JP3114060U - Antibody test kit using Salmonella antigen formulation - Google Patents

Abstract

An antigen formulation comprising a SE FliC9 kDa polypeptide or a complex thereof, and serologically testing SE infected chickens and currently commercially available SE inactivated vaccinated chickens, and a test kit including the same, and the antigen Of SE subunit vaccine with low stress responsiveness.
A support in which an antigen consisting of a SE9 kDa polypeptide in a 53 kDa polypeptide, which is a main antigen of Salmonella flagellar antigen, or a complex thereof is supported on a support in a single container, and various controls. Each reagent container contains serum, serum diluent, conjugate diluent, and orthophenylenediamine (OPD) diluent.
[Selection] Figure 1

Description

  The present invention relates to a test kit for distinguishing between field-infected animals and vaccinated animals using a Salmonella antigen formulation.

  Salmonella bacteria are known to be pathogenic in both humans and animals. In particular, Salmonella enteritidis (hereinafter referred to as SE) has attracted attention as one of the causative bacteria of human food poisoning since the late 1980s. As a result of many studies, it has become known that SE is contaminating shell eggs produced in poultry farms, and SE countermeasures in poultry farms (egg farms) are regarded as important.

  In recent years, in addition to improving general hygiene management as an SE measure for poultry farms, attempts have been made to inoculate SE-inactivated vaccines at an early age of hens. However, this case has the following problems.

(1) When SE-inactivated vaccine is inoculated, SE-specific antibody is produced, but this produced SE-specific antibody cannot be serologically distinguished from natural infection of field SE.
(2) The currently inactivated SE inactivated vaccine uses an antigen in which cells are inactivated, that is, an SE cell that has been killed by formalin as a central antibody. (Enterotoxin), and it is known that vaccination causes various side reactions (eg, inhibiting chicken weight gain). Among inactivated vaccines, oil-based adjuvant vaccines that are known to induce an excellent immune response also have the disadvantage of inducing stress in inoculated chickens.
(3) Currently, poultry farms are contaminated with Salmonella species other than SE, such as Salmonella typhimurium, Salmonella Infantis, etc. Although development is desired, it is difficult to mix many Salmonella cells, giving chickens more stress than necessary and securing the necessary amount of antigen stably. Multivalent vaccines using body antigens are considered difficult to manufacture.

  In order to solve the above problem (1), the present inventors have already made a difference in the types of antibodies produced between SE-inactivated vaccinated chickens and SE-infected chickens against various SE surface antigen substances. It has been clarified that there is a difference between SE inactivated vaccinated chickens and SE infected chickens against flagellar antigen, one of the SE surface antigens.

As surface antigens of SE, 14 kDa, 17 kDa and 23 kDa polypeptides are known as ciliary antigens, which have been studied as antigens of subunit vaccines, respectively, but are still reported to be effective antigens as vaccines. Not.
On the other hand, flagella is regarded as a suitable detectable marker antigen for Salmonella vaccines and retains flagella in the wild-type form, but has the ability to induce antibodies against at least one of flagellin or flagellar antigenic determinants. There is a description of a vaccine that utilizes a Salmonella bacterium that no longer exists (see, for example, Patent Document 1).

On the other hand, with respect to the problem due to the adjuvant of the problem (2), for example, in Patent Document 2, there is an attempt to solve by a vaccine formulation using a hydrophilic oil-based adjuvant to form a water-in-oil-in-water emulsion. Although described, the SE inactivated vaccine contains a large amount of antigens that killed the cells, so as shown in problem (3), it is difficult to produce a multivalent vaccine for the entire genus Salmonella. Has been. As a solution to this, it is considered useful to extract only effective antigens from SE cells and create a multivalent vaccine as a subunit vaccine. However, effective SE antigens as subunit vaccines have been reported so far. It has not been.
JP 2001-186874 A JP 2002-80396 A

  There is a strong desire to develop a vaccine that can easily distinguish between vaccinated chickens and field-infected chickens, and has a low-stress SE-inactivated vaccine or a single-unit or multi-subunit Salmonella vaccine containing an effective antigen of SE. However, no effective one has been confirmed yet. In light of this, the present inventors have conducted extensive research. As a result, a part of the SE53 kDa polypeptide (SEFliC), which is the main flag of SE flagellar antigen, FliC (major flagellar antigen) 9 kDa The peptide was found to be an antigen that can specifically detect antibodies produced by SE inactivated vaccines inoculated into chickens, and using this SEFliC9 kDa polypeptide or complex thereof, currently commercially available SE inactivated vaccinated chickens It is an object of the present invention to provide a test kit using an antigen formulation for serologically detecting.

