JPH0672889B2 - Strain-specific tuberculin diagnostic agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention comprises BCG-derived D substance as an active ingredient,
The present invention relates to a tuberculin diagnostic agent for discriminating between a BCG vaccine-sensitized organism and a human tubercle bacillus-sensitized organism, which induces a specific reaction to a CG vaccine-sensitized organism.

Conventionally, a so-called tuberculin test, which is an intradermal reaction test using purified tuberculin (hereinafter abbreviated as PPD), has been generally performed as an index of sensitization with Mycobacterium tuberculosis. However, although the PPD used in the tuberculin reaction is called purified tuberculin, it has been found to be a complex of various antigens, and it is considered that cross-reactivity is observed in M. tuberculosis and BCG sensitized organisms. ing.

Therefore, when a seroconversion is observed for the first time in a tuberculin reaction test, it is determined whether it is due to infection of human M. tuberculosis, which is dangerous for the human body, or due to BCG vaccination for immunizing anti-tuberculosis. It is extremely difficult to do so, and in this case it is necessary to give due consideration to the subsequent health management.

From these facts, it is strongly expected that highly purified tuberculin antigens, especially bacterium-type specific antigens, which replace PPD will appear, and many efforts have been accumulated. In recent years, with the progress of substance purification methods, some highly purified tuberculin antigens have been reported, but the ones whose properties as bacterium-type-specific antigens have been clarified are extremely rare and have not been put to practical use. . For example, Nagai et al. [Infection and Immunity, 31
, 1152 (1981)], MPB70 is a highly purified tuberculin antigen produced by BCG bacteria and is considered to be a bacterium-type specific antigen that reacts only with BCG-sensitized organisms. It is said that the internal reaction becomes negative after 20 weeks after sensitization, and it is considered to have little practical utility in this field.

The present inventor has previously isolated substance D, which is a novel and potent tuberculin antigen derived from Mycobacterium tuberculosis, and has already filed a patent application (Japanese Patent Application No. 57-186830). After that, as a result of further detailed examination of the properties of substance D, it was confirmed that substance D obtained from BCG bacteria is an extremely potent tuberculin antigen compared to PPD in the BCG vaccine-sensitized body. It has been found that, unlike the BCG vaccine-sensitized organism, the human-type Mycobacterium tuberculosis-sensitized organism exhibits extremely weak activity as compared with PPD. Furthermore, when an amount of antigen sufficient to induce a sufficient skin reaction in the BCG vaccine-sensitized organism was intradermally inoculated into the M. tuberculosis-sensitized organism, it was completely impossible to induce the skin reaction. The present inventor has paid attention to these findings and completed the present invention.

That is, the object of the present invention is to use a substance D derived from BCG bacterium as a tuberculin antigen and utilize the reaction of the BCG vaccine sensitized organism specifically, to obtain a BCG vaccine sensitized organism and a human type Mycobacterium tuberculosis sensitized organism. It is to provide a tuberculin diagnostic agent for discrimination.

Next, a structural example of substance D which is a tuberculin antigen used in the present invention and physicochemical properties of substance D will be shown.

Production Example 1 Production of intermediate purified product from BCG bacteria Mycobacterium bovis BCG (ATCC 19015) meat extract-
Using glycerin medium, static culture was carried out at 37 ° C for 6 weeks.

50 mM to 2 kg of wet cells obtained by filtering the culture with cheese cloth
After adding 10 parts of phosphate buffer (pH 7.0) to suspend the cells, the cells were disrupted with ice cooling with Dyno-Mill.

The obtained crushed bacterial cells were centrifuged under cooling to remove solid components, and a cell-free extract 8.2 was obtained. To this, 24.6 g of streptomycin sulfate as a nucleic acid precipitating agent was added, and the mixture was sufficiently stirred, then allowed to stand overnight at 4 ° C. to generate a precipitate, and centrifuged to obtain a supernatant 7.8.

6 kg of solid ammonium sulfate was added to the nucleic acid-removed supernatant,
After saturation with stirring, the mixture was left standing overnight to generate a precipitate, which was then centrifuged to obtain a precipitate. This precipitate was suspended in a small amount of distilled water, filled in a dialysis tube, and dialyzed against distilled water 10 for 2 days.
Thereafter, the dialyzed external solution was made into a 10 mM acetic acid suspension (pH 4.2), and dialyzed at 4 ° C. for 3 days. The dialysis external solution was replaced with a new solution twice a day. After the completion of dialysis, centrifugation was performed to obtain a supernatant 2.2.

To this supernatant, cold ethanol 1.0 was added little by little with stirring, and the mixture was allowed to stand overnight at 4 ° C. and then centrifuged to obtain a supernatant, which was concentrated under reduced pressure to about 100 ml.

The column was packed with CM cellulose (CM52, manufactured by Wattmann) that had been equilibrated with 70 mM acetate buffer (pH 4.1) in advance.
A 15 x 200 mm bed was created. The supernatant was dialyzed against the same buffer and passed through this column at a flow rate of 30 ml / h, and then eluted with the same buffer. Absorbance 0.05 in eluate
(280 nm) and above was collected. This fraction contained 238 mg of protein.

