JP2000088854A - High sensitive immunological detection measuring method for microorganism (bacteria, fungus, virus, producing substance) and quantitative method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To quantitatively detect a microorganism at a high sensitivity by producing a product by adding dropwise an enzyme substrate to a microorganism dual antibody binding substance remained on a solid phase matrix. SOLUTION: A solid phase matrix requires a pore diameter through which a microorganism can not be passed. For example, the pore diameter is particularly preferably 0.22-0.45 μm in a bacteria inspection and 0.80 μm in the case of a fungus. After the microorganism is filtered and accumulated on the solid phase matrix if necessary, a sample solution remained on the solid phase matrix is washed with a washing liquid. A specific antibody to be detected is added dropwise to the microorganism on the solid phase matrix and a reaction of the microorganism with the antibody is carried out. As the antibody, an antibody against a specific microorganism is sometimes used if desired. A substance necessary for an enzymatic reaction is added dropwise to an enzyme labeled antibody binding microorganism captured and remained on the solid phase matrix and an enzymatic reaction is carried out. A product is finally produced and is measured. A determination of the desired microorganism is carried out by previously preparing a calibration line representing a reaction amount in the case of using a constant amount of antigen and applying it thereto.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION The fields to which the present invention should be applied are mainly in the medical and food industries. The technical features of the present invention are its wide range of application and flexibility as compared with conventional immunological microbiological testing methods. Conventional immunological microbial assays have been developed solely for the detection of specific microorganisms. However, the present invention is not limited to the determination (identification) of bacterial species names. Bacterial species in the test sample using antibodies specific for the antigen (bacterial cell wall peptidoglycan: a component of the bacterial cell wall that is not present in human cells) or an antigen (bacterial cell wall lipoticoic acid) that is common only to Gram-positive bacteria It is possible to determine whether or not is present and whether or not it is a Gram-positive bacterium. (This discrimination method is currently typical of Gram staining, but is more sensitive than this staining. is there). Since fungi also have fungus-specific common specific antibodies (eg, galactomannan), it is also possible to determine whether a fungus is present in a sample. And because the measurement method is quantitative,
The number of bacteria present can be determined. In addition, a screening test can be performed by mixing specific antibodies to determine the presence or absence of several types of specific bacterial species that are significant in sample analysis. Further, the present invention relates to a method and a reagent for detecting or quantifying metabolites (especially toxins) produced by microorganisms, which can be used not only for determination of the number of bacteria and the name of bacteria but also for viral antigens and chlamydia antigens.

2. Description of the Related Art At present, propagation culture methods and specific cell culture methods utilizing various media are basically used for diagnosis of infectious diseases of microorganisms (bacteria, fungi, viruses, chlamydia, etc.). The method is called the standard method (gold standard). Although these culture methods are reliable measurement methods, it takes a long time (number of days) for obtaining results to be said to be a major drawback. Although this temporal problem could not be solved for a long time, a rapid assay method that does not rely on culture has been studied recently, and a typical assay method is a genetic test or an immunological test. Law. Genetic testing method is Polymerase Chai
n Reaction method (PCR method) is a typical measurement method to extract DNA of a microorganism, by amplifying it using a polymerase resistant enzyme in a way that can be detected with high sensitivity, substantially measurement sensitivity is ¹⁰ 1 - 10 ³ CFU (Colon
y forming Unit) / ml. Currently, tuberculosis tests are used mainly in university hospitals and large-scale testing centers, but general hospital facilities and small-scale testing facilities require special measurement equipment and room space to prevent contamination. Properties and the complexity of sample pretreatment. Very few genetic testing methods have been implemented in food microbiology. On the other hand, the immunological test method has already been automated by the serum test department for a wide range of items such as tumor markers, trace hormones, administered drug blood concentrations, and hepatitis virus that can be detected by blood analysis, which has led to the spread of genetic testing. It is much more widespread and is widely used in general laboratories. The immunological test has a major feature in that it is simple and easy to automate as compared with the genetic test. However, it is hardly used for detecting microorganisms (bacteria, fungi, viruses, chlamydia) antigens. Recently, a manual immunological detection method for pathogenic Escherichia coli O-157 has been marketed, and insurance coverage has been determined for the first time. These manual methods are simple and quick, but have a measurement sensitivity of 10 ⁴ to 10 ⁵ CFU / ml or more (on average around 10 ⁵ CFU / ml), which is lower than that of the medium culture method and the genetic measurement method. Low sensitivity. It is recognized that the current limit of the measurement sensitivity of the immunological microorganism detection method is 10 ⁴ / ml or more.

SUMMARY OF THE INVENTION An object of the present invention is to provide a genetic test (PCR) for diagnosing infection of microorganisms.
Method), a highly sensitive enzyme-immunological microbiological test method comparable to that of
An object of the present invention is to provide a reagent used for performing the measurement method.

Means for Solving the Problems In the conventional method for detecting a microorganism antigen immunologically, first, a specific antibody for a microorganism to be detected is stably fixed on the inner wall of a well of a microplate or in a cellulose fiber and is contained in a sample. The method employs a method of selectively binding only the desired microorganism from the various microbial microorganism groups to the antibody and separating the antibody from other microorganisms. Although this means can capture the target bacterium by the specificity of the antibody, a secondary antibody labeled with an enzyme or a colloidal gold substance is used as a means for detecting the captured microorganism, and its target is the first. The only option is to employ a method of binding to the target microorganism itself captured by the antibody. In other words, the conventional immunological microorganism detection method has no choice but to proceed in the order of the first antibody + microorganism + labeled second antibody (microorganism target) stably fixed to the test tube wall etc. It can be said that this is a sandwich detection method in which cells are positioned in the middle. This sandwich measurement means can bind the primary antibody and the labeled secondary antibody only to the antigen portion on the surface of the microorganism. Microorganisms are very large objects compared to protein substances in human blood.However, because there are so many bacterial species, it is necessary to prevent cross-reactivity, and there are only a few unique antigen groups that can bind to the microorganisms themselves. . As a result, the conventional immunological microorganism detection method has only adopted the method of selecting and separating the target microorganism from a sample in which various microbial groups are mixed, and the measurement sensitivity is lower than that of the conventional culture method and genetic test. It is considered that the sensitivity was lower than that of the PCR method. In order to overcome this problem, the present invention provides a newly designed reaction vessel with microorganisms (especially in the case of bacteria or fungi: viruses,
Chlamydia is passed through) by dropping a sample suspected of containing, without forced aspiration, the solution in the sample is absorbed into the lower layer by natural absorption, and the microorganisms are captured on the solid phase matrix without leakage. . After the capture, a step of dropping an antibody (a monoclonal antibody is preferable in terms of specificity) that reacts with the target microorganism and binding the antibody to the microorganism without aspiration. Washing and removing unbound primary antibody (B / F separation); Next, a step of further dropping an enzyme-labeled antibody (preferably a polyclonal antibody in terms of binding strength) targeting the primary antibody bound to the microorganism, not to the microorganism, to the solid phase matrix. A step of preparing a conjugate of a microbial antigen, a primary antibody, and an enzyme-labeled secondary antibody having the primary antibody as an antigen on the surface of the solid phase matrix without aspirating the enzyme-labeled antibody solution. A step of washing and removing (B / F separation) the unbound labeled secondary antibody in the lower absorbent layer. A step of dropping an enzyme substrate onto the microorganism-two-body antibody conjugate remaining on the solid phase matrix to produce a product. The present invention relates to a method for detecting or quantifying a microorganism by a sandwich method (comprising a two step method using two kinds of antibodies) comprising the above steps. On the other hand, it is also possible to use the primary antibody itself which is labeled with an enzyme. Many products in which the primary antibody is labeled with an enzyme are polyclonal antibodies. Conversely, bacterial species-specific monoclonal antibodies with a large number of products have a small number of enzyme-labeled products, so that users often use enzyme labeling, and the degree of enzyme labeling must be carefully monitored. The Onestep method using the primary enzyme-labeled antibody is technically simple, but has the same sensitivity as the current immunological microbiological test method. In fact, it is not known before the test whether the target microorganism to be detected is contained in the sample,
Even if it is included, it is not known whether it is very small or very large. The most confusing problem in immunological detection methods is that it is difficult to detect a reaction at a so-called optimal antigen-antibody ratio, and the prozone phenomenon is likely to occur. In order to cover a wide range of the optimal antigen-antibody ratio, a measurement method in which the primary antibody is enzymatically labeled and directly bound to the target microorganism (mostly polyclonal antibodies)
Is low in measurement sensitivity but strong in binding force, and is particularly important when a large amount of the target microorganism is present. On the other hand, when the target microorganism is present in a small amount, the secondary reaction is accurate and has high measurement sensitivity, but when the target microorganism is present in a large amount, the prozone phenomenon tends to occur easily. Therefore, taking advantage of both characteristics, if the primary antibody is mixed with an enzyme-unlabeled one and an enzyme-labeled one and subjected to an inspection test, the sample will contain a large amount even if the target bacterium is contained in a small amount. In this case, false negatives are not produced, and coloration of the enzyme reaction in proportion to the number of bacteria is easily obtained.