The present invention is used to serologically test field-infected chickens and vaccinated chickens by the enzyme antibody method, and is an antibody against Salmonella in a fluid sample comprising a serum sample, an egg yolk sample or a body fluid and a tissue extract. A support for carrying a test kit for detecting a SE9 kDa polypeptide in a 53 kDa polypeptide as a main antigen of the flagellar antigen of Salmonella species or a complex thereof in a single container And various control sera, serum diluents, conjugate diluents, and respective reagent containers each containing an orthophenylenediamine (hereinafter referred to as OPD) diluent.
This makes it possible to serologically accurately distinguish between field-infected chickens naturally infected with Salmonella and vaccinated chickens infected by SE-inactivated vaccination.

  At this time, the support on which the antigen comprising the SE9 kDa polypeptide or a complex thereof is carried is supported as a fusion protein in which the 9 kDa polypeptide is fused with glutathione-S-transferase (hereinafter referred to as GST). It may be considered to have stabilized on the body.

  According to the present invention, a support carrying a FliC9 kDa polypeptide or a complex thereof confirmed to have a serological difference between SE-inactivated vaccinated chickens and SE-infected chickens is set with each reagent. This makes it possible to easily distinguish between SE-inactivated vaccine-inoculated chickens and SE-infected chickens.

Embodiments of the present invention will be described below with reference to the drawings.
FIG. 1 is an accommodation state diagram of a test kit 1 showing an embodiment of the present invention. 2 is a container, in which a support 3 carrying an antigen for confirming serological differences between SE-inactivated vaccinated chickens and SE-infected chickens, various control sera and serum Each reagent container 4 containing a diluted solution, a conjugate diluted solution, and an orthophenylenediamine (hereinafter referred to as OPD) diluted solution is accommodated.
The SE fliC gene used in the antigen formulation of the present invention encodes FliC (53 kDa), which is the main protein that constitutes the flagella of SE, is composed of 1518 base pairs, and is composed of 505 amino acids. ing. In addition, comparing the amino acid sequence of SE with the amino acid sequences and base sequences of the N-terminal and C-terminal of other Salmonellas, it was confirmed that a fairly high homology exists. Therefore, in the present invention, a specific region (9 kDa polypeptide portion) of SEFliC was isolated and purified by genetic engineering, and the SE9 kDa polypeptide or a complex thereof was purified as follows.

A. Isolation and analysis of SE fliC gene Amplification and cloning of fliC by PCR method PCR primer SEF and primer SER were designed based on the known nucleotide sequence. Genomic DNA was purified from 15 strains of cells and amplified by PCR using this as a template. PCR conditions were as follows: denaturation 94 ° C., 30 seconds, annealing 60 ° C., 30 seconds, amplification 72 ° C., 90 seconds, and 30 cycles. The amplified DNA fragment was confirmed by agarose gel electrophoresis (1% agarose gel) (see FIG. 2). In order to remove and purify the impure DNA in the amplified PCR fragment (DNA), the amplified gene was subcloned into the pCR2.1T vector, replicated in E. coli, and cloning was attempted.

2. Determination of base sequence of fliC gene After cloning, one of the 4 clones isolated was used as a template, and primers SEF, SER, SER3 and SER4 were used to determine the base sequence. The nucleotide sequence was determined by the dye terminator method using a DNA autosequencer ABI310 (Aplied Biological Instrument). The cycle sequence reaction conditions were 25 cycles of denaturation 96 ° C., 10 seconds, annealing 50 ° C., 5 seconds, amplification 72 ° C. and 4 minutes.

B. 2. Expression and purification of the fusion protein Preparation of expression clone An attempt was made to clone a region from 735 bp to 1010 bp in the base sequence of the fliC gene which is specific to SE. Designing attB1SEFepi and attB2SERepi as attB adapter primers for amplifying 733 bp to 1008 bp, and using the clone whose base sequence of the fliC gene was determined as a template, denaturation 94 ° C., 30 seconds, annealing 55 ° C., 30 seconds, amplification 72 ° C., 30 seconds PCR was carried out under conditions of 30 cycles. The obtained DNA fragment was connected to a donor vector (pDONR ^™ ) containing an attP site. Thereafter, E. coli (DH5α) is transformed, it is confirmed that an entry clone has been constructed, this entry clone is transferred to a destination vector (pDEST ^™ 15), and transformed into E. coli (BL21-S1 ^™ ). An expression clone was prepared (see FIG. 3). The above vector and E. coli were from Invitrogen.

