JPH05322897A - Method for measuring enzyme immunity of bacterium using filter - Google Patents

Abstract

PURPOSE:To obtain a bacterium detecting method by which a bacterium can be quickly and easily detected and, at the same time, the quantity of the bacterium can be determined. CONSTITUTION:The title method is used for measuring the enzyme immunity of a bacterium by catching the bacterium on a filter, causing an immunoreaction by adding the primary antibody and enzyme-labeled secondary antibody or an antibody-like substance to the bacterium on the filter. Then, after un-reacted antibodies are washed away from the filter, an enzyme reaction is caused between the composite product of an antigen-antibody reaction and a chromagenic substrate on the filter and the quantity of the bacterium is measured by detecting the chromagenic intensity on the filter. Since the bacterium is concentrated on the filter, the detection sensitivity can be improved and, since no culture is required for cultivating the bacterium, the measurement can be completed within several hours. In addition, no specific utensil, technique, facility, etc., are required.

Description

Detailed Description of the Invention

[0001]

[Industrial application] Legionnaires' disea
se) has been established as a bacterial respiratory infection,
The cause bacteria Legionella pneumophila (Legionella
pneumophila) (hereinafter referred to as Legionella).

The onset form of this disease includes sporadic cases and outbreaks such as nosocomial infections. In both cases, the route of infection is considered to be air infection through soil, river water, air conditioners or circulating water in buildings. Has been. In particular, when a mass outbreak occurs, the cooling tower for air conditioning is regarded as a representative source of infection.

The Legionella retention rate in air-conditioning cooling towers is generally quite high (40% or more) (Riki Nakahama, Infectious Diseases Journal, 57: 643-655 (1983), Naomi Ito, Infectious Diseases Magazine. , 57: 682
-694 (1983), Atsushi Saito et al., Infectious Diseases Magazine, 55: 439-446 (198
1)), it is considered difficult to disinfect all of them.
However, regular cleaning and, if necessary, disinfection are also necessary.

If there is an inspection method capable of promptly checking the degree of contamination of Legionella, it becomes possible to accurately grasp the time when cleaning or disinfection is necessary. The Legionella test to which the present invention is applied does not require a specific instrument, technique, facility, etc., and can rapidly detect Legionella.

[0005]

2. Description of the Related Art As a current method for detecting Legionella,
Culture method (Bueschig, WJ et al., J. Clin. Microbiol., 17,1153
(1983)), direct fluorescent antibody method (Cherry, WB and Mckinne
y, RM, USDepartment of Health, Education, and Well
fare, CDC Georgia, USA, 92-103 (1979)), DNA
Probe hybridization method (Edelstein, PH,
J. Clin. Microbiol., 23 (3), 481 (1986)) and the like are known.

The culture method is as follows: inoculate a sample into a medium to suppress the growth of mixed bacteria, and after culturing for 5 to 7 days, subculture the colony into a confirmation medium, confirm the growth, and if there is growth, This is a method of identifying by performing a slide agglutination reaction with a Legionella antibody. However, it is difficult to set the condition for suppressing the mixed bacteria during the culture for increasing the sensitivity. If the suppression is weak, the growth of mixed bacteria will progress, and fishing of Legionella is impossible,
Strong suppression suppresses not only the growth of mixed bacteria but also the growth of Legionella. Further, it takes 1 to 2 days for the determination.

The direct fluorescent antibody method is used as a rapid diagnostic method. The sample is smeared on a non-fluorescent slide, dried, and fixed. Anti-Legionella antibody was reacted as a primary antibody for 30 minutes, washed with PBS (phosphate buffered saline), and then F
A secondary antibody labeled with a fluorescent substance such as ITC (fluorescein isothiocyanate) is reacted for 30 minutes. Finally, after washing with distilled water, it is sealed with glycerol and observed with a fluorescence microscope. Positive bacteria are judged based on the intensity of fluorescence and the form of the bacteria. The specificity of this method is as high as 99%, but the sensitivity is low, and the number of positive bacteria is often 10 or less in the entire visual field, so careful observation is required.

According to the DNA probe hybridization method, chromosomal DNA extracted from a subject is labeled with photobiotin and is adsorbed on a solid phase chromosomal DNA of Legionella.
And hybridized with peroxidase-labeled streptavidin, (TBZ), and H ₂ O ₂ . It has high specificity and sensitivity, but requires special equipment and technology.

