KR100280332B1 - Production of egg yolk antibody(anti-K99) against fimbrial antigen of E. coli strain K99 - Google Patents

Abstract

In the present invention, egg yolk is isolated from eggs of laying hens immunized with E. coli K99 Phili antigen, diluted 9-fold with distilled water of PH 5.0, frozen at -20 ° C, and then thawed at room temperature and centrifuged to remove lipids and solids. The present invention relates to a method for separating egg yolk antibodies from egg yolk by filtration and lyophilization of the remaining supernatant. The method of the present invention enables the production of a single antibody capable of specifically binding to E. coli K99 Fili antigen. In addition, it is very effective to use yolk antibodies useful for diagnosis and treatment.

Description

Separation method of egg yolk antibody against E. coli K99 phylogeny {Production of egg yolk antibody (anti-K99) against fimbrial antigen of E. coli strain K99}

The present invention relates to a method for isolating yolk antibodies that specifically bind to diarrhea-induced E. coli K99 phylogeny antigen in livestock.

In accordance with the trend of livestock breeding and multi-dose breeding, bacterial and viral diarrheal diseases of livestock are becoming the most common and problematic diseases in livestock farms. The mortality and growth retardation caused by diarrhea in livestock are causing economic losses to breeding farmers. In particular, diarrhea caused by Escherichia coli is known to occur in almost all breeding farms in Korea. The incidence of E. coli diarrheal disease is closely related to the age of newborn livestock. When diarrhea develops, there are two types of diarrhea before and after weaning. The most common causative agents of diarrhea are K88, K99, 987P, and F41, which have K antigens. These cause the diarrheal disease in almost all livestock species, although the causative bacteria differ depending on the breeder. However, of these, K99 is a major pathogen that causes diarrhea in newborn calves. The age of diarrhea occurs in calves up to several days and no later than 1-2 weeks of age. However, diarrhea occurring at 1 to 2 weeks of age is often caused by a mixed infection with pathogenic E. coli, bovine rotavirus, bovine coronavirus and the like. The mortality rate from neonatal diarrhea is very high (10-15%) (Kim et al. 1994). In addition, after weaning, the immune antibodies transferred to the newborn calves through the mother's milk begin to drop rapidly from 2 to 3 weeks of delivery, and are almost lost after 1 month of age. Calf diarrhea caused by Escherichia coli is most common in calves less than 2 months old (Yoon, 1990).

Recently, many vaccines and antibiotics have been used to prevent and treat diarrhea caused by Escherichia coli K99 (Nagy et al., 1978). However, in the case of vaccines, the preventive effect is limited to newborn calves, and antibiotics are currently limited worldwide for antibiotic administration because antibiotics are resistant to young calves and may remain in the body when misused and abused. In this reality, it has been reported that passive immunization using maternally derived antibodies is very effective in preventing diarrhea and various diseases in the neonatal period. In particular, it has recently been reported that diarrhea prevention and treatment can be prevented when oral administration of specific antibodies to toxin antigens of E. coli to newborn calves that have been ingested with colostrum (Ozpinar et al., 1996, Yokoyama et al., 1992). Based on these results, Grand Laboratories Inc. of the United States has come to produce antisera preventive and therapeutic drugs against E. coli and Clostridium perprigens. However, antiserum agents do not have the disadvantages and sufficient amounts of antiserum that are produced at a high cost and for a limited period of time, and have a problem in that a high cost and various techniques are required for separation and purification of immunoglobulins.

Accordingly, it is an object of the present invention to provide a method for separating egg yolk antibodies that can specifically bind to the Fili antigen from egg yolks that are immunized to E. coli K99 Fili antigen using physical methods.

In order to achieve the above object, the present inventors separated egg yolk from eggs of laying hens immunized with E. coli K99 Phili antigen, diluted 9-fold with distilled water of PH 5.0, frozen at -20 ° C, and then melted and centrifuged at room temperature. And by removing the solid and the remaining supernatant was filtered and lyophilized to separate the yolk antibody from egg yolk to complete the present invention.

Hereinafter, the configuration and operation of the present invention.

1 is a photograph showing the SDS-PAGE pattern of K99 Phil in E. coli K99 ETEC.

