JPS62148859A - Immunological measurement method for bacillus retained in bacterial exosporium - Google Patents

Abstract

PURPOSE:To measure all influenza bacilli without overlooking regardless of types and to improve the degree of detection by bringing the antibody obtd. by immunizing the crude exosporium protein of the hemophilus influenza bacillus as an antigen into an animal into an immunological reaction with a specimen. CONSTITUTION:Any influenza bacilli are usable regardless of the types thereof. A specified amt. of the crude exosporium protein is inoculated intracutaneously, subcutaneously, intramuscularly, intraperitoneally and intraveneously to the animal or into the sole of a foot thereof several times, by which the antiserum is obtd. The immunogical reaction which is generally practised may be simply executed in the case of measuring the influenza bacilli in the specimen by using the antiserum (antibody) prepd. in the above-mentioned manner; for example, an enzyme immunoassay, radioimmunoassay, direct fluorescent antibody method, indirect fluorescent antibody method, reverse passive hemagglutination method, reverse passive latex agglutination method and coagulation method are usable.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an immunoassay method for bacteria harboring the outer membrane of a bacterial body, and more specifically, to an immunoassay method for bacteria harboring outer membranes of bacteria, and more specifically, for measuring Haemophilus influenzae (Haemophilus influenzae).
The present invention relates to a method for immunologically measuring Haemophilus influenzae using a polyclonal antibody obtained using the crude membrane protein of Haemophilus influenzae as an antigen.

[Conventional technology and its problems]

Conventionally, the method for measuring Haemophilus influenzae (hereinafter referred to as Haemophilus influenzae) is an immunoassay method using antiserum obtained by immunizing rabbits with the bacteria killed with formalin etc. is being carried out.

However, Haemophilus influenzae is classified into 6 types, A% B, C% D, E, and F, based on the capsular serum type, and 80 to 90% of Haemophilus influenzae isolated from the respiratory tract of actual patients are of the A type. ~ It is a non-type group that does not belong to any of the F stars. Therefore, the conventional measurement method using antiserum is not suitable for measuring all Haemophilus influenzae, and in order to measure all Haemophilus influenzae, the antiserum obtained using the six A-F types as antigens is necessary. They must be used in combination, but in this way the individual antisera are diluted, reducing detection sensitivity, and even with this method, non-tied H. influenzae cannot be detected. Therefore, there has been a desire for a method that can measure all influenza bacteria with high sensitivity.

[Failure to solve the problem]

Under these circumstances, the present inventor conducted intensive research and found that polyclonal antibodies obtained using the crude protein of a single strain of Haemophilus influenzae as an antigen have high specificity for Haemophilus influenzae, and that they may be used against strains of Haemophilus influenzae other than the type used. The present invention has been completed based on the discovery that all types of influenza germs, whether they are type or non-type, can be measured without exception.

That is, the present invention provides a method for measuring Haemophilus influenzae, which is characterized in that an antibody obtained by immunizing an animal with a crude membrane protein of Haemophilus influenzae as an antigen is allowed to react with a subject. It is.

The crude membrane protein of Haemophilus influenzae is already known =
1), for example, Infection and Immunity'
) Vol, 36. No.l, pp. 80-88, 19
It is isolated by the method described in 1982. Any type of Haemophilus influenzae can be used here, regardless of the type.

As a method for preparing antibodies by immunizing animals with crude membrane proteins, methods known per se are employed.

As animals, mammalian animals such as domestic rabbits, goats, sheep, horses, and horses are used. Animal skin, with a certain amount of crude column membrane protein,
Antisera are obtained by several subcutaneous, intramuscular, intraperitoneal, intravenous or foot inoculations.

In order to measure Haemophilus influenzae in a subject using the antiserum (antibody) prepared in this way, it is sufficient to perform an immune reaction that is commonly used in such cases, such as an enzyme immunoassay. , radioimmunoassay, direct fluorescent antibody method, indirect fluorescent antibody method, reverse passive collapsing ball agglutination method, reverse passive latex agglutination method, and coagulation method.

〔Effect of the invention〕

Since the present invention uses antibodies obtained from crude membrane proteins,
It has the advantage of being able to measure all Haemophilus influenzae regardless of type, being specific to Haemophilus influenzae, and having a high degree of detection.

〔Example〕

Next, an example will be given and explained.

(1) Bacterial outer membrane protein extraction method Haemophilus influenzae thailand 7'6 strain 1.
f-s) Extract 5” f/d, Hemin 10 Z
Bacteria were cultured for 18 to 20 hours in plain heart infusion liquid medium 37rK supplemented with 100 μl/- of IP/a% NAD, and the cells were collected by continuous centrifugation, centrifugally washed twice with 150 sd physiological saline, and then incubated at 200 sj. of lithium chloride extraction buffer (200 mM lithium chloride, 100 m
Resuspended in M lithium acetate, pH 6.0), approximately 10 of [l! Stir vigorously for 3 hours at 45C with a rotary stirrer added, 12.
Centrifuge for 000>115 minutes, then 25,000
The supernatant after centrifugation for 120 minutes was dialyzed against purified water and used as crude membrane protein.

(2) Preparation and Purification of Crude Membrane Protein Antiserum 2fng (protein concentration) of the crude membrane protein obtained in the above (1) is used as an inoculation amount, and intravenously administered to domestic rabbits once a week for 4 times. After continuous weekly vaccination, immunization is performed by inoculating once a week for 3 consecutive weeks at intervals of 4 weeks. One week after the final immunization, blood was collected and antiserum was obtained by a conventional method. This antiserum was subjected to ammonium sulfate salting out and ion exchange to prepare an IgG fraction, which was used as an anti-crude outer membrane protein antibody.

