JPH04503114A - Staphylococcal fibronectin receptor antibody detection test - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Test for detecting Staphylococcal fibronectin receptor antibodies Diagnostic test or test for detecting Staphylococcal fibronectin-receptor antibodies Concerning statutory law.

The infectious microorganism Staphylococcus aureus We have been investigating the mechanism by which .us) adheres to host tissues. This feels like it's attached. This is because it is the first stage of dyeing progress. If the bacteria cannot attach to the surface, the bacteria will be swept away by body fluids that normally wash tissues, and no infection will occur. That's it Indeed, attachment is a critical first step in the initiation and spread of infection. Generally, microorganisms It has been found to adhere to surface components of host tissues.

A specific receptor on Staphylococcus aureus is called ``fibronectin.'' was discovered to interact with host proteins and mediate attachment to host tissues. . Fibronectin acts as a "glue" that binds one cell to another It plays an important role in wound healing and is essential for host reproduction.

Fibronectin plays an important role in S. aureus attachment to host tissues. There is some evidence to suggest that There are observations: (i) Staphylococcus aureus has a receptor specific for fibronectin. (ii) Staphylococcus aureus fibronectin-receptor (FN-R) expressed on the bacterial surface where it can interact with host tissues. ), (iii) the fibronectin-receptor is activated by S. aureus that invades the host. expressed in greater numbers on clinical isolates of fungi than on non-invasive isolates, (iv) Purified FN-R reduced the interaction of Staphylococcus aureus with fibronectin. (v) Antibodies against FN-H are based on the association of fibronectin with Staphylococcus aureus. (vi) Fibronectin is often infected with Staphylococcus aureus (vii) Fibronectin, which is found at the site where Staphylococcus aureus adheres, (viil) specific fibroblasts from a complex mixture of host proteins. Removal of cutin weakens S. aureus binding, but removal of other proteins (ix) Antibodies against fibronectin are produced by Staphylococcus aureus. Inhibits binding to host tissues.

Fibronectin receptor polysaccharides play a major role in the initiation of Staphylococcus aureus infection There are also data suggesting that (i) fibronectin receptors Staphylococcus aureus mutants that are isogenic to the parent strain except for the ability to express (ii) yellow, which colonizes the heart valves of rats 120 times less efficiently than the parent strain; Monoclonal antibodies that recognize the most invasive strains of staphylococci preferentially target fibro Interacts with nectin receptors.

Recently, an exogenous protein expressed on Staphylococcus aureus that specifically binds to fibronectin has been reported. A polysaccharide was isolated. It can be highly purified and the fibronectin receptor functions as

The object of the present invention is to improve the ability of Staphylococcus fibronectin receptors. An object of the present invention is to disclose a diagnostic test or assay method for detecting antibodies in a patient.

Additionally, it is possible to quantify antibiotic production in response to purified fibronectin receptors. It is also an object of the present invention to disclose an ELISA test method that can be used for .

The inventor has developed a diagnostic test for the detection of antibodies to the staphylococcal fibronectin receptor. The assay method is based on an antigen expressed on Staphylococcus aureus that specifically targets fibrone. Using purified fibronectin receptor exopolysaccharide that binds to cutin as an antigen We discovered that it can be prepared by

Exopolysaccharides contain no lipids and less than 2% protein, making up exopolysaccharides The sugars that do this are aminohexoses. Purified exopolysaccharide is Staphylococcus aureus cross-reacts with monoclonal antibodies directed against Type 8 capsule membrane polysaccharides. However, the fibronectin receptor polysaccharide of the present invention contains galacturonic acid. , and therefore distinct from type 8 membrane material. Nectin receptor exopolysaccharide has an average molecular weight of ~1000 kdal.

and compete with intact organisms for binding to fibronectin .

Fibronectin receptor exopolysaccharide was expressed from intact S. aureus cells. Surface materials containing fibronectin receptor polysaccharides can be removed using non-destructive methods such as gentle sonication. Gently detaches from cells without destroying or killing them through necrosis treatment The exopolysaccharide is then removed by DEAE ion exchange and and purified by fibronectin affinity chromatography.

