KR100414837B1 - Diagnosis kit for detection salmonella - Google Patents

Abstract

The present invention relates to a diagnostic kit for detecting Salmonella, and to a method for detecting Salmonella comprising an anti-Salmonella antibody purified from serum or eggs of an animal immunized with Salmonella and a diagnostic kit for Salmonella detection. The diagnostic kit of the present invention can detect human or poultry Salmonella infections accurately and simply within a short time, and thus can be effectively used to determine whether Salmonella is infected.

Description

Diagnostic kit for Salmonella detection {DIAGNOSIS KIT FOR DETECTION SALMONELLA}

The present invention relates to a diagnostic kit for detecting Salmonella antigens comprising an antibody against Salmonella antigens. More particularly, the present invention relates to a diagnostic kit comprising an egg obtained by immunizing a Salmonella flagella protein antigen to a laying hen and an antibody contained in the egg, and a method for detecting Salmonella antigen using the same.

Salmonella is still a problem in bacterial infections throughout the world (Foulaki K, Gruber W, and Schlecht S 1989 Infect Immun 57: 1399-1404). Salmonella is a bacillus with enterococci and usually has motility caused by chimeric flagella. . Many Salmonella belonging to the genus Salmonella are pathogenic bacteria that cause food poisoning, fever, sepsis, gastroenteritis. As to the cause of the wave of Latif's (paratyphoid) and the like Salmonella is a type of jangyeol Entebbe utility disk (Salmonella enteritidis) and Salmonella typhimurium (Salmonella typhimurium), which mainly deals with chicks who harm children. In addition, most of the adult systems infected with Salmonella enteritidis carry Salmonella enteritidis, which causes enteritis and food poisoning in humans through the eggs of the system (Altekruse SJ, Koehler J, Hickman-Brenner F, Tauxe RV, and Ferris K 1993, Epidemiol Infect 110: 17-22; Henzler DJ, Ebel E, Sanders J, Kradel D, and Maso J 1994 Avian Dis 38: 37-43)

Recently, the problem of salmonellosis in Korea is serious, and the situation is causing economic losses to the poultry industry.

Flagella (H-antigen) of Salmonella cells is known as a pathogenic factor adherent to host cells. (Ada GL, Nossal GJV, Pye J, and Abbot A 1963 Nature (London) 199: 1257- 1262; Fey H, and Wetzstein HP 1975 J Med Microbiol Immunol 161: 73-78; Lockman HA, Curtiss III R 1990 Infect Immun 58: 137-143). Enables invasion to cause various diseases (Lee CA, and Falkow S 1990 Proc Natl Acad Sci USA 89: 1847-1851; Jones BD, Lee CA, and Falkow S 1992 Infec Immun 60: 2475-2480). Many studies have been actively conducted on vaccines and anti-infectious immunity to prevent and treat them (Germanier R 1982 Development of a new oral attenuated typhoid vaccine. Pages 419-422. In L. Weinstein and BN Fields (ed.), Seminars in infectious disease, vol. 4, Bacterial vaccines.Georg Thieme Verlag, Stuttgart) Salmonella It is about more than 2,000 species so far: that the number is too many preventable by vaccines (Kim CJ, Nagaraja KB, Pomeroy BS 1991 Am J Vet Res 52 1069-1074) there is a limit. Also, even if antibiotics are used to prevent infection and become Salmonella-negative family, stopping antibiotics only increases susceptibility to re-infection by Salmonella.

Diagnosis of diseases caused by external bacteria, such as Salmonellosis, is made by microscopic or culture examination of the presence of bacteria in patients suspected of Salmonellosis or in poultry specimens. However, microscopic examination has a disadvantage in that the results can be obtained quickly, but the sensitivity is low, and the culture examination has a high sensitivity but it takes about 5 to 8 weeks to confirm the result.

In order to solve these drawbacks, many studies have been conducted to extract diseases or components (eg, nucleic acids, antigens, etc.) from patients' specimens and apply them to disease diagnosis. That is, immunodiagnostic methods based on antigen-antibody detection and diagnostic methods by PCR have been developed. Among them, the antigen-antibody detection method can be widely used in clinical laboratories because it is not only excellent in specificity or sensitivity of diagnosis, but also economical because no special equipment or facility is required.

However, these methods are merely examples of methods for detecting bacteria at the laboratory level, and diagnostic kits using these methods have not yet been developed. Therefore, there is an urgent need for the development of a diagnostic kit that can detect bacteria in a patient's specimen accurately and simply in a short time.

Therefore, an object of the present invention is to provide a diagnostic kit capable of detecting Salmonella.

It is another object of the present invention to provide a method for detecting Salmonella using a diagnostic kit.

