WO2011085422A1

WO2011085422A1 - Immunochromatographic device for detecting chlamydiae and gonococci

Info

Publication number: WO2011085422A1
Application number: PCT/AT2011/000013
Authority: WO
Inventors: Kim Scheuringer
Original assignee: Kim Scheuringer
Priority date: 2010-01-13
Filing date: 2011-01-12
Publication date: 2011-07-21
Also published as: AT509276A1; AT509276B1

Abstract

The present invention relates to a device for identifying chlamydiae and gonococci in a sample, comprising a liquid-permeable solid medium, which defines a path for a liquid flow, said medium having regions provided at a distance from each other along the flow path, wherein the regions are divided in the following sequence into a feeding section, a reaction region, a detection region, and an inspection region, wherein a neutralizing buffer is provided in the dry state in the feeding section, wherein the buffer is a basic buffer having a pH of 11 to 13, in particular 12.7, or an acidic buffer having a pH of 2.5 to 6.5.

Description

IMMUNOCHROMATOGRAPHIC DEVICE

FOR DETERMINING CHLAMYDIEN AND GONOKOKKEN

The present invention relates to a device, a method and a kit for the detection of microorganisms.

Microorganisms can be detected by various methods and devices. The most sensitive and exact method for the determination of microorganisms is the polymerase chain reaction, with which even the smallest amounts of microorganisms can be specifically detected. However, such methods require expensive equipment that can be operated only by skilled persons.

Alternative methods for the determination of microorganisms utilize antibodies that are capable of specifically binding to one or more epitopes of a microorganism. Although less sensitive than molecular biological methods (such as polymerase chain reaction), these methods are easier to handle and much faster. In order to allow the use of antibodies for the determination of microorganisms even untrained persons, devices such as test strips have been developed. With the help of test strips on which microorganism-specific antibodies are immobilized, anyone can easily ascertain the presence of microorganisms in a sample.

Depending on the microorganism to be determined, the samples must be processed on a test strip before use. In the course of sample preparation, the sample can be diluted by the addition of basic or acidic solutions, with which the microorganisms, for example, be lysed. As a result, the sample has a pH that does not allow binding of antigen to antibody so that this sample can not be applied directly to a test strip. Therefore must gene of the sample, it can be neutralized by adding a buffer prior to applying, ^¬. By adding solutions to sample the volume thereof changes, so that there is a further dilution of the sample, whereby the sensitivity of the assay is significantly redu ^¬ sheet.

US 5,773,234 discloses test strips for detecting Chlamydia antigens in patient samples. The sample application zone of the test strip is thereby produced. by soaking an absorbent material with a buffer, then frozen at -70 ° C and lyophilized.

US 2005/0130120 A1 discloses test strips which, inter alia, are suitable for detecting a chlamydia infection in a patient. The sample application zone of these test strips may include, among other things, pH controlling or modifying substances.

US 2006/154315 A1 discloses methods and means for the determination of streptococci. The means described therein include, among others, a test strip made of a porous material. The test strip has a sample application zone impregnated with a buffer intended to neutralize nitrous acid present in the sample. The sample application area is prepared by treating the porous material of the test strip with a buffer and other substances and then dried and assembled into the test strip.

It is an object of the present invention to provide a device, which i.a. avoids the disadvantages of the prior art.

The present invention relates to a device for the determination of chlamydiae or gonococci, in a sample comprising a liquid-permeable solid medium, which defines a path for a liquid flow, which medium along the liquid path spaced areas, said areas in the following order in a Task area, a neutralization buffer is provided in the dried state, wherein the buffer is a basic buffer having a pH of 11 to 13, in particular 12.7, or an acidic buffer with a pH of 2.5 to 6.5.