A preferred Staphylococcus aureus strain is called strain 6850.

Indirect ELIS to screen patients for anti-fibronectin receptor antibodies In the A test, the exopolysaccharide acting as a ligand is attached to a solid phase immunosorbent support. Combine with the holding body. The test serum contains zero-order fibronectin that reacts with bound exopolysaccharides. Chin is added and the mixture is incubated and washed. Finally alkaline phos Anti-fibronectin antibody conjugated with fatase is added. The test is in the sample serum Compatible with the label designed and used to measure the amount of antibody bound to exopolysaccharide in The process ends with a detection step. Monochrome for fibronectin receptor exopolysaccharide ELISA test using fibronectin receptor polysaccharide as an antigen for naru antibody used as a standard in

The monoclonal antibody used is the antibody against type 8 membrane polysaccharide mentioned above. Alternatively, fibronectin receptor exopolysaccharide can be injected into mice and fibronectin receptor exopolysaccharide can be injected into mice. Removed mouse spleen showing antibodies that block bronectin binding to FN-R , the antibody was cloned and the antibody was cloned by Kohler and Milstein (Kohler and Milstein). Hybridomas were prepared by the method of Milstein), and fibronectic A monoclonal anti-inflammatory drug that inhibits the binding of FN-R to FN-R. It may also be an antibody obtained by selecting a human body.

Other objects and advantages of the invention will be apparent to those skilled in the art.

In a preferred practice of the invention, the diagnostic test is a fibronectin receptor exopolysaccharide ( FN-R) in a microtiter (a+1 crotiter) plate. play Incubate at room temperature. The test serum is added and the mixture is allowed to stand for approximately 60 minutes. After washing , polyclonal rabbit conjugated with alkaline phosphatase, versus human fibro Add nectin antibody (1:2000 dilution), incubate for 2 hours, and wash. enzyme The substrate is added and the mixture is allowed to stand for 2 hours. Then, using an automated fluorescence spectrophotometer The amount of staphylococcal fibronectin receptor antibodies present in the sample serum is quantified.

The invention is further illustrated by the following example.

Example 1 FProchin 6 Eso・゛ Staphylococcus aureus (strain 6850) was incubated for 16 hours in a chemically defined dialyzed medium. I grew it. Bacteria were removed by centrifugation and added to Hank's balanced salt solution. Washed twice in liquid and then once in sterile distilled water. The organism is then placed in distilled water. resuspended in water and gently sonicated (cup-horn heat system sonication). sonication (vi Determined by no change in ability count) and no DNA release removes cell surface material without killing Staphylococcus aureus. sound The wave-treated mixture is then centrifuged to remove bacteria. Save the supernatant and freeze-dry it. Ru. The lyophilized material was placed on a QAE/Sephadex column and (NaCl) The gradient elution 0 fraction was collected and the presence of fibronectin receptor polysaccharide (FN-R) was detected. Fibronectin radiolabeled using Staphylococcus aureus ATCC25923 Determined by competitive inhibition. Both portions containing FN-R are then treated with G-200 Sefa. Pass through a dex column. Collect both parts coming out of this second column, and F To determine the presence of N-H, those fractions containing FN-R were then frozen together. It freezes and dries. This material is approximately 100 times larger than the starting material made by flushing out the bacteria. Refined. Chemical characterization tests of this substance indicate that it is a polysaccharide with less than 2% protein. is composed of carbohydrates, indicating that it contains no fat and is primarily carbohydrates. The sugars are aminohexoses and contain galacturonic acids. FN -R has an average molecular weight of ~1000 kdal. This polysaccharide is G. aureus It cross-reacts with a monoclonal antibody against the membrane polysaccharide of type 8 of the bacterium; The chemical characterization test was performed on a sample containing only mannosaminouronic acids and fluosamine. This indicates that it is different from the membrane polysaccharide of type 8.