1 is a photograph of electrophoresis of the flagella protein of Salmonella of the present invention on an SDS gel,

2 is a graph showing a standard curve for quantitative analysis of Salmonella enteritidis flagella protein of the present invention,

3 is a graph showing the correlation between Salmonella enteritidis flagella protein and Salmonella enteritidis bacterium concentration (CFU / ㎖) of the present invention,

4 is a graph showing a standard curve for quantitative analysis of Salmonella typhimurium flagella protein of the present invention,

5 is a graph showing the correlation between Salmonella typhimurium flagella protein of the present invention and Salmonella typhimurium bacteria concentration (CFU / ㎖),

Figure 6 is a graph showing a standard curve for quantitative analysis of Salmonella cholera Escherichia flagella protein of the present invention,

Figure 7 is a graph showing the correlation between Salmonella cholera Escherichia flagella protein and Salmonella cholera Escherichia coli concentration (CFU / ㎖) of the present invention,

Figure 8 is a graph showing a standard curve for quantitative analysis of Salmonella Dublin Plasella protein of the present invention,

Figure 9 is a graph showing the correlation between Salmonella Dublin Plasella protein and Salmonella Dublin bacterial concentration (CFU / ㎖) of the present invention,

Figure 10 shows a photograph of the diagnostic kit for Salmonella choleraesuis, Salmonella Dublin, Salmonella typhimurium and Salmonella enteritidis antigen detection of the present invention.

The present invention to achieve the above object

(a) reacting the sample and the control in a reactor coated with an anti-salmonella antibody;

(b) detecting the antigen-antibody reactant produced by the reaction using a secondary antibody-chromatase conjugate and a color substrate solution; And

(c) comparing the detection results for the sample and the control;

It provides a Salmonella infection diagnostic method comprising a.

In addition, the present invention

(a) a reactor coated with an anti-salmonella antibody;

(b) controls comprising Salmonella standard antigen and antiserum of animals not injected with Salmonella antigen;

(c) secondary antibody-chromatase conjugates; And

(d) a substrate-containing solution that reacts with the color developing enzyme;

It provides a diagnostic kit for Salmonella detection comprising a.

Hereinafter, the present invention will be described in detail.

The present inventors have conducted research and efforts to develop a diagnostic kit that can detect Salmonella present in patients or poultry specimens with higher sensitivity and specificity, and use Salmonella enzyme linked immunosorbentassay (SELISA). The diagnostic kit containing the immunized antibody was completed.

The diagnostic kit of the present invention can be applied to both humans and animals, preferably, it is preferably selectively applied according to the infection target of Salmonella.

Salmonella diagnostic kits for humans can be detected in Salmonella enteritidis or Salmonella typhimurium . Salmonella diagnostic kits for pigs are Salmonella choleraesuis , Salmonella Entebbe utility disk (Salmonella enteritidis) and Salmonella typhimurium herd can be detected by targeting been Salmonella selecting Titanium least one member from the group consisting of (Salmonella typhimurium), Salmonella diagnostic kit for cattle Salmonella two Dublin ( Salmonella dubrin), Salmonella Entebbe utility disk (Salmonella enteritidis) and Salmonella typhimurium herd can be detected by the target salmonella been selected help (one or more from the group consisting of Salmonella typhimurium). That is, the diagnostic kit of the present invention can detect Salmonella most infectious to the test object. However, the diagnostic kit of the present invention differs only in the type of antibody included in the diagnostic kit according to a test subject, and all other essential components are the same.

The present invention provides a diagnostic kit for detecting Salmonella present in human or poultry specimens and a method for detecting Salmonella using the same.

The diagnostic kit of the present invention comprises: (a) a polystyrene plate coated with wells of anti-salmonella antibodies purified from antiserum or egg yolk from an animal against Salmonella antigens, (b) a positive control including a Salmonella standard antigen and a negative control. A control, (c) a conjugate in which an enzyme expressing a color by a reaction with a substrate is labeled with a secondary antibody, (d) a substrate-containing solution to be color-reacted with the enzyme, (e) a wash solution for each reaction step, and (f) characterized in that it comprises a solution for stopping the enzyme reaction.

The antibody of the present invention is preferably purified from antiserum obtained by immunizing Salmonella antigens to an animal, and most preferably, purified from serum or eggs obtained by immunizing a laying hen. Antibodies of the invention are preferably coated with 25 ug / 100 [mu] l per polystyrene plate well.

The control included in the kit of the present invention includes a positive control and a negative control. The positive control is a mixture comprising Salmonella or Salmonella antigens and the negative control is a serum from a laying hen not infected with Salmonella antigen.

The enzyme of the secondary antibody is preferably a conventional chromophore for color reaction, HRPO (Horseradish peroxidase) is most preferred. In addition, the chromase is conjugated to the secondary antibody is preferably provided as a yolk antibody-phenoxidase conjugate (Yolk antibody-peroxidase conjugate).

As the substrate for inducing color development, it is preferable to use a conventional substrate according to the color-enzyme to be used. When the color-enzyme is HRPO, TMB (3,3 ', 5,5'-Tetramethyl benzidine) is used as the substrate. More preferably, the substrate is provided in a solution phase consisting of substrate buffer (0.1 M NaAc, pH 5.5).

The wash solution is preferably a buffer solution consisting of 0.02 M phosphate buffer, 0.13 M NaCl, and 5% Tween20, and the wash solution is 100% after reacting the secondary antibody to the antigen antibody conjugate after the antigen antibody reaction. uL is added to the wells of the plate and washed three to six times.

