KR920007205B1 - Wash composition test kit and their use to determine herpes simplex viral antigen - Google Patents

Abstract

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Description

[Name of invention]

Cleaning composition, test kit, and use thereof for the measurement of herpes simplex antigen

Detailed description of the invention

The present invention relates to a cleaning solution and its use for the detection of herpes simplex virus. In particular, this solution is used for detecting herpes simplex virus antigen by separating the non-complex material from the antigen and the complex of the antibody. The present invention is useful in diagnostic procedures. The present invention also relates to a diagnostic test kit comprising a cleaning solution.

[Background of invention]

In recent years, immunoassays have been used to detect the presence of infectious diseases. In order for such an assay to be useful, only certain organisms must be able to be detected with high confidence. In most cases, this requires the separation and reaction of the antigen specific to that organism and the corresponding antibody. For such an assay to be commercially successful, it also needs to be relatively inexpensive, simple and quick to use.

One such organism that can be detected by immunoassay is herpes simplex virus. In spite of the increased control over the various bases by vaccination and treatment with various antiviral agents, infection with herpes simplex virus (hereinafter abbreviated as HSV) remains a significant problem. There are two types of HSV, type 1 mainly around the oral cavity, and type 2 mainly around the vulva of the human body. Skin infections and viral encephalitis are just two significant consequences from HSV infection.

Due to the prevalence of HSV infection, there has been considerable interest in rapid, simple and reliable testing for the detection of causative viruses. However, when using known diagnostic methods, there are often indistinguishable from HSV and some similar viruses. Thus useful diagnostic tests for HSV-1 or HSV-2 should be specific only for these viruses, such as Epstein-Barr viruses, Cytomegalovir-us, and Varicella zoster. ) Should not be sensitive to viruses such as viruses or any other virus.

HSV has been detected using a variety of analytical techniques, including electrophoresis, aggregation and enzyme-linked immunoassays (ELISA). In these techniques, when an immunological complex is formed between an HSV antigen and an antibody against it, the complex is usually separated from the uncomplexed substance to improve detection sensitivity. Separation is accomplished by filtration, centrifugation or affinity chromatography. In most cases, the complexes are insolubilized in some way and washed with a number of solutions including distilled water, phosphate buffered saline solutions or nonionic surfactants in order to deposit the non-composites.

For example, US Pat. No. 4,535,057 discloses a solid phase assay for viral antigens, such as herpes simplex narcissus antigens, wherein the antibody-antigen complex is commercially available under the trademark Tweeen 20. Washed several times with a phosphate buffered saline solution containing a nonionic surfactant (21 columns, lines 66-67), the pH of this solution is generally 7.0-7.5.

While such cleaning solutions are commonly used in immunological methods, there has been a continuing need to improve sensitivity in assays and reduce unwanted background signals in the detection of HSV. .

[Summary of invention]

The present invention solves the disadvantages of using a known cleaning solution in the analysis method by using a water-soluble cleaning solution useful in the assay for herpes simplex virus, wherein the solution has a pH of about 9-11.5. It consists essentially of an alcohol amine or a salt thereof and a nonionic surfactant.

Furthermore, the present invention is directed to contacting a biological sample suspected of containing a herpes simplex virus antigen with an antibody against it, and forming a non-complex from the complex using the aqueous washing solution described above. To isolate C. and to detect the presence of a complex as an indicator for the presence of herpes simplex virus in the sample.

Diagnostic test kits useful for the measurement of herpes simplex virus antigens comprise (a) the above-described cleaning solution and (b) antibodies against herpes simplex nasal antigens.

Taiwanese assays for the HSV of the present invention are fast, reliable and easy to use. For example, this method can be performed within about 30 minutes at room temperature. This method is highly reliable, for example, for the detection of antigens extracted from HSV or HSV infected cells or cell membranes.

This assay is highly sensitive, exhibits a low background, and minimizes nonspecific binding of the antibody to the solid support comprising the antigen-antibody complex. These advantages are obtained using a special water soluble cleaning solution of the present invention having a pH of about 9-11.5 and consisting of alcoholamines or salts thereof and nonionic surfactants.