It has surprisingly been found that it is possible to adjust the pH of an untreated or pretreated sample to a level which allows binding of antigen to antibody in the test strip to provide a buffer in the dried state in the feed area. After contacting the sample with the dried buffer, it is dissolved in the liquid-permeable solid medium, so that its buffer effect can unfold and neutralizes the sample on the medium. The neutralized sample eventually moves due to the capillary action of the medium in this further to the other areas of the device (reaction area, detection area and control area).

The provision of a buffer in the dried state in the application area has the advantage that the user of the device must perform no or a less expensive sample preparation, since an important step (the neutralizing, if necessary) takes place directly in the task area of the device according to the invention.

The device according to the invention may, in principle, be any test strip known in the art.

The liquid-permeable solid medium and the liquid permeable solid support, which respectively can be used OF INVENTION ^¬ dung according to which, may be of any material generally having a certain porosity, where ^¬ at particularly preferred porous carrier comprising Nitrocellulo ^¬ se, nylon, PTFE, polystyrene, latex, glass fibers, silica gel, aluminum ^¬ miniumoxid, cellulose, dextran, agarose, polyvinyl alcohol, come into question. Of course, the device according to the invention can also comprise, in addition to one or more liquid-permeable solid medium (s), other media or materials which have different properties. For example, it is also possible to provide a non-liquid-permeable support in the device on which the liquid-permeable solid medium rests and stabilizes it.

"The task pane", as mentioned above, means that the buffer either directly in liquid-permeable solid medium or that a further liquid ^¬ permeable solid medium is arranged after the area of responsibility.

The term "buffer in dry state" means that the medium in the object area includes a buffer which is ge ^¬ dried. Such a medium is obtainable by contacting the medium with a liquid buffer, followed by a drying process of the task pane be removing ^¬-sensitive water or solvent. By this procedure, a ^¬ object area is available, the a buffer in includes dried state.

It has been found according to the invention that the device according to the invention is particularly well suited to detect chlamydia.

Chlamydia cause various acute and sometimes chronic diseases in humans. Typical diseases caused by chlamydia include venereal genital infections, ocular infections and atypical pneumonia. The genus Chlamydia contains the species Chlamydia trachomatis, Chlamydia pneumoniae and Chlamydia psittaci.

Chlamydia trachomatis is primarily pathogenic to humans. In Chlamydia trachomatis, 18 serotypes are distinguished and it is the most common bacterial agent of sexually transmitted infections.

For example, Chlamydia trachomatis may cause eye infections or even blindness, depending on the manifestation of the serotype.

In order to diagnose a chlamydia infection in good time and, as a result, start therapy early enough to prevent complications, it is crucial to quickly diagnose such an infection.

The detection of chlamydia by means of test strips requires in the prior art to disrupt the microorganisms with an acidic or basic buffer followed by a neutralization step in which a corresponding basic or acidic buffer is used. By neutralizing the pH of the sample, the detection of Chlamydia in a sample is made possible.

In addition to Chlamydia, there are a variety of other microorganisms that are of importance to humans. Examples of such microorganisms are gonococci responsible for gonorrhea and fungi.

The most common diagnostic tests for the determination of microbial infections are kits which have to be used at least partly by qualified personnel or which are used exclusively in the laboratory. Therefore, the rapid determination of whether a microbial infection occurs, for the affected person is not possible.

The device according to the invention can also be part of a kit for the determination of microorganisms, in particular chlamydia, gonococci and fungi, in a sample comprising the following components:

a) at least one sampling means, preferably at least one cotton swab or tampon,

b) a container comprising a buffer for breaking up chlamydiae or gonococci and suitable for receiving the means for sampling, preferably the cotton swab or tampon,

c) a device according to the present invention.

With the kit according to the invention it is possible for the first time due to the simple handling of the individual components to diagnose a microbial infection.

The provided sampling means, preferably a cotton swab or tampon, which are ideally added to the kit in sterile form, serve to remove the sample to be examined. For example, smears such as vaginal smears can be made with these agents.