DETAILED DESCRIPTION OF THE INVENTION As a method for capturing microorganisms on the surface of a solid phase matrix, it is only necessary to add a biological sample, which is obtained by treating a biological sample directly or as required, onto a support. The pore size of the solid phase matrix needs to be a pore size through which microorganisms cannot pass. When the microorganism sample is a bacterium, for example, a pore size of 0.5 μm or less is preferable,
In the case of fungi, a pore size of 5.0 μm or less is preferred. More specifically, 0.22 μm or 0.45 μm for bacterial testing
Particularly preferred is a pore size of 0.8 μm for fungi.
Is particularly preferred. In the case of detecting substances produced by viruses, chlamydia and microorganisms (especially toxins) which cannot be captured by natural filtration with this fixed pore matrix, the pore size does not need to be strictly defined, and antibodies against the toxin stably bound to the fiber. It suffices if the state can be supplied. For this purpose, a dense filter made of glass fiber or cellulose fiber may be used, or an immobilon filter having a fixed pore (it is said that antibodies are easily stably fixed: Millipore) may be used. Naturally, if the drop amount of the sample is increased, the detection sensitivity can be increased in proportion to the drop amount. According to a specific operation method of the present invention, after the microorganisms are filtered and accumulated on the solid phase matrix, the sample solution remaining on the solid phase matrix is washed with a washing solution, if necessary. Next, a specific antibody to be detected is dropped onto the microorganism on the solid phase matrix, and the microorganism is reacted with the antibody. As the antibody, an antibody against a specific microorganism (for example, pathogenic Escherichia coli O-157, Salmonella, Mycobacterium tuberculosis, etc.) is often used depending on the purpose. However, in the present invention, an antibody that reacts with peptidoglycan, which is a main component of the bacterial cell wall, which serves as an antigen common to all bacterial species, or an antibody against lipotic acid that only Gram-positive bacteria have may be used. In addition, if you want to narrow down the search for a specific sample in a specific sample (for example, Salmonella, O-157, Vibrio, etc. are often detected when examining convenience), it is necessary to identify specific multiple bacterial species. By using a mixture of specific antibodies, it is possible to measure whether a screening test is performed to determine whether or not a certain bacterial species is present in a sample. The enzyme-labeled antibody-bound microorganisms captured and left on the solid-phase matrix in the above immunological microbiological test are allowed to drop the substance necessary for the enzymatic reaction to allow the enzymatic reaction to proceed, and finally produce the product. The amount of the target microorganism is detected or quantified.On the other hand, for the quantification of the target microorganism, a calibration curve showing the reaction amount when a certain amount of antigen is used is created in advance, and the quantification is performed by using this. Do.

Exemplification of the flexibility of the present invention An immunological test for a microorganism has been limited to an assay for detecting a specific bacterial species. With this method alone, it is not possible to know the species of the sample in which the presence of the microorganism is suspected, so that various antibodies are used endlessly until the result becomes positive. This is the same in the PCR method, and it is necessary to continuously use a probe for a specific strain endlessly. However, in the measurement of life cycle, for example, meningitis is a fatal infection in children, but it is extremely important to judge whether it is bacterial, viral, or other inflammation. First, it is necessary to determine whether bacteria are present in the cerebrospinal fluid (there is no probe that is common to all bacteria in the PCR method at present). If there is no probe at this time), the test can be narrowed down to about 4 species of Gram-positive bacteria, Streptococcus pneumoniae or Streptococcus group B, Staphylococcus, and Listeria which are frequently detected from cerebrospinal fluid. If the name of the bacterium is quickly found out, the antibiotic to be administered is not blindly used, and a rather localized treatment can be performed. However, there has never been an immunological microbiological test method capable of making such a judgment. In the present invention, a monoclonal antibody against peptidoglycan, which is a main component of the bacterial cell wall, commonly present in all bacterial species can be used, and the presence of several bacteria /
It can be detected with a sensitivity of ml to several tens / ml. Gram-positive bacteria can be distinguished by a monoclonal antibody against lipotic acid present only in the cell wall of the gram-positive bacteria, and monoclonal antibodies against four bacterial species can be easily obtained. As a result, the bacterial species can be determined. For peptidoglycan and lipoticoic acid, human body components include N-acetyl gluctosamine (NAG) and titin body (polymer o).
fNAG, NAG oligomers) and the like exist, but the method involves the use of NA
Since the G-based substance passes through and moves to the lower absorption layer, it has a characteristic that a false reaction can be avoided. In the following example, if the test sample is diarrhea stool suspected of food poisoning with severe abdominal pain,
At present, the only immunological microbiological test method is a kit for detecting pathogenic Escherichia coli O-157 as a target bacterial species. Those with diarrhea are not only O-157, but also Salmonella, Vibrio, Campylobacter and the like are the main bacterial causative bacteria. In the present invention, immunological microbiological tests can be performed in parallel and simultaneously with the same sample pretreated in parallel with the test of O-157.

