JPS62177451A - Red blood cell sensitized with anti redionera polysaccharide antibody - Google Patents

Abstract

PURPOSE:To exactly make early diagnosis of a redionera disease by refining an anti- redionera bacillus polysaccharide antibody (PS antibody) from the redionera bacillus antibody, sensitizing the same with red blood cells and detecting the polysaccharide antigen contained in the redionera bacillus strain. CONSTITUTION:The polysaccharide is added and mixed to ad with the redionera bacillus antibody and the mixture is centrifugally separated and is washed with a physiological salt soln. The antigen and antibody dissociates and become soluble when an acetic acid buffer soln. is added thereto. Only the antibody settles when a satd. aq. ammonium sulfate soln. is added thereto. The antibody is, therefore, collected by a centrifugal sepn. and the ammonium sulfate is removed, by which the PS antibody is refined. The red blood cells are treated by using a coupling agent consisting of a tannic acid, etc., and a liquid contg. the PS antibody is brought into contact therewith to obtain the PS antibody sensitized blood cells. The sensitized blood cells induce an agglutination reaction when the cells contact the redionera bacillus polysaccharide antigen and therefore, the agglutination reaction is caused by a microtiter method and the concn. of the redionera bacillus polysaccharide antigen in the body fluid is quantitatively determined, by which the early diagnosis of the redionera disease is exactly made.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to blood cells sensitized with anti-Legionella polysaccharide antibodies.

More specifically, the present invention relates to anti-Legionella polysaccharide antibody-sensitized blood cells sensitized with specifically purified anti-Legionella polysaccharide antibodies.

[Prior art and its problems] Legionnaires' disease or Legionnaires' disease, also called veterans' disease, is caused by Legionella pneumophila (Legionella pneumophila).
It is an infection caused by Legionella bacteria such as L. lla pneumophila, and is often clinically recognized as pneumonia, accounting for 1-2% of idiopathic pneumonias and 4-2% of difficult-to-diagnose virus-like pneumonias.

This disease was first discovered in America in 1376, but has since spread to Europe, Africa, Australia, and other countries.
It has been discovered in India, Japan, and other countries, and it has become one of the most important diseases among pneumonias, partly because there are few effective treatments, but it is difficult to diagnose because there are no clinically specific symptoms. is based on the isolation and culture of this bacterium. However, since there is no good selective culture method for this bacterium, the method used is to inoculate and infect guinea pigs, then sacrifice and isolate the bacterium. Although this method is excellent in terms of detection rate, it requires a lot of cost, labor, and time, and is not easy to apply to clinical tests.

However, when Legionella bacteria infection occurs in humans, bacterial components are first liberated and released into the blood and urine, causing antigenemia, so bacterial components that serve as antigens must be specifically detected from body fluids. If possible, it would be possible to diagnose Legionella infections at an extremely early stage. In this case, the antigen to be detected must be a stable substance and unique to Legionella bacteria, which is present in all strains.

In addition, the antigen released into the blood due to infection combines with the antibodies present in the patient's blood to form an antigen-antibody combination, that is, an immune complex! Since it rarely exists as an isolated antigen, it is necessary to create a system that can easily dissociate the antigen from this immune complex and use it for measurement.

From this point of view, the present inventor used polysaccharides contained in all Legionella strains as antigens. Legionella bacteria are heat-resistant and extremely stable substances that contain a certain amount of polysaccharide in their cell walls depending on the bacterial species to which they belong, and are solubilized and released into the blood after infection. Since this polysaccharide specifically binds to the polysaccharide, anti-Legionella polysaccharide antibodies (hereinafter referred to as "PS antibodies") are specifically separated and purified from the Legionella cell antibodies produced by immunization. We have established a method for detecting polysaccharide antigens by sensitizing red blood cells and performing a reverse passive hemagglutination reaction, and have completed the present invention.

