[DESCRIPTION] [Invention Title]

SIMULTANEOUS DIAGNOSTIC KIT FOR TRICHOMONAS VAGINALIS AND CHLAMYDIA TRACHOMATIS [Technical Field]

The present invention relates to a simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia trachomatis. More specifically, the present invention relates to a diagnostic kit which can diagnose Trichomonas vaginalis and Chlamydia trachomatis simultaneously with improved specificity and sensitivity at reduced time and cost. [Background Art]

The present invention relates to a simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia trachomatis.

Trichomoniasis is caused by Trichomonas vaginalis infection.

Trichomonas vaginalis is a sexually transmitted protozoan parasite which was discovered by Donne in 1837 and causes trichomoniasis. It is reported that about 170 million persons are newly infected with Trichomonas vaginalis each year.

Trichomoniasis is a sexually transmitted disease (STD) by Trichomonas vaginalis infection which is usually inapparent. For women, symptoms of trichomoniasis include vaginal burning sensation, itching sensation, inflammation and edema in vaginal mucosa, etc. Trichomoniasis can cause premature rupture of fetal membranes, ectopic pregnancy, sterility, low birth weight and cervical cancer (Bowden and Garnett, 1999; Grodstein, et al., 1993; Hardy, et al . , 1984; Minkoff, et al., 1984; MuIler, 1988; Zhang and Begg, 1994). For men, symptoms of trichomoniasis are usually not apparent, however, the male is the most important transmitter of the infection.

Trichomonas vaginalis can be diagnosed via direct microscopy on vaginal fluid specimen, polymerase chain reaction (PCR), immunological approaches, etc.

Chlamydias!s is caused by Chlamydia trachomatis infection.

Chlamydia trachomatis can be diagnosed via various methods such as culture and fluorometry, however, there remain problems such as high cost, long diagnosis time and low accuracy. As specific antigen for diagnosis of Chlamydia trachomatis was developed recently, immunodiagnostic kits such as ELISA kit and rapid kit have been commercially available.

The infection of Trichomonas vaginalis or Chlamydia trachomatis may be inapparent, so people can carry and transmit Trichomonas vaginalis or Chlamydia trachomatis to their sexual partners without knowing it.

Nowadays, trichomoniasis is diagnosed via various methods such as vaginal smear, anaerobic culture and PCR.

The most effective method for diagnosis of trichomoniasis is PCR, while it requires expensive tools and complex procedures. Thus most widely used methods are vaginal smear and anaerobic culture, however, they have low accuracy and are time-consuming. And these methods even require experienced clinical expert for diagnosis.

Therefore it is required to develop a rapid kit method for clinical use which is fast, easy-to-use and sensitive. There are a variety of rapid kits for Chlamydia trachomatis diagnosis, however, there is only one kit for diagnosis of Trichomonas vaginalis. Furthermore, the rapid kit for Trichomonas vaginalis diagnosis, which was developed by an U.S. company, is hard to purchase in South Korea. [Disclosure] [Technical Problem]

Accordingly, the present invention has been made to solve the above- mentioned problems occurring in the prior art, and an object of the present invention is to provide a simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia trachomatis, which can diagnose Trichomonas vaginalis and Chlamydia trachomatis simultaneously using vaginal fluid.

And, it is another object of the present invention to provide a simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia trachomatis, by which diagnosing time can be significantly reduced and

diagnosis can be performed with higher accuracy in a shorter time.

And, it is another object of the present invention to provide a simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia trachomatis, by which Trichomonas vaginalis and Chlamydia trachomatis can be diagnosed with high sensitivity and specificity.

And, it is another object of the present invention to provide a simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia trachomatis, which can be used as a diagnostic tool for Trichomonas vaginalis and Chlamydia trachomatis diagnosis of obstetrics and gynecology patients, and which can be used as one of diagnostic tools for mass screening in medical institutions such as public health centers and general hospitals. [Technical Solution)

In order to accomplish above objects, the present invention provides a simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia trachomatis wherein"- a sample pad which is soaked in buffer solution for calibrating chemical state of a specimen and then dried; a conjugate pad which is soaked in colloidal gold conjugate and then dried; a nitrocellulose membrane pad comprising a first detection zone wherein anti-Chlamydia trachomatis antibodies are fixed, a second detection zone wherein anti- Trichomonas vaginalis antibodies are fixed and a third detection zone wherein anti-mouse immunoglobulin G antibodies are fixed; and an absorbent pad are sequentially attached onto a plastic card.