Detailed description of the invention

The present invention includes methods for determining the presence of HSV in biological samples obtained from patients using aqueous cleaning solutions and standard medical and microbiological techniques. Biological samples include, for example, swab samples taken from the patient's cervix, urethra, eyes, throat or anus and body fluids such as synovial fluid or lesions. The biological sample thus obtained is considered to contain HSV or HSV infected cells or membranes containing the antigen to be measured.

The present invention can be used to detect the entire virus or the entire cell or membrane infected with the virus, but in order to measure with high sensitivity in a relatively short time, it is possible to effectively lyse the virus or virus or virus infected cells to release sufficient antigens. It is preferable.

The present invention is present in either of the detectable antigens HSV-1 or HSV-2, or in both virus strains. Glycoproteins in virions can be extracted and detected according to the present invention.

Extracted antigens can be obtained using any useful method or reagent, including the extraction methods or reagents mentioned in US Pat. No. 4,430,437 and US Pat. No. 4,661,349 and European Patent Publication No. 0 183 383.

Preferred extraction compositions have a pH of about 8.5-12. Appropriate buffers or bases can be used to achieve the desired pH. Some buffers are bases strong enough to provide alkaline conditions as well as buffering capacity. For other buffers that do not, a strong base (a hydroxide such as sodium hydroxide or potassium hydroxide) is used to obtain the pH, which is then used to maintain this pH.

This extract composition contains at least one alcohol amine or salt thereof in an amount of at least about 0.05 mole, preferably about 0.1-1 mole. Useful alcoholamines include ethanolamine, diethanolamine, propanolamine, triethanolamine and salts thereof (such as hydrochloride, sulfate, acetate, piclinate and oxalate). Others will be apparent to those skilled in the art. If necessary, a mixture of alcohol amines or salts thereof may be used.

The composition also contains one or more nonionic surfactants which are condensation products of alkylphenols with ethylene oxide. Preferred alkylphenols have 1-20 carbons in a linear or branched alkyl group on the phenol. Octylphenol is most preferred. In general, these compounds have about 5-35 ethylene oxide groups. These nonionic surfactants are readily prepared using known methods and starting materials, but many can also be obtained commercially.

Other useful nonionic surfactants include TritonX-100 and Triton Polyoxyethylene ethers such as those sold under the trade name Triton or under the trade name Brij, such as N101, polyoxyethylene sorbitan derivatives such as those sold under the trade name Tween (e.g. Tween-20) and the trade name Tergitol (e.g. , Polyglycol ethers such as those sold as Tergitol NPX and NP-7). Other useful materials will be apparent to those skilled in the art, especially after reference to McCutcheon's Emulsifiers and Detergents, 1986 Edition, McCutcheon Division, Publishing Co., Glen Rock, N.J., which is a standard reference for surfactants.

The at least one nonionic surfactant is present in the extract composition in an amount of at least about 1% by weight (based on the total composition weight), preferably about 4-10% by weight.

A third important component of the extraction composition of the present invention is at least one cholic acid, salts thereof or derivatives thereof. Useful materials include, but are not limited to, cholic acid, kenodeoxycholic acid, deoxycholic acid, sodium deoxycholate, potassium kenodeoxycholic acid, ammonium cholate, and others that would be apparent to those skilled in the art. This component is present in the composition in an amount of at least about 0.2% by weight (based on the total composition weight), preferably about 0.5-5% by weight.

The extract composition also contains anionic surfactant in an amount of at least about 0.1% by weight (based on the total composition weight), preferably about 0.2-1% by weight. Useful anionic surfactants include, but are not limited to, water soluble or water dispersible compounds composed of C _6-20 alkyl sulfate anions and alkali metals (eg, lithium, sodium or potassium) or ammonium cations. Preferably, said alkyl has C _6-12 (eg linear or branched hexyl, octyl, decyl, 2-methylhexyl and dodecyl groups), arylsulfonic acid having C _6-10 in the aryl ring or Salts thereof (as described above) are also useful. Representative anionic surfactants include ammonium dodecyl sulfate, sodium dodecyl sulfate, rubidium dodecyl sulfate, sodium decyl sulfate, lithium hexyl sulfate, potassium octyl sulfate and lithium decyl sulfate.