Then, this fraction was concentrated under reduced pressure and then dialyzed against 5 mM Tris-hydrochloric acid buffer (pH 8.0). The dialyzed product was passed through a QAE Sephadex A-25 (Falmacia) column (15 × 160 mm) equilibrated with the same buffer at a flow rate of 30 ml / h, and then washed with 700 ml of the same buffer.

Next, the intermediate purified product was eluted using the same buffer solution to which 0.15 M sodium chloride was added. A portion showing an absorbance of 0.01 (280 nm) or more in the eluate was collected and used as an intermediate purified product. The protein content of the obtained intermediate purified product was 106 mg. The intermediate purified product was dialyzed against distilled water and then freeze-dried.

Production Example 2 Production of substance D from the intermediate purified product Sephadex G100 column (264 × 960, which had been equilibrated with 10 mM phosphate buffer to which 0.15 M sodium chloride was added in advance.
mm), the intermediate purified product of Production Example 1 dissolved in 5 ml of the same buffer was developed with the same buffer at a rate of 11 ml / h according to a conventional method. About 2m by collecting a portion of the effluent volume of 330 to 375ml
It was concentrated under reduced pressure to l.

Then, using a Sephadex G75 Superfine column (16 × 1480 mm) that had been equilibrated with the above buffer in advance, the above concentrate was developed with the same buffer at a rate of 6 ml / h. A portion of the effluent volume of 130 to 160 ml was collected. This fraction contained 11 mg of protein.

Furthermore, this fraction was dialyzed against 5 mM Tris-HCl buffer (pH 8.0) and equilibrated with the same buffer in advance.
After passing through a Sephadex A-25 column (10 × 450 mm) at a rate of 10 ml / h, linear concentration gradient elution with 150 ml of each of the same buffer and a buffer containing 0.12 M sodium chloride was performed. The eluate was measured for protein content and sugar content, and the salt concentration was approximately 0.07.
The peak of glycoprotein eluted in the region of ~ 0.09M was collected. This fraction was packed in a dialysis tube, dialyzed against the above-mentioned buffer solution 1, and then QAE Sephadex A-25 under the same conditions.
Chromatography using a column was performed.

The obtained fraction containing the desired product was dialyzed against distilled water and freeze-dried to obtain 5.4 mg of substance D (weight of freeze-dried product). The amount of protein of substance D thus obtained was 0.74 mg.

The protein amount and sugar amount were measured by the following methods.

(I) Protein: Lowry method using bovine serum albumin as standard (Stauffer, CE, Analytical Biochemistry, 69, 646
P., 1975).

(Ii) Carbohydrate: a phenol-sulfuric acid method using glucose as a standard (Dubois, M. et al, Analytical Biochemistry, 28, 350
P., 1956).

Next, the physicochemical properties of substance D are shown.

(1) It is a glycoprotein and has a white powder appearance.

(2) Solubility in solvent; soluble in water, methanol,
Insoluble in ethanol, ether and acetone.

(3) Molecular weight; 26,000 according to gel filtration method using Sephadex G75 Superfine (4) Ultraviolet absorption spectrum; as shown in FIG.

(5) Infrared absorption spectrum; as shown in FIG.

(6) Amino acid composition (g / D substance 100g) Aspartic acid 0.5, Threonine 9.1, Serine 1.4, Glutamic acid 11.4, Proline 36.4, Glycine 3.1, Alanine
4.0, valine 7.4, isoleucine 24, leucine 1.5 (7) Sugar composition (g / D substance 100g) Mannose 19.0, Glucose 1.2 (8) By the double diffusion method using agar plates, one Settling lines are formed.

(9) The delayed allergic reaction activity in BCG vaccine-sensitized guinea pigs is 8 times or more that of purified tuberculin.

The substance D thus obtained showed about 30 times as much activity per dry weight as that of PPD in the intradermal reaction test in BCG vaccine-sensitized guinea pigs, as shown in the test examples described below. PPD in sensitized guinea pigs
It is about one-fifteenth of the above, and it is judged to have sufficient utility depending on the inoculation amount of the antigen used.

In addition, when the tuberculin reaction response persistence after sensitization with BCG vaccine was measured, it showed almost the same transition as PPD used as a control drug, and it is considered to be highly practical.

In addition, in an in vitro blast transformation test of splenic lymphocytes using spleen cells of M. tuberculosis-sensitized guinea pigs, in the case of BCG vaccine sensitization, a sufficient reaction was observed even at an antigen addition amount of 0.2 μg / ml, but in humans In the case of sensitization with type M. tuberculosis, it is necessary to add 25 μg / ml or more of antigen to obtain a similar reaction. This result shows that the skin reaction of the living body by this diagnostic agent and the in vitro immune reaction typified by the lymphocyte blastogenic reaction are in good correlation with each other. It can be said that the character as a specific antigen is made clearer.