Figure 2 is an ELISA procedure for testing the cross-linking reaction between monoclonal antibody and K99 Phil in order to investigate the antigen specificity of the isolated K99 Phil.

Figure 3 is a photograph showing the results of immunoblotting for the identification of isolated K99 Phili antigen.

Figure 4 is a separation procedure of egg yolk antibody (IgY) isolated from egg yolk produced from laying hens.

Figure 5 is an ELISA experiment procedure for examining the level of antibody production against E. coli K99 Phili antigen.

6 is a graph showing the antibody titer after immunization.

Figure 7 is an ELISA experiment procedure for assaying the specificity of the antibody produced against post-immune E. coli K99 Phili antigen.

8 is an ELISA experiment procedure for confirming the degree of reaction between the produced E. coli K99 yolk antibody and K99 ETEC cells.

The present invention comprises the steps of isolating and identifying a unique antigen from K99 pili; It is composed of the steps of producing an antibody by immunizing it to the scattering step and evaluating the antibody separately. Hereinafter, the specific configuration of the present invention will be described with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1: K99 strain culture

E. coli K99 (field) was used at the ECRC (Escherichia coli Reference Center, the Pensylvania State University, USA) and used for centrifugation in stationary culture at 37 ° C for 72 hours in 9 l of sterile Minca medium for pili separation. (15 degreeC, 5,500 rpm, 20min) collect | recovered.

Example 2 Separation and Purification of K99 Pili

The separation and purification of K99 pili was performed by modifying and supplementing the method of Erickson (1991). The recovered bacteria were suspended in 150 ml of 0.02 M PB-2.0 M urea buffer, followed by heat treatment (70 ° C., 20 min), followed by centrifugation at 20,000 × g for 20 minutes to collect the supernatant. Only the separated supernatant (crude pili) is collected, titrated to pH 4.0 with 2.5% citric acid and stirred at room temperature for 30 minutes. The acid treated solution was left in a refrigerator (4 ° C.) for 2 hours and centrifuged (20,000 g, 20 min, 4 ° C.) to obtain a precipitate containing pili. After the precipitate was suspended in PBS again, the acid treatment was repeated three times to separate and recover purified K99 pilus preparates.

Purity of the isolated K99 pilus preparates was confirmed by SDS-PAGE, and antigenicity was confirmed by Immunoblotting and ELISA reaction with monoclonal antibody.

Example 3 Qualitative Testing of K99 pili

Experimental Example 1: Protein Analysis

In order to measure the content of K99 Pili protein, Akins (1995) microwave BCA method was performed. BCA protein kit (Pierce Chemical Co.) was used as analytical reagent, and 96-well microplate (Falcon) was used.

Experimental Example 2: SDS-polyacrylamide Gel Electrophoresis

The separated K99 pili was performed by SDS-PAGE according to the method of Laemmi (1970). The electrophoresis device was a Mini-Protein II cell Kit (Bio-Rad) and the gel was 4-20% gradient Mini-Protein. II electrophoresis apparatus (Bio-Rad Laboratories, Richmond, CA, USA) was purchased and used. As a marker for measuring the molecular weight of Pili protein, Bio-Rad's Low marker and High marker were purchased and used. Electrophoresis was performed at 200V for 50 minutes and then stained using Silver Stain Plus (Bio-Rad).

The K99 pili antigen was isolated by using the 2.5% Citric acid treatment, the highest in K99 pili antigen separation method, in terms of purity and recovery, and then electrophoresed with SDS-polyacrylamide gel (12%), followed by Coomassie brillient blue R-250. As a result of staining, the main band appeared at 18kDa, and the experimental results are shown in FIG. 1. The purity of the main band was measured with a densitometer and found to be very pure and refined to more than 95%.

Experimental Example 3: Cross Reaction Investigation by ELISA Method

K99 pili isolated from monoclonal antibodies (Mab, CVL, United Kingdom) against pseudo enterococci (K99, K88a, K88b, 987P, F41) purchased to investigate antigenic specificity of isolated K99 pili Investigation of the degree of cross-linking reaction with was carried out by ELISA (Enzyme linked immunosorbant assay) (Fig. 2).

