JP2002181824A - Sugar antigen recognizable endotoxin measurement method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting endotoxin having a specific sugar antigen more accurately and rapidly than a conventional method to find infection caused by a gram-negative bacteria such as enteropathogenic Escherichia coli O157 and Salmonella species. SOLUTION: Focusing on the fact that endotoxin as an exosporium component of the gram-negative bacteria consists of two sites of polysaccharide and lipid A, quantity of endotoxin (bacteria species) having a specific sugar antigen is determined when an endotoxin activity is measured by selecting only the endotoxin originating from a target bacteria by means of a carrier carrying a specific antibody specific to the polysaccharide site of the endotoxin, and then, bringing a limulus reagent into reaction with the lipid A site. Alternatively, instead of the limulus agent, a specific lipid A binding substance (ALF, ENP, tachyplesion, CAP, and the like) labeled with a fluorescent material is used for determining presence of endotoxin (bacteria species) having the specific sugar antigen.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to stool, blood, body fluid,
The present invention relates to a method for recognizing a difference in antigenicity of a sugar chain of endotoxin on the surface of a gram-negative bacterium with a specific antibody in a bacteriological examination of food or the like, and applying a characteristic of lipid A to identify a type of contaminating bacterium.

[0002]

2. Description of the Related Art Infectious diseases caused by pathogenic Escherichia coli O157: H7 are sporadic in Japan, causing frequent watery stool, severe abdominal pain and marked bloody stool. It causes severe complications such as sickness syndrome and encephalopathy and is often fatal in young and elderly people.
For this reason, the Health Sciences Research Project and the Japan Medical Association have officially reported that early diagnosis and the need for antibiotics are necessary.

As a conventional detection method for detecting the presence or absence of infection by pathogenic Escherichia coli O157: H7, a method of detecting the presence or absence of O157 polysaccharide using an antigen-antibody reaction (Escherichia coli immune serum O-157: Denka biopsy) Manufactured by The Rev
EAL for E. E. coli O157: H7: Air Brown), a method for detecting verotoxin released by pathogenic bacteria by applying an antigen-antibody reaction (Line Judge: Cosmo Bio, Ortho VT1 / VT2: Ortho, Novopus Vero Toxin EIA: BioRad) and a method for amplifying and detecting the Vero toxin gene by PCR (O157)
Vero toxin gene One Shot PCRScreen
ing and Detection Kit: manufactured by Takara Shuzo Co., Ltd.). Since both methods have low detection sensitivity, it is necessary to perform a culturing operation for about 8 hours in order to proliferate the amount of bacteria in the sample, except for a sample having a particularly large amount of bacteria.
In particular, in the early stage showing signs such as diarrhea and abdominal pain where the number of bacteria has not been sufficiently increased, long-term culture beforehand is essential for diagnosing an infection caused by pathogenic Escherichia coli O157: H7. Similarly, long-term cultivation was required for detecting verotoxin-producing Escherichia coli other than pathogenic Escherichia coli O157: H7, Shigella, and Salmonella.

[0004] In the field of clinical medicine, by discriminating general enteritis having a favorable prognosis as early as possible from highly pathogenic enteritis such as pathogenic Escherichia coli O157, and promptly starting treatment with an antibiotic or the like, The challenge is to prevent serious complications such as hemolytic uremic syndrome and encephalopathy. It is also necessary to quickly identify suspected infections and non-infected individuals in a swarm of food poisoning patients. However, all of the conventional test methods require more than half a day for determination, and thus have not been able to satisfy the needs of such clinical sites.

[0005]

DISCLOSURE OF THE INVENTION The present invention relates to a gram-negative bacterium which has endotoxin in the outer membrane of its ubiquitous cell, wherein one endotoxin molecule comprises a polysaccharide having an antigenic property inherent to the bacterium, and is identified by a specific antibody. This is a method of quantifying endotoxin specific to a bacterial species by applying what is possible and that the other polar A portion of the same molecule has a common biological activity. Pathogenic E. coli O157, O26, O111, dysentery,
The presence of important intestinal infectious gram-negative bacteria such as Salmonella can be determined for each bacterial species by a factor of more than 1000 times higher than the conventional method and extremely shorter than that of the conventional method by 15-45.
It is determined in minutes. Since it can be detected with high sensitivity, it is not necessary to perform bacterial culture in advance to enrich the bacteria.

