JP2002525605A - Salmonella antigen formulation and kit for detecting Salmonella antibodies - Google Patents

Abstract

  (57) [Summary] PROBLEM TO BE SOLVED: To provide an improved reagent or method for serological or immunological measurement of a Salmonella antibody. SOLUTION: The present invention provides a Salmonella antigen formulation containing at least one lipopolysaccharide protein complex of Salmonella enterica, and a sample of meat juice, serum or egg yolk by an immunoassay method, particularly a heterogeneous ELISA method, Or a kit for detecting a Salmonella antibody in another liquid sample. In the above kit, the solid support carries a Salmonella antigen formulation that immunologically reacts with the antibodies provided by the present invention.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a Salmonella antigen formulation (formulation: Formulalierung) and an antibody against Salmonella in a gravy sample, serum sample or yolk sample or other fluid sample by immunological detection methods, especially heterogeneous ELISA methods. It relates to a kit for detection (Bestimmung).

[0002]

[Prior art]

Diseases caused to humans by Salmonella are transmitted, inter alia, by contaminated food and drink. In addition to pathogens that are compatible with humans or specific animal species, a number of serovars (or serotypes: Serovaren) are present in humans and animals in Salmonellos disease.
en) or can cause infection. In the fields of veterinary medicine and medicine, food and drink management and animal production, there is an increasing demand for rapid and safe detection of Salmonella from the viewpoint of the onset of many diseases and protection of consumers. Various methods are known for detecting and quantifying Salmonella. For example, bacteria can be cultured in various media. Serotyping is possible by selective growth on a solid selective medium. This method takes a total of four or five days.

[0003] It is also known to detect Salmonella antibodies by using an immunological quantification method, especially a heterogeneous ELISA method (Enzyme-linked immunosorbent assay for Salmonella ser).
ology using lipopolysaccharide antigen, J. Vet. Diagn. Invest. 7: 481-487 (1
995); Bradford P. Smith, George W. Dilling, John K. House, Hans Konrad,
Nadia Moore). In this case, a microtitration plate (Mikrotiterpl
atte) is coated with a Salmonella antigen. During the incubation of the sample in the coated well (well), antibodies specific for Salmonella form a complex with the antigen. It is not the bound (complexed) sample that is removed by washing. An enzyme-labeled second antibody (which should recognize the (first) antibody bound to the antigen) binds to the antibody bound to the antigen. Unbound complexes are removed by washing. When a coloring substance (substrate) is further added, color development (substrate conversion) is caused by the enzyme to which the antibody is bound. The color reaction can then be used for spectroscopic determination of the amount of Salmonella antibody in the sample and is proportional to the amount of Salmonella antibody.

In this case, lipopolysaccharide (LPS), in particular cases Salmonella Typhinurium (STM) and Salmonella Choleraesuis (SCS) type Salmonella LPS are used as Salmonella antigens. It is difficult to immobilize this Salmonella antigen on a microtitration plate, especially for automation of the detection method. Due to the poor fixation of this Salmonella antigen, especially its binding and its loss of reactivity, it is often necessary to replace (refresh) the medium of the test plate before performing the test. However, this requires a period of at least two days, even for this detection method. Furthermore, the disadvantage that the positive control of the STM- or SCS-antibody showed a cross-reactivity with SCS- or STM-lipopolysaccharide-antigen of more than 30% was also evident.

[0005] Difficulties arise with conventional diagnostic methods for the following reasons. That is, for example, serological control of, for example, a swine population (bestaenden) from more than 2000 known and classified serotypes (Serovaren) in the Kauffman White antigen analysis table For this reason, the broadest possible spectrum (range) must be obtained, or on the other hand, the emphasis is on the detection of specific serotypes (eg bird populations), here S. Typhimurium and S. Enteritidis and S. Gallin
Detection or elimination of arum-pullorum is required. The lipopolysaccharide-antigen corresponding to the Salmonella O-antigen must be immobilized on a solid support, generally a microtitration plate, for use in the ELISA method. However, the immobilization of lipopolysaccharide (LPS) by ELISA-solid support is much weaker, heterogeneous and unstable than immobilizing proteins. When several lipopolysaccharide-preparations are used for immobilization on a single support, the elimination, competition inhibition, cross-reactivity or unmasking of the antigens further exacerbates the immobilization properties of the lipopolysaccharides which are merely undesirable. And may be difficult to use in a diagnostic test system or almost impossible to use to provide a particular task.