The toxicity of substance D used in the present invention is extremely weak, and when injected subcutaneously into mice, the acute toxicity shows an LD ₅₀ value of 100 mg / kg or more, which is sufficiently safe compared to the inoculation dose.

Tests using this diagnostic agent include the method of directly inoculating the living body to examine the reaction, and the method of in vitro immunoassay using immunocompetent cells, but the skin is usually tested according to the method of the tuberculin test. Internal reaction test is simple.

When this diagnostic agent is used for an intradermal reaction test, it is preferable to use a phosphate buffered sodium chloride solution at a concentration of 50 to 500 ng / ml.
The formulation prepared to give 0.
Inoculate 1 ml and measure local redness and induration 24 or 48 hours later. If the reaction value (mean value of the vertical and horizontal diameters) is 10 mm or more, the reaction with this diagnostic agent is positive, and it is judged as a BCG vaccine-sensitized organism.

In addition, for example, if it is determined to be the first seroconversion by inoculation with PPD, but the reaction with this diagnostic agent is determined to be negative, the trunk line of M.tb. .

Hereinafter, examples and usefulness of the present invention will be shown by test examples.

Example Formulation for Diagnostic Agent 1 mg of BCG cell-derived substance D obtained in Production Example was dissolved in 2 distilled water for injection, and then 10 g of lactose was added to dissolve it, and then Nyukuri Pore filter (0.2 μm; It was sterilized and filtered using Nyukrypoor Corporation. Each 1 ml of the obtained filtrate was aseptically dispensed into a vial bottle and then freeze-dried to produce a diagnostic drug preparation.

Test Example 1 Delayed-type allergic reaction activity test of substance D 9 weeks ago, BCG vaccine (manufactured by Nippon BCG Co., Ltd., the same applies hereinafter), subcutaneous injection of 500 μg of physiological saline suspension,
Liquid paraffin suspension of 200 μg of heat-killed Mycobacterium tuberculosis Aoyama B strain or 200 μg of heat-killed Mycobacterium tuberculosis H37Ra
Using the Hartley fiber guinea pigs, each of which has been delayed sensitized by intramuscular injection of the liquid paraffin suspension of, the substance D obtained in the production example and PPD (purified tuberculin, manufactured by Nippon BCG Co., Ltd.) It was inoculated intradermally on the flank. Table 1 shows the results of measuring the local redness diameter (arithmetic mean value of the longitudinal diameter and the transverse diameter) after 24 hours.

Test Example 2 Sustained test of delayed type allergic reaction in BCG vaccine-sensitized guinea pigs Hartley female guinea pigs that were delayed-sensitized by subcutaneous injection of 500 μg of BCG vaccine in physiological saline were divided into 3 groups, and 1 Group D 6 animals were inoculated intraperitoneally with the substance D obtained in Production Example and PPD (purified tuberculin, manufactured by Nippon BCG KK) at 6, 12 and 24 weeks after sensitization. Table 2 shows the results of measuring the local redness after 24 hours.

Test Example 3 Lymphocyte Juvenile Test BCG vaccine-sensitized guinea pigs and human tubercle bacillus Aoyama B strain-sensitized guinea pigs prepared in the same manner as in Test Example 1 were used. The spleen was aseptically removed from each guinea pig and the method of Onozaki et al.
37, 1977) to prepare a splenocyte suspension. This 10
The cells were suspended in RPM1640 medium containing% fetal bovine serum at 2 × 10 ⁶ / ml and dispensed into a 96-well culture plate to give 3 × 10 ⁵ cells per well. After adding D substance or PPD (P-TB reagent, manufactured by Mitsui Pharmaceutical Co., Ltd.) antigen obtained in Reference Example to this, 3 days for 4 days in a 5% CO ₂ incubator.
The cells were cultured at 7 ° C., and 0.5 μCi of ³ H-TdR was added 6 hours before the end of the culture. Thereafter, the radioactivity incorporated into the cells was measured by the method of Ohno et al.

The results are shown in Table 3.

The reaction value was calculated by the following formula.

Test Example 4 Acute toxicity The substance D obtained in Production Example was dissolved in physiological saline to 100 6-week-old female ddY mice per group, and 100 mg / kg body weight was subcutaneously administered. This substance did not suppress weight gain during the observation period of 1 week after administration, and no death occurred. This result indicates that the LD ₅₀ in subcutaneous administration of substance D is 100 mg / Kg or more.

[Brief description of drawings]

FIG. 1 is an ultraviolet absorption spectrum diagram of substance D (1 mg / ml distilled water), and FIG. 2 is an infrared absorption spectrum diagram of substance D.

Claims (1)

[Claims] 1. A tuberculin diagnosis for distinguishing between a BCG vaccine-sensitized organism and a human tubercle bacillus-sensitized organism, which comprises a substance D derived from BCG bacteria as an active ingredient and causes a specific reaction to the BCG vaccine-sensitized organism. medicine.