In order to be able to better determine the microorganisms possibly occurring in the sample, the sampling means are brought into contact with a buffer which for the most part breaks up the cells. This digestion makes the epitopes of the cells, for example, for antibodies, more accessible, allowing the cells to be detected with increased efficiency.

The container suitable for receiving the sampling means preferably comprises a minimum amount of buffer of 50-1000 μΐ, preferably 100-500 μΐ, in particular 300 μΐ, which is sufficient to disrupt the cells and dissolve them from the agent, so that the cells or cell fragments remain in the solution.

Since digestion of the microorganisms is carried out in either the basic or acidic pH range, it is necessary to neutralize the sample solution with either an acidic or basic buffer prior to contacting it with a microorganism detection agent. For example, if the means for detecting microorganisms is an antibody that is too acidic or too basic, the binding of antibodies to antibodies may gene is greatly reduced, so that no reliable and reproducible determination is possible. It is essential that when using an acidic capping reagent, a basic neutralizing buffer be used, and vice versa.

Means for the determination of microorganisms, in particular chlamydia, gonococci and fungi, are well known in the art. Due to the field of application of the kit according to the invention and in particular due to the group of people applying the kit according to the invention or the device according to the invention, it is of advantage that such means are as simple and uncomplicated as possible and can be used without the use of expensive equipment. Such agents are well known in the art and are already widely used. The majority of these detection means is based on the binding of antibodies to surface antigens of the microorganisms to be determined.

According to a preferred embodiment of the present invention, the container b) comprises a basic buffer having a pH of 7 to 14, preferably 10 to 14, more preferably 11 to 13, in particular 12.7, or an acidic buffer with a pH of Value of 1 to 7, preferably 1 to 6.5, more preferably from 2.5 to 6.5, wherein the kit each comprises a basic and an acidic buffer.

The inventive kit comprises - as mentioned above - a container with a digestion buffer. Depending on the type of digestion selected (acidic or basic), the dried neutralization buffer should be selected (acidic or basic).

The basic buffer preferably comprises 3 to 5 g, preferably 4 g, NaOH, 3 to 4 g of ethylenediaminetetraacetic acid (EDTA), 1 to 3 g, in particular about 2 g, sodium salt of the hydrated acid in 1 l of water.

The acid buffer preferably comprises 3 to 5 g, preferably 4 to 4.5 g, KH2 PO, 5 to 9 g, preferably 7 to 8 g, Na2HPC>, 2 to 8 ml, preferably 3 to 6 ml, especially 5 ml, a mixture of 2-methyl-4-isothiazolin-3-one and 5-chloro-2-methyl-4-isothiazolin-3-one (proclin) in 1 L of water, the pH being at pH 6 to 6.5 is set.

The pH of this buffer can be increased by the addition of acid be adjusted to the appropriate pH, preferably HCl is used.

The acid buffer preferably comprises 3 to 5 g, preferably 4 to 4.5 g, KH2 PO, 5 to 9 g, preferably 7 to 8 g, Na2HPC>, in 1 L of water, the pH being at pH 1 to 3 , in particular to pH 2.5, is set.

The test strip preferably comprises a solid carrier of an absorbent medium, which medium is subdivided into a feed area, into a reaction area, into at least one detection area and into at least one control area.

Preferably in the reaction zone, there are mobile antibodies which are capable of binding to an epitope of a surface antigen of the microorganisms to be determined, the antibodies preferably being labeled with a dye, colored, fluorescent or magnetic particles, in particular latex particles, or with colloidal gold are.

According to a preferred embodiment of the present invention, antibodies are immobilized in the detection region, which are able to bind to an epitope of a surface antigen of the microorganisms to be determined, wherein the epitope of the antigen is capable, or is unable, of the in the Reaction area to bind mobile antibody arranged. According to the invention, antibodies can be immobilized in the detection region which bind to the same or another epitope of an antigen of the microorganisms to be determined. It has been found that the test strips used in the US

5,073,484 and 5,654,162 are most suitable for use in the process of the invention.

