key: cord-0033625-udssuaqp authors: Fan, Jinghui; Zuo, Yuzhu; Li, Tanqing; Zhang, Xiaobo title: Preparation and physicochemical property of chicken yolk immunoglobulin (IgY) against porcine transmissible gastroenteritis virus (TGEV) date: 2009-06-15 journal: Front Agric China DOI: 10.1007/s11703-009-0054-6 sha: 16a25bec69939f0ecf7e6679b9b862fe52b2ddc3 doc_id: 33625 cord_uid: udssuaqp Oral administration of immunoglobulin prepared from the egg yolk of hens immunized with porcine transmissible gastroenteritis virus (TGEV) has been demonstrated to reduce piglets mortality significantly in our previous studies. In the present study, we investigated the stability of chicken yolk immunoglobulin (IgY) specific to TGEV by measuring the remaining activity by ELISA. The results showed that the IgY was stable between pH 4 and pH 11. In the incubation with pepsin at pH 4 and pH 6, about 90% and 100% of the IgY activity remained, respectively. IgY activity could remain approximately 80% at 60°C for 30 min, suggesting that pasteurization can be applied to sterilizing the product. The stability of IgY at 25°C and freezing-thawing treatment indicated that the IgY was easy to be conserved. These results highlight the attractive potential application of IgY as the antibodies of oral administration for treatment of TGEV infections. Transmissible gastroenteritis (TGE) is a highly contagious viral disease of swine characterized by vomiting, diarrhea, and dehydration. The causative agent has been demonstrated to be porcine transmissible gastroenteritis virus (TGEV) . For piglets at the age of younger than two weeks, nearly 100% of them die when exposed to virulent TGEV (Pensaert, 1984; Saif and Wesley, 1999) . TGE is of considerable economic importance to large swine-breeding units. Since swine are born without immunoglobulin, the presence of TGEV-specific antibodies in the colostrum and milk of the sows is critical for the survival of the piglets (Tuboly and Nagy, 2001) . This suggests that oral administration of specific antibodies is effective in preventing intestinal infection. However, oral administration of antibodies is prohibitively expensive when large amounts of antibodies are required (Shin et al., 2002) . Therefore, it is important to search for effective methods as alternatives to maternal antibody for treatment of TGEV infections. In 1960s, a large amount of immunoglobulin was found in egg yolk and later denominated IgY (Hatta et al., 1990) . The IgY antibodies showed similar biological activities to colostral antibodies in neonatal pigs (Yokoyama et al., 1992; Yokoyama et al., 1993; Murata and Namioka, 1977) . Some studies indicated that IgY was effective, safe, and protective in intestinal infection (Sunwoo et al., 1996; Kuroki et al., 1997; Sarker et al., 2001; Hennig-Pauka et al., 2003) . These studies demonstrated that IgY from immunized chickens was an effective means against specific bacterial and viral infections. In the previous study, we produced IgY against TGEV and investigated its immunoprophylactic effect in neonatal pigs (Zuo et al., 2008) . Oral administration of the IgY antibodies has been demonstrated to reduce piglets mortality dramatically. In this study, we evaluated the potential application of IgY as an alternative method against TGEV in piglets by investigating its stability. Isolation and purification of IgY were previously described in detail by Zuo et al. (2008) . Briefly, the white leghorn hens, 25 weeks old, were immunized intramuscularly with TGEV emulsified with Freund's adjuvant. The crude antibody from yolk was extracted by the water-soluble fraction and further purified using 40% ammonium sulfate precipitation followed by dialysis. Purified IgY was filtered through 0.2 μm membrane filter and stored at -20°C before further experimentation. To assess the titers of IgY specific to TGEV, we performed the enzyme-linked immunosorbent assay (ELISA). Ninetysix-well plates were coated overnight at 4°C with purified TGEV antigen and blocked with 0.5% poly vinyl alcohol. After incubation for 2 h at 37°C, the plates were washed, and then, 100 μL volumes of appropriately diluted IgY preparations were added. After incubation, the plates were washed and incubated with HRP-conjugated goat antichicken IgG (invitrogen) for 1 h. The plates were washed again, with substrate solution added (0.04% 3, 3′, 5, 5′tetramethylbenzidine in phosphate-citrate buffer containing 0.02% H 2 O 2 ). After 10 min incubation at room temperature, the reactions were stopped with 50 μL of 2 mol$L -1 H 2 SO 4 per well, and the absorbance at 492 nm was read with an Immunoreader BIO RAD 680. The antibody titer was expressed as P/N values, where P represents the OD 492 nm of IgY from immunized hens at a given dilution and N the OD 492 nm of IgY from nonimmunized hens at the same dilution. IgY solutions were incubated at 4°C, 10°C, 25°C, 37°C, 60°C, 70°C, 80°C, and 90°C for 30 min. The heat treated IgY was cooled in a water-ice bath. The remaining antibody activity was measured by ELISA. Antibody activity was represented as a percentage of the untreated control. For the pH stability test, the pH of IgY solutions was modulated to the desired pH 2 to 12 with NaOH or HCl. After being incubated at 37°C for 2 h, each IgY solution was neutralized. The remaining antibody activity was measured by ELISA. For the pepsin stability test, the pH of IgY solution was adjusted to 2, 4, and 6, respectively. IgY solution of each pH was mixed with pepsin at a rate of 20∶1 (m/m). The mixtures were incubated at 37°C for 1, 2, 3, and 4 h. After the incubation, each IgY solution was neutralized to inactivate the pepsin. The procedure of trypsin stability test has no difference with the pepsin stability test but with the pH of IgY solution. In our test, the IgY solution was adjusted to pH 7.5. The IgY solution was treated with five cycles of freezing and thawing. The remaining antibody activity was