DETAILED DESCRIPTION OF THE INVENTION Pretreatment of Sample The sample used in the present invention is a sample suspected of containing microorganisms, and in the medical field, includes biological samples collected from various infectious disease patients. Examples include blood, urine, sputum, cerebrospinal fluid, stool, and pus. On the other hand, in the field of food production, there are raw materials such as meat, eggs, vegetables, and milk, and various processed products.
Since the fixed pores of the solid phase matrix have a very small pore size of the order of μm, they have a disadvantage that they are easily clogged. For example, when testing for Mycobacterium tuberculosis, the most common sample is sputum, but sputum has a large amount of mucus and a high degree of viscosity, and pneumonia often has a large number of purulent cells. Moreover, since the interstitial substances are also various, pretreatment of sputum is indispensable. In the case of a genetic test method, several steps more complicated pretreatment than in the present invention are required, and the test does not start unless the cells are destroyed and the nucleic acid in the cells is extracted. Simple pretreatment is sufficient, and no nucleic acid extraction is required. The only point is that a uniform bacterial suspension is prepared and the antigen site on the bacterial surface is treated so that it can easily bind to the antibody. Specifically, the sputum was collected with a cotton swab to fill a cotton ball portion, put into a test tube containing 2.0 ml of a diluent containing one or two glass beads, and mixed vigorously with a vortex mixer. And the sputum is physically homogenized by rotating glass beads. After squeezing the sample from the swab, 2500 rotations, 3
After centrifugation for 5 minutes, put the sputum residue on
A sample obtained by filtering an omnipore membrane (millipore) having an m-pore diameter and a cotton ball through a pretreatment container (FIG. 2) improved by connecting two pipette tips to each other is used as a sample.
The time required is about 5 minutes. When urine is a sample, the presence or absence of a certain amount of bacteria is defined as a cutoff value, as in the case of a bath or pool water. In the case of urine, the concentration is 10 ⁵ / ml, so use a 20 μl sample of urine diluted 500 times with a diluent. Next, when feces is a sample, as in the case of sputum, there is a marked difference in sample properties such as stool, loose stool, nausea, and mucous stool, as in the case of sputum. Collect with a cotton swab. The stool sample is soaked evenly in the swab swab. 1.5m of this swab
l into a test tube containing 0.025 ml of chloroform and mixed vigorously with a vortex mixer.
Iron the swab with the test tube wall to create an even stool suspension. When it is centrifuged at 2,500 rpm for 3 minutes, the fat protein component in the stool is transferred to chloroform and precipitates in the form of a spot on the bottom of the test tube. The sedimentation supernatant becomes more transparent and the bacteria are evenly suspended in the supernatant. As in the case of Mycobacterium tuberculosis, a sample that has been filtered through a pretreatment container (FIG. 2) to which a cotton ball and an omnipore membrane having a pore size of 5.0 μm are connected is used as a sample.

Detailed Description of the Invention: Reaction vessel The solid phase matrix used in the present invention is composed of a dense fiber material or a porous material. Materials constituting the fiber material or the porous material include glass, quartz, ceramic, paper, and resins such as nylon, polystyrene, nitrocellulose, cellulose mixed ester, cellulose acetate, polyvinylidene difluoride, polytetrafluoroethylene, and polycarbonate. Filter paper, glass fiber filter paper, cellulose filter paper, cellulose mixed ester filter paper, cellulose acetate filter paper, hydrophilic polyvinylidene difluoride filter paper, hydrophobic polyvinylidene difluoride filter paper, hydrophilic polytetrafluoroethylene filter paper, polycarbonate filter paper Are preferred. In the present invention, a solid support having a fixed pore size is preferred. The pore size is 0.1 to 5.0 μm, preferably 0.1 to 5.0 μm.
2 to 1.0 μm. As a specific solid support,
Bacteria include membrane filters with a pore size of 0.22 μm or 0.45 μm. On the other hand, fungi include membrane filters having a pore size of 0.80 μm. As this membrane filter, a commercially available one can be used. Particularly preferred membrane filters include bacteria,
ADVA for accumulating and attaching microorganisms such as fungi
NTTEC Toyo's cellulose mixed ester membrane, cellulose acetate membrane, polycarbonate membrane, Millipore's cellulose mixed ester membrane, hydrophilic polyvinylidene difluoride membrane, hydrophilic polytetrafluoroethylene membrane, polycarbonate membrane Is raised.
On the other hand, in order to detect or quantify toxins produced by bacteria, fungi, etc., preferred porous filters for stably fixing antibodies to filter fibers are the above-mentioned various membrane filters, Immobilon P filter manufactured by Millipore, and Examples include a glass fiber filter and a cellulose fiber filter manufactured by TOYOBO. The solution absorbing means includes, as a porous material having a sufficient absorbing power to absorb the liquid flowing into the test apparatus during the detection or quantitative operation, for example, absorbent cellulose paper, glass fiber paper, and the like. . The shape of the reaction vessel is not particularly limited as long as it has a solution absorption means in a lower layer of a porous matrix having a certain pore size and a solid phase matrix,
For example, there can be mentioned those having a cylindrical shape and a circular shape which can be used in multiple layers. (Details of the container configuration are shown in Fig. 1.)

Detailed Description of the Invention: Diluent, Washing Solution Since monoclonal antibodies react only with a single antigen group, they have extremely high specificity and little cross-reactivity. However, the ability to precipitate or aggregate soluble antigens and cellular antigens is generally weak, and the binding ability of polyclonal antibodies containing various antibody groups is overwhelmingly stronger. And when targeting microorganisms such as bacteria, the surface structure on the microorganism side (the outermost membrane of Gram-negative bacilli is covered with a mucous substance)
Must also be considered. At present, only reagents or kits using polyclonal antibodies are commercially available because these stand out before the immunological microbiological test as a complicated factor. Monoclonal antibodies do not easily bind to specific bacteria even in a pure series in which only pure cultures of specific bacteria are suspended, or when a commonly used phosphate buffer or Tris buffer is used as a sample diluent. . However, a strong binding reaction occurs with polyclonal antibodies. In order for the monoclonal antibody to properly bind to the microbial antigen, the optimal pH, ionic strength, and reaction volume are significantly affected as measurement conditions.
It was necessary to devise a method of adding a certain amount of protein to the mixture.
As a result of adding bovine serum albumin, fetal bovine serum, newborn calf serum, goat serum, rabbit serum, horse serum, pig serum, and chicken serum to the added protein, the addition of bovine serum albumin showed a clear antigen-antibody reaction. Has been slightly improved. The addition amount is 30% product, Bu
The amount may be 0.05 to 2.5 ml (the optimal amount is 0.2 ml) per liter of ffer. However, the protein substance that showed the most significant improvement was the addition of mouse serum producing monoclonal antibodies. The addition of mouse serum is good for the identification of specific strains, Mycobacterium tuberculosis, Salmonella, and pathogenic Escherichia coli O-157. Although antibody binding was not successful, it was found that a clear reaction was obtained with mouse serum. The cause is that the bacterial cell surface is electrically negatively charged by the carboxyl group when the buffer alone is used, but when suspended in phosphoric acid or Tris buffered saline, the NaCl in the saline is dissociated and Na + and Cl− This is because the minus charge on the bacterial surface pulls Na +, and the bacterial cell surface is eventually covered with plus + charge. On the other hand, since the antibody protein is similarly brass + charged, the antigen portion on the bacterial surface and the antibody binding site are electrically repelled and are hardly bound. Although polyclonal antibodies have binding ability to overcome this charge situation, monoclonal antibodies are presumed to be weak enough to bind unless animal proteins are added and the charge + power is reduced. Although there are many animal species, it is considered that mouse serum can minimize the charge state among them. The amount of mouse serum to be added may be very small. The amount of addition may be 0.01 to 1.0 Mol Tris NaCl buffer or 0.01 to 1.0 Mol NaCl phosphate buffer.
0 L, 0.1 mg / 1 L to 1000 mg / 1 L if used for suspension of pure culture bacteria, 1.0 mg / L
mg to 1000 mg / dl (the optimal amount is 5.0 mg to 10 mg / dl).
0 mg / dl). However, since mouse serum is derived from the same animal as the monoclonal antibody, strict attention is required since the amount of addition affects the blank value. On the other hand, the composition of the washing solution has no influence on the antigen-antibody binding substance, and a solid color matrix having a low color blank value must be selected. The less salt contained in the washing solution, the stronger the washing effect. Therefore, as a result of the examination, a 0.01 Mol phosphate buffer or a 0.01 Mol Tris buffer (both of N
aCl is excluded) Surfactant Tween 8 for 1.0 L
0 to 1.0 ml to 10.0 ml (the optimal amount is 2.0 to 6.0 ml).
0 ml), containing 22% sodium alkyl ether sulfate (Family: Kao) 5.0 ml to 600 ml
(The optimal amount was 50 to 300 ml), the background coloring was low and the effect of mouse serum addition was low.