An optional component in the cord composition is one or more inorganic salts such as alkali metal, ammonium or alkaline earth salts. Representative salts include, but are not limited to, sodium chloride (most preferred), potassium ammonium chloride scale, calcium chloride, ammonium sulfate, barium sulfate, and others apparent to those skilled in the art. The salt is preferably present in an amount of at least about 0.3 moles, more preferably about 0.5-2 moles.

Antigen extraction can be performed using any suitable method. Preferably, the extraction is carried out in a suitable extraction device designed specifically for this purpose. Many such devices are known in the art and include, for example, US Pat. No. 4,746,614.

Biological samples (filtered or unfiltered) may be treated with any of a number of assay methods to determine the presence of herpes antigens (either extracted or infected whole cells or virions). Such methods include, but are not limited to, culture techniques, counterimmunoelectrophoresis, and serological tests, which are undesirable and can only be selected in some cases.

Preferably, the HSV antigen is detected using an immunoassay, wherein the antigen is immunologically reacted with one or more suitable antibodies to form an immunological complex. Antigens from either or both of HSV-1 or HSV-2 can be detected. Preferably, both are detected simultaneously. After the non-composite material is separated from the complex using the cleaning solution of the present invention, the complex is detected using a suitable radiometric reagent, colorimetric reagent, fluorescent reagent or reagent labeled enzyme. do. In some cases, the reagent is a labeled antibody against the antigen, and in another case, the labeled anti-antibody is against an unlabeled antibody that reacts with the antigen.

In a preferred embodiment, the complex is immobilized on any type of solid support, with or without coating, and then treated with a suitable extraction method. Other assays include agglutination of immunological complexes when at least one reactant (such as an antibody) of the complex is attached to a labeled or unlabeled particle in a mask form that aggregates together during complex formation. Such agglutination assays are described in European Patent Application Publication No. 0 183 215.

Examples of useful assays are competitive immunoassay, radioisotope labeled immunoassay (including radioisotope labeled immunoprecipitation) or enzyme-linked immunosorbent assay (or so-called "ELISA"). Such analytical methods are generally described in U.S. Patent No. 4,430,437 and are described too much in others and cannot be mentioned. The HSV antibodies used may be against either antigen or several antigens, preferably against antigens extracted from virions or cells. In one embodiment, the antibody is against a single glycoprotein of either HSV-1 or HSV-2. In other embodiments, the mixture of different antibodies is against several antigens, such as glycoproteins derived from both HSV-1 and HSV-2. In a third preferred embodiment, a single antibody is used that is reactive with specific glycoproteins from both HSV-1 and HSV-2.

Antibodies used in the assay may be polyclonal or monoclonal antibodies which may be purchased or prepared using known methods. Preferred antibodies are monoclonal antibodies. One such monoclonal antibody is obtained from hybridoma cell line 283--2A1-lD4-2C3 (ATCC deposited HB-9684) using standard methods.

In useful solid phase immunoassays, the estimated glycoprotein antigens are "captured" on small polymeric particles by covalently reacting or adsorbing with reactive groups on the particles that react with free aman or sulfhydryl groups. The captured antigen then reacts with the appropriate antibody to form a bound immunological complex. Uncombined diseased material is separated using a microporous membrane filter.

Preferred immunoassays are performed by capturing the extracted antigen on a coated or uncoated microporous membrane filter that is also used to separate the uncomplexed material from the resulting immunological complex. Other immunoassays are performed using surfactant-coated microporous membranes. Most preferably, the microporous membrane is an uncoated or untreated nylon material as indicated in Example 2 below.

Generally, the gemstones in a preferred embodiment of the present invention using any form of solid support (preferably the membrane or beads as described above) are done as follows: The extracted antigen is free, cellulose, Contact with a solid support, such as a ceramic or polymeric material, Preferably, the support consists of any natural or synthetic polymeric material with which the extracted antigen will bind rapidly without controlling excessive incubation or other conditions. Addition polymers made from polyesters, polyamides, polycarbonates, polyethyleneamines, cellulosic materials and ethylenically unsaturated vinyl monomers, and others known in the art.In general, if the membrane is positively charged, the cationic group is Quaternary ammonium salts, quaternary phosphonium salts, quaternary sulfonium salts, quaternary pyridinium salts, quaternary pyrimidium salts or quaternary imides Dazolium salts and quaternary ammonium salts are preferred.