The conventionally published FN-R purification method involves removing bacteria from a complex medium and destroying and cleaving them. This was due to the use of bacteria. This means that the effectiveness of the extracted material is The first reason that causes problems regarding the interpretation of sex is that bacteria are It is capable of absorbing complex medium components on top. Media used in previous reports is made from mammalian tissue-crude digest, and is made from fibrous tissue. Ingredients known to interact with nectin (e.g. collagen and fibrin) There was a high possibility that it contained phosphorus). Chemically undefined medium is yellow grape can attach to the surface of cocci and produce fine particles as if they were produced by bacteria. Contains macromolecules produced by Second, using cleaved Staphylococcus aureus As a result, many intracellular proteins are detached, and some of them become fi can interact with bronectin, but they are expressed on the surface of microorganisms. There are only a few things. Destruction-cleaved Staphylococcus aureus is carried by intact bacteria have been observed to contain a larger number of fibronectin receptors. Ta Antibodies produced against the protein fibronectin receptor The fact that these proteins do not block binding to Staphylococcus aureus Proteinaceous substances are critical for promoting fibronectin-Staphylococcus aureus interaction. This indicates that it is not a ligand. This reacts with the carbohydrate FN-R of the present invention, In contrast to antibodies that almost completely block fibronectin binding. in this way , bacterial cleavage releases fibronectin receptor substances that are not normally expressed. Ru. These "eliminated" substances are different from FN-R, which is expressed on the outside of the bacteria. follow Therefore, it is possible that a new technology for extracting surface substances without destroying and cleaving bacteria may be developed. Prevents removal of non-surface fibronectin receptor material. in this way This extraction technique is unique and allows for the removal of carbohydrates, which are important in the pathogenesis of Staphylococcus aureus infections. This results in the isolation of the compound FN-R polysaccharide.

Example 2 Monoclonal against fibronectin receptor exopolysaccharide Using the FN-R polysaccharide obtained by the above method as an antigen, a monoclonal anti- body was prepared. 10074 Freund's adjuva with complete composition of blackcrumb antigen (Freund's adjuvant) and injected into BALB/C mice. (injected subcutaneously at the site of the mouse. 0 μg was injected intraperitoneally. The mice were injected 100 μg intravenously in the mouth for 6 weeks. . Two to three days later, mice were injected intraperitoneally with 100 ug of antigen. Collect blood from mice, Blocking antibody (antibody that blocked the binding of fibronectin to FN-R in an ELISA test) The spleen was removed from the mouse that showed the body. Screened about 800 clones After that, some clones are added to the Koran and Milstein (Kohler a). Cancerous tumors derived from myeloid tumors of the bone marrow according to the method of M.D. Milstein Hybridomas were created by fusing cells and their clones. the Monoclonal antibodies are produced by screening hybridomas to produce the desired antibodies. We selected hybridomas that grow in a medium that produces . 7 species that inhibit activity Monoclonal antibodies were generated. , two types of monoclonal antibodies make it 1: When diluted to 100%, the binding of fibronectin to FN-R was determined by ELISA assay. It showed an inhibition rate of 70-92%.

Example 3 Rin 4 ELISA to determine the presence of anti-FN-R antibodies in patient serum The FN-R exopolysaccharide used was derived from a large number of FN-R expressing strains (Staphylococcus aureus strains). 6850) (ATCC No. 53657). This stock was discovered by screening hundreds of species of Staphylococcus aureus. FN-R polysaccharide is Purified as described above. Steam the freeze-dried polysaccharide to a concentration of 1 mg/ml. Resuspended in distilled water. It was then tested for inhibitory activity in a standard binding assay and of 3 μg of III-labeled fibronectin at a concentration of 5xlO@yellow binding to Daphylococcus ATCC 25923 (which is typically about 1 μg of polysaccharide) This FN-R polysaccharide to act as an antigen was then adjusted to a concentration that inhibited 50%. Add 10 μg to each of 96-well (we 11) microtiter plates. Serum from the patient or as a control was placed in the well and allowed to dry overnight. Either antibody was added to the wells. Serum is gelatin-cephalometric 1 microgram of primary fibronectin that had been pretreated with Fibronectin was added to each well. Incubate at room temperature for 60 minutes and wash wells. , alkaline phosph conjugated with rabbit anti-fibronectin monoclonal antibody. Atase was added. After the greenhouse, the wells were washed again and appropriate media added.