[0006]

[Problems to be solved by the invention]

It is an object of the present invention to provide improved reagents (formulations: Mittel) and methods for serologic or immunological detection of various Salmonella serotypes, especially for quantification of anti-Salmonella antibodies. That is.

[0007]

[Means for Solving the Problems]

According to the present invention, this object is achieved by an antigen formulation according to claim 1 and a method for immunological detection (quantification), in particular a heterogeneous ELISA method according to claim 9, a gravy sample, an egg yolk sample or The problem is solved by a test kit for detecting antibodies to Salmonella in a serum sample. In this kit, the support carries a Salmonella antigen formulation of the invention for effecting an immune reaction with the antibody to be detected.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION

Further advantageous embodiments of the invention are described in claims 2 to 8 and in claims 10 to 19.

With the Salmonella antigen formulations of the invention, very good binding properties can be obtained on a solid support for diagnosis. Binding to the protein transfers the favorable binding properties of the protein to Salmonella-lipopolysaccharide, which is originally only suitable for a limited extent due to its unfavorable binding properties on the diagnostic solid support. This makes it possible to produce a stable, uniform and highly sensitive lipopolysaccharide antigen coating on microtitration plates, and to meet the traditional requirements of Salmonella serology, ie, antibodies against different specific Salmonella serotypes according to each task. A diagnostic system suitable for the detection of chromosomes can be developed. In addition to using only one of the modified Salmonella lipopolysaccharide conjugated to the protein, in combination with other Salmonella-lipopolysaccharide-protein conjugates and / or Salmonella antigen preparations, and at the same time other bacterial preparations, For example, a combination with a lipopolysaccharide-protein complex of another Gram-negative bacterium is also possible. Due to the large number of Salmonella O-antigens found in the Kaufmann-White-Schema and the diagnostic targeting resulting therefrom (targeting), a suitable antigen combination (the Individual components or multiple components can be used in diagnostic systems by binding to a protein of the invention and binding to a solid support). The lipopolysaccharide conjugate conjugated to the protein, due to its advantageous properties, allows for the binding, uniformity, high sensitivity and storage stability of solid support coatings for immunoassay testing systems.

According to an advantageous embodiment of the invention, the support comprises a complex comprising STM-lipopolysaccharide and bovine serum albumin (BSA) and Salmonella cholera suis (Salmon
ella cholera suis (SCS) type, and according to the present invention, STM lipopolysaccharide and BSA form a complex in a ratio of 1: 0.5 to 1: 3 and S
The best binding properties were obtained when the ratio of CS to STM-lipopolysaccharide-BSA conjugate was 1:10 to 1: 200.

It was surprising that it was confirmed that the Salmonella antigen formulations of the present invention also exhibited high stability in addition to good binding properties. The Salmonella antigen formulations of the invention are non-cross-reactive, for example, Salmonella-lipopolysaccharide-serum albumin conjugates based on Salmonella antigens of the Salmonella typhimurium (STM) type and Salmonella of the Salmonella cholerae swiss (SCS) type. At least 90% of the most frequently detected serotypes can be detected in an antigen-mix formulation comprising the antigen. The Salmonella antigen formulation of the present invention binds very well to a support, such as a microtitration plate used in an ELISA method. Therefore, the support can be prepared in advance and prepared for analysis. Therefore, there is no need for the user to perform the coating.

According to another advantageous embodiment of the invention, the support is provided with a complex of STM-lipopolysaccharide and bovine casein and STM-Salmonella-lipopolysaccharide, according to the invention: Very good binding properties when the STM-lipopolysaccharide and casein form a complex in the range of 1: 0.5 to 1: 3 and the ratio of STM-lipopolysaccharide complex to STM-lipopolysaccharide is 1: 1 was gotten. Due to the particular composition of this antigen formulation, the serotypes Salmonella enteritidis, Salmonella typhimurium and Salmonella galinarum-prolum associated with the avian industry
Pullorum) antibodies can be detected simultaneously, in which case also the binding of the microtitration plate is very good and the storage stability of the microtitration plate prepared in advance is also good.