4). Next, we tried to express a large amount of the fusion protein and purify it.
1 ml of an overnight culture of the transformant is prepared, added to 100 ml of LB liquid medium, and cultured at 37 ° C. for about 6 hours until the OD ₆₀₀ becomes 0.5, and then the final concentration becomes 0.3M. Then, sodium chloride was added and cultured at 30 ° C. for 12 hours.

5. Purification of fusion protein Collect the above culture broth, add 250 μl of protease inhibitor (manufactured by SIGMA), add Triton X-100 to a final concentration of 0.2%, ethanol bath and water bath (60 ° C.) The cells were frozen and thawed three times to disrupt the bacteria. The disrupted bacteria were centrifuged (13,500 rpm, 10 minutes, 4 ° C.), and the centrifuged supernatant was passed through a GSTrap FF 5 ml column (Amersham Pharmacia Biotech) to bind the fusion protein to the carrier in the column. Elution was performed with 5 ml of 10 mM reduced glutathione (50 mM Tris-HCl, pH 8.0). The eluted fusion protein was dialyzed with PBS to obtain a purified sample.

  The test kit 1 in the present invention is for detecting antibodies against Salmonella in a serum sample, egg yolk sample or other fluid sample by enzyme antibody method, and the above Salmonella antigen prescription is contained in one container 2. In addition to the support 3 coated with a product, it comprises a reagent container 4 containing various control sera, serum dilution, conjugate dilution and OPD dilution, respectively. As the control serum, Salmonella positive and Salmonella negative non-cross reactive sera are used. Particularly advantageous for Salmonella antibody quantification are control sera obtained from Salmonella positive animals immunized by field infection or vaccination.

The subunit vaccine in the present invention is composed of an antigenic substance comprising the FliC9 kDa polypeptide antigen or a complex thereof and a pharmaceutically acceptable carrier.
Hereinafter, each reagent and sample will be described in more detail on the assumption that the above Salmonella antigen formulation is used in the ELISA method, but the present invention is not particularly limited thereto, and may be used in other enzyme antibody methods. .

  The support 3 (hereinafter referred to as ELISA plate) is obtained by coating a GST9 kDa polypeptide antigen on a microplate. That is, the ELISA plate was prepared by thawing the GST9 kDa polypeptide purified as described above, diluting it with a coating buffer so that the protein concentration was 0.05 μg / well, and coating the microplate well at 2 to 5 ° C. overnight. It was prepared by blocking with 1% bovine albumin-PBS (sterile phosphate buffered saline) at room temperature for 1 hour, and then removing the blocking solution and sufficiently drying and sealing the well. The coating buffer used contained 2.93 g of sodium bicarbonate, 1.59 g of sodium carbonate and 0.2 g of sodium azide in 1000 ml, and was adjusted to pH 9.4 to 9.6.

  At this time, by using an antigen formulation in which a FliC 9 kDa polypeptide is fused with GST, the antigen formulation can be easily coated on a microplate, and the prepared ELISA plate has good stability and uniformity. A highly sensitive coating was obtained. In order to maintain detection accuracy, the ELISA plate is preferably stored in a dark place at 2 to 5 ° C. until use.

  Salmonella positive serum is prepared from chickens immunized with SE inactivated vaccine (Layer Mun SE). 21 to 70-day-old SPF chickens are inoculated with 1.0 dose subcutaneously of layer Mun SE and raised in an isolator. Blood will be collected 4 weeks after vaccination, serum will be separated and immobilized at 56 ° C for 30 minutes. An agglutination reaction (PD-RPA) is performed with a commercially available chick dysentery diagnosis antigen, and serum showing an aggregation value of 8 times is diluted 500 times. This is preferably stored at −40 ° C. until use, but may be freeze-dried.

  Salmonella negative serum is prepared from 21-70 day old SPF chickens. After blood collection, sera are separated and immobilized in the same manner as the above Salmonella positive serum, confirmed to be negative with PD-RPA, pooled, and diluted 10-fold with fetal bovine serum. 100 μl of this is dispensed into 5 ml vials and lyophilized. Moreover, it is preferable to preserve | save in a 2-5 degreeC dark place until use.