And, the Trichomonas vaginalis antibodies are characterized by being produced by steps comprising: inoculating Trichomonas vaginalis protozoa into a capped glass tube culture containing 5ml of TYM broth to final cellular concentration of 2X10 cell/ml and culturing them at 37°C in an atmosphere containing 5% of CO2 for 24 hours in CO2 incubator; centrifuging the cultured trichomonads at 1500rpm for 15 minutes and washing them twice with sterilized phosphate buffer saline (PBS) of pH 7.2 after the disposal of the supernatant liquid; resuspending the sediments from the previous step in ImI of RPMI 1640

7 serum-free media, inoculating 1X10 of the trichomonads into an Eppendorf tube culture containing ImI of RPMI 1640, then culturing them at 37°C in an atmosphere containing 5% of CO ₂ in CO2 incubator, and then extracting excretory-secretory products (ESP); mixing 500 _/ ^ of Trichomonas vaginalis ESP (TvESP) with concentration of lmg/m£ with the same volume of complete Freund's adjuvant (CFA) and injecting the mixture into the skin of a female rabbit, and after two weeks, mixing 5Q0μ£ of TvESP with concentration of lrag/ mi with the same volume of incomplete Freund's adjuvant (IFA) and injecting the mixture into the skin of the rabbit, and separating the serum from the blood which is extracted from the ear blood vessel of the rabbit before each injection, and after another two weeks, mixing 5OO//45 of TvESP with concentration of lmg/mϋ. with the same volume of IFA and injecting the mixture into the abdominal cavity of the rabbit, thus immunizing the rabbit; and dialyzing the immune rabbit serum against Ii of loading buffer three times for three hours and centrifuging at 12,00Og for 20 minutes, and then collecting the supernatant liquid and filtering it through 0.45jωn filter, thus producing anti-Trichomonas vaginalis antibodies.

And, the colloidal gold conjugate is characterized by the colloidal gold which is conjugated with anti-Trichomonas vaginalis antibody and anti- Chlamydia trachomatis antibody.

And, the colloidal gold conjugate is characterized by the colloidal gold particles of which average diameter is 20 to 40nm.

And, the nitrocellulose membrane pad is characterized by comprising the first detection zone, the second detection zone and the third detection zone, wherein respectively anti-Chlamydia trachomatis antibody, anti-Trichomonas vaginalis antibody and anti-mouse immunoglobulin G antibody are detection antibodies.

And, the anti-Chlamydia trachomatis antibody of the first detection zone is characterized by reacting with a specimen and antibody part of the anti-Chlamydia trachomatis antibody-colloidal gold conjugate to develop

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color, and the anti-Trichomonas vaginalis antibody of the second detection zone is characterized by reacting with a specimen and antibody part of the anti-Trichomonas vaginalis antibody-colloidal gold conjugate to develop color, and the anti-mouse immunoglobulin G antibody of the third detection zone is characterized by directly reacting with antibody part of the anti- Chlamydia trachomatis antibody-colloidal gold conjugate to develop color.

And, the antibodies mentioned above are characterized as monoclonal or polyclonal. [Advantageous Effectsl

Accordingly, the present invention provides a simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia trachomatis, which can diagnose Trichomonas vaginalis and Chlamydia trachomatis simultaneously using vaginal fluid.

And, the present invention provides a simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia trachomatis, by which diagnosing time can be significantly reduced and diagnosis can be performed with higher accuracy in a shorter time.

And, the present invention provides a simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia trachomatis, by which Trichomonas vaginalis and Chlamydia trachomatis can be diagnosed with high sensitivity and specificity.

And, the present invention provides a simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia trachomatis, which can be used as a diagnostic tool for Trichomonas vaginalis and Chlamydia trachomatis diagnosis of obstetrics and gynecology patients, and which can be used as one of diagnostic tools for mass screening in medical institutions such as public health centers and general hospitals. [Description of Drawings]

The features of the invention will be better understood by reference to the accompanying drawings which illustrate presently preferred embodiments of the invention. In the drawings:

FIG. 1 is a perspective view of a simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia trachomatis according to an embodiment of the present invention!

FIG.2 is a cross-sectional view of the simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia trachomatis according to an embodiment of the present invention)

FIG. 3 is a reference drawing which is showing the detection principle employed in each detection zone of the simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia trachomatis according to an embodiment of the present invention;

FIG. 4 is a reference drawing which is showing a sensitivity test result of the simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia trachomatis in EXAMPLE 2 according to an embodiment of the present invention; and,

FIG. 5 is the protein band patterns of extracted ESP according to an embodiment of the present invention. description of the elements in the drawings>

100^ simultaneous diagnostic kit according to the present invention

110: sample pad

120: conjugate padl30-' nitrocellulose membrane pad

132: first detection zonel34: second detection zone

136'- third detection zonel40: absorbent pad [Best Model

Detailed description is made hereinafter for a preferred embodiment of a simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia trachomatis according to the present invention with reference to the attached drawings.