In another aspect, the present invention provides a method for detecting Salmonella antigens using the diagnostic kit as follows. The method of detecting Salmonella antigens is to react the anti-Salmonella antibodies and the control specimens to the control, respectively, wash with a wash solution, and then add a secondary antibody labeled with an enzyme that displays color by reaction with a substrate, and then wash with wash solution. After the addition of the substrate-containing solution of the enzyme, the color reaction was carried out, characterized in that the absorbance at 450 nm is measured. The patient's specimen is preferably used by sweeping feces and using supernatant by centrifugation (1500 rpm, 5 min) prior to use in diagnostic kits.

The sensitivity in methods using a diagnostic kit of the present invention can be measured Plastic gelatinase protein concentration or Salmonella concentration of Salmonella in a sample, can be measured in the case of salmonella, 10 ⁵ to 10 ⁹ CFU / ㎖, such as the Have Since the concentration of the bacteria causing clinical diarrhea is 10 ⁷ to 10 ⁸ CFU / ㎖, the diagnostic kit of the present invention can accurately diagnose whether Salmonella infection, and the measurement process is simple, can be directly used in the field.

Preferred embodiments are presented to aid in understanding the invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Example 1

Acquisition of antibody against Salmonella

1, strain

Salmonella Entebbe utility disk (S. enteritidis) and standard strains of Salmonella typhimurium (Salmonella typhimurium) strains were used under pre-sale in Canada (Economic innovation and Technology Canada, Winnipeg , Canada) antibody yield process for each Salmonella, respectively The same experimental procedure was carried out, but the following is indicated as Salmonella.

2. Isolation of Flagella Protein

Strains were inoculated with 100 μl per 1 L of Trypticase Soy Broth (Difco) and incubated in large quantities at 4 L. Strains were recovered by stationary incubation at 37 ° C. for 48 hours and centrifugation (3,000 × g) at 4 ° C. for 30 minutes. The recovered strains were obtained by Ilahim et al. (Ibrahim GF, Fleet GH, Lyons MJ, Walker RA 1985 J). Plasella protein was isolated by a modified and supplemented method of Clin Microbiol 22: 1040-1044). The recovered strain was suspended in 0.15 M PBS (0.02 M phosphate buffer, 0.13 M sodium chloride, pH 7.2), pH 2.0 was adjusted with hydrochloric acid, and stirred for 30 minutes. After stirring, the supernatant was obtained by centrifugation (14,000 × g) at 4 ° C. for 15 minutes, and the pH 7.4 was titrated with 1 M sodium hydroxide, and the solution was added at a temperature of 2.67 M and the solution was allowed to stand at 4 ° C. overnight. The supernatant was again centrifuged (20,000 × g, 15 minutes), and the precipitate was dissolved in 50 ml of 0.15 M PBS and placed in dialysis tubing (Sigma Chemical Co.) for 18 hours on 0.15 M PBS, followed by fresh 0.15 M PBS. Dialysis was again for 4 hours.

3. Protein Analysis

In order to confirm the size and purity of Plastic gelatinase protein obtained in the experiment referred to emri (Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature (London) 227:. 680-685) by the method SDS -PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) was performed. The electrophoresis device was a small protein kit (Mini-Protein II Cell Kit, BIO-RAD). Electrophoresis was performed at 200 V for 50 minutes and gel staining was performed with Coomassie Brilliant Blue R-250. In order to investigate the purity of the isolated flagella protein, the results of electrophoresis based on the SDS-PAGE method are shown in FIG. 1. Lane 1 of Figure 1 is 97, 66, 31 kDa in order from the top of the protein macro, lane 2 is the flagella protein of Salmonella choleraesuis of 53.4 kDa, lane 3 of the flag of Salmonella Dublin 55.6 kDa Gel protein, lane 5 is Salmonella typhimurium flagella protein of 50.7 kDa, and lane 6 is Salmonella enteritidis flagella protein of 54.6 kDa. The size of the isolated proteins was the same as the known flagella protein.

In addition, the concentration of the flagella protein obtained above was measured by a modified and supplemented microwave BCA (bicinochoninic acid) method of Robert et al. (Robert EA, Rocky ST 1995 Molecular Biotechnology . Humana Press Inc. 17-24). Salmonella antigens obtained in this experiment were used as the following antibody preparation and standard antigen.

4. Antibody Production

(1) Public animals and test site

Animals used for the production of antibodies to the flagella protein obtained in the experiment was used 15 eggs of 36 weeks-old ISA-brown hens laying eggs, breeding was carried out in the animal breeding dong of Dankook University animal resources science.

(2) antibody formation

Flagella protein (200 μg / ml) and Freund's complete adjuvant (Sigma Chemical Co.) were respectively mixed in equal amounts to emulsify. 1 ml of the emulsion was inoculated first by intramuscular injection into 0.25 ml of each of the laying muscles. Booster injection was performed every two weeks after the first inoculation, and immunized with Freund's incomplete adjuvant (Gibco) in 1998. The test was run for 12 weeks from September 16 to December 9, 1998.

(3) Blood collection and egg recovery

Blood was collected at 2 weeks intervals from the lower vein (翼 下 靜脈) and separated serum and stored at -20 ℃ was used for various experimental measurements. Eggs were collected daily and stored at 8 ° C. for use in experiments.