Serum dilution is 1000 ml by adding 10 ml of fetal bovine serum, 1 ml of polysorbate 20, 0.02 g of sodium azide, 0.3 ml of 1% phenol red and 1 ml of gentamicin to sterile phosphate buffered saline (hereinafter referred to as PBS). , Dissolved using a stirrer, filtered and sterilized, then divided into sterilized containers and stored at 2-5 ° C.
The conjugate diluted solution is adjusted to 1000 ml by adding 2 ml of polysorbate 20 to PBS, 1 ml of fetal calf serum and 0.05 g of malachite green, dissolved using a stirrer in the same manner as the serum diluted solution, sterilized by filtration and sterilized. Store at 2-5 ° C.

  The OPD diluent was adjusted to 1000 ml by adding citric acid-hydrate 21.01 g, anhydrous phosphoric acid-sodium hydrogen phosphate 28.40 g, 30% hydrogen peroxide water 1 ml to distilled water, and after dissolution, 2-5 ° C. Save with.

The inspection procedure by the ELISA method using the reagent adjusted as described above is shown below. At this time, each reagent is returned to room temperature before use.
First, an inspection sample is prepared.
As a sample, chicken serum or egg yolk is used. Take 0.6 ml of the sample with a 1 ml syringe in a micro test tube, add an equal volume of serum diluent, mix well, and make a primary dilution of the sample (2-fold dilution). Next, remove the sample dilution microplate (FALCON tissue culture microtest plate flat bottom, low-evaporation type, with 96-well lid) in rows 1 to 8 and remove serum dilution at 250 μl / well. Dispense and dispense 4 μl / well of the primary dilution of the sample and thoroughly agitate with a plate mixer for 20 minutes to prepare the secondary dilution of the sample (125-fold dilution).

1. Reaction with primary serum (reaction with sample)
Salmonella-negative serum (500-fold dilution) is dispensed in rows 1 to 4 of stage A of the dilution microplate, and Salmonella-positive serum (1000-fold dilution) is dispensed in rows 5 to 8. On the other hand, 75 μl of serum dilution was dispensed into all wells except the A-stage 1st to 8th rows of the ELISA plate carrying the GST9 kDa polypeptide antigen, and 25 μl of the secondary dilution of the above sample was added (final dilution). Becomes 500 times). Further, 100 μl each of Salmonella negative sera in rows A to 1-4 of the ELISA plate and Salmonella positive sera in rows 5 to 8, is allowed to react with the antigen for 30 minutes at room temperature.

2.2 Reaction with secondary serum (reaction with conjugate)
After the reaction of 1 was completed, the reaction solution was discarded, the wells were washed 3 times with distilled water to remove water, and then the enzyme-labeled anti-chicken IgG (H + L) rabbit serum was diluted 3000 times with conjugate diluent. Dispense 100 μl into all wells and allow to react at room temperature for 30 minutes.

3. Reaction with substrate (color development)
After the reaction in step 2 is completed, the reaction solution is discarded, the well is washed three times with distilled water, water is removed, and orthophenylenediamine is appropriately diluted with an OPD diluent so that the concentration becomes 0.05 mg / well. Dispense into each well and allow to react for 10 minutes at room temperature.

4). Stopping the reaction After the reaction in 3 is completed, 100 μl of 3N sulfuric acid is dispensed into all wells to stop the color development.

5. Determination of antibody titer After stopping the reaction in 4, the absorbance (OD) of each sample is measured at 490 nm with a plate reader, and the m-OD value (corrected OD value) is calculated by the following equation.
m-OD value = (S-NC) / (PC-NC) (S: OD value of sample, PC: OD value of Salmonella positive serum, NC: OD value of Salmonella negative serum)
Hereinafter, the present invention will be described in detail by way of examples.

-Trial as marker detection system and setting of positive standard in m-OD value Three SE inactivated vaccination groups were used, and samples were collected as follows.
(1) Eighteen eggs from a 192-day-old chicken group inoculated with a commercially available SE inactivated vaccine (Layer Mune SE-NB) at 81-93 days of age, (2) 49-54 days and 70-89 days of age 19 chicken eggs from a 420-day-old chicken group inoculated with Layer Mun SE, and (3) 22 chicken eggs from a 690-day chicken group inoculated with Layer Mun SE at 57, 58 days and 96-99 days old. Collected. Conventional CAF-SE Elisa (manufactured by our company) used as a conventional SE antibody detection test method and rapid plate agglutination reaction (PD-RPA) using chick dysentery diagnosis antigen coated with 9 kDa polypeptide I went with the law. The results are shown in Tables 1 to 3 (Table 1: 192 days old, Table 2: 420 days old, Table 3: 690 days old).