Enzyme antibody methods have been reported as a method for detecting Legionella antigens present in body fluids, but this method requires complicated procedures and a lot of labor, and is not easy to apply to clinical tests. However, by using the reverse passive hemagglutination method, Legionella infection can be reliably diagnosed at the early stage of onset with extremely simple operations.

[Structure of the Invention] The present invention relates to PS antibody-sensitized blood cells, which are made by binding a PS antibody to red blood cells via a coupling agent.

The red blood cells used for the sensitized blood cells of the present invention are live red blood cells or fixed red blood cells, but from the viewpoint of preservation of the sensitized blood cells, it is preferable to use fixed red blood cells. Live red blood cells are obtained from sheep, goats, chickens, turkeys, humans, etc. according to conventional methods. Fixed red blood cells are prepared as follows. That is, formalin is added to live red blood cells suspended in Alsever solution for fixation. As the coupling agent, tannic acid, glutaraldehyde, chromium chloride, etc. can be used, and among them, tannic acid is preferred.

Legionella bacteria were grown at 37℃ using B-CYE agar medium etc.
If cultured with carbon dioxide gas, bacterial cells can be easily obtained in about 2 days. Anti-Legionella antibodies can be easily obtained by washing the cells with physiological saline, heating them at 100° C. for about 30 minutes, and immunizing rabbits, goats, etc. in the usual manner.

Since this antibody also contains the PS antibody, the Ps antibody is specifically separated from this antibody.

Several methods are known for extracting polysaccharide antigens, such as phenol water extraction, trichloroacetic acid extraction, and ethylenediaminetetraacetic acid (EDTA) extraction. Among these, the phenol water extraction method is easy to operate and is suitable for this purpose. That is, after washing the cultured bacterial cells, suspend them in boiling water and keep at 88°C, add the same amount of 90% phenol at 68°C, keep at this temperature for 10 minutes, cool it, and then heat it at 3000 rpa+. After centrifuging for about 15 minutes, it will separate into an upper layer and a lower layer. Take the upper layer and dialyze it against distilled water.
Ultracentrifugation at 00,000X g for 2 hours yields the polysaccharide as a clear precipitate. By repeating suspending this precipitate in water and ultracentrifuging it several times, a sample with few impurities can be obtained.

Next, in order to separate and purify PS antibodies from anti-Legionella antibodies, follow the following method.First, add a polysaccharide antigen to the antibody and mix well to form an immune complex, centrifuge and fractionate. Wash thoroughly with saline. When acetic acid buffer (p) 12.8 is added to this, the antigen and antibody dissociate and become soluble. When a saturated ammonium sulfate aqueous solution of pH 2.8 is added to this, the polysaccharide does not precipitate, but only the antibody precipitates, so this is collected by centrifugation, washed with a saturated ammonium sulfate aqueous solution, dissolved in water, and then dissolved in physiological saline etc. The PS antibody is purified by dialysis to remove ammonium sulfate.

The sensitized blood cells of the present invention are produced as follows. That is, red blood cells are treated with a coupling agent to obtain coupling agent-treated blood cells (red blood cells with a coupling agent bound to the surface of the red blood cells), which are then brought into contact with a solution containing a PS antibody to produce PS antibody-sensitized blood cells. obtain.

Diluents include bovine serum albumin (hereinafter referred to as rBSAJ) in glycine buffered saline, phosphate buffered saline, etc.
About 0.1% is used, and 0.01 to 0.5% of sodium azide (NaN3) is added.

Since the PS antibody-sensitized blood cells of the present invention are agglutinated by the Legionella polysaccharide antigen, first, body fluids such as human serum and urine are diluted with distilled water, and this is heated at 100°C for about 10 to 20 minutes. When the antibody portion constituting the immune complex is inactivated to liberate the polysaccharide antigen, and PS antibody-sensitized blood cells are brought into contact with this solution or its diluted solution, the antigen is present in the heated body fluid or its diluted solution. The sensitized blood cells then undergo an agglutination reaction.