In the control region, preferably antibodies are immobilized which are capable of binding to the mobile in the reaction region arranged antibodies.

According to a preferred embodiment of the present invention, the container b) comprise 50-1000 μΐ, preferably 100-500 μΐ, in particular 300 μΐ, of the respective buffer.

To test whether the means for detecting the microorganisms are functioning properly, the kit preferably further includes a positive control.

According to a particularly preferred embodiment of the b) comprises a pH indicator, preferably bromothymol blue, preferably in an amount of 0.001 to 0.005 g, in particular in an amount of 0.003 g.

The addition of an indicator allows the user to determine if the pH of the reagent meets the requirements and can be considered neutralized.

A further aspect of the present invention relates to a method for the diagnosis of a microbial infection, in particular a chlamydia, gonococcal or fungal infection, comprising the steps:

a) providing a means for sampling, preferably at least one cotton swab or a tampon,

b) sampling,

c) transferring the sample to the sampling means into a container comprising a buffer for the digestion of chlamydia or gonococci,

d) incubating the sample with the buffer for 5 seconds to 30 minutes, preferably for 30 seconds to 15 minutes, more preferably for 1 to 10 minutes, especially for 5 minutes,

e) transferring the neutralized buffer to a device according to the present invention for detecting

Chlamydia or gonococci; and f) diagnosing a chlamydial or gonococcal infection when in step e)

Chlamydia or gonococci are detected.

The buffers and agents used in the process according to the invention have already been described above.

Another aspect of the present invention relates to the use of a kit for the determination of a chlamydial or gonococcal infection.

The object of the present invention will be explained in more detail with reference to the following examples, but without being limited thereto.

EXAMPLES:

B e i s p i e l l:

This example demonstrates the detection of Chlamydia trachomatis, one of the three known species of the family of

Chlamydia bacteria, infections with Chlamydia trachomatis are the most common sexually transmitted infections. For Germany alone, the number of new infections to 300,000 estimated at 500,000 a year. Younger adults (15-25 years), persons with frequently changing sexual partners, sexual partners (also unnoticed and asymptomatic) of infected persons as well as newborns of infected mothers are most frequently affected. Infections without sexual contact (eg in the swimming pool) are rare but can not be ruled out.

Chlamydia trachomatis infections often cause no symptoms. However, Chlamydia trachomatis can also cause discomfort such as eye inflammation, arthritis and inflammation of the urinary tract. Furthermore, untreated infections often lead to infertility and increase the risk of ectopic pregnancies and premature births. In developing countries, infections with Chlamydia trachomatis are the most common cause of neonatal blindness.

Of particular importance is the treatment of chlamydial infections during pregnancy. Around 50% of infected mothers pass the infection on to the newborn. Common neonatal complications include conjunctivitis and pneumonia.

preparations

Test components: Sample container with 300 μΐ buffer A, small bottle with 400 μΐ buffer B, swab (= SWAB) and foil packaging with test strips and desiccant

Buffer A

NaOH 4g

EDTA 3.722 g

Sodium cholhydrate 2 g

Water 1 L

pH 12.7

Buffer B

KH ₂ P0 ₄ 4.47 g

Na ₂ HP ₄ 7.87 g

Proclin 5 ml

pH adjustment with HCl to pH 6.4

The swab (= SWAB) was cautiously mother neck (end of the vaginal canal) introduced. The swab was evenly rotated for about 30 seconds and slowly pulled out.

Since there is little fluid in the buffer bottle, it had to be collected by shaking it on the bottom of the bottle.

The swab was dipped with the sample to the bottom of the bottle. The swab was rotated in the liquid and squeezed several times on the sidewall to allow its sample material to disperse well in the buffer solution.

The swab was left in the bottle for about 5 minutes.

From the small buffer bottle B 6 drops were dropped into the large buffer bottle A.

The buffer bottle A was closed and pivoted.