Detailed Description of the Invention: Primary Antibodies: Antibodies specific to microorganisms (bacteria, fungi, chlamydia, rickettsia, viruses), as well as antibodies to many toxins produced by microorganisms, are numerous in Japan. In addition, manufacturers around the world (eg, Chemicon in the United States, Bioline in Italy, Baylostat in the United States,
Biogenesis, UK, etc.), developed and purified, and commercially available. These various types of commercially available antibodies can be used in the present invention. An example of a typical antibody used to determine a specific bacterial name is Mycobacterium tuberculosis (Mycobacter).
erium tuberculosis) mouse monoclonal antibody (hereinafter abbreviated to anti-M. tuberculosis), mycobacterium avian (Mycobacterium)
avium) for Streptococcus pneumoniae
cus pneumoniae), anti-Strepto
anti-Streptoc for coccus A group
Occus B group, anti-Streptoco
For Ccus D group, anti-caries streptococci (St
anti-Staphylococcus aureu for S. reptococcus mutans
se), anti-Staphylococcus
cus epidermidis), anti-E. coli (Es)
Herechia coli, anti-enterohemorrhagic pathogenic Escherichia coli O-15 (Echerichia coli O-15)
7: H7), anti-Salmonella (Salmonella)
sp. ), For anti-dysentery (Shigella sp.),
Anti Campylobacter (Campylobacter)
sp. ), Anti-vibrio (Vibrio choler)
ae, parahemolyticase, anti-Klebsiella sp., anti-Brotheus (Proteus sp.), anti-rayionella (Legionella pneumophila)
For use, anti-Helicobacter p
ylori), anti-Pseudomonas
aeruginosa), anti-haemophilis (Heam)
opilisinflenzae, Heamorphi
lis sp. ), Anti-pasteurella (Pasteur)
ella sp. ), For anti-meningococci (Neisseri)
a meningitidis group A, B,
C, X, Y, Z, W, E), anti-phosphorus bacteria (Neisse)
ria gonorrhea, anti-Listeria monocytogenes (L
isteria sp. ), Anti-botulinum (Clo
Clostridium difficill for Strium botulium
e) and others. Antibodies to fungi include, for example, anti-Candida albican
se Candida tropicals Candi
da stlatoidea). In addition, several types of antibodies against antigens common to all bacterial species are commercially available, not for searching for specific bacterial species, but they can also be used, and typical ones are antibacterial cell wall peptidoglycan (Bacterial peptidoglyca).
n), anti-gram-positive bacterial cell wall lipoticoic acid (Bac
terminal lipoteichoic acid) and the like. Antibodies that can be classified into large groups include antibodies for anti-enterobacteria (Enterobacteriaceae α, β) that can be classified into bacteria that inhabit the intestinal tract. Examples of common antibodies to fungi include anti-mannan, anti-galactomannan, and 1-3β-D glucan. On the other hand, antibodies against toxins produced by bacteria and fungi are for enterotoxins A, B, C, D and TSST-1 produced by Staphylococcus aureus, enterotoxins produced by Vibrio cholerae, and verotoxins VT1 produced by pathogenic Escherichia coli. VT2, Clostridu
D1 toxin, toxin A, Clostridium perfringe produced by m difficile
s produced enterotoxin. On the other hand, since viruses and chlamydia are originally a group of bacteria that are difficult to culture, there are great expectations for antigen-antibody immunoassay, and thus commercially available antibodies are more common than bacterial species. On the other hand, a primary antibody labeled with an enzyme (peroxidase, alkaline phosphatase, etc.) is also commercially available, although it is a typical example.
sherichia coli) O-157: for H7,
Anti-Salmonella sp., Anti-Listeria sp., Anti-Campylobacter sp.
Etc. However, there is definitely more enzyme-unlabeled antibody.
Therefore, when only unlabeled antibodies are commercially available, peroxidase is labeled by the NAKONE method when labeling is desired. On the other hand, many labeled secondary antibodies are commercially available, and many are polyclonal antibodies. Since the polyclonal antibody has a strong ability to bind to a large number of antigen groups, it is highly useful because the number of epitopes that can be bound to a mouse-derived monoclonal antibody is large. The most frequently used is a peroxidase-labeled anti-mouse-goat and rabbit immunizing antibody, but when the primary antibody is other than a mouse, the peroxidase-labeled anti-rabbit-antibody is used depending on the animal species. Goat immunized antibody, peroxidase-labeled anti-rabbit -sheep immunized antibody, peroxidase-labeled-
Rat immune antibodies and the like. It is necessary to change the working concentration of the above antibodies according to the concentration of the obtained antibody and the reaction binding capacity, but it does not greatly deviate from the instruction manual of each antibody, and most commercial antibodies are liquid Is frozen, but when a frozen product is used as a stock solution, the solid-phase matrix method of the present invention often uses a 3 × to 60 × dilution. However, a sufficient reaction can be obtained with a very small amount of about 2.0 μl for both the primary antibody and the labeled secondary antibody.

Detailed Description of the Invention: Reaction time In the present invention, a certain amount of a sample suspected of being a microorganism is dropped on a solid phase matrix, so that the microorganism is not sucked by a solution absorption system below the solid phase matrix. The solution is removed and the microorganisms are filtered and collected on the solid phase matrix. At this point, the water around the microorganism becomes extremely small, the surface of the microorganism is almost bare, the distance between the primary antibody dropped in the next stage is very short, and the antigen group on the microorganism side and the primary antibody Are in a state where they can be easily bound. Therefore, the reaction time until a sufficient antigen-antibody conjugate is obtained may be as short as several minutes (2 to 5 minutes). The unbound primary antibody is also absorbed by the dropping of the washing solution without being sucked into the absorption layer below the matrix, so that the contact distance between the labeled secondary antibody and the primary antibody bound to the target microorganism is extremely high. Because it is in a close state, it takes several minutes (2-5
Minutes), the binding reaction time with the primary antibody ends. After the unbound labeled secondary antibody has been removed to the lower layer by the washing solution, the color can be formed in an enzymatic reaction as long as the matrix surface exhibits a color tone. These series of reaction steps have a speed of completion in 6 to 15 minutes.