The pre-made test system of the present invention can be procured at any time by storage, can be used as needed, and can be used for a wide range of tests (screening) for a large number of samples as required in the fields of medicine and agriculture. )It can be performed.

The kit of the present invention comprises, in addition to the support coated with the Salmonella antigen formulation of the present invention, various control sera, washing concentration buffer, dilution buffer and anti-antibody enzyme complex, It consists of a corresponding substrate solution and a stop solution. Anti-antibody enzyme conjugates are preferably used for the detection of antibodies against Salmonella in porcine serum, preferably the anti-porcine-immunoglobulin-peroxidase conjugate, and preferably for the detection of antibodies against Salmonella in chicken serum. -Chicken-immunoglobulin-peroxidase complex, and the substrate solution is tetramethylbenzidine (TMB)
Dilute hydrochloric acid is used as the stop solution.

[0015] The STM / SCS-Salmonella antigen is described in Appelmelk, BJ et al ;, J. of Immun.
olog, Methods, 82 (1985): 199-207 "An Enzyume-Linked Immunosorbent Assay
(ELISA) for the measurement of Antibodies to different Parts of the gra
m-negative Lipopolysaccharide Core Region ", prepared from Salmonella cultures. The Salmonella-lipopolysaccharide of that type is then combined with serum albumin to form a Salmonella-lipopolysaccharide-serum albumin complex. .this is,
Eur.JB in 1972 by Galanos, C., ETRietschel, O. Luederitz, O. Westphal
This is performed by the method described in iochem.

For example, in the form of a microtitration plate, coating of the support is carried out by incubation with the antigen formulation of the invention, blocking of non-binding sites with a protein solution in the presence of a buffer solution. The support thus produced is washed and then dried.

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 sample to be tested is contacted with a support coated with a Salmonella antigen formulation of the invention. Antibodies specific for Salmonella are coated
Form a complex with the antigen during the incubation of the sample in the well. Unbound material is removed by blotting or tapping (Ausschlagen) and washing with washing buffer. Thereafter, the anti-antibody enzyme conjugate is added. Unbound antibody enzyme complex is removed by blotting or tapping and washing with a washing buffer. When TMB is added as a substrate, color development (substrate conversion) occurs due to the enzyme bound to the antibody. The color-forming effect can be used for spectral photometric (spectrophotometric) determination of the amount of Salmonella-antibody in the sample, and is proportional to the amount of antibody.

For comparison, a test with a control serum is performed, and the measured absorbance of the control serum is compared to its given concentration. A regression line is determined based on this, and the concentration of the Salmonella antibody in the sample can be determined from the absorbance of the sample measured using the regression line. Alternatively, based on available control sera,
Using the pre-given cut-off formula, the antibody content of the sample to be ranked as positive, negative or unknown (three) is determined.

[0020]

【Example】

 Hereinafter, embodiments of the present invention will be described in detail.