The test was analyzed using a fluorescence analyzer. If the patient has no antibodies in the serum, the maximum value will be recorded. Let's go. However, if a specific antibody is present, it is likely that fibronectin binding to the FN-R polysaccharide, giving a low fluorescence optical reading. Antibody concentration and fireflies It can be used to determine antibody titer values from a direct inverse relationship with luminosity. You can get the results you want. FN-H antibodies are placed on fibronectin bound to the wells. of all receptors present in the wells and a fibronectin concentration of 1 μg. It was found to be sufficient to saturate the body.

Example 4 Grape production Kuching fl Nondestructively perform crude exopolymer extraction from intact living stocks as described above. This was done by sonication. obtained after pelleting and discarding the sonicated bacteria. The supernatant was sterilized by filtration, dialyzed against distilled water, and lyophilized to produce purified yellow from strain 0 6850. color Staphylococcus fibronectin receptor exopolysaccharide sostaphin (lysos taphin) treated bacteria, anion exchange chromatography and gel filtration method Prepared using

Example 5 , ± I ELISA Fibronectin receptor exopolysaccharide from strain 6850 was added to a 96-well polystyrene plate. adsorbed onto a microtiter plate, washed, and left to air dry. series In the control plate, fibronectin and amino- and carboxy- Terminal fibronectin fragments (50 μg/ml in TBS) were added to the plate. Soaked overnight, washed and air dried. Each plate was rinsed with buffer A() before the assay. 50mM NaC, 1150mM NaC, 20mM MgClt, 3% bovine serum alkaline Bumin, and 10 μg/ml rabbit polyclonal IgG [Sigma Chemica Sigma Chemical Co., St., Louig.

MO)) pH 7,4). All preparations were performed at room temperature, and dilution of reagents was Dilution was performed using buffer A, and washing was performed using buffer A excluding BSA and rabbit IgG. I used it. In fibronectin binding and control assays, 100 μl of fibronectin (0.01.0.1.1.0.10 per ml of buffer A) ．． 100 μg), 180 kilodalton fibronectin fragment (10 g g/ml) and 27 kilodalton fibronectin fragment (10 μg/ml) ml) was added to the wells and incubated for 60 minutes. For inhibition assays, wells are diluted in a series of Pre-incubate for 60 minutes with 100 μl of diluted serum or fibronectin fragment. After that, add 40μl of fibronectin (2.0μg/ml) and incubate for 60 minutes. I continued to feel warm. After washing, polyclonal rabbits conjugated with alkaline phosphatase Add anti-human fibronectin antibody (1:2000 dilution) and incubate for 2 hours. Placed and washed. Substrate, 4-methylumbelliferyl phosphate (IM 2 -0°2 mg/ml in amino-2-methyl-1-propanol, pH 9,9,2 5mM ZnC1m, supplemented with 1.0mM MgC1□) was added to each well, After incubating the plates for 2 hours, readings were taken on an automated fluorescence spectrophotometer. All samples was tested in triplicate. Control wells with no fibronectin added Subtract the average value of the readings from the sample well from that of the sample well and make that measurement between 10' and 1 Expressed in 0.04 standard fluorescence units (SFU). Inhibits fibronectin binding to wells. Harm rate (%) is 100x [(average 5FU of FN well) ~ (serum or 27kdF Average of wells pre-populated with N fragment 5FU)]/(Average of FN wells 5FU).

Generated against membrane serotypes 5 (MAb831) and 8 (MAb828). The monoclonal antibody was bound to a microtiter coated with purified FN-R. inhibit. High concentrations of rabbit Ig resulted in only a slight decrease in FN binding and MA The specificity of inhibition by b. Type 8 serotype is better than type 5 serotype. was also active. Relatively high concentrations of protein were required to obtain inhibition However, this indicates that common epitopes were only partially cross-reactive. Ru. This is consistent with the data obtained by the inventors regarding the chemical composition of FN-H.