One preferred embodiment uses particles on microporous membranes, where both the particles and the membrane capture antigens deduced.

The support may be configured in any suitable form, such as beads, gels, films or membranes. Preference is given to the microporous membranes described herein. Generally, this membrane has an average pore size of about 0.1-20 μm.

The supports described herein can be used with other instruments (bottles, test tubes, swabs, beakers, or cups) to perform this assay. Optionally and preferably, the support is a microporous membrane that fits into a disposable test device capable of carrying out the assay and containing all liquids. Useful test devices and forms are U.S. It is known from literature in the art, such as patents 3,825,410, 3,888,629, 3,970,429 and 4,446,232. Particularly useful devices are described in European Patent Publication No. 280 558.

Almost immediately after the antigen contacts the support, the antigen binds to the support. The binding can be done in a direct manner, meaning that the antigen is not bound through a biologically binding compound (such as an antibody) attached to the support, or indirectly through this binding compound. Unbound materials can be cleaned out using the cleaning solution of the present invention (described below).

Therefore, within about 10 minutes, preferably within 10-120 seconds of contact time, the bound antigen contacts the appropriate antibody (or mixture thereof) against the HSV antigen to form an immunological complex on the support. If the analysis is done using a disposable test device, the support is a microporous membrane that can flow out of the liquid and the uncombined material in the sample when the antigen binds to the membrane.

In a preferred embodiment, the antibody against the antigen is labeled for testing. Useful labels are known in the art and can be detected by other chemical or specific binding reactions that provide testable species as well as chemical or biological compounds that can be tested directly or indirectly using appropriate methods and instruments. There is a compound. Examples of useful labels include radioisotopes, enzymes, coenzymes, enzyme inhibitors, fluorescent compounds, cofactors, chemiluminescent compounds, phosphorescent compounds, biotin or derivatives thereof, avidin or derivatives thereof, ferritin, magnetizable particles , Dyed particles, and others are apparent to those skilled in the art. Radioisotopes or enzymes are the preferred labels. Such labels can be attached to antibodies using known techniques. When the label cannot be detected directly, another reagent or compound is needed to obtain a detectable reaction product or specific binding product. For example, when the label is biotin, it can react with aberdela, which is associated with an enzyme, to provide a detectable species. When the label is an enzyme such as glucose oxidase, urease, peroxidase, alkaline phosphatase and the like, a substrate and salt providing reagent are also needed. Peroxidases and alkaline phosphatases are particularly useful.

In a particularly preferred embodiment, the label is peroxidase, and at any stage of the assay, hydrogen peroxide and the appropriate dye forming reagent in the dye providing composition are added to provide a detectable dye. Useful dye providing reagents include, for example, tetramethylbenzidine and derivatives thereof, and leuco dyes such as triarylimidazole leuco salts (described in US Pat. No. 4,089,747) or peroxidase and hydrogen peroxide. Other compounds that react in the presence of a dye to provide a dye (ie, compounds that react after the catalytic action of peroxidase to provide a dye).

In another embodiment, the detection of an antibody-antigen complex formed with the herpes simplex non-prepared antibody bound to a support is suitably labeled for detection (as above) and is specific for the HSV antibody (described below). Is made).

The antibodies used in the assay can be supplied in a mixture with one or more blocking proteins that reduce nonspecific interactions with the support. Useful proteins are known and include, for example, casein, α-casein, serum of fetal bovine and gamma globulin of swine. One useful blocking composition contains a non-immunological blocking protein and an amphoteric surfactant.

To promote the formation of immunological complexes bound to the support, antibodies and antigens are generally incubated at a temperature of about 15-30 ° C. for 10 minutes. Preferably, the incubation is carried out at room temperature (ie 1-8-25 ° C.) for 5 minutes.

After incubation and within about 10 minutes of antigen-antibody contact, the bound complex is washed one or more times with an aqueous wash solution. The cleaning solution of the present invention is used for at least one such cleaning (described below).

In the embodiments described above, when the HSV antibody is labeled, the assay procedure after washing is to detect the label directly or indirectly after adding the appropriate field of view. This is done relatively quickly after washing the bound complex. If necessary, label detection can be facilitated by incubation if the reagents ensure this label detection. The label is then detected after a suitable time using standard equipment and methods.