Example 1 Method for Producing Antigen Formulation and Solid Support and Detection of Porcine-Antibodies to Salmonella
Production of Salmonella-lipopolysaccharide-antigen for use in an on- site test kit The required Salmonella serotypes S. thyphimurium and S. choleraesuis (these are Robert Koch-Institut, Bundesinstitut fuer Infektionskrankheiten un
d nicht uebertragbare Krankheiten, Nationales Referenzzentrum fuer Salmo
nellen und andere Enteritiserreger, available from Postfach, 38843 Wemigerode) in 50 l fermenters and incubated at 37 ° C. for 8 hours. Thereafter, 505 ml of a 36% formaldehyde solution is added to the culture so that the final concentration of formalin in the culture is 1% in order to kill the pathogenic bacteria. Incubate at room temperature for at least 4 hours. 2. Prepare sterile controls (sterilized) for each culture and incubate for 24-48 hours. 3. Tangential filtration of culture medium
To concentrate. 4. The cell suspension is centrifuged at 8000 rpm for 30 minutes. 5. The cell pellet is washed three times with a phosphate buffer solution (PBS) and centrifuged at 8,000 rpm for 30 minutes. 6. Next, the cell pellet is resuspended in sterile distilled water at a ratio of 1: 1. 7. The precipitated cells were transferred to three 2000 ml lidded flasks (approximately 300 ml per flask) and kept overnight in the refrigerator. 8. Preparation of acetone for next operation step to be performed the next day: 22 pieces of 500 ml of acetone overnight in a freezer at -22 ° C. 9. 1500 ml of cold acetone is added to each flask containing the cell suspension. 10. Cap the flask, shake well, and store in a freezer at -22 ° C for 2-3 hours. 11. Carefully aspirate the supernatant and discard. Next, 500 ml of cold acetone is added to each flask. The flask is again stored in the freezer for 2-3 hours. 12. After repeating step 11 two more times, the flask is kept overnight in the freezer. 13. Carefully aspirate the supernatant again and discard. 14． The precipitate is then separated into several petri dishes and left overnight under aeration so that residual acetone can be evaporated off. 15. The dried precipitated cells are ground in a mortar, ground into a weighed Erlenmeyer flask (Erlenmeyer flask), and placed overnight under aeration. 16. The powdered bacterial cells are dissolved in sterilized distilled water at a ratio of 1: 1. 17． The cell suspension is incubated in a 65 ° C. water bath for 5 minutes. 18. The same amount of 90% phenol is required depending on the total amount of the cell suspension. To make this, the phenol is dissolved directly in a flask with distilled water. 90%
The phenol solution is also heated to 65 ° C. and incubated for 5 minutes. 19. The phenol solution and the cell suspension were mixed in a 1: 1 ratio in a 2000 ml round bottom flask, shaken vigorously, and further shaken at 65 ° C. for 20 minutes at 65 ° C. (Schuettelwasserb).
Incubate at ad). 20. Cool the round bottom flask to 4 ° C. in an ice water bath. 21. 250 ml (centrifugation) sample container (Becher) of the cell / phenol mixture
And centrifuged at 13000 rpm for 40 minutes. 22. The supernatant is collected, put into 3 to 4 dialysis tubes (for 15 kD), and dialyzed against VE-water (deionized water) overnight. 23. The contents of the dialysis tube are collected again, inspected for clarity, and centrifuged at 5000 xg for 20 minutes if particles can still be identified. 24. The obtained supernatant or dialysate was washed with 0.1% sodium azide (Natriumazid).
) And save. 25. Confirmation of antigen-dilution at the time of use

Preparation of Salmonella-lipopolysaccharide-BSA-conjugate. 1. The obtained Salmonella-lipopolysaccharide-antigen is dissolved in distilled water containing 0.5% TEA (triethanolamine). 2. After resuspension, incubate for 30 seconds in an ultrasonic bath. 3. The final concentration of Salmonella-lipopolysaccharide antigen is adjusted to 1 mg / ml. 4. BSA (Bovine Serum Albumin) suspension in 0.5% TEA prepared in advance (
10 mg / ml) and the Salmonella-lipopolysaccharide-antigen suspension prepared in step 3 are mixed in equal amounts. 5. The mixture obtained is (dispensed) into 1.0 ml aliquots and lyophilized (lyophilisieren). 6. The freeze-dried Salmonella-lipopolysaccharide-BSA-mixture is 0.5% TEA1m
Resuspend in 1 l. 7. Step 5 is repeated three times and Step 6 is repeated twice. 8. The thus obtained Salmonella-lipopolysaccharide-BSA-conjugate is prepared at 2 to 8 ° C.
To save.