Although FN-R is a large exopolysaccharide, it is similar to type 5 and type 8. The chemical structure is clearly different from that of the capsular polysaccharide. Therefore %ELISA assay method was able to identify antibodies that can inhibit FN binding to FN-R on S. aureus. It has been demonstrated that it is possible and useful in the following three clinical areas: (i) ELISA screens antibodies that protect against staphylococcal adhesion to host tissues. Worth cleaning. (if) if these antibodies are protective in animal models; If it is proven that it can be controlled, it can be tested to prevent human disease (vaccination). (possibly due to anti-tin or passive antibodies), (iii) EL I SA is not Which patients are at high risk for developing invasive staphylococcal infections? This will be useful in predicting what will happen.

Human plasma normally contains 300 to 600 μg of FN per microtiter. Ru. Blood clots consume large amounts of FN, but anti-F Enough FN remains in the serum to compete with the NR antibody. highly refined Using FN, an FN of 1100n can be detected by the assay. ELISA Sensitivity is due to the specificity of FN by interaction through its gelatin-binding domain. Treat the serum with gelatin-Sepharose, which removes the antibodies but leaves the antibodies intact. It can be increased by

The number of binding sites expressed on various S. aureus strains and the number of binding sites extracted from those strains. A positive correlation was observed between the reaction intensities obtained in ELISA from non-protein antigens. It was done. However, since this substance is not highly purified, It is extremely difficult to maintain quantification during the numerous steps required to make a product. However, since FN-R is somewhat unstable, no quantitative conclusions can be drawn from these data. Some genes that are not expressed, probably involved in the expression of capsular exopolysaccharide, are F As mentioned above, a portion of FN-H (chemically Although distinctly distinct from type 8 membrane material) There are suggestive cross-reactive epitopes. A more important observation is that the membrane serotype and FN- R expression can be isolated in some strains. This is FN-R An analogy showing that capsulation is an independent variable in the pathogenesis of Staphylococcus aureus. For example, FN-R may be important for adhesion to host tissues, whereas the capsule Anticapsular antibodies may be important in counteracting the protection of human neutrophils. A concept supported by the discovery that it enhances the phagocytosis of Staphylococcus aureus. be. Because FN-R is expressed on a wide variety of staphylococcal serotypes, anti-FN-R The R antibody not only has anti-adhesion properties against a very wide range of staphylococci but also has opsonic anti- It should also be physically active.

2 Smith Compact (SLllith Compact) 390 1 1 53 7-de 4 (Mardi) 4105 E-3003560 E-50563760 E-30582330 E-597560 502A 30 0 6826 21.50000 472 6 C2380 879R455, /1536 20 0ENDO7400842 685049,0001000 8ATCC259237500±3200io Storis h) Washed and lyophilized for use in 2330 assay (1xlO” cells/assay). *Water: A strain grown in a log phase in a chemically defined medium, lightly sonicated. lyophilized sonicated supernatant for ELISA. As the above antigen adsorbed (50 μg/ml) into microtiter wells, Competitive ELIS using FN-R exopolysaccharide or FN-H monoclonal antibodies The A test and similar diagnostic tests determine whether a patient has developed an intrusive Staphylococcus aureus infection. It can be used to predict when you are exposed to a high degree of danger.

The microorganism Staphylococcus aureus is a powerful infectious agent. host fibronectin and Interactions between S. aureus play an important role in the initiation of infection. FN-R polysaccharide Antibodies against fibronectin are monoclonal or polyclonal antibodies. can be used in methods of preventing infections caused by Staphylococcus aureus.