(3) ELISA method 1 using anti-crude outer membrane protein antibody
) Preparation of immobilized antibody Dilute the anti-crude outer membrane protein antibody (IgG fraction) obtained in item (2) above with 0.05 M carbonate buffer (pH 9,6) to 50%
A μi/− antibody solution was prepared, and 200 μj of this was dispensed into each well of an EIA microplate. After the microplate was left at 4-10C for 24 hours, the contents of the wells were washed with distilled water. Then in each well 2%
Bovine albumin-containing phosphate buffered saline (p[(7,2) 2
00 pJ and left at 4 to 10 C for an additional 24 hours to obtain a homophasized antibody.

Similarly, this immobilized antibody is washed with distilled water before use.

2) Preparation of labeled antibody Alkaline phosphatase (derived from calf small intestine, manufactured by Boehringer) 3-Misaki was added to 0.1M N containing 1mM magnesium chloride.
, N-bis(2-hydroxyethyl)-glycine 1
(pH 8.5) was added 1 at a time to dissolve, and then 20 μm of potassium periodate was added and left at 37° C. for 2 hours. Thereafter, the mixture was centrifuged at 800 rpm for 5 minutes to remove insoluble materials, and the supernatant was dialyzed overnight against 0.1 M carbonate buffer (pH 9.5) containing 1 mM magnesium chloride. Next, 15 μl of the anti-crude outer membrane protein antibody (IgG fraction) according to item (2) above was added, left at 4 to 10 tZ'' for 14 days, and diluted 20 times with phosphate buffered saline containing 2% bovine albumin. Stored frozen.

When used, dilute it to the required dilution rate with phosphate buffered saline containing 2% bovine albumin.

3) Assay method Anti-crude outer membrane protein antibody (Ig
A microplate on which G fraction was immobilized was washed with distilled water, and the crude membrane protein itself obtained in item (1) above or bacteria cultured in the liquid medium used in item (1) above 200 μ of culture solution containing the body! Dispense to address. After reacting this microplate at room temperature for 2 hours, washing with distilled water,
Diluted solution of labeled antibody prepared in (3)-2) above 200
After adding μl and reacting for 1 hour at room temperature, the contents are washed with distilled water. Next, a substrate solution for measuring alkaline phosphatase activity (45 mM disodium phenylphosphate and 2
0.025M carbonate buffer containing mM 4-aminoantipyrine, pH 10.2) at 200μ! Add and react for 20 minutes at room temperature. After that, 100μ of coloring solution (0,8 sodium periodate)! is added to stop the enzymatic reaction and generate color, and the activity of alkaline phosphatase involved in the antigen-antibody reaction is measured by absorbance at a wavelength of 500 rLtn using an ELISA analyzer.

(4) Results 1) Before conducting an investigation using clinically isolated strains, six strains of Haemophilus influenzae capsular serotypes A-F and non-type influenza strains that do not belong to any of the capsular serotypes A-F' were detected. 8 strains of bacteria, and 4 strains of capsular type B (clinical isolates), including the bacteria from which crude membrane protein was obtained in section (1) above, were described in "Rill's standard method" in section (31-1) above. Measurements were carried out using the ELTSA method in accordance with the operating procedure described in Table 1, and the results shown in Table 1 were obtained.

That is, in the ELISA method using an anti-crude outer membrane protein antibody, even when measuring the bacterial cell culture fluid in parentheses, the crude membrane protein of the p18 strain itself can be detected by the method described in (1) above. Positive results were also obtained in all cases of measurement. As a control, ELISA was conducted using an antibody obtained by immunizing rabbits with a formalin-killed strain of Haemophilus influenzae type B in the same manner as the conventional commercially available antiserum for capsular typing.
When the method was performed, only type B bacteria reacted, similar to the commercially available antisera for different capsular types.

This also shows that the method according to the present invention is effective in measuring all Haemophilus influenzae.

Table 1: Absorbance values measured for crude outer membrane protein of the jar Chestnut *Absorbance values measured for bacterial cell culture 11) Regarding the 179 clinical isolates identified as Haemophilus influenzae by biochemical properties,
Regarding the culture solution containing the bacterial cells cultured in the liquid medium used in section 1), the results shown in Table 2 were obtained according to the operating procedure described in "Measurement method" in section (31-1) above. That is, in the ELISA method using an anti-crude outer membrane protein antibody, 1 out of 179 strains was detected.
Two strains (96%) yielded positive results. As a control, we performed the ELtSA method using an antibody obtained by immunizing a rabbit with a formalin-killed strain of Haemophilus influenzae type B using the same method as a conventional commercially available antiserum for capsular typing. However, in a commercially available live bacteria agglutination test, only the products from the two companies that were identified as type B tested positive. Similar measurements were also performed on 111 strains of bacteria other than Haemophilus influenzae, and negative results were obtained for all strains.

This also shows that the method according to the present invention has high specificity for Haemophilus influenzae and is effective in measuring all Haemophilus influenzae.

※l Antiserum for capsular serotyping made by Denka Main Light ※※II
Difco-produced antiserum for plasticizing the capsule serum (voluntary) April 25, 1986

Claims (1)

[Claims] 1. A method for measuring Haemophilus influenzae, which is characterized by making an antibody obtained by immunizing an animal with the crude outer membrane protein of Haemophilus influenzae Haemophilus influenzae as an antigen react with the subject.