Two drops of the sample were applied to the sample application field of the test strip (US 5,073,484, US 5,654,162, Glad et al., Acta Chem. Scand. 6: 449-450 (1980), Glad et al., Anal. Bioch. 85: 180 (1978). , Norgard-Pederson, Clinica Chemica Acta 48: 345 (1973) and Laureil et al., Methods in Enzymology, 73, Part B, 339 et seq. (1981)).

After about 15 minutes, the test result on the test strip could be read.

FIG. 2: Comparison of the standard cassettes chlamyCARE-C (see example 1) with modified cassettes, which comprise an additional membrane soaked in buffer B from example 1.

To perform a chlamydia test as described in Example 1, it is necessary to use buffers A and B in 2 steps.

It is now being tested whether a prepared and soaked in buffer B membrane can be installed in the cassette and thus a step in the test implementation can be spared.

It is compared in this example if the results of the newly prepared cassettes are the same as those of the normal cassettes. 4 different batches are compared.

First, a membrane was prepared which was immersed in Buffer B and allowed to dry overnight in the dry room becomes. The thus-prepared membrane was attached to and fixed to the sample application area of the test strip.

The unmodified test strips were marked with letter N + number and the prepared with letter P + number.

The positive control used was Chlamydia Trachomatis Serovar LGV (4.21 mg / ml) from BIODESIGN.

For the conventional test strips were first the

Stock solutions (SL) were prepared as follows: 84.2 g of antigen were dissolved in 500 μL of distilled water

Then the controls were prepared:

Negative control: Buffer A + B for conventional cassettes and for prepared cassettes Buffer A diluted 1: 1 with distilled water.

Control 1: SL 1: 1 + buffer A + B

Control 2: SL 1: 2 + buffer A + B

Control 3: SL 1: 4 + buffer A + B

Control 4: SL 1: 8 + buffer A + B

Control 5: SL 1:16 + buffer A + B

For the prepared cassettes, the stock solution (SL) was prepared as above and then the positive controls were used according to the scheme as above, but instead of buffer A + B, only buffer A (diluted 1: 1 with distilled water).

Results :

Sample lot. : 08619 Lot. : 01720 Lot. : 03722 Lot. : 06723 herkomm prepahergere prepahergere prepahergere prepaküppiert kömmiert kömmiert kömmiertlich

Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg.

control

Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. control

1 Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos.

control

2

Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. control

3

Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos.

control

4

Pos. Weakly Weakly Weakly Weakly Weakly Weakly Weakly Weakly KonPos Pos Pos Pos Pos Pos Pos Pos

5

Results:

All cassettes showed the same results. For the prepared cassettes, the lines were a little bit stronger.

Claims

Claims :

A device for the determination of chlamydiae or gonococci, in a sample comprising a liquid-permeable solid medium defining a path for a liquid flow, the medium having areas spaced apart along the liquid path, said regions being arranged in the following order into a task area Reaction area, a detection area and a control area are divided, characterized in that the task area a neutralization buffer is provided in the dried state, wherein the buffer is a basic buffer having a pH of 11 to 13, in particular 12.7, or an acidic Buffer with a pH of 2.5 to 6.5.

2. Device according to claim 1, characterized in that in the reaction region are arranged mobile antibodies which are capable of binding to an epitope of a surface antigen of the microorganisms to be determined, the antibodies preferably with a dye, colored, fluorescent or magnetic particles, especially latex particles, or are marked with colloidal gold.

3. A device according to claim 1 or 2, characterized in that antibodies are immobilized in the detection area, which are capable of binding to an epitope of a surface antigen of the microorganisms to be determined, wherein the epitope of the antigen is capable or not in a position is to bind to the mobile in the reaction area arranged antibody.

4. Device according to one of claims 1 to 3, characterized in that antibodies are immobilized in the control region, which are capable of binding to the mobile in the reaction region arranged antibodies.