(4) Isolation of Egg Yolk Antibody (IgY) Mixture

Kim JW, Cho SH, Koh SY, Kim C 1997 The isolation of pili from enterotoxigenic E. coli (K99) and its production of egg yolk antibody.Korean Federation of the Societies in Animal Sciences, Proceedings 130 P97040) It separated according to the method of. Eggs produced from the immunized laying hens were collected and egg yolks were separated and diluted 10-fold with distilled water (pH 5.0). The diluted yolk solution was adjusted to pH 5.0 and frozen overnight at -20 ° C. Frozen yolks were thawed at room temperature and then centrifuged (10,000 × g) at 15 ° C. for 30 min to collect only the supernatant. The supernatant was filtered through filter paper (Whatman No. 1) to separate antibodies. The antibody did not cause an immune response to other species when examined for antibody specificity.

Salmonella determination by ELISA

(1) Salmonella Enteritidis

The competitive sandwich ELISA method was used to confirm the Salmonella bacterium measurement of the antibody prepared above.

Dilute Salmonella enteritidis antibodies in a 96-well immuno-microplate (NUNC, Denmark) with carbonate-bicarbonate buffer (pH 9.6) at 10 μg / 100 μl per well. The wells were coated at 4 ° C. overnight. The coated plate was washed three times with washing solution (0.02 M phosphate buffer, 0.13 M NaCl, pH 7.2, 0.05% Tween20) and then left at room temperature for 2 hours with 5% skim milk solution (pH 7.3, Difco). . Secondary antibodies, flagella protein and whole cells were prepared by diluting 5% skim milk solution and PBS-T (PBS containing 0.05% Tween-20) in the same amount. The standard concentration range of Salmonella enteritidis flagella protein was set at 10, 3.333, 1.111, 0.37, 0.123, 0.041 μg / ml and the reaction was performed in each well. After washing the plate, the secondary antibody was placed in the well and reacted at 37 ° C for 2 hours. The secondary antibody (rabbit anti- S. Enteritidis antibody; 1: 2,000) is mixed with AP-Donkey anti-rabbit IgG (Jackson; 1: 1,250), it is prepared at 37 ℃ 2 hours before use. After the secondary antibody reaction, the substrate solution was placed in a well and subjected to enzymatic reaction for 20 minutes. The substrate solution is a solution in which 1 tablet of phosphate substrate tablets (ρ-nitrophenyl phosphate, SIGMA-104) is dissolved in 5 mL of 10% diethanolamine (0.5 mM MgCl ₂ pH 9.8). The washing of the plate during each process was performed six times. After the enzyme reaction, the reaction was stopped with 5 M sodium hydroxide, and the color development was measured at 405 nm on a microplate reader (Molecular Devices; E Max).

2 is a graph showing the color development according to the amount of Salmonella enteritidis flagella protein, Salmonella enteritidis antibody of the present invention is to measure Salmonella enteritidis flagella protein up to 10 ㎍ / ㎖-0.041 ㎍ / ㎖ It can be shown. The regression equation of the graph was SQRT (Y) = 0.93266 * Ｘ + 1.03948 and the coefficient of determination (R ² ) for the regression equation was 0.95.

In addition, in order to check the accuracy and reproducibility of the measurement in the quantitative analysis of Salmonella enteritidis flagella protein according to the standard curve shown in FIG. 2, the intra-assay and inter-assay were repeated six times. Intra-assay confirms Salmonella flagella protein quantitative error between 96-well plates subjected to 6 ELISA assays, and inter-assay confirms inter-assay protein quantification errors on the same 96-well plate. The coefficient of variation (C.V.%) for intra-assay and inter-assay differed according to the level of standard concentration. Intra-assay was 1.21 to 12.52% and for inter-assay 3.01 to 13.35%. The results are shown in Table 1.

TABLE 1

Way Standard (µg / mL) No. O.D. C.V. (%) intra-assay 10.00 6 2.370 ± 0.028 1.21 3.333 6 1.346 ± 0.028 2.09 1.111 6 0.826 ± 0.042 5.09 0.370 6 0.414 ± 0.023 5.63 0.123 6 0.136 ± 0.010 8.01 0.041 6 0.062 ± 0.008 12.52 inter-assay 10.00 6 2.363 ± 0.071 3.01 3.333 6 1.407 ± 0.048 3.42 1.111 6 0.698 ± 0.045 6.43 0.370 6 0.390 ± 0.024 6.25 0.123 6 0.151 ± 0.161 10.70 0.041 6 0.062 ± 0.008 13.35

Therefore, this method shows high reproducibility when measuring high concentration of Salmonella enteritidis flagella protein, and can be used as a basis for judging Salmonella infection.