measured by ELISA. The IgY solution was preserved at 25°C for 1, 2, 3, 4, 5, and 6 month(s), respectively. The remaining antibody activity was measured by ELISA. Eggs were produced up to three months after the first inoculation of hens with TGEV. The IgY was extracted by the water-soluble fraction (WSF) and purified by ammonium sulfate precipitation. The titer of IgY solution was examined by ELISA. The result showed that the specific antibody started to increase in the egg yolk at the 10th day and became higher after each boosting. The IgY reached its peak at the eighth week and kept at a high level until the last week during our test (Fig. 1 ). The IgY solution was stable at 37°C. However, at 60°C for 30 min, the IgY lost approximately 20% of its antibody activity. The antibody activity significantly decreased at 70°C and was almost completely lost at 80°C (Fig. 2) . IgY, specific to TGEV, showed a broad stability between pH 4 and pH 11. However, the antibody activity significantly decreased at pH 3 and pH 12. IgY lost 70% of its initial activity at pH 2 (Fig. 3) . The antibody activity of IgY was decreased rapidly when incubated with pepsin at pH 2. After 4 h of incubation, no active IgY almost existed. It can be deduced that the pepsin could facilitate the denaturation of IgY in an acidified environment (Figs. 3, 4) . By contrast, about 90% and 100% of the activity remained at pH 4 and pH 6, respectively (Fig. 4) . In trypsin stability test, IgY also showed a broad stability to trypsin. Although the antibody activity of IgY was somewhat decreased, approximately 80% of the antibody activity was still remained after 4 h of incubation (Fig. 5) . These results accorded with those obtained by Jaradat and Marquardt (2000) , who claimed that the remained IgY activity was 82%. The IgY solution was treated with five cycles of freezing and thawing. The antibody activity of IgY, as determined by ELISA, had no change. It has been found that IgG from Remaining activities after the treatments were measured using ELISA and were expressed as a percentage of the initial activity. Fig. 3 Acid stability of IgY Note: IgY was treated at various pH values for 2 h. Remaining activities after the treatments were measured using ELISA and were expressed as a percentage of the initial activity. Fig. 4 Pepsin stability of IgY Note: IgY was treated with pepsin at pH 2, 4, and 6 for 1 h, 2 h, 3 h, and 4 h. Remaining activities after the treatments were measured using ELISA and were expressed as a percentage of the initial activity. Fig. 5 Trypsin stability of IgY Note: IgY was treated with trypsin at pH 7.5 for 1 h, 2 h, 3 h, and 4 h. Remaining activities after the treatments were measured using ELISA and were expressed as a percentage of the initial activity. mammals serum was significantly affected by freezing and thawing (Na et al., 1998) . The result achieved in this test suggested the particular character of IgY. After being preserved at 25°C for six months, the remaining antibody activity of IgY was measured by ELISA. The results showed that antibody activity had no change compared with the initial activity. Similar results were obtained by Larsson et al. (1991) , suggesting that the IgY was quite stable at normal temperatures. Transmissible gastroenteritis (TGE) is an acute enteric disease of swine, most often fatal to newborn piglets (Bohl et al., 1975) . Economic loss in the swine industry resulted from intestinal diseases is extremely high. Since piglets can not mount efficiently active immunity, immunoprotection for newborn piglets is largely dependent on passive immunity through colostral immunoglobulin from the immunized dam (Cox et al., 1993; Lanza et al., 1995; Roth, 1999; Kweon et al., 2000) . However, the maternal antibody level in piglets markedly reduced at three weeks old (Zhang and Lu, 2000) . Therefore, it is necessary to enhance the passive immunity by administrating exogenous antibody (Li, 2003) . Native IgG purified from immunized mammal serum is of great cost, and its physicochemical property is unstable, which may limit their clinical application (Wang et al., 1997; Zhao et al., 2003) . Tremendous efforts have been made to seek alternatives to IgG for a more widely available means of treating, suppressing, or preventing TGEV infections. IgY is the immunoglobulin that differs from IgG. Since it was found by Williams in 1962 (Williams, 1962 , many researchers have paid special attentions to the advantages of IgY and made progress in IgY research (Hatta et al., 1993; Long et al., 1997; Na et al., 1998; You et al., 2006) . However, conflict reports are also available about the stablity of IgY. Indeed, some are contrary to others, especially the stability toward heat, pepsin, and trypsin. In order to evaluate the potential use of IgY in the prevention and treatment of TGEV infection, the stability of IgY was investigated by measuring the remaining activity by ELISA. The results showed that the IgY was stable between pH 4 and pH 11. When we tested the stability toward pepsin and trypsin, it was demonstrated that the IgY stay active when incubated with pepsin at pH 4 and pH 6 and showed a broad stability to trypsin. These results indicated that IgY could increase in its resistant to the gastric acid of piglets and the digestion of pepsin and trypsin, and it could be used as the antibodies for oral administration. IgY activity could be remained approximately 80% at 60°C for 30 min, suggesting that pasteurization can be applied to sterilize the product. The stability of IgY at 25ºC and freezing-thawing treatment indicated that the IgY was easy to be conserved. All these encouraging results together with its higher productivity and effectiveness would make the IgY be a novel approach to the management of TGEV infection in piglets. 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The authors wish to thank Doctor Rongyan Zhou for her assistance of data analysis.