[Enzyme Reaction Coloring Solution] As a substrate for enzyme coloring, for example, when the labeling enzyme is peroxidase, a benzidine derivative or a triphenylmethane derivative is preferable. Examples of the benzidine derivative include 3,3 ′, 5,5 ′,-tetramethylbenzidine (Ultra Troy). Examples of the triphenylmethane derivative include tri [4-N, N-di (3-sulfopropyl) aminophenyl] methane hexasodium salt. ULTRAVO is available from Intergen. If this reagent is shielded from light, it can be stably coexisted even if it is mixed with aqueous hydrogen peroxide, so that there is no need to make necessary adjustments and it can be used as one reagent. Since the coloring color tone of the present dye is blue, the measurement wavelength is a long wavelength range of 675 nm, so that the influence of bilirubin, hemoglobin, and the like mixed in the biomaterial can be easily avoided. The triphenylmethane derivative can be obtained from Fujirebio.

Example 1: Measurement with Mycobacterium tuberculosis Mycobacterium strain used: Mycobac isolated from patient sputum and stored
terium tuberclosis strain Antibody used: antituberculosis (Mycobacterium)
0.1 ml of mouse monoclonal antibody (Chemicon MAB-738)
Thaw the product (frozen) with 2.0 ml of 0.01 Mol NaCl phosphate buffer and stock. One case /
Add 2.0 μl dropwise. Anti-mouse / goat immunoglobulin / peroxidase-labeled antibody IgG (H + L) (Caltag Labor
attories INC. CodeNo. M3000
7): 2.0 ml of the product was added with 18.0 ml of the same buffer and stocked, and 2.0 μl of a × 3 dilution diluted at the time of use was dropped. Blocking solution: Block Ace (Dainippon Pharmaceutical cat. No.
-UK-B25) diluted 4 times with deionized water. Bacterial suspension diluent: As described above, mouse serum is added. Washing liquid: As described above, a solution to which two kinds of surfactants are added Bacterial solution preparation: Mycobacterium tuberculosis has a rough growth type colony in a separation medium, and a uniform bacterial suspension cannot be created from colony collection. Then, inoculate Mycobros (Far East Pharmaceutical Co., Ltd.), which can produce a suspension of equivalent bacteria, and incubate for 14 days in static culture (shaking mixed once a day to obtain a stock suspension). A serial dilution series was prepared and used for the experiment.
In addition, in order to grasp the accurate number of bacteria, 1% of Ogawa medium was inoculated from the prepared bacterial solution series, and the number of bacteria that grew 4 weeks later was calculated. ◎ The present invention; solid matrix measurement method Blocking: blocking solution (20.0 μl) Sampling: suspension of diluted bacteria (25.0 μl) Membrane washing: twice washing solution (8.0 μl) Primary antibody: Chemicon: MAB-738 (2.0 μl) Reaction time 2.5 minutes Membrane wash: Wash solution (8.0 μl) Repeated 4 times Labeled secondary antibody: above Caltag (2.0 μl) Reaction time 2.5 minutes Membrane wash: Wash solution (8.0 μl) ) Repeat 5 times Enzyme reaction solution: Ultra Trouble solution (8.0 μl) Reaction time 1 minute Visual judgment: Control blank is diluted solution without bacterial inoculation Result: The number of bacteria in the stock solution grown on mycobros is 4.3 × 1
It was 0 ⁸ co / ml. A dilution series was prepared from this stock solution, but the experiment was repeated five times. The same results were obtained in all of the five experiments. Positive values were obtained in the minimal bacterial suspension at 4.3 cells / ml, and positive values appeared twice out of 5 times, and 43 cells / ml were strongly positive. Thereafter, as the number of bacteria increased, the color of the enzyme became stronger. As a result, prozone phenomenon haunt the immunoassay method is weak, 10 ⁵ U / ml
In strong coloration was almost equal color tone with 10 ⁷ U / ml. The filtration capacity of the solid phase matrix is 10 ⁷ cells / m
but in more than 100μl l was able filtration, bacteria stock 10 ⁸
In the case of ko / ml, the bacterial concentration was too high, and the sample amount was 25.0μ.
1 was the limit. When calculated based on the minimum bacterial concentration positive value, the measurement sensitivity of the present invention is 4.3 cells / ml. Since a positive result is obtained with a bacterial concentration sample, if the number of bacteria captured on the solid phase matrix is 1 to 2 cells, It turned out that a positive value was obtained. In order to perform each dilution of the bacteria under the same measurement conditions, the sample volume was standardized to 25.0 μl. However, when the bacterial concentration is low, the experiment can be performed with a sample volume of 200 μl. Additional test: Since a matrix having a constant fine diameter is used in the present invention, clogging of the matrix occurs in proportion to the number of filtered bacteria, and there is a concern that tailing in each step of the immunological test may affect the test results. Therefore, the results of the present invention were examined using bacterial species other than Mycobacterium tuberculosis. The strain used was Staphylococcus aureus, S
stepococcus featurealis, Cory
nebacterium sp. , Escherici
a coli, Klebsiellapneumoni
ae, Pseudomonas aeruginosa
6 species. These bacterial species were subjected to pure culture, serial dilutions of the bacterial suspension were prepared using a diluent to which mouse serum was added, and the above-described technique for the tuberculosis solid phase matrix measurement method was carried out as it was.
As a result, 6 when the sample volume of 25.0μl in bacterial species, person other than bacteria stock 10 ⁸ U / ml sample Na' observed effects. I.e. in the following cell concentration 10 ⁷ U / ml was the same as the negative control of tuberculosis uninoculated, 1
If there 0 ⁸ U / ml, were found to color equivalent coloration when M. tuberculosis count co / ml exits. It was also found that the sensitivity of detection and measurement of the present invention was 10 cells / ml or more, and when the number of cells was less than this, the influence of other bacterial species had to be considered.