In addition, the degree of infection of Salmonella was determined to indirectly measure the concentration (CFU / ml) of Salmonella enteritidis from the specimen using the standard concentration curve for Salmonella enteritidis flagella protein. The concentration of Salmonella enteritidis flagella protein (µg / ml) and the concentration of Salmonella enteritidis (CFU / ml) assuming that the concentration of Salmonella enteritidis flagella protein will increase with the number of Salmonella enteritidis bacteria. ), And the correlation with Correlation studies were performed using ELISA, and standard concentration ranges of Salmonella enteritidis flagella protein were 10 μg / ml, 3.333 μg / ml, 1.111 μg / ml, 0.37 μg / ml, 0.123 μg / ml, and 0.041 μg /. It was set to ㎖, the concentration of Salmonella enteritidis bacteria (CFU / ㎖) was carried out by experiment using a diluted 10 times from 3.4 × 10 ⁹ (CFU / ㎖) to 3.4 × 10 ⁴ (CFU / ㎖), The results are shown in FIG.

As a result, the correlation between the concentration of Salmonella enteritidis flagella protein (µg / mL) and the concentration of Salmonella enteritidis (CFU / mL) was very high at 0.979. It can accurately measure the concentration of Salmonella enteritidis on the basis of a standard curve made of Salmonella enteritidis flagella protein when measuring the concentration of Salmonella enteritidis in feces and specimens (CFU / mL). That is, the measuring method of the present invention can measure Salmonella enteritidis bacteria up to 10 ⁵ to 10 ⁹ (CFU / mL). This indicates that it is useful as a diagnostic method, including the intestinal bacterial concentration 10 ⁶ to 10 ⁷ (CFU / ㎖) that causes clinical diarrhea.

(2) Salmonella typhimurium

In the same manner as described above with Salmonella typhimurium antibody, Salmonella typhimurium flagella protein and Salmonella typhimurium bacteria, the standard graph of Figure 4 was obtained. The Salmonella typhimurium antibody of the present invention has been shown to be able to measure Salmonella typhimurium flagella protein from 10 μg / ml to 0.041 μg / ml. The regression equation of the graph is SQRT (Y) = 1.28995 * Ｘ + 1.47646 and the coefficient of determination (R) for the regression equation is²) Was 0.95. In addition, in order to check the accuracy and reproducibility of the measurement in the quantitative analysis of Salmonella typhimurium flagella protein according to the standard curve shown in FIG. The coefficient of variation (C.V.%) for intra-assay and inter-assay differed according to the level of standard concentration. Intra-assay, 3.47 to 9.76% and for inter-assay, 3.32 to 15.44%. The results are shown in Table 2.

TABLE 2

Way Standard (µg / mL) No. O.D. C.V. (%) intra-assay 10.00 6 3.246 ± 0.113 3.47 3.333 6 2.040 ± 0.010 4.89 1.111 6 1.129 ± 0.058 5.13 0.370 6 0.580 ± 0.057 9.87 0.123 6 0.253 ± 0.012 6.56 0.041 6 0.114 ± 0.011 9.76 inter-assay 10.00 6 3.299 ± 0.110 3.32 3.333 6 2.478 ± 0.172 6.96 1.111 6 1.275 ± 0.057 4.48 0.370 6 0.637 ± 0.031 4.92 0.123 6 0.254 ± 0.015 6.09 0.041 6 0.087 ± 0.013 15.44

In addition, the correlation between Salmonella typhimurium flagella protein and Salmonella typhimurium bacteria was also measured and shown in FIG. 5. Correlation was performed using ELISA method and the standard concentration range of Salmonella typhimurium flagella protein was 10 μg / ml, 3.333 μg / ml, 1.111 μg / ml, 0.37 μg / ml, 0.123 μg / ml, 0.041 μg / It was set to ㎖, and the concentration of Salmonella typhimurium (CFU / ㎖) was tested using a 10-fold dilution from 2.8 × 10 ⁹ (CFU / ㎖) to 2.8 × 10 ⁴ (CFU / ㎖). As a result, the correlation between the concentration of Salmonella typhimurium flagella protein (µg / ml) and the concentration of Salmonella typhimurium bacteria (CFU / ml) was very high at 0.983. It can accurately measure the concentration of Salmonella typhimurium bacteria based on a standard curve made of Salmonella typhimurium flagella protein when measuring the concentration of Salmonella typhimurium bacteria in feces and specimens (CFU / mL). That is, the measuring method of the present invention can measure Salmonella typhimurium bacteria up to 10 ⁵ to 10 ⁹ (CFU / ㎖). This indicates that it is useful as a diagnostic method, including the intestinal bacterial concentration 10 ⁶ to 10 ⁷ (CFU / ㎖) that causes clinical diarrhea.

(3) salmonella cholera

The standard graph of FIG. 6 was obtained in the same manner as described above with Salmonella choleraescis antibody, Salmonella choleraescis flagella protein and Salmonella choleraescis bacteria. The Salmonella choleraescis antibodies of the present invention have been shown to measure Salmonella choleraescis flagella protein from 10 μg / ml to 0.041 μg / ml. The regression equation of the graph is SQRT (Y) = 0.69962 * Ｘ + 0.87786 and the coefficient of determination (R) for the regression equation is²) Was 0.95. In addition, in order to check the accuracy and reproducibility of the measurement in the quantitative analysis of Salmonella cholera Escherichia flagella protein according to the standard curve shown in FIG. The coefficient of variation (C.V.%) for intra-assay and inter-assay differed according to the level of standard concentration. Intra-assay, 2.78 to 16.60%, and for inter-assay, 2.95 to 14.18%. The results are shown in Table 3.