Coating of microtitration plate Salmonella antigen mixture was 0.04 mg / l SCS-lipopolysaccharide and 4.0 mg / l
It is made from an aqueous solution of the STM-lipopolysaccharide-BSA conjugate. Nunc / Nunc Polysorb microtitration plates are coated by adding 100 μl of the Salmonella antigen mixture per well and incubating at 7 ° C. for 12 hours. After the incubation, aspirate the unfixed Salmonella antigen mixture and wash the plate three times with 250 μl of distilled water for each well. Next, blocking of the site where the antigen is not bound is performed using a carbonate buffer (Karbonatpuffer).
(0.1 M, pH 9.6) as a solvent in 200 μl of a 1% serum albumin solution for 15 minutes at room temperature. Next, 250 μl of washing buffer (Waschpuffer
) And then the coated and blocked microtitration plates are dried under aeration (Luftstrom) for 12-14 hours.

Use in Test Kit The kit for detecting swine antibodies to Salmonella and the quantification method kit are comprised of the following reagents: Reagents: Amount: Concentration buffer for washing-phosphate buffered saline solution containing Tween 20, concentrated 10-fold, sterilized with Thimerosal 30 ml 2. 2. Sample dilution buffer, protein-containing buffer, sterilized with sodium azide 2 x 30 ml 3. TMB substrate solution, usable as it is 12 ml Stop solution-1M hydrochloric acid 12ml
5. Anti-antibody complex (rabbit (Kaninchen) -anti-porcine-horseradish (Meerrettich) -peroxidase complex) in buffer containing protein stabilizer, concentrate, sterile with Tylomesal 50 μl
6. Control serum No. 1, Salmonella positive, serum obtained from hyperimmunized pigs, freeze-dried 300 μl
7. Control serum No. 2, Salmonella positive, serum obtained from hyperimmunized pigs, freeze-dried 300 μl
8. Control serum No.3, Salmonella positive, serum obtained from hyperimmunized pigs, lyophilized 300 μl
9. Control serum No. 4, Salmonella positive, serum obtained from hyperimmunized pigs, lyophilized 300 μl
10. Control serum No.5, Salmonella negative, serum obtained from pig (low antibody titer), freeze-dried 300 μl
11. Salmonella antigen-coated test plate (inactivated), 96 wells / plate 1

Sample preparation-Juice-usually obtained by freezing and thawing the muscle tissue (Muskulatur) of the diaphragm column (Zwerchfellpfeiler)-is diluted 1:30 in sample dilution buffer. • Dilute serum samples 1: 400 in sample dilution buffer.

Test Procedure: Place all reagents at room temperature (18-23 ° C.) before use, shake gently and mix well. 1. Duplicate according to the inspection plate division on the worksheet (Arbeitsblatt)
Column 2) Record the control and sample positions (well numbers) in the quantification (Doppelbestimmung), eg K1 = A1 / A2; K2 = B1 / B2; K3 = C
1 / C2; K4 = D1 / D2; K5 = E1 / E2; remaining positions (wells)
Is the position for the sample in the double (2 rows) quantification. 2. Pipette 100 μl each of the dissolved control and the pre-diluted sample into the wells of the test plate, and cover the test plate with a cover. 3. Incubate at room temperature for 60 minutes. Then, suck or dump (Auss
Empty the wells by chlagen). 4. Wash (each well) three times with 200 μl of the previously prepared washing buffer. Buffer is removed (from each well) after each washing step. 5. 100 μl of the previously prepared rabbit-anti-pig-horseradish-peroxidase-conjugate is added to each well. 6. Incubate at room temperature for 60 minutes. Each well is then emptied by blotting or tapping. 7. Wash (each well) three times with 200 μl of the previously prepared washing buffer. The buffer is removed (from the wells) after each washing step. 8. 100 μl of TMB-substrate solution is added to each well. 9. Incubate at room temperature for 7 minutes. 10. The reaction is stopped by adding 100 μl of a stop solution to each well. 11. Calibrate the photometer against air as a blank value. The photometer measures at 450 nm and a reference wavelength of 630 nm.

Evaluation The absorbance of the control serum to be measured is compared with the given concentration, and a regression line is determined based on it. The concentration of the Salmonella-antibody in the sample is obtained from the absorbance of the sample measured using it.

