract, Fab and Fab′2 fragments still have the activity to bind antigen; thus, they show neutralizing properties in the GI tract and are detectable in stool samples (Carlander et al., 2000). In this study, it was hard to detect the antibody activity of nonencapsulated egg yolk in the small and large intestines; however, high levels of activity were observed with encapsulated egg yolk. In the large intestine, encapsulated samples showed relatively higher antibody activity than the nonencapsulated ones. Non-encapsulated groups showed low antibody activity, similar to the negative controls. Interestingly, relatively big differences between the encapsulated and nonencapsulated samples were observed with colostrum administration, and time-dependent increases in activity were also observed with the encapsulated egg yolk administration (Figs. 2C and 3C). In conclusion, our results demonstrate that encapsulation of immunoglobulin can successfully protect the antibodies from gastric inactivation. The encapsulated samples retained more activity than the non-encapsulated ones, indicating that encapsulation may be an effective method for protecting immunoglobulin from gastrointestinal inactivation, enabling its use in oral passive immunization techniques for the widespread prevention of enteric pathogens.
Acknowledgments This work was supported by a grant from the BioGreen21 Program (No. PJ007176), Rural Development Administration, and the Korean Research Foundation grant (no. KRF-2008-313E00622), Republic of Korea (to J. L.). This work was funded by a research grant from Korea Yakult Co., Ltd. (to H.-J. W.).
References Carlander, D., Kollberg, H., Wejaker, P.E., Larsson, A., 2000. Peroral immunotherapy with yolk antibodies for the prevention and treatment of enteric infections. Immunol. Res. 21, 1. Chalghoumi, R., Beckers, Y., Portetelle, D., Thewis, A., 2009. Hen egg yolk antibodies (IgY), production and use for passive immunization against bacterial enteric infections in chicken: a review. Biotechnol. Agron. Soc. 13, 295. Cho, Y.H., Lee, J.J., Park, I.B., Huh, C.S., Baek, Y.J., Park, J., 2005. Protective effect of microencapsulation consisting of multiple emulsification and heat gelation processes on immunoglobulin in yolk. J. Food Sci. 70, E148. Dohler, J.R., Nebermann, L., 2002. Bovine colostrum in oral treatment of enterogenic endotoxaemia in rats. Crit. Care 6, 536. Gapper, L.W., Copestake, D.E., Otter, D.E., Indyk, H.E., 2007. Analysis of bovine immunoglobulin G in milk, colostrum and dietary supplements: a review. Anal. Bioanal. Chem. 389, 93.
J. Lee et al. / Journal of Immunological Methods 384 (2012) 143–147 Hilpert, H., Brussow, H., Mietens, C., Sidoti, J., Lerner, L., Werchau, H., 1987. Use of bovine milk concentrate containing antibody to rotavirus to treat rotavirus gastroenteritis in infants. J. Infect. Dis. 156, 158. Hiraga, C., Kodama, Y., Sugiyama, T., Ichikawa, Y., 1990. Prevention of human rotavirus infection with chicken egg yolk immunoglobulins containing rotavirus antibody in cat. Kansenshogaku Zasshi 64, 118. Horie, K., Horie, N., Abdou, A.M., Yang, J.O., Yun, S.S., Chun, H.N., Park, C.K., Kim, M., Hatta, H., 2004. Suppressive effect of functional drinking yogurt containing specific egg yolk immunoglobulin on Helicobacter pylori in humans. J. Dairy. Sci. 87, 4073. Korhonen, H., Marnila, P., Gill, H.S., 2000. Milk immunoglobulins and complement factors. Br. J. Nutr. 84 (Suppl. 1), S75. Kuroki, M., Ikemori, Y., Yokoyama, H., Peralta, R.C., Icatlo, F.C., Kodama Jr., Y., 1993. Passive protection against bovine rotavirus-induced diarrhea in murine model by specific immunoglobulins from chicken egg yolk. Vet. Microbiol. 37, 135. Kuroki, M., Ohta, M., Ikemori, Y., Peralta, R.C., Yokoyama, H., Kodama, Y., 1994. Passive protection against bovine rotavirus in calves by specific immunoglobulins from chicken egg yolk. Arch. Virol. 138, 143.