If the HSV antibody is not labeled, after washing the bound complex, it is contacted with the antibody against the unlabeled antibody. This second antibody (ie anti-antibody) is suitably labeled with any of the labels described above and can be supplied with the blocking composition described above. Antibodies can be monoclonal or polyclonal and can be prepared using commonly purchased or known methods.

After this contact, the resulting antigen-antibody-antibody complex bound to the support is incubated at a temperature of about 15-30 ° C. for about 10 minutes. Preferably, the incubation is performed at room temperature for about 5 minutes.

Further washing is performed to remove the unconjugated material, and an appropriate enzyme substrate or other necessary reagents are added to provide detectable species. The bound antigen-antibody-labeled antibody complex is then detected on the support using standard radiometric, colorimetric, fluorometric or other detection techniques.

The cleaning solution of the present invention may be used at least once throughout the method of the present invention to improve the low background and high sensitivity as described above. In addition, as long as the cleaning solution of the present invention is used at least once, a standard cleaning solution can also be used in the process, preferably it is used in the first cleaning step.

The aqueous cleaning solution of the present invention has a pH of about 9-11.5, preferably about 10-11. Such pH may be obtained using one or more suitable buffers or strong bases. Useful bases include, but are not limited to, alkali metals or ammonium hydroxides, such as sodium hydroxide, potassium hydroxide or ammonium hydroxide, and others known in the art. In some cases, only a buffer is sufficient to raise the pH to the desired level, but in other cases a strong base is needed to raise the pH, and then a buffer solution is needed to maintain the pH. E.g. If alcoholamine or salts thereof are used and a pH of about 9.5 or more is desired, a strong base such as nitrile hydroxide is needed to raise the pH of the solution.

This cleaning solution consists essentially of at least about 0.05 moles of alcoholamine or salts thereof, preferably about 0.1-1 moles. Useful alcoholamines include ethanolamine, diethanolamine, propanolamine, triethanolamine and salts thereof (such as hydrochloride, sulfate, acetate, picrinate, oxalate). Others will be readily apparent to those skilled in the art. Alcoholamines or mixtures of salts thereof may also be used if desired. Ethanolamine or its salts are particularly preferred.

The cleaning solution also includes a nonionic surfactant that is water soluble or dispersible in one or more water present in an amount of at least about 0.05% by weight (based on the total weight of the solution), preferably about 0.1-2% by weight. Useful surfactants include the trade name Triton (eg, Triton). X-100), or polyoxyethylene ethers such as those sold under the trademark Brij (e.g., Brij 30), and polyoxyethylene sorbitans such as those sold under the trademark Tween (e.g., Tween 20, Tween 21, Tween 40). Commercially available polyglycol ethers, and polyethylene glycols under the trade name Tergitol (eg, Tergitol NP-7), but are not limited thereto.

Particularly useful nonionic surfactants are the aforementioned polyethylene ethers and polyoxyethylene sorbitan, in particular Tween 20 (tradename). Other useful surfactants may be determined by those skilled in the art by reference to McCutcheon's Emulsifiers and Detergents, 1986, McCutcheon Division Publishing Co, Glen Rock, N.J., which is a standard reference for surfactants.

Representative cleaning solutions of the present invention and their preparation are described in Example 1 below.

A preferred method for measuring herpes simplex virus is

A. The solid support in the disposable test device is contacted with a solution presumed to contain HSV antigen, which is an antigen extracted from a biological sample, for a time sufficient to bind the antigen to the solid support,

B. Washing unbound material from the bound antigen using the aqueous washing solution of the present invention,

C. To form an immunological complex bound to a solid support, the bound antigen is contacted with an antibody against it,

D. Using a water soluble cleaning solution (preferably, the cleaning solution of the present invention) to separate the non-compound material from the complex,

E. consists of measuring the presence of bound complex as an indicator for the presence of herpes simplex virus in the sample.

If desired, the cleaning solution of the present invention may be supplied as part of a diagnostic test kit that also consists of one or more other reagents, diagnostic pieces or other useful materials. Generally, such kits include at least antibodies (labeled or unlabeled) to the antigens of herpes simplex virus, which also include anti-antibodies (if desired), extraction compositions, extraction devices, test devices, dye providing reagents or compositions. , Ingredients, instructions, swabs, and other ingredients useful for conducting the analysis, which may be packaged in any suitable manner and provided in one or more packages or containers.