TABLE 3

Way Standard (µg / mL) No. O.D. C.V. (%) intra-assay 10.00 6 1.870 ± 0.052 2.78 3.333 6 1.087 ± 0.031 2.85 1.111 6 0.743 ± 0.043 5.82 0.370 6 0.415 ± 0.054 13.03 0.123 6 0.228 ± 0.034 15.30 0.041 6 0.112 ± 0.018 16.60 inter-assay 10.00 6 2.239 ± 0.066 2.95 3.333 6 1.165 ± 0.022 1.93 1.111 6 0.639 ± 0.015 2.38 0.370 6 0.361 ± 0.020 5.61 0.123 6 0.155 ± 0.018 11.64 0.041 6 0.064 ± 0.009 14.18

In addition, the correlation between Salmonella cholera Escherichia flagella protein and Salmonella cholera Escherichia coli was also measured and shown in FIG. 7. Correlation studies were performed using ELISA, and the standard concentration ranges of Salmonella choleraescis flagella protein were 10 μg / ml, 3.333 μg / ml, 1.111 μg / ml, 0.37 μg / ml, 0.123 μg / ml, and 0.041 μg. It was set to / ml, the concentration of Salmonella cholera Escherichia coli (CFU / ㎖) was tested using a 10-fold dilution from 0.5 × 10 ¹⁰ CFU / ㎖) to 3.4 × 10 ⁶ CFU / ㎖).

As a result, the correlation between the concentration of Salmonella choleraescis flagella protein (µg / ml) and the concentration of Salmonella cholera Escherichia coli (CFU / ml) was very high at 0.996. It can accurately measure the concentration of Salmonella cholera Escherichia coli on the basis of a standard curve made of Salmonella cholera Escherichia flagella protein when measuring the concentration (CFU / mL) of Salmonella cholera Escherichia coli in feces and specimens. . That is, the measuring method of the present invention can measure Salmonella choleraesche bacteria up to 10 ⁵ to 10 ⁹ (CFU / ㎖). This indicates that it is useful as a diagnostic method, including the intestinal bacterial concentration 10 ⁶ to 10 ⁷ (CFU / ㎖) that causes clinical diarrhea.

(4) Salmonella Dublin

In the same manner as described above with Salmonella Dublin antibody, Salmonella Dublin Plasella protein and Salmonella Dublin bacteria, the standard graph of Figure 8 was obtained. The Salmonella Dublin antibody of the present invention has been shown to be able to measure Salmonella Dublin flagella protein from 10 μg / ml to 0.041 μg / ml. The regression equation of the graph is SQRT (Y) = 1.17044 * Ｘ + 1.34891 and the coefficient of determination (R) for the regression equation is²) Was 0.96. In addition, in order to check the accuracy and reproducibility of the measurement in the quantitative analysis of Salmonella Dublin Plasella protein by the standard curve shown in FIG. 8, the intra-assay and inter-assay were repeated six times. The coefficient of variation (C.V.%) for intra-assay and inter-assay differed according to the level of standard concentration. Intra-assay, 4.02 to 14.33%, and for inter-assay, 2.72 to 14.52%. The results are shown in Table 4.

TABLE 4

Way Standard (µg / mL) No. O.D. C.V. (%) intra-assay 10.00 6 2.907 ± 0.119 4.02 3.333 6 1.882 ± 0.123 6.52 1.111 6 1.042 ± 0.064 6.10 0.370 6 0.604 ± 0.061 10.08 0.123 6 0.225 ± 0.027 11.92 0.041 6 0.076 ± 0.011 14.33 inter-assay 10.00 6 2.837 ± 0.077 2.72 3.333 6 1.591 ± 0.048 3.03 1.111 6 0.855 ± 0.035 4.07 0.370 6 0.519 ± 0.088 16.88 0.123 6 0.284 ± 0.051 18.05 0.041 6 0.010 ± 0.014 14.52

In addition, the correlation between Salmonella Dublin Plasella protein and Salmonella Dublin bacteria was also measured and shown in FIG. 9. Correlation studies were performed using ELISA, and standard concentration ranges of Salmonella Dublin Plasella protein were 10 μg / ml, 3.333 μg / ml, 1.111 μg / ml, 0.37 μg / ml, 0.123 μg / ml, and 0.041 μg / ml. Salmonella Dublin concentration (CFU / ㎖) was tested using a 10-fold dilution from 2.4 × 10 ¹⁰ (CFU / ㎖) to 2.4 × 10 ⁶ (CFU / ㎖). As a result, the correlation between the concentration of Salmonella Dublin Plasella protein (µg / ml) and the concentration of Salmonella Dublin bacteria (CFU / ml) was very high at 0.975. It can accurately measure the concentration of Salmonella Dublin on the basis of a standard curve made of Salmonella Dublin Plasella protein when measuring the concentration of Salmonella Dublin (CFU / ml) in feces and specimens. That is, the measuring method of the present invention can measure Salmonella Dublin bacteria up to 10 ⁵ to 10 ⁹ (CFU / ㎖). This indicates that it is useful as a diagnostic method, including the intestinal bacterial concentration 10 ⁶ to 10 ⁷ (CFU / ㎖) that causes clinical diarrhea.