All of these methods require specific equipment, technology and facilities. Contamination of Legionella in actual cooling towers is frequent and it is difficult to disinfect all of them. If the effect of the disinfectant disappears, it will spontaneously grow again in a few weeks at the earliest. Therefore, it is necessary to carry out rapid inspections on a regular basis, which is not possible with current methods.

[0010]

In the conventional bacterial detection methods, it often takes several weeks to make a determination in order to maintain a certain level of sensitivity and specificity, and it is also necessary to use a specific instrument, technique and facility. is necessary. In the case of infectious diseases such as Legionella whose condition worsens rapidly, it is necessary to have a bacterial detection method that has both rapidity, simplicity, and quantitativeness.

[0011]

Means for Solving the Problems The present invention comprises (1) capturing a bacterium on a filter, and (2) a primary antibody against the bacterium and a secondary antibody or antibody-like substance labeled with an enzyme for the antibody. Then, the antigen-antibody reaction is carried out by sequentially adding it onto the filter, (3) unreacted primary antibody and secondary antibody or antibody-like substance on the filter are washed, and (4) secondary enzyme or antibody-like substance labeling enzyme. The enzyme immunoassay method for bacteria is characterized in that a substrate that develops a color upon reacting with is added onto the filter to develop a color, and (5) the intensity of the color development is detected. Furthermore, the present invention is an enzyme immunoassay method in which the bacterium is a bacterium of the genus Legionella.

[0012] The present invention is intended to capture the amount of bacteria by the strength of color tone in combination with concentration of bacteria by using a filter, fixation, and enzyme immunoassay. That is,
Using the filter as a support for the reaction, collecting the bacteria on the filter and directly performing the antigen-antibody reaction and the enzyme reaction,
This is a method of measuring the amount of bacteria. In this method, the sensitivity can be increased by collecting and concentrating the bacteria on the filter. Therefore, culturing is not required, determination can be performed in a few hours, and no specific equipment, technique, facility, etc. necessary for measurement are required, so that bacteria can be detected quickly and easily. Moreover, since the basic principle is the same as the direct fluorescent antibody method, the specificity is high.

A method for detecting Legionella to which the present invention is applied will be described as an example. First, the impurities contained in the material need to be removed by a prefilter. The pre-filter used has the ability to retain fine particles, but subtle differences in retention interfere with subsequent operations. For example,
If the retention capacity is weak, fine contaminants will pass through, and there is a strong possibility that clogging will occur during filtration with the reaction filter. On the contrary, if the retention capacity is strong, not only fine contaminants but also target bacteria are captured, and bacteria do not collect in the reaction filter. The prefilter used for detection of Legionella preferably has a particle retention capacity of 1.2 to 1.6 μm and is made of glass fiber.

In order to collect the bacteria contained in the sample after prefiltration, filtration is performed using a reaction filter, and the bacteria are collected on the filter. The filter used here is required to have a specific pore size capable of collecting bacteria to be measured and a material compatible with enzyme immunoreaction. In this measurement, a pore size of 0.45 μm is used. If the pore diameter is smaller than this, the passage speed of the sample becomes slow, and eventually clogging occurs. In addition, in order to wash the unreacted antibody when performing the enzyme immunoreaction on the filter,
A material that absorbs proteins as little as possible is preferable, and one made of cellulose acetate or polyvinylidene difluoride (PVDF) is preferable.

Next, the Legionella remaining on the reaction filter is adjusted to around pH 7.0 with a buffer solution for the reaction with the antibody. For pH adjustment, phosphate buffered saline (10 mM PB
S, pH 7.2), Tris buffered saline (10 mM TB)
S, pH 7.2) can be used. The anti-Legionella antibody that specifically reacts with Legionella is preferably a monoclonal antibody in order to prevent non-specific reaction, for example, MAB-830.
(Chemicon company), 69-955 (ICN company) etc. can be used. Any enzyme-labeled secondary antibody that can be usually used can be applied to this method. Further, as the enzyme-labeled antibody-like substance, it is necessary to select a substance having low nonspecific reactivity, and peroxidase-labeled protein-G is used.

In order to remove unreacted primary antibody, enzyme-labeled secondary antibody or non-specifically adsorbed substance of antibody-like substance on the filter, the filter is washed with a specific washing solution. For this purpose, Triton X- which is a nonionic surfactant
A buffer containing 100, for example, PBS / 5% Triton X-100 can be used. Since the adsorption of the antibody protein and the enzyme-labeled antibody-like substance on the filter is strong, the surfactant Triton X-100 used here needs to have a high concentration. Also, since Triton X-100 at a concentration of 5% suppresses the enzymatic reaction, it is necessary to replace with PBS after washing with PBS / 5% Triton X-100, for example.

