Set immunoassay for retrospective diagnosis of infectious bovine rhinotracheitis animals

 

(57) Abstract:

The invention relates to veterinary Virology and biotechnology. The kit contains a 96 well microplate, sensitised afinno purified virus antigen RTI, antiart (positive) and normal (negative) native control sera, conjugate individualo immunoglobulin class G with peroxidase, a 20-fold concentrate califofnia buffer, non-ionic detergent, 10-fold concentrate of substrate buffer, orthophenylphenol, hydrogen peroxide and stop reagent. The kit increases the sensitivity of the immunological reaction and reduces the time to diagnose. table 2.

The invention relates to biotechnology and can be used for large-scale retrospective immunoassay in the diagnosis of infectious bovine rhinotracheitis animals.

Known kit for the diagnosis of infectious bovine rhinotracheitis animals enzyme-linked immunosorbent assay (ELISA), with panels for the production of ELISA, the antigen of the virus of infectious bovine rhinotracheitis in cattle, antiart serum control serum, individuai immunoperoxidase conjugate, californiakitty one-deputizing, califas the (Technical conditions 19.10.97-89 "Kit for immunoassay in the diagnosis of infectious bovine rhinotracheitis in cattle", approved BS Gosagroprom USSR 01.08.89). (Prototype) (1). However, this set has a lower sensitivity, high cost and requires more time for the given reaction.

The aim of the invention is to increase the sensitivity, the shortening of the setting reaction and lowering the cost of the kit immunoassay for retrospective diagnosis of infectious bovine rhinotracheitis animals by identifying specific antiart antibodies in the blood sera and other clinical material. This goal is achieved by the fact that the set consists of: 96 well polystyrene microplate, sensitised affine purified antigen of the virus of infectious bovine rhinotracheitis, strain TC-a (VIEW) B2 (2), in the amount of 0.075 - 0.125 mg per well, antiart (positive) and normal (negative) control sera, conjugate individualo immunoglobulin class G with peroxidase, a 20-fold concentrate califofnia buffer, non-ionic detergent (tween-20, -80 or Triton X-305), 10-fold concentrate of substrate buffer, orthophenylphenol, hydrogen peroxide and stop reagent.

The kit includes the following biological and chemical components:

1. 96 hole mikta, strain TC-a (VIEW) B2 in the amount of 0.075 - 0.125 mg per well 2 pcs.

2. Antiart (positive) serum of cattle 1 amp. (PL. ), the amount of 0.005 cm3, working dilution 1:200, titer in ELISA least 1:12600.

3. Normal (negative) serum 1 amp. (PL.), the amount of 0.005 cm3working dilution of 1:200.

4. Individuai immunoperoxidase conjugate lyophilized amp. (PL. ), volume 1 cm3, working dilution 1:20.

5. 20-fold concentrate of the incubation buffer (0.2 M califofnia buffer containing ZM sodium chloride, pH 7,3) 1 FL. volume 50 cm3.

6. Tween-20, -40, -80, or Triton X-305, 1 FL. - 1 cm3.

7. 10-fold concentrate of substrate buffer (1.5 M nitrate phosphate buffer solution, pH 5.0) 1 FL. volume 5 cm3.

8. Orthophenylphenol (OFD), 1 FL. - 20 mg.

9. Gidroperit (hydrogen peroxide), 1 table. - 0,5 g

10. Stop reagent 1M solution of sulfuric acid 1 FL. volume 10 cm3.

Preparation of kit components.

1. Preparing working buffers

1.1. Incubation of phosphate-saline tinby buffer solution for dilating control, analyzed sera and individualo conjugate, th water up to 1000 cm3and dissolved in this volume, 1 cm3Tween-20, -40, -80, or Triton X-305. The incubation buffer is a clear solution with a pH of 7.2 to 7.4.

1.2. Substrate citrate phosphate buffer solution pH 5.0-5.2 to prepare by bringing the contents of the vial with a 10-fold citrate phosphate buffer up to 50 cm3with distilled water.

The substrate solution is prepared not earlier than 10 minutes prior to the introduction into the wells of the panels. For this, 20 mg of orthophenylphenol dissolved in 2 cm3rectified spirit, add 48 cm3substrate buffer and contribute 0.8 cm3the hydrogen peroxide solution obtained by dissolving tablet hydropenia 10 cm3of distilled water. The solution should be transparent and should have a light yellow color.