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Larsson, A., Balow, R.M., Lindahl, T.L., Forsberg, P.O., 1993. Chicken antibodies: taking advantage of evolution—a review. Poult. Sci. 72, 1807. Lee, J., Woo, H.J., 2010. Antigenicity of partial fragments of recombinant Pasteurella multocida toxin. J. Microbiol. Biotechnol. 20, 1756. Marnila, P., Rokka, S., Rehnberg-Laiho, L., Karkkainen, P., Kosunen, T.U., Rautelin, H., Hanninen, M.L., Syvaoja, E.L., Korhonen, H., 2003. Prevention and suppression of Helicobacter felis infection in mice using colostral preparation with specific antibodies. Helicobacter 8, 192. Sarker, S.A., Casswall, T.H., Juneja, L.R., Hoq, E., Hossain, I., Fuchs, G.J., Hammarstrom, L., 2001. Randomized, placebo-controlled, clinical trial of hyperimmunized chicken egg yolk immunoglobulin in children with rotavirus diarrhea. J. Pediatr. Gastroenterol. Nutr. 32, 19. Schwarz, R., Kaspar, A., Seelig, J., Kunnecke, B., 2002. Gastrointestinal transit times in mice and humans measured with 27Al and 19F nuclear magnetic resonance. Magn. Reson. Med. 48, 255. Thapa, B.R., 2005. Therapeutic potentials of bovine colostrums. Indian J. Pediatr. 72, 849.
Contents lists available at SciVerse ScienceDirect
Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim
Technical note
Stability of orally administered immunoglobulin in the gastrointestinal tract Jeongmin Lee a, b, Hae-Eun Kang a, 1, Hee-Jong Woo a,⁎ a b
Laboratory of Immunology, College of Veterinary Medicine, Seoul National University, Seoul 151‐742, Republic of Korea Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151‐742, Republic of Korea
a r t i c l e
i n f o
Article history: Received 22 March 2012 Accepted 1 June 2012 Available online 9 June 2012 Keywords: Oral administration Encapsulation Immunoglobulin Passive immunization
a b s t r a c t Oral administration of immunoglobulin in the colostrum or egg yolk has been considered an effective tool for preventing enterobacterial infection via passive immunization. During this process, the transmission and residence of the active immunoglobulin are the most important conditions for successful protection. We investigated the stability of encapsulated colostrum and egg yolk immunoglobulin for the effective transmission of immunoglobulin in the gastrointestinal (GI) tract. First, we measured GI transit time. Contrast media passed through and reached the stomach within 10 min, the small intestine within 3.5 h, and the cecum within 5 h. Both the encapsulated colostrum containing anti-hepatitis A virus (HAV) antibody (IgG) and egg yolk with anti-rotavirus antibody (IgY) showed lower antibody activity than the nonencapsulated colostrum did in the stomach after administration; however, significantly higher antibody activities were observed in the encapsulated groups than in the non-encapsulated groups in the small intestine 3.5 h after the administration. In the large intestine, the antibody activities of the encapsulated groups were maintained or slightly increased in a timedependent manner; however, the titers of each non-capsulated control were as low as the negative controls. Therefore, this encapsulation is considered a useful tool for the delivery of active antibody through the GI tract. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Passive transfer of immunoglobulin is considered an important process that protects newborns against endobacterial infection. Colostrum, a milky fluid secreted on the first day or early period after parturition, can protect newborn mammals against environmental pathogens; in particular, bovine colostrum contains high concentrations of immunoglobulins, mainly immunoglobulin G (IgG), which has a pivotal role in passive immunization (Korhonen et al., 2000; Marnila et al., 2003). Bovine colostrum also has a large number of immuneregulating constituents—antimicrobial compounds, including
⁎ Corresponding author at: Laboratory of Immunology, College of Veterinary Medicine, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151‐742, Republic of Korea. Tel.: +82 2 880 1262; fax: +82 2 877 8284. E-mail address: [email protected] (H.-J. Woo). 1 Present address: Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado 80523-1619, USA. 0022-1759/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2012.06.001