Comparative tests were performed on the coated and blocked microtitration plates over a period of 6 months. Then, for 94 / plate / month, the coefficient of variation CV of 2.29-8.7% with respect to the measured sample volume
Was required. This result confirms that the coating plate of the present invention has high long-term stability. Control tests confirm the accuracy of the quantification performed. The control used was tested for cross-reactivity and found to be non-cross-reactive.

Example 2 Preparation of Antigen Formulation and Solid Support and Gefluegel against Salmonella
) Use in antibody detection kit

Production of Salmonella-lipopolysaccharide-antigen A lipopolysaccharide antigen is produced in the same manner as in Example 1 from the required Salmonella serotype S. Typhimurium.

Production of Salmonella-lipopolysaccharide-casein-conjugate The obtained Salmonella-lipopolysaccharide-antigen is dissolved in distilled water containing 0.5% TEA (triethanolamine). 2. After resuspension, incubate for 30 seconds in an ultrasonic bath. 3. The final concentration of Salmonella-lipopolysaccharide-antigen is adjusted to 1 mg / ml. 4. A casein suspension (derived from milk) in 0.5% TEA prepared beforehand (10 mg / m
l) and an equal amount of the Salmonella-lipopolysaccharide-antigen-suspension prepared in step 3 are mixed. 5. The resulting mixture is dispensed into 1.0 ml aliquots and lyophilized. 6. The lyophilized Salmonella-lipopolysaccharide-casein mixture was again added to 1 ml of 0.5 ml.
Suspend in% TEA. 7. Step 5 is repeated three times and Step 6 is repeated twice. 8. The thus obtained Salmonella-lipopolysaccharide-casein-conjugate was used for 2 to 8
Place in ° C.

Preparation of Solid Support First, a mixture of Salmonella-lipopolysaccharide-casein / Salmonella-lipopolysaccharide in a 1: 1 ratio of 1.0 mg / l STM-lipopolysaccharide-casein and 1.0 mg / l STM-
It is made from an aqueous solution containing lipopolysaccharide. The coating of the microtitration plate of Nunc / Nunc Polysorb is carried out by adding 100 μl per well of Salmonella-lipopolysaccharide-casein / salmonella-lipopolysaccharide-mixed antigen and incubating at 7 ° C. for 12 hours. After the incubation, aspirate the Salmonella antigen mixture and wash the plate three times with 250 μl of distilled water per well. Next, the site of the well to which no antigen was bound was replaced with a carbonate buffer (Carbonatpuffer) (0.1 M
, PH 9.6) with 200 μl of a 1% casein solution at room temperature for 15 minutes. Next, 250 μl of washing buffer (Waschpuffer) (PBS-
After washing twice with Tween, the microtitration plate coated and blocked with the antigen mixture is aerated for 12 to 14 hours (Luftstrom).
) Dry under.

Kit for Detecting Avian Antibody Against Salmonella and Method for Quantification Kit comprises the following reagents: Test plate coated with Salmonella antigen (inactivated) 96 wells per plate 2 2. 2. Positive control, 10-fold concentrated antibody, chicken anti-Salmonella serum in buffer containing protein stabilizer, used after dilution with dilution buffer 100 μl 3. Negative control, 10-fold concentrated antibody, chicken serum non-reactive with STM and SE in buffer containing protein stabilizer, diluted with buffer for dilution 100 µl 4. Sample dilution buffer, protein-containing buffer, sterilized with sodium azide 2 x 60 ml 5. Concentration buffer for washing, phosphate buffered saline solution containing Tween 20, concentrated 10-fold, sterilized with Thiomersal 60 ml 6. 6. Concentrate in buffer containing antibody conjugate (anti-chicken-horseradish peroxidase conjugate), protein stabilizer, use after dilution with dilution buffer 100 μl 7. TMB (Tetramethylbenzidin) substrate solution, can be used as is 24 ml Stop solution-1M hydrochloric acid 24ml

Sample preparation Serum sample Dilute with a dilution buffer at a ratio of 1: 400 and mix.・ Egg yolk Dilute with a dilution buffer to a ratio of 1: 400 and mix.