The following materials, compositions and solutions were used in the following examples and the examples are provided to illustrate the invention but do not limit the scope of the invention.

Antibody Preparation

Hybridoma cells producing monoclonal antibodies against HSV were prepared using known methods described by and colleagues (Nature, 256, pp 495-497, 1975). Hybridoma cell lines were produced that produce monoclonal antibodies that respond to epitopes on the common glycoprotein antigens of both HSV-1 and HSV-2. This hybridoma cell line was deposited as ATCC HB-9684.

[Antigen preparation]

To prepare antigens for use as positive controls, HSV-1 strain F and HSV-2 strain G were incubated in HEP-2 cells (ATCC CCL-23, respectively.) Infected cells were subjected to low speed centrifugation. Pelletize and resuspend the pellet with 15 ml volume of phosphate in saline solution in 50 ml Corex tube Resuspended cells were sonicated and subjected to aminomethyltrioxalene (500 mg / ml) for 15 minutes. It was then exposed to ultraviolet light for 15 minutes under constant stirring.

A positive control well of the test apparatus containing HSV-1 and HSV-2 antigens (inactivated by UV and dissolved by detergent) was added to bovine serum albumin (0.1% by weight) and hydrophilic polymer (5% by weight). %) Was immersed in the filtration membrane of the test well.

[Antibody conjugate preparation]

Yoshitake and his colleagues. Monoclonal antibodies against HSV were conjugated to horseradish peroxidase using the method described in Eur. J. Biochem., 101, 395 (1979). 0.5% by weight of α-casein), Tween 20 nonionic surfactant (0.1% by weight), thimerosal preservative (0.01% by weight) and p-methoxyphenol (100 mmol) mixed with the resulting conjugate And then filtered. The final antibody concentration in the solution was 1.5 μg / ml. It was stored with bovine serum albumin (1% by weight). The conjugate used in the negative control wells of the test device was an antibody against creatine kinase prepared using the method described above and labeled with peroxidase.

[Luco dye providing composition]

Hydrogen peroxide (10 mmol), 2- (4-hydroxy-3-methoxyphenyl) -4,5-bis (4-methoxyphenyl) imidazole leuco dye (0.005% by weight), poly (vinyl) Pyrrolidone) (1% by weight), 4'-hydroxyacetanilide (5 mmol) and diethylenetriaaminepentaacetic acid (10 mmol).

Hydrogen Peroxide Solution

An aqueous solution containing hydrogen peroxide (10% by weight), diethylenetthiaminepentaacetic acid (0.005% by weight) and preservative (0.01% by weight) was prepared.

Extraction Composition

Extract compositions were prepared by mixing the following components in water: Nonidet NP-40 nonionic surfactant (5 wt.%, Trade name), sodium deoxycholate (0.75 wt.%), Sodium dodecyl sulfate anionic surfactant (0.4 Weight percent) and movie sodium (1 mole). The pH of this composition was adjusted to 10.75 using 12N sodium hydroxide.

[Phosphate Buffered Saline Solution]

Sodium chloride (0.15 mol). The solution (0.05 mol) was prepared from sodium deuterium phosphate (0.01 mol) and sodium hydrogen phosphate (pH 7.2, 0.04 mol).

[Blocking composition]

An aqueous blocking composition was prepared containing α-casein (0.5 wt%), Tween 20 (tradename 0.1 wt%), p-methoxyphenol (100 mmol) and preservative (0.01 wt%). A disposable test device with three test wells was used for the analysis. This test apparatus is a microporous nylon membrane (Pall Corp. Biodyne) that is uncoated in each test well. Had A).

Example 1

[Washing solution]

The cleaning solution of the present invention was prepared by mixing the following components in water: Tween 20 nonionic surfactant (tradename: 0.1 wt%), thimerosal preservative (0.01 wt%), ethanolamine hydrochloride (0.26 mole) and thimero Flesh preservative (0.01% by weight). The pH of this solution was raised to 10.75 by addition of 12N sodium hydroxide.