Example 2: Results with pathogenic Escherichia coli O-157: H7 (Results compared with Osou O-157 detection kit) Escherichia coli O-
157: H7 stock strain was used. Antibody used: Peroxidase-labeled Escheric
ia coli O-157: H7 goat polyclonal antibody (KIRKEGAD and PERRY L)
ABRATORIES, INC. Code No. 04
-95-90): 0.1 mg product to 1.0 mol Tr
Dissolve with 1.0 ml of is Buffer (TAKARA), dilute × 30 times before use, and drop 2.0 μl / 1 container. Anti-O-157: H7 mass monoclonal antibody Im
munoprecipitate LPS of O-
157: H7 (ViroStat INC. Code N)
o. 1031): 1.0 ml product was converted to the same buffer 2.0 ml
And stock it, and drop 3.0 μl / 1 container. Anti-mouse / goat immunoglobulin / peroxidase-labeled antibody IgG (H + L) (Caltag Labor
attories INC. CodeNo. M3000
7): A 2.0 ml product was added with 18.0 ml of the same buffer solution, stocked, and 2.0 μl of a × 3 dilution diluted at the time of use was dropped. Block solution; Block Ace (Dainippon Pharmaceutical cat. No.
-UK-B25) diluted 3 times with deionized water. Bacterial suspension diluent; as described above, to which bovine serum albumin has been added. Cleaning solution; as described above, a solution to which three kinds of surfactants are added. Comparison method; Ortho Escherichia co.
li O-157 detection kit for 96 times (preparation for experiment);
The operation of culturing on a Trisoi sheep blood agar plate medium at 35 ° C. for 20 hours was subcultured three times, and the grown pure moss was subjected to the experiment. A portion of the bacterial moss was collected and evenly suspended in a diluent to which mouse serum had been added to obtain a stock solution, which was serially diluted with the same diluent by 10-fold to prepare a serially diluted bacterial solution series, which was used as a sample.
(Specifically, the concentration that can be recognized by the naked eye to the extent that the bacterial suspension turbidity is slightly clouded (Mac Farland 0.5 =
1.0-2.0 × 10 ⁸ cells / ml). Using this bacterial solution as a stock solution, dilution by a factor of 10 was repeated to prepare a serially diluted bacterial solution series. The bacterial concentration at each stage is n
× 10 ⁸ cells / ml, n × 10 ⁷ cells / ml,...
× was 9 levels from 10 ⁰ U / ml. Here, for confirmation of n pieces, 0.05 ml was added to n × 10 ³ pieces / ml from n × 10 ³ pieces / ml.
^{From 2} colonies / ml, 0.1 ml was collected, applied to a blood plate agar medium, cultured at 35 ° C. for 20 hours, and the number of colonies that grew was calculated and n was divided. As a result, this time n =
1.7. Therefore, the stock solution prepared first was 1.7.
This means that × 10 ⁸ cells / ml were suspended. ◎ The present invention; solid-layer matrix measurement method (two-antibody method) Blocking: blocking solution (20.0 μl) Sampling: diluent bacterial solution (25.0 μl) Membrane washing: washing solution (8.0 μl) repeated twice Primary Antibody: Virostat above (3.0 μl) Reaction time 2.5 min Membrane washing: Washing liquid (8.0 μl) repeated 4 times Labeled secondary antibody: Caltag above (2.0 μl) Reaction time 2.5 min Membrane washing: Washing liquid ( 8.0μl) Repeat 5 times Enzyme reaction solution: Ultra-low solution (8.0μl) Reaction time 1 minute Visual judgment: Control blank is diluent without bacterial inoculation Results; As compared with the control blank, the minimum bacterial concentration at which a color change was clearly observed due to the enzyme reaction was 17 cells / ml. Back calculation from the sampling amount revealed that a positive reaction could be obtained if 1-2 were captured on the solid phase matrix. In the stepwise bacterial concentration suspension, the color intensity is proportional to the bacterial concentration, but at high bacterial concentration, the color appears to be slightly degraded, but the prozone phenomenon is a small phenomenon. It seems. Examination of Ortho kit reagents Ortho detection kits were used according to the instruction manual.
The time required for the test until a result is obtained is about 60 minutes. The measurement principle is that a primary antibody (anti-O-157: H7 antigen rabbit polyclonal antibody is immobilized on a microplate well and O-157: H7 in the sample is selectively bound. After the sample is discarded from the well, strictly The wells were washed several times, and the peroxidase-labeled anti-O-157: H7 goat polyclonal antibody was added as a secondary antibody to the O-157 cells themselves bound and captured by the primary antibody on the well tube wall. A concentration of pure moss (O-157: H7) in the sample treatment solution attached to the kit and a level at which white turbidity can be recognized by the naked eye (Mac Farland 0.5 = 1.0). to to 2.0 × 10 ⁸ U / ml equivalent) was uniformly suspended. the bacterial solution as a stock solution, as with the solid phase matrix method described above, repeated × 10-fold dilution, creating serial dilutions bacterial suspension series Was. A portion of this dilution series bacterial solution was applied to a blood agar plate medium, and calculates the colonies has been developing and cultured 35 ° C. 20 h, adjusted stock was determined the correct number of bacteria 2.1 × 10 It was ⁸ cells / ml.
Ortho microplate solid phase (ELISA) using the diluted bacterial solution of the step and the sample treatment solution without bacteria as blanks
The method was experimented. Results: The ELISA method was 2.1 × 10 ⁴ cells / ml, a weak color was recognized, the absorbance of the microplate reader (450 nm single wavelength measurement) was 0.150, and the kit negative control limit absorbance was 0. The absorbance was slightly higher than .140. 2.1 × 10 ³ cells / ml was negative and the absorbance was 0.140, which was the same as the blank value.
At 2.1 × 10 ⁵ cells / ml, the absorbance suddenly increased,
0.850. The grades approximate results noted in Ortho's instructions, the kit measurement sensitivity is 10 ⁴ U / m
It was determined to be around l.固 相 Solid phase matrix (first antibody labeling method) Using the same serially diluted bacterial solution series used for the experiment in the solid phase matrix two antibody method, experiments were carried out with goat polyclonal labeled antibodies. In this assay, the primary antibody is bound to the bacteria captured on the solid phase matrix. Since the antibody is labeled with an enzyme, the result can be obtained by a one-step method. The minimum bacterial concentration positive result in this assay is 1.7 × 10
It was ⁴ cells / ml. Although the measurement sensitivity is almost the same as that of the kit of Ortho, the enzyme-labeled antibody used in the solid phase matrix measurement method is the same as the second antibody of Ortho, a peroxidase-labeled O-157: H7 antigen goat polyclonal antibody. When an antibody directly bound to a microorganism (O-157) is labeled with an enzyme, ELI using a microplate is used.
It is SA method, any solid matrix method, 10 ⁴ U /
Bacterial concentration around ml was the result which can be said to be the minimum measurement sensitivity. The solid-phase matrix measurement method using two kinds of antibodies is ELISA method of Ortho and One Ste
The reason for the higher sensitivity compared to the p method is that it is important for bacterial immunoassay. ELI
In both the SA method and the One Step method, enzyme coloration is detected by labeling an enzyme with a bacterial species-specific antibody protein, but this measurement method has a limit of about 10 ⁴ to ⁵゜ / ml measurement sensitivity. . After binding the specific monoclonal antibody to the target microorganism, the secondary heterologous antibody (monoclonal antibody) obtained by immunizing other animals with the primary antibody (monoclonal antibody) bound to the bacterial cells instead of the bacterial cells themselves (Preferably polyclonal antibody)
It has been found that extremely high sensitivity can be obtained.