Test Procedure: Place all reagents at room temperature (18-23 ° C.) before use, shake gently and mix.
1. Pipette 100 μl of dilution buffer as a blank value into well A1. 2. Pre-prepared positive controls were added to wells B1 and C1, respectively.
Pipette 0 μl each. 3. Pre-prepared negative controls were added to wells D1 and E1, respectively.
Pipette 0 μl each. 4.1 100 μl of sample diluted 1: 400 or 90 μl of dilution buffer
Then, 10 μl of the sample diluted 1:40 is pipetted into the remaining wells, and the test plate is covered with a cover. 5. Incubate at room temperature for 60 minutes, after which each well is emptied by blotting or tapping. 6. Wash (each well) three times with 200 μl of the previously prepared washing buffer. Remove buffer (from wells) after each wash. 7. 100 μl of the prepared complex is added to each well. 8. Incubate at room temperature for 60 minutes, after which each well is emptied by blotting or tapping. 9. Wash (each well) three times with 200 μl of the previously prepared washing buffer. Remove buffer (from wells) after each wash. 10. Add 100 μl of TMB-substrate solution to each well. 11. Incubate at room temperature for 7 minutes. In order to perform a precise test, each well of the test plate must be reacted for exactly 7 minutes. That is, the substrate solution and the stop solution must be pipetted in this order. 12. The reaction is stopped by adding 100 μl of a stop solution to each well. 13. The photometer is calibrated against the blank value at the position A1. 450nm with photometer
And a measurement at a reference wavelength of 630 nm.

Evaluation The evaluation is performed by obtaining a cut-off value (Cut-Off-Wert) from the measured values of the positive and negative controls included in the test kit. In order to perform an effective test, the negative control (NK) had an average (center) value (MW) of OD0
0.3 or less, the positive control (PK) must have an average (center) value of OD 0.6 or more. The value of the blank at position A1 should be less than OD0.1, but may be less than OD0.2.

A. Field Infection (Feldinfektion) To detect a negative sample, the cutoff value is determined from the OD value according to the following equation. -Negative cutoff: (MWPK-MWNK) / 5 + MWNK Samples with OD values below the negative cutoff are considered negative if the test is valid. 1. In order to detect a positive sample, the cutoff value can be determined from the following equation. -Positive cutoff: (MWPK-MWNK) / 3 + MWNK Samples with an OD value greater than or equal to the positive cutoff are considered positive if the test is valid. 1. Samples with OD values between the negative and positive cutoff values should be tested again, possibly some time after taking a new sample.

B. Vaccination (Impfung) Inoculation method (Adsorbatvakzinen), live inoculum (Lebendim)
pfstoffe)), and there are special factors related to stock (preservation), so no limit values can be provided. The cutoff value for field infection is only a guide. To monitor vaccine stock, experimenters should determine their own parameters.

[0039]

【The invention's effect】

The use of a Salmonella antigen formulation results in a homogeneous, stable and highly sensitive coating of the antigen in the experimental results. Improved shelf life and homogeneity of microtitration plates coated with Salmonella antigen formulations,
Therefore, it becomes possible to manufacture a standardized (standardized) ELISA inspection system. The shelf life of a microtitration plate coated and blocked with a Salmonella antigen formulation reaches at least 7 months when the coefficient of variation CV is less than 10%. A test system manufactured based on the antigen formulation of the present invention, STM-lipopolysaccharide-casein complex / STM-lipopolysaccharide-mixed antigen, is suitable for Salmonella enteritidis, Salmonella typhimurium and Salmonella galinarum-prolum. Enables simultaneous detection of antibodies.