active complement and lactoferrin, and other bioactive substances like growth factors and oligosaccharides; these bioactive molecules can be used for the protection of the gastrointestinal mucosa (Korhonen et al., 2000; Thapa, 2005). Immunoglobulin Y (IgY) is the major antibody in birds, reptiles, and lungfish blood. It is also found in chicken egg yolk, and has functional similarities with mammalian IgG, although their physical and chemical properties are different (Larsson et al., 1993). Because IgY is extracted from the yolks of laid eggs, the method of antibody production is non-invasive, and large quantitative production is possible without having to bleed the animal. Therefore, its application to specific foods or drugs for protection from enteric pathogens such as Salmonella, Helicobacter pylori, enterotoxigenic Escherichia coli (ETEC), and Campylobacter jejuni has been investigated (Horie et al., 2004; Chalghoumi et al., 2009). In addition, because the abuse of medical drugs, including antibiotics, has been considered a serious threat to public health as well as animal husbandry, practical uses and trials of IgY are becoming a more attractive
144
J. Lee et al. / Journal of Immunological Methods 384 (2012) 143–147
and alternative approach for accomplishing protective immunity in newborn or immunologically weak individuals (Chalghoumi et al., 2009). Regarding the application of both colostrum and egg yolk for passive immunization, the critical limitations of oral administration are mainly due to the degradation and inactivation of antibodies by digestive enzymes and lytic molecules in the alimentary canal. Specific antibody properties are gradually decreased in the gastric juice and proteolytic enzymes in the intestine; therefore, for safe and effective transmission of antibodies in the colostrum or egg yolk, protecting antibodies from these unfavorable environments should be considered for establishing passive immunity via oral administration. In a previous study, anti-hepatitis A virus antibody and antirotavirus antibody were obtained from bovine colostrum and from hen egg yolk, respectively. Then the encapsulation process for these antibodies was developed and patented (Cho et al., 2005) (Korea patent numbers 1005431100000, 1005503740000, and 1007488420000). In this study, we investigated the stability of encapsulated immunoglobulin of the colostrum and yolk for effective transmission of immunoglobulin during digestion in the mouse alimentary canal. The encapsulation efficacy for transmission was calculated and compared with respect to time.
Table 1 Experimental groups for investigation of antibody activity in the alimentary canal. Experimental Oral administration group
Number of mice
Group 1
15
Group Group Group Group
2 3 4 5
Bovine colostrum with IgG (nonencapsulated)a Encapsulated bovine colostrum with IgGa Egg yolk IgY (non-encapsulated)b Encapsulated egg yolk IgYb PBS (negative control)
15 15 15 15
The samples for oral administration were supplied by Korea Yakult Co., Ltd. a Anti-hepatitis A virus (HAV) antibodies (IgG) from bovine colostrum were raised from cows immunized with HAV antigen. b Anti-rotavirus antibodies (IgY) from egg yolk were raised from hens with rotavirus antigen.
(group 5) (Table 1). Three hundred microliters of each sample was orally administered to the animals. After oral administration, contents in the stomach, small intestine, and large intestine were recovered by adding 1 mL
2. Materials and methods 2.1. Sample preparation The samples for oral administration were obtained from Korea Yakult Co., Ltd. Non-encapsulated and encapsulated antihepatitis A virus (HAV) antibodies (IgG) in bovine colostrum were raised from cows immunized with HAV VP1 antigen; non-encapsulated and encapsulated anti-rotavirus antibodies (IgY) in egg yolk were raised from hens immunized with rotavirus VP5 and VP8 antigens. The procedures for immunization, antibody quantification, and encapsulation have been patented and described in previous studies partially (Cho et al., 2005). 2.2. Estimation of transit time in the mouse alimentary canal To estimate the transit time in the mouse digestive tract, mice were fed with 300 μL of 40% barium sulfate after a 24-h starvation, and were then examined by radiography every 10 min after oral administration. 2.3. Oral administration to mice Animal experiments were performed in a closed system under the control of the Animal Welfare Committee of Seoul National University Institutional Animal Care and Use Committee (SNUIACUC) in accordance with the laboratory's animal ethics guidelines. Seven-week-old female BALB/c mice (Charles River Laboratories, MA, USA) were randomly selected and divided into the following 5 groups, 15 animals each: non-encapsulated bovine colostrum with anti-HAV IgG (group 1), encapsulated bovine colostrum with anti-HAV IgG (group 2), non-encapsulated egg yolk with anti-rotavirus IgY (group 3), encapsulated egg yolk with anti-rotavirus IgY (group 4), and PBS for negative control
Fig. 1. Estimation of transit time in the alimentary canal by using contrast medium in mice. After administration of 40% barium sulfate, mice were examined by radiography after 10 min (A), 20 min (B), 30 min (C), 3.5 h (D), and 5 h (E).