Washing buffer was used by adding 0.05% Tween20 (Sigma) to 0.15M PBS (0.2M Phosphate Buffer, 0.13M NaCl, pH 7.2), and coating was performed using Bicarbonate buffer capsules (Sigma). Blocking buffer was used 5% skim milk (pH 7.4, Gibco). Dilution buffer was mixed with PBS-T (0.05% tween 20) and 5% skim milk in the same amount, and substrate buffer was titrated to pH 5.5 with saturated citric acid in 0.1M sodium acetate trihydrate (NaAc.3H ₂ O). Used. As the primary antibody, K88a, K88b, 987P, F41, and K99 monoclonal antibodies were used at a level of 0.25 µg / µl. The secondary antibody was diluted 1,000-fold by Sheep Anti-mouse IgG (whole molecule) peroxidase conjugate (sigma). TMB (3,3 ', 5,5'-Tetramethyl benzidine, Merck) was used as a substrate for the enzyme. 3% hydrogen peroxide (Sigma) was used as a reducing agent, and 4N H ₂ SO ₄ was used as a reaction terminator. The plate was a microtest III flexible assay plate (Falcon).

In order to examine the cross-reaction between the isolated K99 pili antigen and other monoclonal antibodies (CVL, United Kingdom) of other ETEC strains (K88a, K88b, 987P, F41), the degree of binding was examined using ELISA. Likewise, K99 pili antigen showed a strong specificity only in K99 monoclonal antibody and did not react at all with other ETEC strains.

The ELISA results of cross reaction among K99 pilus protein and the monoclonal antibodies of the different E. coli strains. Fimbrial adhesin Monoclonal antibodies (CVL ^* ) K88a K88b 987P F41 K99 K99 (field) pilus adhesin - - - - +++

Co., Ltd. *: Purchased from CVL (United Kingdom)

Experimental Example 4: Immunoblotting

After electrophoresis of the isolated K99 pili antigen with SDS-PAGE (12%), Gershoni and Palade (1983) using Mini Trans-Blot Cell of Bio-Rad to confirm whether the band shown at about 18 KDa is K99 pili Immunblotting was performed by the method of. Membrane was used as nitrocellulose paper (Bio-Rad, Trans-Blot Transfer Medium), and 200-fold dilution of K99 monoclonal antibody (CVL, England) as the primary antibody. Secondary antibody was used by AP-conjugated anti-mouse IgG (Jackson, USA) at 1,000 times. NBT (5-bromo-4chloro-indolyl phospate, Sigma) and BCIP (ρ-nitro blue tetrazolium chloride, Sigma) were used as colorants.

As a result of immunoblotting, it was confirmed that the main band was K99 pili as shown in FIG. 3.

Example 4 Production of K99 pili Antibodies

The animals used for antibody production were two rabbits of Newzealand white species weighing 2.5 kg and a five-week-old Hy-line brown laying hen. Immunity method is as follows. 150 µg of the isolated K99 pilus antigen was mixed with 0.5 ml of Saline and emulsified with 0.5 ml of Freund's complete adjuvant (FCA). The emulsion was injected into the rabbit's back and hind limbs with a total of 1 ml subcutaneous and intramuscularly injected. The laying hens were inoculated first with 4 ml of 0.25 ml each to the pectoral muscle, and booster injection was given 2 weeks after the first vaccination. At intervals up to 12 weeks after emulsification with Freund's incomplete adjuvant (FIA), the same procedure as the first inoculation was performed.

Blood sampling was done weekly. Rabbits were used for experiments by collecting blood from the vein of the vein and laying hens, separating blood serum, and storing it at -20 ° C. Eggs were collected every day and stored at 8 ℃ was used for the experiment.

Egg yolk antibodies were isolated weekly from egg yolk produced from laying hens. Separation of the IgY mixture was performed by modifying and supplementing the method of Akita and Nakai (1993) and Jensenius et al. (1993). The modified separation method was performed as shown in FIG. 4 based on the physical method excluding chemicals as much as possible.