Claims (19)

[Claims] At least one Salmonella enterica
(Rica) lipopolysaccharide-protein-conjugate. 2. The Salmonella antigen formulation of claim 1, comprising at least one Salmonella enterica lipopolysaccharide-serum albumin complex. 3. The Salmonella antigen formulation according to claim 1, comprising at least one lipopolysaccharide-casein complex of Salmonella enterica. 4. A composition comprising at least one lipopolysaccharide-protein conjugate of Salmonella enterica and at least one other antigenic or lipopolysaccharide preparation of Salmonella enterica or other bacterium. A Salmonella antigen formulation according to claim 1. 5. A Salmonella antigen mixture comprising a Salmonella-lipopolysaccharide-serum albumin-conjugate based on Salmonella typhimurium (STM) type Salmonella antigen and a Salmonella antigen of Salmonella cholerae suisse (SCS) type. The Salmonella antigen formulation according to claim 4, characterized in that: 6. A Salmonella antigen mixture comprising a lipopolysaccharide-casein complex of Salmonella typhimurium and a lipopolysaccharide preparation of Salmonella typhimurium.
Or the lipopolysaccharide-casein complex of Salmonella typhimurium. 7. A Salmonella-lipopolysaccharide-serum albumin complex based on the Salmonella cholerae suisse (SCS) type Salmonella antigen and the Salmonella typhimurium (STM) type Salmonella antigen is 1:10 to 1: 200. The Salmonella antigen mixture according to claim 5, wherein the mixture comprises: 8. The salmonella-lipopolysaccharide-serum albumin according to claim 5 or 7, wherein the Salmonella-lipopolysaccharide-serum albumin complex is a Salmonella lipopolysaccharide-bovine serum albumin complex. Complex. 9. A kit for detecting an antibody against Salmonella in a gravy sample, serum sample, yolk sample or other fluid sample by an immunological detection method, particularly a heterogeneous ELISA method, comprising: Contains an antigen for immunoreaction with the antibody, and further contains various control sera, washing concentration buffer, dilution buffer and anti-antibody enzyme complex, and corresponding substrate solution and stop solution. A kit for detecting an anti-Salmonella antibody, wherein the support is provided with a Salmonella antigen preparation comprising at least one lipopolysaccharide-protein-complex of Salmonella enterica. 10. The kit of claim 9, wherein the support is provided with a Salmonella antigen formulation comprising at least one lipopolysaccharide-serum albumin complex of Salmonella enterica. 11. The kit of claim 9, wherein the support is provided with a Salmonella antigen formulation comprising at least one Salmonella enterica lipopolysaccharide-casein complex. 12. The salmonella comprising at least one lipopolysaccharide-protein conjugate of Salmonella enterica and at least one other antigenic or lipopolysaccharide preparation of Salmonella enterica or other bacteria. The kit according to claim 9, wherein an antigen formulation is provided. 13. The Salmonella antigen mixture comprising a Salmonella-lipopolysaccharide-serum albumin complex based on Salmonella typhimurium (STM) type Salmonella antigen and a Salmonella cholerae suis (SCS) type Salmonella antigen. The kit according to claim 12, wherein is provided. 14. The support is a lipopolysaccharide of Salmonella typhimurium.
The kit according to claim 12, wherein a Salmonella antigen mixture comprising: a casein complex and a lipopolysaccharide preparation of Salmonella typhimurium, or a lipopolysaccharide of Salmonella typhimurium-casein complex is disposed. 15. The support comprises a Salmonella-lipopolysaccharide-serum albumin-conjugate based on Salmonella cholerae suisse (SCS) type Salmonella antigen and Salmonella typhimurium (STM) type Salmonella antigen. The kit according to claim 13, further comprising a Salmonella antigen mixture containing the cells in a ratio of 1:10 to 1: 200. 16. The kit according to claim 13, wherein the Salmonella-lipopolysaccharide-serum albumin complex is a Salmonella-lipopolysaccharide-bovine serum albumin complex. 17. The method according to claim 9, wherein the anti-antibody-enzyme conjugate is an anti-porcine-immunoglobulin-enzyme conjugate for detecting an antibody against Salmonella in a pig sample. The kit according to any one of claims 1 to 4. 18. The method according to claim 9, wherein the anti-antibody-enzyme complex is an anti-chicken-immunoglobulin peroxidase complex for detecting an antibody against Salmonella in a chicken sample. A kit according to any one of the preceding claims. 19. The kit according to claim 9, wherein control sera from salmonella positive and negative animals from veterinarily controlled components are used as control sera.