J. Lee et al. / Journal of Immunological Methods 384 (2012) 143–147
145
Fig. 2. Antibody activity after oral administration of non-encapsulated (□) and encapsulated (■) colostrum in the gastrointestinal contents (A), small intestinal contents (B), and large intestinal contents (C), respectively. Active antibody was measured by indirect ELISA for 10 min, 3.5 h, and 5 h, and then calculated using the following equation: [OD value of experiment] / [OD value of positive control]. Values are presented as a percentage. The statistically significant difference between encapsulation and non-encapsulation is indicated with an asterisk (p b 0.05).
of PBS from 5 animals per group, respectively, with intervals of 10 min, 3 h 30 min, and 5 h. These intestinal fluids were administered to measure antibody activity.
2.4. Indirect ELISA for antibody titration Active antibodies in intestinal fluid were titrated by indirect ELISA. One microgram of each protein (HAV VP1 antigen for the colostrum and rotavirus VP5 and VP8 antigens for yolk IgY) was used as antigens. The procedures were described previously (Lee and Woo, 2010). All samples were subsequently tested in duplicate. The antibody activity was calculated by the following equation: [OD value of experiment] / [OD value of positive control]. The value was presented as a percentage.
3. Results and discussion Bovine milk and colostrum contain abundant bioactive components, including growth factors, immunoglobulin, lactoperoxidase, lysozyme, lactoferrin, cytokines, nucleosides, vitamins, peptides, and oligosaccharides, which are of increasing relevance to animal and human health (Gapper et al., 2007). Moreover, oral administration of bovine colostrum has been proved an effective method in the treatment of intestinal pathogen infections; the biological activity of bacteria and endotoxins was reduced by immunoglobulin in the colostrum (Dohler and Nebermann, 2002). In hens, IgY is passively transferred from their serum to the egg yolk, which protects the chicks from various infectious diseases (Sarker et al., 2001). Moreover, the amount of IgY
Fig. 3. Antibody activity after oral administration of non-encapsulated (□) and encapsulated (■) egg yolk in gastrointestinal contents (A), small intestinal contents (B), and large intestinal contents (C), respectively. Active antibody was measured by indirect ELISA for 10 min, 3.5 h, and 5 h, and then calculated and presented by the equation in the legend of Fig. 2. The statistically significant difference between encapsulation and non-encapsulation is indicated with an asterisk (p b 0.05).