[0006]

Means for Solving the Problems The outermost layer of Gram-negative bacteria is called endotoxin and is composed of a water-soluble polysaccharide portion and a hydrophobic lipid A portion. The polysaccharide portion greatly differs in structure (antigenicity) depending on the bacterial species, and specific antigens that react only with the polysaccharide of each bacterial species have been obtained. On the other hand, the lipid A portion has a small difference depending on the bacterial species, and applies that the lipid A of endotoxin derived from any bacterial species has a common biological activity, that is, a limulus activity and a binding property to an endotoxin binding protein. .

By selecting specific antibodies against the polysaccharide portion of endotoxin of various Gram-negative bacteria, it is possible to theoretically identify all Gram-negative bacteria including pathogenic bacteria such as pathogenic Escherichia coli O157, O26, O111, dysentery and Salmonella. Becomes possible.

First, a specific endotoxin sugar chain antibody (prior art) is fixed to an antibody-binding carrier (prior art). The material and shape of the carrier to be fixed are not limited as long as a sufficient coupling constant is secured. The antibody is immobilized for the purpose of selecting only specific endotoxins and washing away others.

Next, both the polysaccharide antigen-specific antibody bound to the carrier and the specimen are mixed with a surfactant (polyoxyethylene glycol-t-octylphenyl ether, polyoxyethylene glycol sorbitan monoalkyl ester, etc.) Surfactants and “solid surface treatment agents” (including Japanese Patent Application No. 10-532723)), mixed with shaking, and endotoxin derived from the target bacterial species by using a polysaccharide antigen-specific antibody as a bridge. Only to the carrier.

Next, all the liquid phases other than the carrier were discarded, and the solution was newly added to a surfactant solution, washed with shaking and stirring, and the endotoxin directly non-specifically bound to the carrier was eluted. The operation of discarding the washing liquid is repeated.

Thus, the lipid A of the endotoxin molecule captured on the solid phase by the polysaccharide moiety via the specific polysaccharide recognizing antibody exists in a spatially free state, and retains the biological reactivity unique to lipid A. I have.

When a Limulus reagent is added to a carrier on which specific endotoxin has been captured, Factor C in the Limulus reagent is activated by lipid A, and a multi-step enzymatic reaction proceeds while being amplified (cascade reaction). When coagulogen is used as a suitable substrate, a gel turbidity reaction is caused, and when a synthetic substrate is used as a final substrate, a color development reaction is caused.

Whereas the measurement of polysaccharide antigen using an antigen-antibody reaction binds at a ratio of 1: 1 to 2, the application of the Limulus activity of lipid A improves the detection sensitivity of polysaccharide antigen several thousand times.

As an alternative to the method of detecting endotoxin captured on a carrier, a labeled lipid A binding protein group can be used instead of the Limulus reagent. Lipid A
The binding substance group includes endotoxin lipid A, such as proteins such as ALF and ENP, peptides such as tachypressin and polyphemsin, and basic proteins (peptides) such as CAP.
Substances that specifically bind to the moiety are included. In particular, when the captured endotoxin is detected by the fluorescently labeled lipid A-binding substance group, an enzymatic reaction is not required, and thus there is an advantage that the measurement time is further shortened and the detection is simplified.

[00015]