  As a comparative example, antibody detection was performed in the same manner as in Example 1 on 100 yolks collected from a farm where SE was separated from the environment about one year ago. The results are shown in Table 4.

  Here, the positive standard in the above-mentioned m-OD value is a positive rate of 50% or more in a test using egg yolk derived from a 650-720 day-old chicken group in the SE inactivated vaccination group. In addition, all the tests were negative in the test using egg yolk derived from an SE-contaminated farm and egg yolk derived from a purchased SPF chicken group (collected from eggs purchased from SPAFAS).

・ Results and discussion It became clear that the SE specific antibody detection rate in egg yolk using CAF-SE Eliza decreased with age, and in PD-RPA method, one case was positive in egg yolk derived from a 192-day-old flock. However, in the ELISA plate of the present invention, when the m-OD value is 0.1 or more positive, the egg yolk derived from 192-day and 420-day-old flocks is used. The positive rate of 95% was positive, and the positive rate was about 70% even for egg yolk derived from a 690-day-old chicken group. In addition, in the yolk from the SE isolation farm, there was no sample showing an m-OD value of 0.1 or more in the ELISA plate of the present invention, and the maximum m-OD value of 100 samples was 0.07. It was. Further, although not shown in the table, the purchased SPF chicken-derived egg yolks were all negative in the ELISA plate of the present invention. Similar results were obtained when serum was used instead of egg yolk.
Based on the above, the ELISA plate of the present invention is considered to enable use as marker detection, and an m-OD value of 0.1 or more is set as a positive reference.

  Cloacas swab, blood and eggs were collected in about 10 times the volume of Hanatetrathionate basal medium (HTB), cultured at 37 ° C. for 48 hours, smeared on MLCB, and after 48 hours of culture, SE positive was determined. The colonies thus obtained were transplanted to tryptosoya agar medium, cultured for 24 hours, and then reacted with Salmonella immune serum O9 group to confirm the presence or absence of aggregation. The oviducts collected aseptically at the time of necropsy were ligated on one side and the eggs being formed were removed, then HTB was added from the other side and the contents were well taken out, and the washing solution was used as a sample. The liver and spleen were collected aseptically from 5 g each, and then homogenized by adding about 10 times the amount of HTB. These were enriched and cultured at 37 ° C. for 48 hours, then streaked on MCLB, and after 48 hours, SE positive was determined. The colonies thus obtained were transplanted to tryptosa agar medium, cultured for 24 hours, and then reacted with Salmonella immune serum O9 group to confirm the presence or absence of aggregation.

Results and Discussion As a result of SE separation from each sample, as shown in Table 5, test No. In 1 and 2, SE isolation was observed at least once from all the chickens in the inoculated group, but an individual in which SE isolation was not observed after 2 weeks or later was tested. In No. 1, two birds, test no. In 2, there was one bird.
From the egg contents and blood, as shown in Tables 6 and 7, SE separation was not observed at any time.
From the cecum contents, liver, spleen, and oviduct, as shown in Table 8, only from the cecal contents, Test No. SE separation was observed in 2 out of 5 of 1. In the control group, all samples were negative.
Further, as a result of examining antibodies in blood and egg yolk, as shown in Tables 9 to 12, in CAF-SE Eliza, two groups of sample Nos. Blood samples 13, 14 and 15 were positive at 1, 2, and 4 weeks later, and sample No. 11, 14 and 15 yolks were positive at 2, 4, and 6 weeks later, slightly later than positive in blood. On the other hand, in the ELISA plate coated with the 9 kDa polypeptide of the present invention, all specimens were negative.
From the above, the antibody detection method of the ELISA plate coated with the GST9 kDa polypeptide in SE-administered laying hens is negative for both serum and egg yolk. It is judged that it is suitable for the marker detection method in the examination using egg yolk.