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obtained from immunized hen eggs is about 18 times higher than that which can be obtained from immunized rabbit serum via conventional methods. It is, thus, much easier to produce desired antibodies in a large scale within a limited time because of high percentages of IgY in egg yolk (~1%). It is also more hygienic to produce IgY from egg yolk than from animal serum or colostrum. Several animal studies have shown effective protection against rotavirus by oral IgY in mice, calves, and cats (Hiraga et al., 1990; Kuroki et al., 1993, 1994). During oral administration of immunoglobulins, the most critical and considerable factor that influences induction of successful passive immunity is the delivery of large amounts of naïve and active antibodies to overcome the unfavorable digestive environment in the alimentary canal. Therefore, in this study, we investigated the stability of encapsulated immunoglobulins in the colostrum and egg yolk for effective transmission in the alimentary canal. First we estimated the transit time in the alimentary canal in mice (Fig. 1). The contrast medium was found in a connecting area between the stomach and the small intestine 10 min after administration, and then, in the small intestine 20 min, 30 min, and 3 h after administration, and finally, in the cecum 5 h after administration. During oral administration of protein-based compounds, GI residence and transit times have been recognized as crucial factors, because precise information regarding the location of compound release in situ allows the optimization of the pharmaceutical potential of oral administration (Schwarz et al., 2002). For evaluating the GI transit time, motility, and compound release, a large number of methods have been applied. Most prominent are the X-ray and scintigraphic techniques, which have been used to monitor orally ingested capsules containing radio opaque material or gamma-emitters. Noninvasive techniques, including ultrasound, metal detectors, magnetic field detectors, and dyes, have been used to avoid the adverse effects of ionizing radiation (Schwarz et al., 2002). In this study, we used radiography involving 40% barium sulfate for measuring GI transit time and it has been proven that this is an easy and non-laborious way of time-dependent tracking in small animals. The acidic environment in the stomach is a harmful condition that decreases antibody activity. For quantitative measurements of active colostrum antibody or egg yolk antibody in the alimentary canal, encapsulated colostrum with anti-HAV IgG or encapsulated egg yolk with anti-rotavirus IgY was administered to mice, and then chased along the digestive tract, respectively. The colostrum or egg yolk sample reached the stomach, the first organ in the GI tract, within 10 min after oral administration, and the antibody activity of the non-encapsulated samples was higher than that of the encapsulated ones; however, the activity levels were not maintained, and sharply decreased to levels comparable to those of the negative controls within 3.5 h and 5 h after administration (Figs. 2A and 3A). However, in the small intestine, encapsulated samples showed higher and more constant levels of antibody activity than the non-encapsulated ones (Figs. 2B and 3B). These results are in accordance with other studies. It has been reported that only 3% of antibodies were active after a 30-min incubation when the specific antibody was tested in a simulated gastric juice containing HCl with pepsin (1100 units/mL) at pH 2.0. At pH 3.0, 22–28% of specific antibodies were intact, and at pH 4.0, 25–39% were still active (Marnila et al., 2003). In the stomach, pepsin can digest
IgG into an F(ab′) fragment; however, colostrum IgG1, which is the most abundant immunoglobulin in colostrums, is resistant to this proteolytic cleavage, and can remain active in the intestine (Korhonen et al., 2000). Relatively high activity of non-encapsulated colostrum in the small intestinal tract can be explained on the basis of these previous reports. The active antibodies surviving in the stomach are usually degraded proteolytically in the small intestine by trypsin and in the large intestine by bacterial flora. Thus, the remnant activity of the antibody is undetectable in stool samples (Hilpert et al., 1987). In the case of IgY, the level of IgY activity is dependent on the amount administered, pH, enzyme activity, and passage time in the GI tract. Although IgY is proteolytically digested to Fab, Fab′2, and Fc fragments by pepsin and trypsin in the GI tract, Fab and Fab′2 fragments still have the activity to bind antigen; thus, they show neutralizing properties in the GI tract and are detectable in stool samples (Carlander et al., 2000). In this study, it was hard to detect the antibody activity of nonencapsulated egg yolk in the small and large intestines; however, high levels of activity were observed with encapsulated egg yolk. In the large intestine, encapsulated samples showed relatively higher antibody activity than the nonencapsulated ones. Non-encapsulated groups showed low antibody activity, similar to the negative controls. Interestingly, relatively big differences between the encapsulated and nonencapsulated samples were observed with colostrum administration, and time-dependent increases in activity were also observed with the encapsulated egg yolk administration (Figs. 2C and 3C). In conclusion, our results demonstrate that encapsulation of immunoglobulin can successfully protect the antibodies from gastric inactivation. The encapsulated samples retained more activity than the non-encapsulated ones, indicating that encapsulation may be an effective method for protecting immunoglobulin from gastrointestinal inactivation, enabling its use in oral passive immunization techniques for the widespread prevention of enteric pathogens.
Acknowledgments This work was supported by a grant from the BioGreen21 Program (No. PJ007176), Rural Development Administration, and the Korean Research Foundation grant (no. KRF-2008-313E00622), Republic of Korea (to J. L.). This work was funded by a research grant from Korea Yakult Co., Ltd. (to H.-J. W.).
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