BEST MODE FOR CARRYING OUT THE INVENTION

[Particle Method] An antibody capable of specifically binding to a target bacterial or serotype polysaccharide antigen is bound to a gel or solid particulate carrier on which the antibody can be covalently immobilized. Glycosyl hard gel (manufactured by Seikagaku Corporation) or the like can be used as the carrier. A small amount of a sample (rectal swab, stool juice, body fluid, blood, squeezed solution of food, etc.) is dispersed in a container containing a surfactant solution and the cells are destroyed. The particles to which the specific antibodies prepared above are bound are added to the container, and mixed by shaking. By this operation, an endotoxin aggregate recognized by the specific antibody is captured on the particle surface. Next, the container is centrifuged, the carrier particles are sedimented, the supernatant is discarded or the solution is discarded by a pipette, a new surfactant solution is added, and after shaking, the solution is discarded again to wash the carrier particles. . This washing operation is repeated several times to sufficiently remove endotoxin nonspecifically bound to the carrier without passing through the antibody. Finally, a Limulus reagent (Endospecy: Seikagaku Corporation) solution is added to the particles to start a Limulus reaction, and endotoxin is measured. Detection of endotoxin means that the target bacterial species is contained in the specimen. Instead of adding Limulus reagent in the final operation,
A fluorescently labeled lipid A binding substance (ALF, ENP, tachyplesin, polyphemsin, CAP, etc.) is added to bind to the captured endotoxin. When particles having fluorescence are detected by a fluorescence detector or a fluorescence microscope, it is determined to be positive. Alternatively, instead of fluorescent labeling, an enzyme-labeled lipid A-binding protein can be bound, and a chromogenic substrate of the enzyme can be added to visualize the reaction.

[00016]

[Method of Using Particles Encapsulating Magnetic Substance] A magnetic substance is encapsulated in the center of a gel or solid particulate carrier to which an antibody can be covalently immobilized on the surface. Next, an antibody capable of specifically binding to the polysaccharide antigen of the target bacterial species is bound. A small amount of a specimen (rectal swab, stool juice, body fluid, blood, squeezed solution of food, etc.) is dispersed in a container containing a surfactant solution and the cells are destroyed. The magnetic substance-enclosed particles to which the specific antibody prepared above is bound are added to the container, and mixed by shaking. By this operation, an endotoxin aggregate recognized by the specific antibody is captured on the particle surface. Next, a magnet is brought into close contact with the outside of the container, and the magnetic substance encapsulated particles are collected at one end of the container. Discard the solution, add fresh surfactant solution, release the magnet and shake to wash the carrier particles. This washing operation is repeated several times to sufficiently remove endotoxin nonspecifically bound to the carrier without passing through the antibody. Finally, a Limulus reagent (Limulus ES-II Test Wako: Wako Pure Chemical Industries) solution is added to the particles, a Limulus reaction is started, and endotoxin is measured. Detection of endotoxin means that the target bacterial species is contained in the specimen. Instead of adding the Limulus reagent in the final operation, a fluorescently labeled lipid A packing material (ALF, ENP, tachyplesin, polyphemsin, CAP, etc.) is added to bind to the captured endotoxin. When particles having fluorescence are detected by a fluorescence detector or a fluorescence microscope, it is determined to be positive. Alternatively, instead of fluorescent labeling, an enzyme-labeled lipid A-binding protein can be bound, and a chromogenic substrate of the enzyme can be added to visualize the reaction.

[00017]

[Microplate Method] An antibody capable of specifically binding to a polysaccharide antigen of a desired bacterial species is bound to a microplate coated with a polymer having a reactive group capable of covalently immobilizing the antibody on the inner surface. Small samples (rectal swabs,
Stool juice, bodily fluids, blood, squeeze of food, etc.) are dispersed in a container containing a surfactant solution and the cells are destroyed. Add a portion of the solution to the microplate and mix by shaking. By this operation, the endotoxin aggregate recognized by the specific antibody is captured on the inner surface of the plate. next,
Discard the solution, add fresh surfactant or distilled water with endotoxin eluent and shake to wash the carrier particles. The solution disposal and washing operations are repeated several times to sufficiently remove endotoxin nonspecifically bound to the carrier without passing through the antibody. Finally, a Limulus reagent (Endospecy) solution is added to the plate to start a Limulus reaction, and endotoxin is measured. Detection of endotoxin means that the target bacterial species is contained in the specimen.

[00018]

[Test Paper (Carrier Sheet) Method] An antibody capable of specifically binding to a polysaccharide antigen of a target bacterial species is bound to one corner of a strip-shaped test paper (carrier sheet). A small amount of a specimen (rectal swab, stool juice, body fluid, blood, squeezed solution of food, etc.) is dispersed in a container containing a surfactant solution and the cells are destroyed.
The test paper (carrier sheet) is immersed in the sample solution, and the endotoxin aggregate recognized by the specific antibody is captured on the particle surface. The test paper (carrier sheet) is pulled up, and the operation of immersion and washing in a new surfactant solution is repeated several times. After pulling up the test paper (carrier sheet), a Limulus reagent (Endospecy) solution is added dropwise, and a color developed within a certain time is regarded as positive.