Test using commercially available SE-inactivated vaccine-inoculated chicken-derived serum It was examined whether or not a commercially available SE-inactivated vaccine has the ability to produce antibodies against a 53 kDa polypeptide.
Commercially available SE inactivated vaccine A was inoculated at 0.5 ml / five in five 26-day-old SPF chickens, B was inoculated at 0.5 ml / fowl in 10 31-day-old SPF chickens, and C was in 33-day-old SPF chickens. 5 inoculate 0.5 ml / wing, D inoculate 0.25 ml / wing into 5 33-day-old SPF chickens, E inoculate 0.25 ml / wing into 4 33-day-old SPF chickens, and inoculate each. Using the serum obtained after 4 weeks, ELISA reaction using CAF-SE Elisa and the ELISA plate of the present invention, and Western Blotting (WB) reaction using 53 kDa polypeptide and GST9 kDa polypeptide as antigens, respectively, antibodies against the antigens Productivity was confirmed. In the WB method, after performing SDS-PAGE using 10% polyacrylamide gel using two kinds of polypeptides, it was transferred to a nitrocellulose membrane and reacted with 1 μl of each test serum for 90 minutes at room temperature. As the next serum, an anti-chicken IgG rabbit serum diluted 2000 times was reacted at room temperature for 90 minutes. Finally, 4-chloro 1-naphthol was used as a substrate. The results are shown in Table 13.

・ Results and discussion In the ELISA method, SE inactivated vaccines C, D, and E were inoculated with half the amount prescribed in the dosage regimen, so there were some chicken groups that showed low antibody titers, but in the WB method Since bands were observed in all specimens in both cases using 53 kDa polypeptide and GST9 kDa polypeptide as the antigen (see FIGS. 4 to 6), the commercially available SE inactivated vaccine produced antibody against 53 kDa polypeptide and 9 kDa polypeptide. It is thought to induce.
From the above, SE-inactivated vaccinated chickens could be distinguished from SE-infected chickens by the detection method (ELISA method) using an ELISA plate coated with a 53 kDa or 9 kDa polypeptide and the WB method using these antigens. .

Test for confirming adhesion of GST9 kDa polypeptide to cells It was confirmed whether 9 kDa polypeptide, which is a part of SE fliC, has an adhesion action to epithelial cells, which is the first step of SE colony formation.
A plate coated with a GST9 kDa polypeptide, a commercially available SE Elisa plate (CAF-SE Elisa) as a control antigen, ND Elisa plate and 1% bovine albumin PBS solution were dispensed at 0.1 ml / well and left at room temperature for 1 hour. Then, HeLa cells adjusted with MEM to 1.9 × 10 ⁴ cells / well each were dispensed to the plate in which the solution was discarded. After reacting at 37 ° C. for 1 hour, the solution was discarded and wells were removed with MEM. The cells were washed and observed for cell adhesion.

Results and discussion The reaction between HeLa cells and each plate was coated with a GST9 kDa polypeptide (-) in a plate dispensed with bovine albumin alone (+) in a commercially available SE Eliza plate (CAF-SE Eliza). The plate was (++) and the commercial ND Eliza plate was (++++), indicating that the GST9 kDa polypeptide is an antigen that adheres to HeLa cells.
Therefore, GST9 kDa polypeptide is considered to be an antigen that plays an important role in colony formation by adhering to epithelial cells among SE fliC.

It is a perspective view which shows the accommodation state of the test | inspection kit which concerns on this invention. It is a figure which shows the PCR image of the 733-1008 bp area | region by the agarose gel electrophoresis method of the sample concerning this invention. It is a figure which shows the confirmation image of the molecular weight of GST9kDa polypeptide concerning this invention. It is a figure which shows the WB reaction result of SE inactivation vaccine A in this Example, and the serum derived from E inoculation chicken. It is a figure which shows the WB reaction result of SE inactivation vaccine B in this Example, and the serum derived from E inoculation chicken. It is a figure which shows the WB reaction result of SE inactivation vaccine D in this Example, and the serum derived from E inoculation chicken.

Explanation of symbols

1 Test Kit 2 Container 3 Support 4 Reagent Container

Claims (2)

  A test kit for serologically testing field-infected chickens and vaccinated chickens by the enzyme antibody method, and detecting antibodies against Salmonella in fluid samples consisting of serum samples, egg yolk samples or body fluids and tissue extracts A support in which an antigen comprising a SE9 kDa polypeptide or a complex thereof in a 53 kDa polypeptide that is the main antigen of Salmonella flagellar antigen is supported on a support, and various control sera, An antibody test kit using a Salmonella antigen formulation comprising a reagent container containing a serum diluent, a conjugate diluent and an orthophenylenediamine diluent, respectively.   A support carrying an antigen comprising an SE9 kDa polypeptide or a composite thereof is characterized in that a fusion protein obtained by fusing the 9 kDa polypeptide with glutathione-S-transferase is carried on the support. An antibody test kit using the Salmonella antigen formulation according to claim 1.

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