2. Preparation of the antigen of the virus of infectious bovine rhinotracheitis in cattle.

A method of obtaining antigen of the virus of infectious bovine rhinotracheitis in cattle for the reaction ELISA (specifications 19.10.97-89 "Kit for immunoassay in the diagnosis of infectious bovine rhinotracheitis in cattle", approved BS Gosagroprom USSR 01.08.89) that includes a reproduction of the virus RTIs in cell culture PT-80, the concentration of the SAI PT-80 ultrasound and clarification of the suspension by low-speed centrifugation. However, the known method is tedious and allows you to get an antigen highly contaminated cell fragments, which affects the sensitivity of the reaction. In the present set of antigen of the virus of infectious bovine rhinotracheitis prepared as follows: to do this, the monolayer of cells PT-80 after the reproduction of the virus of infectious bovine rhinotracheitis, strain TC-a (VIEW) B2, precipitated by centrifugation and resuspended in 0.01 M califorina buffer containing 0.15 M sodium chloride, 1% Triton X-100, pH of 7.2 to 7.4. The resulting suspension is treated with ultrasound apparatus of the type UZDN-21 power 22 kHz impulsive 6 times in 30 seconds. Then disintegrate centrifuged at 5 thousand rpm for 25-30 minutes. The formed precipitate is discarded, and the supernatant diluted in 10 volumes califofnia buffer containing 0.15 M sodium chloride, pH of 7.2 to 7.4. Received antigenaemia suspension is applied on a column immunosorbent assay (Enrichment agarose with immobilized as a ligand monoclonal antibodies to various glycoproteins of the virus of infectious bovine rhinotracheitis). After an hour of incubation in the mode of the reactor column was washed with 10 volumes califofnia buffer containing 0.15 M sodium chloride, 0.1% of Triton X-100, pH of 7.2 to 7.4, and 5 volumes of the t 0.1 M glycine buffer, pH 2.0. Obtained in this way, the antigen of the virus of infectious bovine rhinotracheitis is a complex of glycoproteins with a molecular mass of from 54 to 180 kilodaltons. The optimal concentration of antigen for sensitization micropanel investigated checkerboard titration with antient serum in ELISA (table 1).

Example 1. The antigen at a concentration of 0.75 ág/cm3califofnia buffer, pH 7,2 - 7,4 poured into 0.1 cm3to the wells of the microplate for setting ELISA, after an hour incubation at +37oWith five times washed with the same buffer, and dried for half an hour at room temperature. Then in the wells of the microplate was added 0.1 cm3positive control serum in dilutions from 1: 200 to 1:3200 in the incubation buffer, prepared according to p. 1.1. After an hour incubation at +37oAnd five wash incubation buffer to each well was added 0.1 cm3working dilution individualo immunoperoxidase conjugate and incubated for one hour at +37oC. Then the microplate five times washed with incubation buffer and show the reaction of substrate buffer, prepared as described in paragraph 1.2. The reaction of the account after 30 minutes spectrophotometrical the Egan at a concentration of 1.0 microgram/cm3califofnia buffer, pH 7,2-7,4 poured into 0.1 cm3to the wells of the microplate for setting ELISA, after an hour incubation at +37oWith five times washed with the same buffer, and dried for half an hour at room temperature. The reaction of the ELISA results carried out as described in Example 1.

Example 3. The antigen at a concentration of 1.25 µg/cm3califofnia buffer, pH 7,2-7,4 poured into 0.1 cm3to the wells of the microplate for setting ELISA, after an hour incubation at +37oWith five times washed with the same buffer, and dried for half an hour at room temperature. The reaction of the ELISA results carried out as described in Example 1.

Example 4. The antigen at a concentration of 0.50 mg/cm3califofnia buffer, pH 7,2-7,4 poured into 0.1 cm3to the wells of the microplate for setting ELISA, after an hour incubation at +37oWith five times washed with the same buffer, and dried for half an hour at room temperature. The reaction of the ELISA results carried out as described in Example 1.

Example 5. The antigen at a concentration of 1.50 mg/cm3califofnia buffer, pH 7,2-7,4 poured into 0.1 cm3to the wells of the microplate for setting ELISA, after an hour inquietude. The reaction of the ELISA results carried out as described in Example 1.

According to the results of a checkerboard titration of antigen and serum in the reaction ELISA optimal working concentration of affinity purified antigen of the virus of infectious bovine rhinotracheitis - mcg/cm3adsorption buffer during the breeding antiart serum 1:200 in incubation buffer and shows a higher sensitivity of the proposed collection is compared to the prototype (table 1).

3. Preparation of control sera.

As a positive control, use antiart sera obtained from naturally recover from infectious bovine rhinotracheitis animals with titers in ELISA is not lower than 1:12600. To do this, animals spend a sterile blood sample from the jugular vein in a volume of 300-400 cm3from each animal. The blood is incubated at +37oWith, then make the stroke and to defend serum incubated for 12-14 h at 4oC. Then the serum separated from the blood clot, clarify by centrifugation for 15 minutes at about 2.5 thousand. /min, to inactivate a water bath at 56oWith 40 minutes Then serum preserved with 0.02% sodium azide and pour 0,005 cm3in ampoules.

Rosecchi quarantine for two months and negatively reactive in ELISA with antigen of the virus of infectious bovine rhinotracheitis, spend a sterile blood sample from the jugular vein in a volume of 300-400 cm3from each animal. The blood is incubated at +37oWith, then make the stroke and to defend serum incubated for 12-14 h at 4oC. Pooled serum is poured into vials under aseptic conditions and treated according to p. 3.