[00019]

【The invention's effect】

[Sensitivity] The present invention is characterized in that the presence of endotoxin captured by a specific antibody against a polysaccharide is detected not by the polysaccharide portion but by the lipid A portion. By adding the Limulus reagent, the sensitivity becomes several thousand times higher than the conventional detection method based on the conventional antigen-antibody reaction. The method of observing with a fluorescence detector or a fluorescence microscope using a lipid A binding substance labeled with a fluorescent dye also has the same high sensitivity. For this reason, the operation of pre-culturing specimens such as stool, blood, body fluids, foods and the like and enriching them becomes unnecessary.

Further, the measurement time is within 45 minutes by the method using the Limulus reagent, and about 15 minutes by the method using lipid A binding substance labeled with a fluorescent dye, which is 3 hours to 8 hours, which is the time required by the conventional method. This can be significantly reduced.

[0002] The present invention is highly sensitive and rapid, so that in clinical practice, the pathogenic Escherichia coli O15 is present in the early stages with only common signs such as diarrhea and abdominal pain.
7, early detection of serious intestinal infections such as O26, O111, dysentery, salmonella, etc., and provide useful information for initiating early treatment. Further, in a patient who has started treatment, the therapeutic effect can be determined in real time by tracking the disappearance of endotoxin derived from infectious bacteria. Furthermore, there is a risk of outbreaks such as food poisoning, which is an extremely powerful tool for rapidly screening large numbers of patients.

[0002] In addition, a specific antibody recognizes and sorts the polysaccharide portion of endotoxin specific to the strain, and prevents non-specific endotoxin derived from a heterologous bacterium from binding to a carrier. Is ensured. The non-specifically bound protein does not have lipid A, and therefore does not cause false positives.

[Brief description of the drawings]

FIG. 1 Binding of a particulate carrier in which endotoxin to be detected is bound to a specific antibody

FIG. 2: Binding relationship between endotoxin to be detected, carrier and lipid A binding protein

FIG. 3 shows carrier particles having magnetic particles embedded therein.

FIG. 4 Measurement method using test paper (carrier sheet) to which specific antibody is bound

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 Endotoxin molecule 1a Lipid A part of endotoxin 1b Polysaccharide part of endotoxin 2 Carrier particle 3 Specific antibody bound to carrier 4 Binding of specific antibody to carrier particle 5 Endotoxin molecule captured by polysaccharide part recognized by specific antibody 6 Lipid A binding protein 7 Label 8 Specific binding of lipid A binding protein to lipid A 9 Magnetic particles 10 Support 11 Test paper (carrier sheet to which specific antibody is bound)

Claims (5)

[Claims] 1. An endotoxin having a saccharide antigen of a specific bacterial species from a group of endotoxins derived from polybacterial species contained in a sample by a specific antibody against a polysaccharide portion of endotoxin (lipopolysaccharide) bound to a carrier. A method of measuring the limulus activity of the lipid A portion present on the opposite side of the polysaccharide portion of the endotoxin molecule after selecting only the endotoxin molecule, and measuring endotoxin having a polysaccharide antigen derived from a specific bacterial species. 2. An endotoxin-binding substance (A) labeled with a fluorescent dye or an enzyme for measuring endotoxin fractionated by a specific antibody against the polysaccharide moiety according to claim 1.
LF, ENP, tachyplesin, polyphemsin, CAP
And the like, and applying specific binding of lipid A to detect endotoxin having a polysaccharide antigen derived from a specific bacterial species. 3. A method for causing a binding reaction between the polysaccharide portion of endotoxin according to claim 1 and a specific antibody in a surfactant solution. 4. An endotoxin derived from another bacterial species non-specifically bound to the carrier to which the specific antibody according to claim 1 has been bound without being bound to the specific antibody by a surfactant solution. how to. 5. A particle having a reactive group covalently bonded to an amino group, a carboxyl group or a hydroxyl group of a protein as a carrier to which the specific antibody according to claim 1 is bound, and a magnetic substance embedded therein.