Patients at high risk for developing Staphylococcus aureus infection should be treated with the above-mentioned diagnostic tests. It can be predicted by law. Therefore, a monocloner to compete with FN-H antibodies in safe and effective amounts in ways that produce passive immunization in highly at-risk patients. It could be administered with

Anti-FN-R monoclonal antibodies are recommended for patients with the following clinical conditions: Motion immunization may be beneficial: 1) Prosthetic joint attachment is a procedure, especially when attaching a hip prosthesis. patient.

2) Patients with heart valves or other implanted devices.

3) Patients undergoing hemodialysis or peritoneal dialysis.

4) Patients with recurrent Staphylococcus aureus infection, especially those with diabetes, rheumatoid arthritis, and antibody deficiency. Patients with oligosyndrome.

5) Patients with severe burns.

6) Immunosuppressed patients, especially those with long-term neutropenia.

7) Patients with internal intravascular catheters, e.g. high nutritional supplements or chemotherapy Patients receiving drugs.

8) Patients undergoing major orthopedic procedures, such as open fracture reduction procedures.

To determine the level of antibodies directed against pre-existing FN-R polysaccharide, The effectiveness of a diagnostic test using FN-R4 polysaccharide as an antigen in an ELISA test will help determine which patients will benefit from this passive immunization. be. However, monoclonal antibodies are also effective in treating Staphylococcus aureus infections. It can be. FN-R polysaccharide is predominantly expressed on noxious Staphylococcus aureus Because the FN-R polysaccharide is present on the surface of bacteria, this immunogenic Monoclonal antibodies directed against can act as opsonins as well as blocking antibodies. So Therefore, passive immunization with this antibody prevents S. aureus from spreading to new sites in the host. limit the likelihood of infection and reduce the severity of Staphylococcus aureus infection progression it is conceivable that. Therefore, another use of anti-FN-R monoclonal antibodies is Patients who are already infected with Staphylococcus aureus, especially those with bacterial infections There are methods for treating patients with or whose screen syndrome is about to progress.

As already mentioned, in addition to monoclonal antibodies against FN-R polysaccharide, the present inventors A monoclonal antibody to the type 8 membrane polysaccharide of Staphylococcus coloris was prepared using the above method and It has been discovered that it can be used for diagnostic tests. These antibodies are known and described by Nels et al. Infection and Immunity, Volume 49 (Nelles, et al. and Io+o+unity, Vol. 49) No. 1. 1985 7 May, pp. 14-18 and Hochkerapel et al. Journal of Clinical ・Microbiology Volume 25 (Hochkeppel et al, Jour n+al.

f C11nical Microbiology, Vol, 25) No. , 3. March 1987, pages 52B-530.

However, the use of monoclonal antibodies in the above methods and diagnostic tests is new. That's a good thing.

The monoclonal antibody is used to block or treat staphylococcal infections. Injecting a sterile pharmaceutical preparation containing noclonal antibodies directly into the animal's bloodstream It is administered to animals by The amount to be injected depends on the size and condition of the animal and This is a safe and effective amount determined by the degree of infection.

It will be appreciated by those skilled in the art that modifications and alterations may be made without departing from the spirit and scope of the invention. It is clear that change is possible. Therefore, except as defined in the claims, the present invention is There is no equivalent restriction.

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Claims (3)

[Claims] 1. 1. A method for detecting fibronectin receptor antibodies in a test sample, the method comprising: (a) Microtiter plate purified fibronectin receptor exopolysaccharide antigen. (b) the test sample and the labeled reagent; mixing and incubating a portion of the mixture using antigen coated wells; (c) separating the bound and free labeling reagent mixtures; (d) Fibronectin acceptance in the test sample by detecting the amount of bound labeling reagent. Measure the amount of body antibodies A method comprising steps. 2. Fibronectin receptor exopolysaccharide obtained from Staphylococcus aureus was used as an antigen. A diagnostic test that tests a patient's fibronectin receptor antibodies using 3. A test kit for detecting fibronectin receptor antibodies, comprising: (a) an insoluble surface or support containing the wells; (b) a fluoride attached to the wells; fibronectin receptor and (c) in a test sample that binds to the fibronectin receptor. means of detecting the amount of antibodies in A kit that includes.