Example 2 Diagnostic Kit

Diagnostic Kits for Humans

In order to prepare a Salmonella diagnostic kit for humans, IgY of the yolk antibody against Salmonella enteritidis or Salmonella typhimurium was first purified by ion exchange chromatography (Ion Exchange Chromatography). After equilibrating the resin with 0.025 M phosphate buffer, 0.2 g of lyophilized Salmonella enteritidis or Salmonella typhimurium egg yolk antibody powder was dissolved in 4 ml of 0.025 M phosphate buffer, and centrifuged at 2,500 rpm for 5 minutes. 2 ml was taken as a sample. The sample was eluted with 0.25 M phosphate buffer (pH 8.0) on ion exchange chromatography to obtain Salmonella enteritidis or Salmonella typhimurium IgY fraction.

0.25 ml of NaIO _{4 and} 0.25 ml of HRPO solution dissolved in carbonate-bicarbonate buffer (pH 9.2) were mixed and reacted in the dark for 2 hours, and then separated Salmonella enteritidis or Salmonella typhimurium IgY 1 The mixture was mixed with ㎖, placed in a Pasteur pipette, and 0.25 g of Sephadex G-25 was added thereto, and left in a dark place for 3 hours. Then, 150 μl of NaBH ₄ was added to the sample eluted with 0.1 M carbonate-bicarbonate buffer, left for 1 hour and 30 minutes, and then 0.9 ml of saturated ammonium sulfate was added for 30 minutes. Stirred slowly at 4 ° C. and centrifuged at 10,000 rpm for 15 minutes. A precipitate, ie HRPO-IgY, was obtained, dissolved in TEN (Tris / EDTA / NaCl) buffer solution, and then added to the same amount of glycerol and stored at -20 ° C.

A diagnostic ELISA kit was prepared using HRPO-IgY obtained in the above experiment, and the components of the kit are as follows.

(1) 96 well microplate coated with 25 μg per well of HRPO-IgY (IgY to Salmonella enteritidis)

(2) 96 well microplates coated with 25 μg per well of HRPO-IgY (IgY to Salmonella typhimurium)

(3) Washing solution (0.02 M phosphate buffer, 0.13 M NaCl, pH 7.2, 0.05% Tween 20)

(4) Yolk antibody-peroxidase conjugate diluted in PBS buffer

(5) 5% skim milk solution, PBS-T (PBS, 0.05% Tween-20)

(6) Color substrate solution-300 μl of TMB (3,3 ', 5,5'-Tetramethyl benzidine) and 35 mL of substrate buffer (0.1 M NaAc, pH 5.5) and 150 μl of 1% H ₂ O ₂

(7) 5 M sodium hydroxide

Diagnostic Kits for Pigs

A diagnostic kit was prepared by separating egg yolk antibodies against Salmonella choleraescis, Salmonella enteritidis and Salmonella typhimurium.

(1) 96 well microplate coated with 25 μg per well of HRPO-IgY (IgY to Salmonella enteritidis)

(2) 96 well microplates coated with 25 μg per well of HRPO-IgY (IgY to Salmonella choleraescis)

(3) 96 well microplates coated with 25 μg per well of HRPO-IgY (IgY to Salmonella typhimurium)

(4) Washing solution (0.02 M phosphate buffer, 0.13 M NaCl, pH 7.2, 0.05% Tween 20)

(5) Yolk antibody-peroxidase conjugate diluted in PBS buffer

(6) 5% skim milk solution, PBS-T (PBS, 0.05% Tween-20)

(7) Color substrate substrate-300 μl of TMB (3,3 ', 5,5'-Tetramethyl benzidine) and 35 mL of substrate buffer (0.1 M NaAc, pH 5.5) and 150 μl of 1% H ₂ O ₂

(8) 5 M sodium hydroxide

Diagnostic kits for cattle

The yolk antibody was isolated for Salmonella Dublin, Salmonella enteritidis and Salmonella typhimurium and the diagnostic method was prepared in the same manner as described above.

(1) 96 well microplate coated with 25 μg per well of HRPO-IgY (IgY to Salmonella enteritidis)

(2) 96 well microplates coated with 25 μg per well of HRPO-IgY (IgY to Salmonella Dublin)

(3) 96 well microplates coated with 25 μg per well of HRPO-IgY (IgY to Salmonella typhimurium)

(4) Washing solution (0.02 M phosphate buffer, 0.13 M NaCl, pH 7.2, 0.05% Tween 20)

(5) Yolk antibody-peroxidase conjugate diluted in PBS buffer

(6) 5% skim milk solution, PBS-T (PBS, 0.05% Tween-20)

(7) Color substrate substrate-300 μl of TMB (3,3 ', 5,5'-Tetramethyl benzidine) and 35 mL of substrate buffer (0.1 M NaAc, pH 5.5) and 150 μl of 1% H ₂ O ₂

Experimental Example

The sensitivity of the measurement kit of Example 2 was confirmed using diluted Salmonella bacteria.