When this reaction is carried out by the microtiter method, it can be seen on the microplate as an aggregated image at the bottom of the tube. That is, a certain amount of diluted liquid is dripped onto the plate, then a certain amount of heated body fluid is added to the first hole, and the mixture is heated with a gilutator.
Dilute 110 times. Sensitized blood cells are dripped and dispensed into this, and the image of aggregation at the bottom of the tube is determined after a certain period of time. In this case, if a standard polysaccharide whose concentration is known accurately is used, it is easy to calculate the concentration in the unknown body fluid by proportional calculation from the agglutination value for this standard substance. Polysaccharide concentration can be determined. That is, quantifying polysaccharide antigens is extremely easy and simple, and does not require any special techniques. Moreover, since the antibody has been specifically purified, it is extremely specific, and has sufficient sensitivity for measurement in human body fluids, and can perform qualitative and/or quantitative determination of a large number of specimens at the same time.

By using the sensitized blood cells of the present invention, the concentration of Legionella polysaccharide antigen in body fluids can be easily and conveniently quantified in a short time, which has not been easily performed in the past, and the early diagnosis of Legionnaires' disease can be made accurately. This will enable appropriate treatment to be provided from an early stage.

Currently, there is no known method for diagnosing Legionnaires' disease using reverse passive agglutination, and there are no documents describing PS antibody-sensitized blood cells, and the agglutination reaction of Legionella antigens using PS antibody-sensitized blood cells is completely new. be.

In addition, this sensitized blood cell reacts only to Legionella polysaccharides and does not react at all to polysaccharides or plasma proteins of other bacterial species;
Since unsensitized blood cells do not react at all to the Legionella polysaccharide, it can be said that these sensitized blood cells are bound to the PS antibody.

[Example 1 of the Invention] Next, the present invention will be explained in more detail with reference to Preparation Examples and Examples, but the present invention is not limited thereto unless it exceeds the gist thereof.

Legionella pneumophila (Legionella pneumophila)
neumophila) AT CC33152 to B-
Inoculated onto CYE agar medium and incubated in a carbon dioxide chamber for 3
The cells were cultured at 7°C for 48 hours. The bacterial cells on the agar were collected by scraping with a Conlage rod, washed three times with physiological saline, suspended in physiological saline, and heated in a water bath at 100°C for 30 minutes to kill the bacterial cells. .

The bacterial cells of Preparation Example 1 were suspended in physiological saline to a concentration of approximately 10 8 /rnl, and this bacterial solution was injected into the ear vein of a rabbit weighing approximately 2.5 kg at a dose of 1.0 − 10 times every 2 days. One week after the last immunization, whole blood was collected from the carotid artery, serum was separated in a conventional manner, and heated at 56°0 for 30 minutes to obtain anti-Legionella bacterial antibodies.

Suspend 50 g of Legionella cells prepared in Preparation Example 1 in water to make 200-g, boil in hot water to 100°C, and then heat to 68°C.
It was kept warm in a constant temperature water tank. 90% of this was preheated to 68℃
200 d of phenol was added and treated for 10 minutes. After cooling in an ice chamber, the mixture was centrifuged at 3000 rpm for 15 minutes to separate the upper layer, which was then placed in a dialysis tube and dialyzed against distilled water overnight. This was centrifuged for 2 hours at 100.0 OOX g using an ultracentrifuge to collect a transparent precipitate, suspended in water, and centrifuged in the same manner. This centrifugation was repeated three times to obtain a Legionella polysaccharide.

To the anti-Legionella bacterial antibody 40d prepared in Preparation Example 2, 44 g of the polysaccharide prepared in Preparation Example 3 was added.