The color-developing substrate is preferably one having a maximum absorption wavelength in the visible region, high sensitivity, easy discrimination, and high stability over time. In order to satisfy the condition, a peroxidase-based color-developing substrate is preferable, and examples thereof include tetramethylbenzidine (TMBZ) and orthophenylenediamine (OP
D) or the like can be used.

The amount of bacteria is judged from the degree of color tone of the dye developed on the filter. When negative, almost no color is seen on the filter.

[0019]

As described above, the method for measuring bacteria according to the present invention is a method in which bacteria collected on a filter are directly detected on the filter by an enzyme immunoassay. Since the bacteria can be concentrated on the filter, the detection sensitivity can be increased, and since the culture is not necessary, the determination can be performed in 2 to 3 hours.

This is a method of detection with an antibody that specifically reacts with the target bacterium. Since the non-specifically adsorbed substances are removed by washing, the specificity to the target bacterium is high.

Since no specific equipment, technique, facility, etc. are required, it can be applied as a simple test kit by which the parties can directly judge the degree of bacterial contamination. The application of the present invention can be applied to the detection of water-borne bacteria such as Escherichia coli, Vibrio parahaemolyticus, Shigella, typhoid, Salmonella, and cholera.

[0022]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 shows a flowchart of a method for detecting Legionella to which the present invention is applied. As a sample
The cooling water for the air conditioning cooling tower was collected from 21 facilities. 20 ml
Sample water was taken in a disposable syringe for use, and a prefilter (glass fiber, pore size 1.2 μm) was connected to the tip and filtered. 100 ml of sample water was used for each sample.

Filtered water was taken in a disposable syringe for 10 ml, and a reaction filter (cellulose acetate, pore size 0.45 μm) was connected to the tip and filtered. As standard, 1 × 10 ⁶ CFU / formalin sterilized and fixed
ml, 1 × 10 ⁵ CFU / ml, 1 × 10 ⁴ CFU / ml, Legionella pneumophila , ATCC
33152) Suspension, and as a negative control, 1 ml of sterile distilled water was placed in a disposable syringe for 1 ml and filtered with a separate reaction filter.

P to a disposable syringe for 2.5 ml
The sample, positive control, and negative control reaction filters were filled with BS and washed twice. Anti-Legionella antibody (× 500 dilution) and peroxidase-labeled protein-G (× 40
The diluted solution (diluted with 00) was taken in a disposable syringe for 1 ml, and about 0.5 ml was injected into each reaction filter, and the mixture was reacted at room temperature for 1 hour.

The PBS / 5% Triton X-100 solution was placed in a disposable syringe for 2.5 ml, and each reaction filter was washed 5 times, and further washed twice with PBS.
The color-developing substrate (TMBZ solution) was taken in a disposable syringe for 2.5 ml, and about 0.5 ml was injected into each reaction filter, and the reaction was carried out at room temperature for 30 minutes.

When judged by comparison with the negative control, among the standards, 1 × 10 ⁶ CFU and 1 × 10 ⁵ CFU clearly showed a difference, but 1 × 10 ⁴ CFU did not show a difference. .. From this, the minimum detection sensitivity of the reaction system is 1 × 10
It was estimated to be ⁵ CFU. 9 out of 21 samples showed a difference compared to the negative control, and at least 1 x 1 in 100 ml of sample water.
0 ⁵ shows that there is a fungus of higher CFU.

[Brief description of drawings]

FIG. 1 is a flowchart of a method for detecting Legionella to which the present invention is applied.

Claims (2)

[Claims] 1. (1) Bacteria are captured on a filter,
(2) A primary antibody against the bacterium and a secondary antibody or antibody-like substance labeled with an enzyme against the antibody are sequentially added onto the filter to cause an antigen-antibody reaction, and (3) unreacted primary on the filter. After washing the antibody and the secondary antibody or antibody-like substance, (4) a substrate that reacts with the labeling enzyme of the secondary antibody or antibody-like substance to develop color is added on the filter to develop color, and (5) the intensity of color development is detected. An enzyme immunoassay method for bacteria, which comprises: 2. The bacterium is a bacterium of the genus Legionella.
The method described.