Example 2

[Analysis of HSV-1 and HSV-2dp]

This example describes the method of the present invention using a sample of a patient containing both or both HSV-1 and HSV-2. Samples were obtained from several patients in several clinics and hospitals using two swabs for each patient. Surecell in a clinic or hospital One swab was used to implement the present invention with the test apparatus and a second swab was used for the confirmation test using standard culture techniques.

The first swab obtained from each patient was placed in an extraction tube and the above extraction composition (1μ1) was added. This swab was added to the extraction solution for 1 to 2 minutes, followed by swirling, and the resulting extract was filtered. 10 μm HDC (trade name Pal1 Corp.) in the middle layer and 5 μm Loprodyne in the lower layer Filter (consisting of Pall Corp.) in advance.

Pre-filtered extracts (200 μl) were then placed in each well of each test device to ensure that any HSV antigen was adsorbed to the membrane in the well.

Test wells were rinsed with the rinse solution (l20 μl) of Example 1 and the hydrogen peroxide solution (120 μl) described above was added to each to remove any nonspecific oxidase. Wells were washed again with washing solution (120 μl). Samples (40 μl) of labeled anti-creatin kinase conjugates (3 μl / ml) in the blocking compositions described above were added to the negative preparation wells of each test device. Samples of anti-HSV conjugate (40 μl) were added to the remaining two wells of each test device.

After incubation at room temperature for 5 minutes to make an antibody-antigen complex, the wells were washed twice (200 μl each) with the wash solution of Example 1.

The above-mentioned luco dye composition (40 μl) was added to each test well and incubated at room temperature for 5 minutes, after which the presence of reddish dye on the membrane as an indicator of the presence of HSV antigen in the sample was evaluated. For the sample of patients tested, the intensity (true positive divided by the sum of true positives and false negatives) was 83% and the specificity (real negative divided by the sum of real and false positives) was 100%. . All positive results of the above method were confirmed by the culture results.

Example 3-5

Comparative Example Three cleaning solutions of the present invention were compared with three cleaning solutions outside the scope of the present invention for herpes simplex virus.

[material]

Example 3: A cleaning solution was prepared as in Example 1.

Example 4: The cleaning solution was the same as that in Example 1 except that Tergitol 7 (trade name, 0.1 wt%) was used instead of Tween 20.

Example 5 Cleaning Solution with Triton Instead of Tween 20 It was the same as in Example 1 except that X-100 (0.1% by weight) was used.

Control A: This cleaning solution was an aqueous solution of sodium hydroxide (1 mol, pH6.86).

Control B: This wash solution contained ethanolamine hydrochloride (0.1 mole), sodium chloride (0.26 mole) and TWeen 20 nonionic surfactant (trade name, 0.1 wt%), pH was 8.

Control C: This cleaning solution was treated with sodium chloride (1 mole) and Triton X-100 nonionic surfactant (0.1 wt.%) Was contained and the pH was 6.86.

[analysis]

Test samples were prepared as follows: HSV cell lysate (described above) at 1:40 dilution containing antigen and cells (40 μl) was mixed with the extraction composition (3960 μl) described above and incubated for 2 minutes at room temperature. It was. The mixture was filtered through a filtration device [upper layer of polystere plug, middle 10 μm HDC (trade name Pall Corp.) and lower 5 μm Loprodyne. Pre-filtration through a filter (pall Corp.), and the sample (200 μl) Each test well of the test apparatus was added. One well was used as a negative control and the other two wells were used as test wells for analysis.

A background sample (200 μl each) containing only the extract composition was also added to the test apparatus.

Each wash solution (120 μL) was added to the test well of the device, followed by hydrogen peroxide solution (120 μL) and a second wash solution (120 μL). A solution (40 μl) containing the peroxidase labeled anti-HSV conjugate (1.5 μg / ml) in the blanking solution described above was then added to the test wells of each device. Solutions (40 μl, 3 μg / ml) containing peroxidase labeled anti-creadin kinase conjugates were added to each negative control well. After 5 minutes incubation at room temperature, each well was washed twice with a wash solution (120 μl each time). The above-described luco dye providing composition (40 μl) was added to each well and then incubated again for 5 minutes at room temperature. The color density on the test well membrane was visually observed, and the color concentration was rated as 0-10 when no color concentration could be observed and 0 when the highest color density was observed. The results of this analysis represent the average of two test wells for each device.