FIG. 1 is a perspective view of the simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia trachomatis according to an embodiment of the present invention, and FIG. 2 is a cross-sectional view of the simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia

trachomatis according to an embodiment of the present invention.

Referring to FIG. 1 and FIG. 2, the simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia trachomatis (100) according to the present invention comprises a sample pad (110), a conjugate pad (120), a nitrocellulose membrane pad (130) and an absorbent pad (140). And, the nitrocellulose membrane pad (130) comprises a first detection zone (132), a second detection zone (134) and a third detection zone (136).

It is preferred that the conjugate pad (120) is soaked in colloidal gold conjugate which comprises colloidal gold particles conjugated with anti- Trichomonas vaginalis antibodies and anti-Chlamydia trachomatis antibodies and then dried.

It is preferred that the first detection zone (132) is wherein anti- Chlamydia trachomatis antibodies are fixed.

It is preferred that the second detection zone (134) is wherein anti- Trichomonas vaginalis antibodies are fixed.

It is preferred that the third detection zone (136) is wherein anti- mouse immunoglobulin G antibodies are fixed.

And, the anti-Trichomonas vaginalis antibodies are produced from antibodies of animals immunized with ESP. The animals that can be used for antibody production include rabbits, sheep and goats.

FIG. 3 is a reference drawing which is showing the detection principle employed in each detection zone of the simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia trachomatis according to an embodiment of the present invention.

Referring to FIG. 3, the detection principle of each detection zone is explained.

Referring to FIG. 3, conjugate of anti-Chlamydia trachomatis antibody and colloidal gold is namely anti-Chlamydia trachomatis antibody-colloidal gold conjugate. Conjugate of anti-Trichomonas vaginalis antibody and colloidal gold is namely anti-Trichomonas vaginalis antibody-colloidal gold conjugate.

FIG. 4 is a reference drawing which is showing a sensitivity test result of the simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia trachomatis in EXAMPLE 2 according to an embodiment of the present invention.

Referring to FIG. 4, Tl, T2 and C means respectively the first detection zone (132), the second detection zone (134) and the third detection zone (136).

Referring to FIG. 3 and FIG. 4, in line Tl, the first detection zone (132), fixed anti-Chlamydia trachomatis antibody develops a color when reacting with a specimen and antibody part of the anti-Chlamydia trachomatis antibody-colloidal gold conjugate.

In line T2, the second detection zone (134), fixed anti-Trichomonas vaginalis antibody develops a color when reacting with a specimen and antibody part of the anti-Trichomonas vaginalis antibody-colloidal gold conjugate.

In line C, the third detection zone (136), fixed anti-mouse immunoglobulin G antibody develops a color when directly reacting with antibody part of the anti-Chlamydia trachomatis antibody-colloidal gold conjugate, without a specimen.

The simu11aneous diagnostic kit for Trichomonas vagina] is and Chlamydia trachomatis according to the present invention can diagnose Trichomonas vaginalis and Chlamydia trachomatis simultaneously using vaginal fluid from patients, and thus can be widely used for early detection of trichomoniasis and chlamydiasis.

A detailed description is given for a simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia trachomatis according to the present invention. It should be noted that the present invention is not limited to the embodiments described below.

[STEP 1] Culture of Trichomonas vaginalis

In order to culture Trichomonas vaginalis, firstly Trichomonas vaginalis protozoa were inoculated into a capped 15ml glass tube culture containing 5ml of TYM broth (TABLE 1) to final cellular concentration of 2X

10 cell/ml, and cultured at 37 ^° C in an atmosphere containing 5% of CO ₂ for 24 hours in CO ₂ incubator, and checked by microscope. Then, Trichomonas vaginalis protozoa were inoculated into a capped 50ml glass tube culture containing

20ml of TYM broth to final cellular concentration of 2X10 cell/ml, and cultured under the same condition for 24 hours in CO ₂ incubator. Finally, for mass culturing of Trichomonas vaginalis, Trichomonas vaginalis protozoa were inoculated into an IL Erlenmeyer flask culture containing 400ml of TYM broth to the same final cellular concentration, and cultured under the same condition for 24 hours in CO2 incubator. The number of trichomonads was examined by microscopy at 100 times magnification after mixed 1 ^; 1 with trypan blue. [Table 1]