On each plate coated with Salmonella enteritidis, Salmonella typhimurium, Salmonella choleraesuis or Salmonella dubrine, each Salmonella bacterium was treated with 5% skim milk and PBS-T (PBS, 0.05% Tween 20) 10 ¹⁰ , Diluted with 10 ⁹ , 10 ⁸ , 10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ CFU / ml, and put in each well was reacted for 2 hours at 37 ℃. The solution of the plate was removed, washed six times with a washing solution, and then 100 μl of the secondary antibody was added to the well and reacted at 37 ° C. for 2 hours. Then, 100 μl of the chromogenic substrate solution was added to each well for 15 minutes. Thereafter, 100 μl of 5 M sodium hydroxide as a reaction inhibitor was placed, and the color development was measured at 405 nm on a microplate reader. As a result, Salmonella enteritidis bacteria concentration was more than 10 ⁶ (CFU / ㎖) and the color reaction can be easily identified with the naked eye.

Figure 10 shows the color reaction of the plate measured using the Salmonella bacteria kit of the present invention, a is Salmonella enteritidis, b is Salmonella typhimurium, c is Salmonella choleraesuis, d is This is a kit kit for Salmonella Dublin. Salmonella measuring kit of Example 2 Salmonella enteritidis bacteria from 10 ⁵ CFU / ㎖ to 10 ¹⁰ CFU / ㎖, Salmonella typhimurium bacteria from 10 ⁵ CFU / ㎖ to 10 ¹⁰ CFU / ㎖, Salmonella cholera Shuis bacteria can be measured from 10 ⁵ CFU / mL to 10 ¹⁰ CFU / mL, and Salmonella Dublin bacteria can be measured from 10 ⁵ CFU / mL to 10 ¹⁰ CFU / mL, which is the concentration of bacteria that cause clinical diarrhea. 10 ⁷ CFU / mL to 10 ⁸ CFU / mL are included to indicate utility as a kit.

As mentioned above, the present invention provides a diagnostic kit capable of measuring Salmonella infection in humans or poultry. The diagnostic kit of the present invention can detect human or poultry Salmonella infections accurately and simply within a short time, and thus can be effectively used to determine whether Salmonella is infected.

Claims (11)

(a) reacting the sample and the control in a reactor coated with an anti-salmonella antibody; (b) detecting the antigen-antibody reactant produced by the reaction using a secondary antibody-chromatase conjugate and a color substrate solution; And (c) Salmonella infection diagnostic method comprising the step of comparing the detection results for the sample and the control, The anti-Salmonella antibody of step (a) is Salmonella infection diagnosis method of animals other than human, characterized in that separated from egg yolk of the laying hens immunized against Salmonella flagella protein. delete The method according to claim 1, wherein the Salmonella is one species from the group consisting of Salmonella enteritidis , Salmonella typhimurium , Salmonella dubrin , and Salmonella choleresuis . A method for diagnosing Salmonella infections in animals other than humans, which is selected as above. (a) a reactor coated with an anti-salmonella antibody; (b) positive and negative controls; (c) secondary antibody-chromatase conjugates; And (d) Salmonella detection diagnostic kit comprising a substrate-containing solution that reacts with the chromophore, The anti-salmonella antibody of step (a) is Salmonella detection diagnostic kit, characterized in that separated from egg yolk of the laying hens immunized against the Salmonella flagella protein. delete The diagnostic kit for Salmonella detection according to claim 4, wherein the anti-Salmonella antibody is coated with 25 ug per well in a 96 well plate which is a reactor. [Claim 5] The diagnostic kit of claim 4, wherein the chromophore is HRPO (Horseradish peroxidase). The method of claim 4, wherein the substrate-containing solution is a color substrate solution, TMB (3,3 ', 5,5'-Tetramethyl benzidine), substrate buffer (0.1 M NaAc, pH 5.5), and 1% H ₂ O ₂ Salmonella detection diagnostic kit comprising a. The method according to claim 4, wherein the diagnostic kit is used for a human sample. Wherein the Salmonella is Salmonella Entebbe utility disk (Salmonella enteritidis) or Salmonella typhimurium (Salmonella typhimurium) is. The Salmonella antibody is Salmonella diagnostic kit for Salmonella characterized in that the antibody to Salmonella enteritidis or an antibody against Salmonella typhimurium. According to claim 4, wherein the diagnostic kit is used for pigs, Salmonella is Salmonella choleraesuis ( Salmonella choleraesuis ), Salmonella enteritidis ( Salmonella enteritidis ) or Salmonella typhimurium ( Salmonella typhimurium ) The Salmonella antibody is an antibody against Salmonella choleraescis, an antibody against Salmonella enteritidis or an antibody against Salmonella typhimurium. Salmonella detection diagnostic kit characterized in that. The method of claim 4, wherein the diagnostic kit is used for cattle, Wherein the Salmonella is Salmonella Dublin two (Salmonella dubrin), Salmonella Entebbe utility disk (Salmonella enteritidis) or Salmonella typhimurium (Salmonella typhimurium), and The Salmonella antibody is an antibody against Salmonella Dublin, an antibody against Salmonella enteritidis or an antibody against Salmonella typhimurium. Salmonella detection diagnostic kit characterized in that.