Example 3 Quantification of bacterial count in liquid sample (particularly in urine) Liquid sample. 14 urine cases of patients were not pre-treated and 500
The doubled product was used as a sample. The reason is that in the case of urine material, contamination in the vulva often occurs at the time of urine collection at 10 ³
To about コ / ml. If true urinary tract infection is due to the number of bacteria is more than 10 ⁶ U / ml has been proposed a medically significant number of bacteria. If the test sample is cerebrospinal fluid and the presence of the bacterium is significant, it is necessary to come to the test without diluting the sample. Antibody used: Antibacterial cell wall (peptidoglycan) mouse monoclonal antibody (Chemicon MAB-
997: IgM fraction) 0.1 ml product (frozen) in 0.0
Thaw with 2.0 ml of 1 Mol NaCl Buffer. Anti-bacterial cell wall (peptidoglycan) mouse monoclonal antibody (Chemicon MAB)
-983: IgG3 plane) 0.1 ml product (frozen) was added to 0.01 Mol NaCl phosphate buffer 2.0
Thaw with ml. An equal mixture of the two lysed antibodies is stocked at -80 ° C. One case / 3.0μ when used
1 is added dropwise. Anti-mouse / goat immunoglobulin / peroxidase-labeled antibody IgG (H + L) (Caltag Labo)
ratories INC. CodeNo. M3000
7): A 2.0 ml product was added with 18.0 ml of the same buffer solution, stocked, and 2.0 μl of a × 3 dilution diluted at the time of use was dropped. Blocking solution: Block Ace (Dainippon Pharmaceutical cat. No.
-UK-B25) diluted 3 times with deionized water. Diluent: A solution to which the above-mentioned mouse serum is added. Washing liquid: A solution obtained by adding a surfactant to the above-mentioned phosphate buffer. Coloring solution: The above-mentioned urethane solution. Measurement method: The measurement was performed in the following operation order. Blocking: blocking solution (20.0 μl) Sampling: diluent urine (20.0 μl) Membrane washing: washing solution (8.0 μl) repeated twice Primary antibody: peptidoglycan antibody (3.0 μl) Reaction time 2.5 minutes Membrane washing: Repeat washing solution (8.0 µl) 4 times Labeled secondary antibody: Caltag above (2.0 µl) Reaction time 2.5 minutes Membrane washing: Washing solution (8.0 µl) Repeat 5 times Enzyme reaction solution: Ultra trouble solution ( 8.0 μl) Reaction time 1 minute Visual judgment: Control blank was diluted 100 times with raw urine and sterile physiological saline according to the standard method of sampling diluent for diluent.

[Table 1]

According to the present invention, microorganisms (bacteria, fungi, produced substances) can be detected or quantified with high sensitivity by immunological testing. The feature of the present invention is not only high sensitivity, but also wide applicability and flexibility. Therefore, the possibility of automatic instrumentation as well as the use method is extremely high.

[Brief description of the drawings]

FIG. 1 shows a detailed view of a reaction vessel. An absorbent is placed on the lower surface of the reaction membrane.

FIG. 2 is a detailed view of a material collecting device.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) G01N 33/543 521 G01N 33/543 521 525 525C 33/577 33/577 B

Claims (8)

