Purified IB-IgY. == Inhibition on Hp urease activity by IgY in vitro == Color reaction experiment for urea reagent to detect Hp urease activity showed that two specific IgY antibodies all had inhibitory effect on Hp urease activity in vitro. vivofound that when IgY-Hp or IgY-IB with sucralfate dual oral therapy was used to treat Hp infected mouse model, the cure rate all could reach 83.3%. Conclusion: According to immunization procedure, high titer specific IgY antibody (1:12800) can be obtained in 49 days and its titer remains stable. Oral administration of the specific IgY antibodies in Hp infected mice can reach a cure rate of 83.3%, and the antibodies are expected to become new drugs and therapeutic methods of targeted therapy against Hp infection. Keywords:Helicobacter pylori, specific IgY, antibacterial effect == Introduction == Helicobacter pylori(Hp) is Gram-negative Helicobacter colonizing on the human gastric mucosa and has now been confirmed as the main pathogenic factor of chronic active gastritis, peptic ulcer, gastric mucosa-associated lymphatic tissue lymphoma and gastric cancer. In 1994, world Health Organization/International Agency for Research on Cancer (WHO/IARC) listed Hp as Class I carcinogen. Hp infection rate in the population is above 50% [1]. At present, clinical main treatment of Hp infection is combination therapy of multiple antibiotics and acid suppression drugs [2]. Although a certain treatment effect has been achieved, there are many problems, such as patients have long-term dependence on drugs, complete cure is difficult, cost is expensive, and drug-resistant strains become more and more. Therefore, development of specific targeted drugs for treating Hp infection becomes particularly important. Egg yolk immunoglobulin (IgY) is the major immunoglobulin of poultry and can be specifically gathered in the yolk through the hen. It has been reported that the specific antibody can be used to prevent dental caries, diarrhea and other infectious diseases [3]. Based on the previous research achievements [4], we prepared the anti-Hp whole bacteria IgY (SS1-IgY) and anti-recombinant Hp specific antigen IB IgY (IB-IgY), and we studied inhibition effects of the two specific antibodies on Hp growth and on Hp urease activityin vitro. After Hp infected BALB/c mouse model orally received the two kinds of specific IgY antibodies, respectively, the antibodies treatment effects on Hp were studiedin vivoso as to provide a theoretical basis for therapeutic antibody drug development of Hp infection targeted therapy. == Materials and methods == == Materials == Helicobacter pyloriSydney strain 1 (Hp SS1) came from Hebei Medical Tirbanibulin Mesylate University. Escherichia coli and Staphylococcus aureus were stored in our lab. Six-week-old SPF level BALB/c mice and 25-week-old hens were provided by Huaxi Experimental Animal Center of Sichuan University. Hp whole bacterial antigen, IB antigen and CTB adjuvant were provided by Sichuan Vaccine Technology Co., Ltd. Omeprazole was purchased from Youcare Pharmaceutical Group Co., Ltd. Sucralfate was purchased from Huanan Pharmaceutical Group Co., Ltd., Guangdong. Clarithromycin was purchased from Wang Lin Tang Pharmaceutical Co., Ltd., Sichuan Province. Canine parvovirus IgY (IgY-CPV) was provided by Sichuan Agricultural University. == Immunization == Intramuscular injection of rCTB by 400 g/hen was used as basic immunization; 7 days later, hens Rabbit Polyclonal to FOXD4 received intramuscular injection of SS1, IB antigens, 300 g/hen; then, every 14 days the additional immunization was performed up to day 91. == Preparation == SS1-IgY and IB-IgY were purified by water Ammonium sulfate precipitation. Indirect ELISA assay was used to detect titers of specific antibodies, SDS-PAGE electrophoresis was used to detect antibody purity and KD5600 UV spectrophotometer was used to detect IgY protein concentration [5]. == IgY inhibited Hp urease activity == 50 l 106CFU/ml Hp bacterial liquid was added into wells on a polyethylene plate, and then 50 l IgY antibody and negative, positive controls were added. Incubation was performed at 37C for 24 h. The 50 l rapid urea reagent was added, and the reaction lasted for 30 min at 37C. OD Tirbanibulin Mesylate values were measured at 578 nm by a microplate reader [6]. == Inhibition on IgYs Hp growth and its bacterial inhibitory specificity == 106CFU/ml Hp was uniformly smeared on the Colombia solid medium; 106CFU/ml Escherichia coli and Staphylococcus aureus were smeared on the LB solid mediums, respectively. A puncher with diameter of 6 mm was used to punch, and agar in the holes was Tirbanibulin Mesylate removed. 50 l specific IgY antibody and positive and negative controls were added to each well, respectively. After above solutions were added, Hp was placed in an anaerobic jar at 37C and was cultured for 72 h for observing inhibition ring size; plate mediums with Escherichia coli and Staphylococcus aureus.