That is, egg yolk was isolated from eggs of laying hens immunized with E. coli K88 Phili antigen, diluted 9-fold with distilled water of PH 5.0, frozen at -20 ° C, and then thawed at room temperature and centrifuged to remove lipids and solids. The supernatant was filtered and lyophilized to separate the yolk antibody from the yolk.

Example 5: Antibody Determination and K99 Antibody Specificity Test

Experimental Example 5: Antibody Value Measurement

Antibody production level investigation for K99 pilus antigen was carried out by ELISA (Fig. 5). Washing buffer was used by adding 0.05% Tween 20 (sigma, P-1379) to 0.15M PBS (0.2M Phosphate Buffer, 0.13M NaCl, pH 7.2) and Bicarbonate buffer capsules (Sigma). Blocking buffer was used 5% skim milk (pH 7.4, Difco). As dilution buffer, PBS-T (0.05% tween 20) and 5% skim milk were mixed in the same amount, and substrate buffer was added to 10% Diethanol-Amine (C ₄ H ₁₁ NO ₂ , Sigma) with 0.5 mM MgCl ₂ . Using a solution titrated to pH 9.8.

Enzyme was diluted 5,000 times with Alkaline Phosphate-conjugated AffiniPure Rabbit anti-chicken IgY (IgG) (Jackson Immuo.) And Alkaline Phosphate-conjugated AffiniPure Donkey anti-rabbit IgG (Jackson Immuno.). Phosphate substrate tablets (Phosphatase, Sigma-104) were used. 5 M NaOH was used as a reaction inhibitor. The plate was a microtest III flexible assay plate (Falcon 3991). IgY samples isolated from laying hens and rabbit antiserum (IgG) or egg yolk were used for the antibody measurement, diluted from 200 to 3 times to 145,800 times and the antibody was used for the measurement.

As shown in FIG. 6, the serum of rabbits increased rapidly at 10 weeks of age and maintained. Laying egg serum gradually increased from 4 weeks of age to the highest antibody titer at 10 weeks of age, and showed a tendency to maintain. The yolk antibody titer gradually increased from 4 weeks of age to 16 weeks, and from 12 weeks of age, the yolk antibody titer maintained higher serum serum levels. The formation of antibodies to the K99 antigen began to occur at 2 weeks of age in the serum of laying hens, and then continued to increase. Antibody formation in egg yolk began to occur 4 weeks after immunization 2 weeks later in serum and continued to increase. On the other hand, after 8 weeks of age, the antibody titer of egg yolk was maintained at the same level or higher than that of serum.

Experimental Example 6: Specificity test of the produced K99 antibody

To investigate the specificity of antibodies against K99 pilus antigen produced in post-immune rabbit serum, laying hen serum (IgG) and laying hen egg yolk (IgY), K99 (field), K12: K99, K88 +, K88ab, 987P pilus antigen The reaction degree was observed using. The buffer used was the same as the antibody used for the measurement, the plate was used 96-well immuno-microplate (NUNC, Denmark) (Fig. 7).

987P, K88 +, K88ab, K99 (field), K12: K99 pili antigen-coated plate, egg anti-K99 antiserum and egg yolk antibody (di-K99 yolk antibody) diluted 20,000 times Alkaline Phosphate-conjugated Affini Pure Rabbit anti-chicken IgY (IgG) (Jackson Immuo.) And Alkaline Phosphate-conjugated Affini Pure Donkey anti-rabbit IgG (Jackson Immuno.) Were used as a secondary antibody. Phosphate substrate tablets (Phosphatase, Sigma-104) were used as the substrate for the enzyme. 5 M NaOH was used as a reaction inhibitor.

As a result, the serum antibody and yolk antibody produced in the present invention showed specific reaction to K99 and K99ab strains as shown in Table 2, and the specificity for K99 antigen was confirmed.

Specificity of chicken serum, rabbit serum and yolk anti-K99 antibody against the fimbrial antigen of the different E. coli strains by ELISA. Fimbrial antigen Host antibody Rabbit serum Chicken serum Egg yolk K88 (field, Manitoba *) K88ab (ECRC **) 987P (ECRC) K99 field (ECRC) K12: K99 (ECRC) --- ++++++ --- ++++++ --- ++++++

Host antibodies were diluted to 1: 20,000.