[table]

These results indicate that cleaning solutions with low pH (ie, less than 9) provide an unacceptably high background as well as a high signal. The cleaning solutions of Examples 3-5 all provided acceptable color concentrations and preferably low backgrounds.

More experiments, Triton It has been shown that cleaning solutions containing X-100 have exceptionally good storage stability.

While the invention has been described in detail with particular reference to the preferred embodiments thereof, it will be understood that modifications and variations can be made to the invention within the spirit and scope of the invention.

Claims (20)

A water-soluble cleaning solution useful for the analysis of Herpes simplex virus having a pH of about 9-11.5 and consisting of an alcoholamine or a salt thereof and a nonionic surfactant. The aqueous cleaning solution of claim 1 having a PH of about 10-11. The aqueous cleaning solution of claim 1 wherein the nonionic surfactant is polyoxyethylene ether, polyoxyethylene sorbitan, polyglycol ether, or polyethylene glycol. 2. The composition of claim 1 having a pH of about 10-11 and composed of ethanolamine or a salt thereof and polyoxyethylene (20) sorbitan monolaurate or octylphenoxy polyethoxyethanol as a nonionic surfactant. , Aqueous washing solution. Method for measuring herpes simplex virus, consisting of: A. To form an immunological complex, a biological sample presumed to contain the herpes simplex virus antigen is contacted with an antibody against it, and B. A pH of about 9-11.5 And separating the non-composite material from the complex using an aqueous amine solution consisting of an alcohol amine or a salt thereof and a nonionic surfactant, and C. presence of the complex as an indicator of the presence of herpes simplex virus in the sample. Detect. The method of claim 5, wherein the herpes simplex virus antibody is labeled for detection. The method of claim 6, wherein the herpes simplex virus antibody is labeled with an enzyme. 8. The method of claim 7, wherein the enzyme label is peroxidase or alkaline phosphatase. The method of claim 5, wherein the herpes simplex virus antibody is not labeled and the antibody-antigen complex is measured using a labeled anti-antibody for detection. The method of claim 5, wherein the measuring method is performed in whole or in part using a disposable test device consisting of a microporous membrane to separate the non-composite material from the composite. A method for measuring the herpes simplex virus antigen, which is composed of the following: A. The microporous membrane in the disposable test apparatus and the solution supposed to contain the herpes simplex virus antigen, which is an antigen extracted from a biological sample that is supposed to contain herpes simplex virus, The antigen is contacted for a time sufficient to allow the antigen to bind to the membrane, and B. has a pH of about 9-11.5, using an aqueous amine cleaning solution consisting of an alcoholamine or a salt thereof and a nonionic surfactant, to which the antigen is bound To wash away the unbound material from C. to form an immunological complex bound to the membrane, the bound antigen is contacted with an antibody, and D. the non-complexed material is separated from the complex using an aqueous washing solution, E. The presence of bound complexes is detected as an indication of the presence of herpes simplex virus in the sample. The method of claim 11, wherein the water soluble cleaning solution used in step D has a pH of about 9-11.5 and consists of an alcoholamine or a salt thereof and a nonionic surfactant. The method of claim 12, wherein the cleaning solutions in steps B and D are identical. The method of claim 11, wherein the nonionic surfactant in the cleaning solution is polyethylene ether, polyoxyethylene sorbitan, polyglycol ether, or polyethylene glycol. The method of claim 14, wherein the nonionic surfactant is polyoxyethylene (20) sorbitan monolaurate or octylphenoxy polyethoxy ethanol. The method of claim 11, wherein the antibody is labeled with a peroxidase and the measurement of the complex is achieved by adding a dye providing composition that provides a dye in the presence of peroxidase and hydrogen peroxide. Diagnostic test kits useful for the determination of herpes simplex virus, consisting of: (a) a cleaning solution having a pH of about 9-11.5 and consisting of an alcoholamine or a salt thereof and a nonionic surfactant, and (b) Antibody against herpes simplex antigen. The kit of claim 17, wherein the antibody is labeled for detection. 18. The kit of claim 17, further comprising a disposable test device for analysis. 18. The kit of claim 17, further comprising an antibody against herpes simplex virus antibody.