5. Device according to one of claims 1 to 4, characterized in that the basic buffer 3 to 5 g, preferably

4 g, NaOH, 3 to 4 g of ethylenediaminetetraacetic acid (EDTA), 1 to 3 g, in particular about 2 g, of the sodium salt of cholic acid rats in 1 L of water.

6. Device according to one of claims 1 to 5, characterized in that the acidic buffer 3 to 5 g, preferably 4 to 4.5 g, KH2PO4, 5 to 9 g, preferably 7 to 8 g, Na2HPC>, 2 to 8 ml, preferably 3 to 6 ml, especially 5 ml, of a mixture of 2-methyl-4-isothiazolin-3-one and 5-chloro-2-methyl-4-isothiazolin-3-one (Proclin) in 1 L of water , wherein the pH is adjusted to pH 6 to 6.5.

7. Device according to one of claims 1 to 5, characterized in that the acidic reagent 3 to 5 g, preferably 4 to 4.5 g, KH2PO4, 5 to 9 g, preferably 7 to 8 g, Na2HPC> 4, in 1 L comprises water, wherein the pH value is adjusted to pH 1 to 3, in particular to pH 2.5

8. Kit for the determination of chlamydiae or gonococci, in a sample comprising:

a) at least one sampling means, preferably at least one cotton swab or tampon,

b) a container comprising a buffer for digesting chlamydiae or gonococci and suitable for receiving the sampling means, preferably cotton swabs or tampons,

c) a device according to one of claims 1 to 7.

9. Kit according to claim 8, characterized in that the container b) a basic buffer having a pH of 10 to 14, preferably 11 to 13, in particular 12.7, or an acidic buffer having a pH of 1 to 6.5, preferably from 2.5 to 6.4, the kit each comprising a basic or an acidic buffer for digesting chlamydiae or gonococci.

10. Kit according to claim 8 or 9, characterized in that the basic buffer 3 to 5 g, preferably 4 g, NaOH, 3 to 4 g ethylenediaminetetraacetic acid (EDTA), 1 to 3 g, in particular about 2 g, the sodium Salt of cholic acid hydrate in 1 L of water.

11. Kit according to claim 8 or 9, characterized in that the acidic buffer 3 to 5 g, preferably 4 to 4.5 g, KH2PO4, 5 to 9 g, preferably 7 to 8 g, Na2HPC> 4, 2 to 8 ml , preferably 3 to 6 ml, especially 5 ml, of a mixture of 2-methyl-4-isothiazolin-3-one and 5-chloro-2-methyl-4-isothiazolin-3-one (proclin) in 1 L of water, wherein the pH is adjusted to pH 6 to 6.5.

12. Kit according to claim 8 or 9, characterized in that the acidic buffer 3 to 5 g, preferably 4 to 4.5 g, KH2PO4, 5 to 9 g, preferably 7 to 8 g, Na2HPC> 4, in 1 L of water wherein the pH is adjusted to pH 1 to 3, in particular to pH 2.5

13. Kit according to any one of claims 8 to 12, characterized in that the container b) 50-1000 μΐ, preferably 100-500 μΐ, in particular 300 μΐ, of the buffer comprises.

14. Kit according to any one of claims 8 to 13, characterized in that the kit further comprises a positive control.

15. Kit according to any one of claims 8 to 14, characterized in that buffer b) comprises a pH indicator, preferably Bromthy- molblau, preferably in an amount of 0.001 to 0.005 g, in particular in an amount of 0.003 g.

16. A method of diagnosing chlamydial or gonococcal infection, comprising the steps of:

b) sampling,

e) transferring the neutralized buffer to a device A device according to any one of claims 1 to 7 for detecting chlamydiae or gonococci, and

(f) diagnosing a chlamydial or gonococcal infection if chlamydiae or gonococci are detected in step e).

17. Use of a kit according to any one of claims 8 to 15 for determining a Chlamydia or gonococcal infection.