+++: 50,000 <antibody titer <100,000

500: antibody titer

Manitoba: Animal health center, Veterinary Sevices branch, Manitoba

Agricultrue, MB, Canada

ECRC: E. coli reference centre, the pensylvania state university,

USA.

Example 6: Investigation of antigen binding capacity of yolk antibody (IgY)

To indirectly investigate the effect of K99 yolk antibodies in vivo, the reaction of K99 ETEC cells with yolk antibodies was performed by ELISA.

10 ml of K99 ETEC cells diluted to 1 × 10 ⁹ (CFU / mL) concentration were dispensed into sterile test tubes, and then added so that the IgY concentrations were 10, 20, 30, 40, 60, 80, and 100 mg, respectively. The group without addition of IgY was used as a control. After addition, shaking culture (60rpm / min) at 37 ℃, after addition, shaking culture (60rpm / min) at 37 ℃, 1mL every 2 hours, 4 hours, 8 hours, 12 hours, 24 hours was collected and 70 ℃ After heat treatment for 20 minutes at refrigerated storage was used in the experiment. The degree of reaction was carried out as shown in Figure 8 using a competitive sandwich ELISA method.

As shown in Table 3, the 10 mg (IgY) treatment group showed a sharp decrease after 2 hours of incubation, and then gradually decreased. In the 20 mg (IgY) treatment group, 2 hours after incubation From then on, it decreased sharply and did not decrease any more. On the other hand, in the 20 mg (IgY) or more treatment group was found to be almost no difference with the 20 mg (IgY) treatment group, which was also effective at the level of about 2 mg / ㎖. As a result, it is thought that the activity of ETEC cell can be reduced in the amount of 2 mg (IgY) in vivo.

The changes of K99 ETEC cell concentration according to different treatment levels and Incubation time. Treatment Cell concentration (CFU / mL) 2 hours 4 hours 8 hours 12 hours 24 hours Control10mg of IgY20mg of IgY30mg of IgY40mg of IgY60mg of IgY80mg of IgY100mg of IgY 1.9 × 10 ⁹ 6.9 × 10 ⁶ 3.3 × 10 ⁶ 3.5 × 10 ⁶ 3.5 × 10 ⁶ 3.4 × 10 ⁶ 3.6 × 10 ⁶ 3.6 × 10 ⁶ 1.9 × 10 ⁹ 5.4 × 10 ⁶ 3.3 × 10 ⁶ 3.4 × 10 ⁶ 3.4 × 10 ⁶ 3.3 × 10 ⁶ 3.7 × 10 ⁶ 3.5 × 10 ⁶ 1.6 × 10 ⁹ 4.9 × 10 ⁶ 3.5 × 10 ⁶ 3.8 × 10 ⁶ 3.4 × 10 ⁶ 3.3 × 10 ⁶ 3.6 × 10 ⁶ 3.9 × 10 ⁶ 1.8 × 10 ⁹ 3.3 × 10 ⁶ 3.5 × 10 ⁶ 3.5 × 10 ⁶ 3.3 × 10 ⁶ 3.5 × 10 ⁶ 3.9 × 10 ⁶ 3.6 × 10 ⁶ 1.9 × 10 ⁹ 3.7 × 10 ⁶ 3.6 × 10 ⁶ 3.1 × 10 ⁶ 3.6 × 10 ⁶ 3.1 × 10 ⁶ 3.4 × 10 ⁶ 3.4 × 10 ⁶