[Claims] 1. A newly-developed reaction vessel ==== A porous solid matrix (including a membrane filter) having a fixed pore size and a natural solution absorbing means below the solid phase matrix are provided to remove microorganisms. When a sample suspected of being contained was dropped, a reaction vessel was devised, comprising a step of naturally absorbing the solution in the sample without forcibly aspirating and capturing microorganisms in the sample in a solid phase matrix. The microorganisms captured on the solid phase matrix of the present reaction vessel are subjected to immunological microorganism detection means, but the separation of the microorganism-bound antibody and the unbound antibody (B / F separation) is not performed with the solid phase matrix as the boundary. The bound antibody can be conveniently obtained by natural absorption means. All of the sample solution and the reagent solution passing through the solid phase matrix are completely absorbed by the absorber under the matrix and do not go out of the reaction vessel. Since infectious samples are handled, the reaction container is useful in terms of safety such as sanitation and epidemics. In the final phase of the immunoassay, a colored product is produced by an enzymatic reaction, which is used in a reaction vessel that can be applied to manual measurement by visual observation and instrumental measurement by an integrated reflectometer. The pore diameter of the solid phase matrix for performing an immune reaction composed of a dense fiber or a porous material is 0.1 to 5.0 μm (the optimal pore diameter is 0.22 μm).
m to 0.65 μm, the more optimal pore size is 0.45 μm)
Claims regarding certain measurement methods. The configuration of the reaction vessel is shown. 1 is shown. 2. Binding target of enzyme-labeled secondary antibody =
=== Conventional immunological microorganism detection and measurement methods use a primary reaction stably fixed to the tube wall of a microplate or special test paper fiber, and selectively target microorganisms in a sample with antibody specificity. It just captures. With that means, the only secondary reaction is to bind the secondary antibody to the captured microorganisms (though this development would make conventional measurement methods less sensitive). In the method of the present invention, microorganisms (such as bacteria and fungi) in a sample suspected of containing microorganisms are dropped into the solid phase matrix, and the sample solution is naturally absorbed by the absorber at the lower layer of the reaction vessel without forced aspiration, The microorganisms in the sample are accumulated and attached to the solid phase matrix, and an antibody (primary antibody: preferably a mouse monoclonal antibody) specific to the microorganism to be detected is dropped and bound to the target microorganism (primary antibody). reaction). Unbound antibody is washed off the lower layer of the matrix. The target microorganism is not targeted to the microorganism + primary antibody conjugate remaining on the solid-phase matrix (the focus of this secondary reaction is greatly different from the conventional method). An enzyme-labeled antibody (secondary antibody: preferably polyclonal antibody) specific to the primary antibody specifically bound to the microorganism is dropped (secondary reaction). Next, the unbound enzyme-labeled secondary antibody is washed off the lower layer of the solid phase matrix, and an enzyme coloring means is applied to the remaining microorganisms + primary antibody + secondary enzyme-labeled antibody conjugate on the solid phase matrix. Describes a method for detecting or quantifying microorganisms (such as bacteria and fungi), which is characterized by detecting or quantifying a dye produced by exercise. On the other hand, the present assay can also measure a primary antibody that directly binds to a microorganism by enzyme labeling. Claims relating to a simpler method for detecting and quantifying microorganisms whose measurement sensitivity is equivalent to that of the conventional method. 3. A method for avoiding the prozone phenomenon and an efficient screening method ==== It is not known before a test whether or not a target microorganism to be detected is contained in a sample.
Even if it is included, it is not known whether it is very small or very large. The most confusing problem in immunological detection methods is that it is difficult to detect a reaction at a so-called optimal antigen-antibody ratio, and the prozone phenomenon is likely to occur. In order to widely cover the optimal antigen-antibody ratio, a measurement method in which the primary antibody is enzymatically labeled and directly bound to the target microorganism has low measurement sensitivity, but is preferable when a large amount of the target microorganism exists. On the other hand, when the target microorganism is present in a small amount, the secondary reaction is accurate and has high measurement sensitivity, but when the target microorganism is present in a large amount, the prozone phenomenon tends to occur easily. Therefore, taking advantage of both characteristics, if the primary antibody is mixed with an enzyme-unlabeled one and an enzyme-labeled one and subjected to an inspection test, the sample will contain a large amount even if the target bacterium is contained in a small amount. There are no false negatives. On the other hand, a measurement method capable of efficiently screening for the presence or absence of several types of significant microorganisms by mixing specific antibodies of significant microorganisms frequently detected from a specific sample is also feasible. Specifically, Salmonella is the most common bacterial species causing bacterial food poisoning, followed by pathogenic Escherichia coli (especially O-157), Vibrio and Canbylobacter. When searching for the convenience of a large number of patients when food poisoning occurs in a large number of people, if screening for the presence of these four bacterial species can be performed first, the focus will be on the four bacterial species, and then the scrutiny of the four bacterial species will be conducted. The stepwise refinement that is performed to determine the causative organism is beneficial and efficient. It is possible to determine within a short time whether to determine the name of bacteria using antibodies of individual strains or to bring them to culture tests only for samples that have obtained positive results for four strains even in sporadic minor food poisoning. it can. Claims relating to the method according to claims 1, 2 and 3, which are operable for this immunological microorganism screening. 4. Detection of virus, chlamydia, and toxin in a new reaction vessel ==== not a microorganism itself,
Various types of substances produced by microorganisms (particularly toxins and bacterial metabolites harmful to the human body), viral antigens, viral antibodies,
Claim 1 for the detection of chlamydia antigens and chlamydia antibodies.
Since it is impossible to physically capture the solid phase matrix having a fixed pore as described in a few items, microorganisms (such as bacteria and fungi) are produced in the solid phase matrix fiber as in the conventional immunological detection method. Stably binds specific antibodies to substances, virus antibodies, chlamydia antibodies, etc., and stably fixes the target substance to the fiber while passing the sample suspected of containing microbial substances, viruses, and chlamydia in the sample. Capture with the allowed antibody. After the other substances coexisting in the sample are washed and removed to the lower absorbent layer, an antibody specific to the target substance and labeled with an enzyme are bound (unbound labeled antibodies are washed and removed to the lower layer of the matrix). A substance required for the enzyme reaction is dropped onto the antibody + target substance + target substance-labeled antibody conjugate remaining in the matrix to produce the product. Toxins, etc.) and viruses are detected or quantified, but the claim relates to the fact that the reaction vessel according to claim 1 is used, so that the reaction waste liquid does not come out of the vessel. 5. Enhancement of binding strength of monoclonal antibody ==
== Compared with various protein substances in human blood, kits and set reagents using monoclonal antibodies for immunologically detecting bacteria and fungi, which can be called giant cells, are hardly available on the market. The cause is that the binding ability of the monoclonal antibody to the microorganism is not as strong as the polyclonal antibody. However, monoclonal antibodies have higher specificity than polyclonal antibodies, and the product Lot. There are many products that have the characteristic that there is little variation and can respond to various bacterial species. It was necessary to prepare a reaction environment in which various types of monoclonal antibodies could strongly and stably bind to each specific bacterial species. When the antigen site of microorganisms (bacteria, fungi, viruses, chlamydia, and microorganism-producing substances) and a specific antibody (especially mouse monoclonal antibody) against microorganisms obtained by immunizing various animals react under the optimal measurement conditions, the sample The monoclonal antibody does not bind to the microorganism at all if the treatment solution or the bacterial suspension is a simple phosphate buffer solution or Tris buffer solution. For binding, the addition of animal sera, especially mouse sera producing monoclonal antibodies (in the buffer described above) gives the strongest binding. Serum components from other animal species also increase binding slightly, but not as much as mouse serum.
From the principle of antigen-antibody reaction, the addition of mouse serum, in particular, lowers the electric double layer on the surface of the microorganism and the electric double layer interfacial potential (ζ-potential) in addition to the electrostatic potential of hydrophobic bonds, hydrogen bonds, and Coulomb. Power, Van der
It is thought to enhance the power of Waals. The solid-phase matrix has a great effect on the support surface that brings out the enhancement of the antibody binding ability. The moisture on the surface of the microorganisms captured by the solid phase matrix is extremely low due to the absorber under the matrix. This state means that there is almost no distance to the next antibody to be dropped, and the antigen-antibody binding is performed at a short distance, and the binding reaction is surely completed in a short time. The amount of mouse serum to be added may be very small and may be 1.0 to 1000 mg / dl (the optimal amount is 5.0 to 10 mg / dl).
0 mg / dl). 6. Invention of a strong washing solution ==== In order to ensure the binding of the monoclonal antibody, mouse serum of the same animal as the monoclonal antibody itself is used as a bacterial suspension or a sample pretreatment solution. It was found that the binding became clear when the enzyme was added, but the background color of the enzyme color reaction on the solid phase matrix was significantly higher due to the addition of the same species serum. Therefore, the washing solution used in the conventional immunological test method reported up to the present time has a poor background removal and is not usable, so that a new washing solution composition having a strong washing power is required. The washing solution composition should contain as few salts as possible, but it is necessary to maintain the pH and buffer capacity so as not to affect the antigen-antibody conjugate. The composition of the cleaning solution was 0.01 Mol. Phosphoric acid solution or 0.01 Mol. A large amount of surfactant is added to 1.0 L of Tris buffer (excluding physiological saline).
4. 22% sodium alkyl ether sulfate.
0-600ml (optimal amount is 50ml-300ml) and T
When a washing solution containing 1.0 ml to 10.0 ml of ween80 (the optimal amount is 2.0 ml to 6.0 ml) is used, the background coloration of the solid phase matrix can be obtained even if a microorganism suspension containing mouse serum is used. Claims that the binding reaction by the monoclonal antibody can be clearly observed while holding it low. 7. A device for pretreatment of a test sample ==== When testing bacteria, fungi, viruses, chlamydia and substances (especially toxins) produced by these, materials derived from living bodies (stool, sputum, urine) , Pus, etc.) are often mixed with miscellaneous substances and interfere with the antigen-antibody reaction. In addition, the same applies to food materials (materials such as meat, eggs, milk, and processed processed foods).
In order to carry out the detection or quantification method described in 6, it is necessary to pay attention to the pretreatment of the test sample. Large insoluble substances are left to wait for sedimentation or centrifugal sedimentation, but stool or sputum of highly contaminated medical samples absorbs soluble food residues such as fat proteins, glycolipids, and glycoproteins into chloroform, which has a high specific gravity. The supernatant is used as a pretreatment preparation sample. However, in the medical test sample, leukocytes, erythrocytes, hemoglobin, squamous epithelial cells, and the like are mixed in the human body fluid, so that the antigen-antibody inhibitor cannot be completely removed only by centrifugation. So we remove large particles with a cotton ball and do not filter finer insoluble substances (larger than bacterial species),
3.0 to 5.0 μm where microorganisms can pass
Claims relating to the contents of a sample pretreatment container (tip shape) in which a filter having a pore size of 2 and a cotton ball are integrated in two layers in a double-layered manner. The details of the sample pretreatment container are shown in the figure. 2 is shown. 8. Highly sensitive enzyme reaction coloring reagent ==== The antibody labeling enzyme is peroxidase, and the substrate for the enzyme reaction is a benzidine derivative (among others, 3,3 ′, 5,5′-tetotamethylylbenzidine) Is preferable) or a triphenylmethane derivative (preferably tri [4-N, N-di (sulfopropyl) aminophenyl] methane 6 sodium), and the detection of a product by coloration with an enzyme reaction is preferred. And a method for measuring colorimetrically with a naked eye and a method for measuring colorimetrically with an integrating reflectometer.
The present invention relates to the measurement methods described in 4, 5, 6, and 7. In addition, the present invention relates to a kit reagent capable of immunologically detecting products such as bacteria, fungi, viruses, and toxins according to claims 1, 2, 3, 4, 5, 6, and 7.