The immune complex formed by the binding of the antigen and antibody precipitated immediately, so it was centrifuged at 3000 rpm for 30 minutes and fractionated.
Washed three times with physiological saline. pH 2.8゜0.1
lods of a saturated ammonium sulfate aqueous solution dissolved in M. acetate buffer 104 and adjusted to pH 2.8 were added. After mixing with a stirrer for 1 hour at room temperature, 10.00 Orpm
The mixture was centrifuged for 60 minutes to precipitate only the antibody, and this precipitate was washed three times with a saturated aqueous ammonium sulfate solution. This was dissolved in 5++ J of distilled water and dialyzed against physiological saline for 18 hours to remove ammonium sulfate. During this period, the external solution was changed twice. After dialysis, insoluble matter was removed by centrifugation at 10.00 rpm for 60 minutes, and the supernatant was used as anti-glycoside antibody. This antibody produced a single sedimentation line with the polysaccharide by the Ochterlony method, proving that it was a single antibody.

Alsepar was added to fresh blood obtained from chickens.
ver) solution, and to this add fixative solution [physiological saline + 3
Add equal volume of 7% formalin (volume ratio 29:1, 37°
The mixture was left in an incubator at C for 24 hours, with occasional shaking during this time. Thereafter, it was washed 5 times with pure water and 15 times with physiological saline.

Fixed red blood cells were suspended in phosphate buffered saline at pH 7.2 containing 0.1% NaN3 to a concentration of 10% and stored in an ice chamber. This fixed red blood cell was stable for over 1 year.

Phosphate buffered saline (pH 7.2) containing the fixed red blood cells obtained in Preparation Example 5 of 2.5% of -1 PBSJ (rPBSJ)
1:100,000 tannic acid/PBS
After adding an equal amount of the same amount to the cells, the cells were treated with tannic acid at 37°C for 15 minutes, washed once with PBS, and suspended in Kojo's PBS to prepare a tannic blood cell solution.

An equal amount of the 1:IEiO PS antibody obtained in Preparation Example 4 was added to this tannin blood cell solution, treated at 37'C for 30 minutes, and then treated with PBS once, and added with BSA to PBS at pH 7.2.
It was washed once with a diluted solution containing 1% O and 0.1% NaN3, and then suspended in 5 times the volume of the diluted solution.

These sensitized blood cells aggregated with Legionella polysaccharide on a microplate, but tannin blood cells, which are not sensitized to PS antibodies, did not aggregate even when Legionella polysaccharide was added under the same conditions. That is, it was confirmed that the sensitized blood cells specifically reacted to the Legionella polysaccharide and aggregated.

2 ψ^ - The reaction was carried out by the microtiter method, and first, the diluted solution was dispensed into the plate in 0.025 J portions using a dropper. 0.025 J of l:4 heated serum or urine was added to the first well. It was diluted 2n with Guiluta. Standard polysaccharides were also diluted in the same way. The sensitized blood cell solution was added dropwise in 0.025-minute increments, placed in a mixer, and allowed to stand at room temperature for 1 hour or more, and then evaluated. The polysaccharide concentration was calculated by comparing the aggregation end point of the standard polysaccharide and the agglutination end point of the sample.

The results of measuring polysaccharide concentrations in human serum and urine using this method are shown in the following table.

As is clear from the above results, polysaccharides in blood and urine were detected only in patients with Legiones disease. That is, it can be said that measuring the polysaccharide antigen concentration in body fluids is extremely useful for early diagnosis of Legionnaires' disease. It can also be applied to determining the extent of lesions.

[Effects of the Invention] By using the sensitized latex of the present invention, polysaccharide antigens can be extremely easily quantified by simply heating the sample with a WL amount of about 0.025, making it particularly suitable for clinical diagnosis. It can be said that

Claims (3)

[Claims] (1) Anti-Legionella polysaccharide antibody-sensitized blood cells comprising an anti-Legionella polysaccharide antibody bound to red blood cells via a coupling agent. (2) The sensitized blood cells according to claim 1, wherein the red blood cells are fixed red blood cells. (3) The sensitized blood cells according to claim 1, wherein the coupling agent is tannic acid.