5. Getting individualo conjugate. Individule immunoglobulins IgG cattle labeled with the enzyme horseradish peroxidase, prepared in accordance with the "Provisional instructions for the manufacture and control antivitamin immunoglobulins labeled with the enzyme horseradish peroxidase approved the BS of the Ministry of agriculture of the USSR 9.12.85 THE 46-21-78-85 (3) and complete their set.

6. Illustration of use of the Toolkit.

6.1. Preparation of biological components of the kit immunoassay for retrospective diagnosis of infectious bovine rhinotracheitis animals.

The contents of each ampoule or vial 4 individuum immunoperoxidase conjugate was dissolved in 20 cm3the incubation buffer, receiving a working concentration of the conjugate. Positive and negative (ampoules or vials). 2 and 3 serum dissolved in 1 cm3the incubation buffer, receiving a dilution of 1: 200. Dissolved serum and EN is Fermentas reaction (ELISA).

6.2.1. Titration of sera: sera titrated on long row of panels. All wells of the panel, excluding the hole of a number of panels 1A, is used to control the substrate mixture and the output of the device to zero, contribute to 0.1 cm3the incubation buffer. In the wells of row a (from 2A) contribute to 0.1 cm3positive sera prepared according to p. 6.1., the number In (from 1B) - negative serum, prepared in a similar way according to p. 6.1. In the 1st hole of the other rows contribute to 0.1 cm3analyzed serum samples, pre-diluted 1:200 in incubation buffer in a separate test tubes or tablets. The contents of all wells titrated in a long line for each serum, using interchangeable tips to micropipettes and get the dilution of sera from 1:200 to 1:409600. Of the last holes of each row 0.1 cm3content removed. Microarrays close the lid, and incubated at 370,5oC for one hour and then washed five times with incubation buffer.

In the wells washed micropanel contribute to 0.1 cm3working solution individualo conjugate, prepared according to p. 6.1, incubated in a thermostat at 370,5oWith 1 hour and washed five times the incubation BU is ribaut in the dark at room temperature for 30 minutes After 30 min the reaction is stopped by adding to each well of 0.05 cm3stop reagent 10.

The results take into account spectrophotometric no later than 30 minutes after the response and expressed in units of optical density (OD492). The titer in ELISA accept the serum dilution giving an OD value of4922 times the OD value492control sera.

6.2.2. When epidemiological survey of livestock in order to identify seropositive and seronegative animals serum not titrated and used at the same dilution 1:200, but in two replications, using for each serum 2 holes die. The setting reaction and the preparation of sera carried out, as described in paragraph 6.2.1. The panel closed with a lid and incubated at a temperature of 370,5oWith thermostat in an hour.

Washing micropanel, the introduction of the conjugate and the manifestation of the reaction carried out as described in paragraph 6.2.1.

The results take into account not later than 30 minutes after the response visually or spectrophotometrically.

Visual accounting: in the presence of specific antibodies in the wells with the test sera are orange-brown staining in the serum dilution 1: 200, comparable weak yellowing, similar to the color of the negative control.

Spectrophotometric accounting: recording the results of the reaction conducted at a wavelength of 492 nm. The results are expressed in units of the optical plane (OP492). Positive consider samples, OP492.which in a dilution of 1:200 in 2 and more times higher than the optical density of the negative control. Dubious consider samples, OP492which is 0,150-0,199 O. that is, the Level of optical density below 0,150 O. E. indicates a negative reaction.

The proposed set was tested in a comparative titration of the investigated sera by ELISA and PH. (Table 2.).

Found that in 86% of cases, positive results were obtained in both methods. All negative sera in ELISA were negative and PH. However, in 14% of cases of infectious bovine rhinotracheitis in cattle diagnosed only by using the proposed ELISA test.

Sources of information

1. Specifications 19.10.97-89 "Kit for immunoassay in the diagnosis of infectious bovine rhinotracheitis in cattle", approved BS Gosagroprom USSR 01.08.89 (prototype).

2. The Russian passport controlrole PI and control antivitamin immunoglobulins, labeled with the enzyme horseradish peroxidase", approved BS Ministry of agriculture of the USSR 09.12.85,

Set immunoassay for retrospective diagnosis of infectious bovine rhinotracheitis animals, characterized in that it contains a 96 well polystyrene microplate, sensitised affine purified antigen of the virus of infectious bovine rhinotracheitis, strain TC-a (VIEW)B2 in the amount of 0.075 - 0.125 mg per well, antiart (positive) and normal (negative) native control sera, conjugate individualo immunoglobulin class G with peroxidase, a 20-fold concentrate califofnia buffer, non-ionic detergent (tween-20, -80 or Triton X-305), 10-fold concentrate of substrate buffer, orthophenylphenol, the hydrogen peroxide, and stop reagent.

 

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