Example 7 Purity of Antibodies (IgY) Isolated from Egg Yolk

Purity of the yolk antibody was carried out through freeze drying and spray drying depending on the physical condition and purpose of the pretreated antibody material. According to the process of Figure 4 from the yolk yolk protein containing egg yolk protein fraction (yield yolk antibody) was separated and used for lyophilization while frozen and stored at -20 ℃. The frozen yolk antibody was mounted in a freeze dryer (ILSIN Engineering; NCFD 5508) adjusted to -50 ° C. and powdered by drying for 2 to 3 days in a vacuum state (10 micron Hg). In order to measure the purity of IgY in lyophilized powder, IgY content and antibody titer before and after lyophilization were measured. The immunological component test was performed by measuring the concentration of IgY by RID (Radial Immuno-Diffusion) method, the protein test was performed by the BCA method, and the antibody titer was measured using the ELISA method of FIG. The powdering of whole egg yolk by spray drying method is not only egg yolk itself can be used as a rich source of nutrients for young livestock, but also it is time and economical by omitting many steps in the process of separating the antibody. It was carried out under the hypothesis that it would have the advantage of simultaneously utilizing the effect of the antibody contained in egg yolk while minimizing the burden. The volume of egg yolk and the capacity of distilled water were diluted at a ratio of 2.5: 1.0 before the spray dryer, and the conditions of the spray dryer were adjusted as follows. The input temperature of the diluted yolk solution was 140 ° C., the injection temperature was 72 ° C., the dosage per hour was 5 L, and the pumping speed was 22,000 rpm. IgY antibody concentrations, antibody titers and protein concentrations before and after spray drying of egg yolk were measured to investigate the contents and titers of antibodies lost during spray drying.

As shown in Table 4, the IgY purity and the antibody of the lyophilized powder showed a protein content of 7.2 mg in 10 mg of the lyophilized powder, and the content of IgY was 3.3 mg, representing 45.8% of the total protein as IgY. . The purity of IgY in the total dry powder was 33%.

Antibody values of yolk antibodies before and after lyophilization were 750,000, indicating no difference between these levels. Therefore, the titer of specific antibody following lyophilization was found to be unchanged. Considering the industrial level, the frozen antibody powder prepared in this experiment was very high in IgY and high antibody value. In addition, the possibility of industrialization in the future is very bright because it is an antibody material necessary for the diagnosis of the presence or absence of K99 antigen from various specimens and the production of ELISA Kit for quantitative measurement of K99 coli.

Meanwhile, as shown in Table 4, the purity and antibody value of the spray-dried powder were obtained from 186.5 g of yolk powder from 900 mL of whole egg yolk, and the protein content of 70 mg of yolk powder was 20.9 mg, of which The content of IgY was 3.45 mg, 16.5% of the total protein was IgY. The purity of egg yolk powder was 4.6%. The antibody titers of egg yolk powder before and after spray drying were 750,000 and 620,000, respectively, and the titers of antibody after drying showed about 130,000 lower titers than before drying. % Was found to be lowered. However, in view of the industrial considerations, the egg yolk powder can be used as a feed additive for livestock to obtain a nutritional effect as well as to obtain a complex effect with an antibody effect as a diarrhea preventive agent. The possibility of industrialization is very bright considering that the cost of separation can be greatly reduced. Once industrialization is in full swing, lyophilization can be used efficiently to reduce production of antibody titers, depending on the purpose of use.

The recovery contents of egg yolk protein, IgY and antibody titer treated by different drying methods. Items Methods of drying Freeze drying Spray drying Initial volume of Egg yolk 15 ml 900 ml Weight of yolk powder produced 460 mg (100%) ^a 186.5 g (100%) Protein content 331.2 mg (72%) 52.2 g (28.0%) IgY content 151.8 mg (33.0%) 8.4 g (4.6%) Antibody titer (K99ETEC) Before treatment 750,000 (100%) b After treatment 750,000 (100%) 620,000 (82.7%)

A: Recovery rates of protein and IgY contents over the weight of yolk

powder

b: Percent changes of the antibody titer levels before- and after

treatment

As described in accordance with the above embodiments, the present invention provides an economical method for isolating egg yolk antibodies against K99 strainer-specific pili antigen, one of the enterocoliform Escherichia coli, which is useful for preventing, diagnosing and treating diarrhea in livestock. It has a very excellent effect to provide a very useful invention in the animal medicine industry.

Claims (1)

Egg yolk was isolated from eggs of laying hens immunized with E. coli K99 Phili antigen, diluted 9-fold with distilled water of PH 5.0, frozen at -20 ° C, and then thawed at room temperature and centrifuged to remove lipids and solids. Separation of the yolk antibody, characterized in that to separate the yolk antibody from the yolk by filtration and lyophilization.

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