Ingredients of TYM broth

[STEP 2] Extraction of ESP

The trichomonads cultured for 24 hours in the above STEP 1 were centrifuged at l,500rpm for 15 minutes, and then washed twice with sterilized

phosphate buffer saline (PBS) of pH 7.2 after disposal of the supernatant liquid. And the sediments were resuspended in ImI of RPMI 1640 serum-free

7 media. Then, 1X10 of the trichomonads and ImI of RPMI 1640 were added into an Eppendorf tube and cultured at 37°C in an atmosphere containing 5% of CO2 in CO2 incubator, for respectively 60 minutes, 90 minutes, 120 minutes, 150 minutes and 180 minutes.

To asses the amount of ESP secretion by Trichomonads concentration, by the same method mentioned above, trichomonads were added into an Eppendorf

6 tube and mixed with RPMI 1640 in the ratio of, respectively, 1X10 cells, 5X

10 cells, 1X10 cells, 5X10 cells and 1X10 cells to 1 ml , and cultured for 120 minutes.

And, after the cultured trichomonads were centrifuged at 10,000 rpm for 30 minutes, the supernatant liquid was collected and moved into another tube, mixed with lOOul solution made of 0.15% deoxycholic acid in ethanol , and then vortexed and set aside for 10 minutes at room temperature. And after 50 μl of 100% tricholoroacetic acid was added, the tube was centrifuged at 10,000 rpm for 5 minutes and the supernatant liquid from the tube was disposed of, the sediment from the tube was dissolved by adding 40 μl of 0.1N NaOH. To examine it by SDS-PAGE electrophoresis method, it was mixed thoroughly with 1Ou 1 of 5x loading buffer and boiled at about 100°C for 5 minutes, and then SDS-PAGE was conducted in 12% polyacrylamide gel. STEP 2 is a preliminary experiment for determining appropriate conditions for experiment of STEP 3.

[STEP 3] Purifying ESP by centrifugalion

The trichomonads cultured in an IL Erlenmeyer flask with 400ml of TYM broth for 24 hours were centrifuged at 1,500 rpm for 15 minutes and washed twice with PBS after the disposal of the supernatant liquid, and then

7 resuspended in RPMI 1640 serum-free media. Then, 2X10 of the trichomonads

and ImI of RPMI 1640 were added into an 1.5L Eppendorf tube and cultured at 37 ^° C in an atmosphere containing 5% of CO2 in CO2 incubator for 2 hours. And after the cultured trichomonads in the Eppendorf tube were centrifuged at 10,000 rpm for 30 minutes, the supernatant liquid was collected and moved into a Centriprep 1OkDa device, mixed with Complete Protease Inhibitor Cocktail (Roche, Germany), and then centrifuged at 3,00Og for about an hour at 4 ^° C. And after the liquid transferred to a supporting membrane was discarded, the rest was centrifuged twice at 3,00Og for about 30 minutes each at 4 ⁰ C. Finally, concentration of remaining proteins was measured by Bradford assay. [Table 2]

The amount of ESP proteins produced by Trichomonas vaginalis cultured in 400ml of TYM media for 24 hours

[STEP 4] Immunizing a rabbit with ESP

500μϋ of Trichomonas vaginalis ESP (TvESP) with concentration of lmg/mi was mixed with the same volume of complete Freund's adjuvant (CFA) and injected into the skin of a female rabbit. After two weeks, 500βt of TvESP with concentration of lmg/mβ was mixed with the same volume of incomplete Freund's adjuvant (IFA) and injected into the skin of the rabbit. And after another two weeks, 50OjLtC of TvESP with concentration of lmg/m£ was mixed with the same volume of IFA and injected into the abdominal cavity of the rabbit. Before each injection, the serum was separated from the blood which is extracted from the ear blood vessel of the rabbit.

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[STEP 5] Purifying anti-ESP antibody

ESP-Immune rabbit serum was dialyzed against Il of loading buffer three times for three hours and centrifuged at 12,00Og for 20 minutes. Then the supernatant liquid was collected and filtered through 0.45μm filter. And the dialyzed serum was loaded onto a column packed with Affi-Gel resin which was conjugated with ESP, then flown through the column by gravity. After washing resin with HM of loading buffer, the resin was washed again with 10 ml of wash buffer (pH 7.5, 1OmM Tris). After adding elution buffer (pH 2.5, 100 mM glycine), ImI each of the extract from the column was transferred into each fraction tube. Then, 1 M Tris was added to neutralize the elution buffer to reach final pH of 7.5. Anti ^~ rTvAP33 antibody elution fraction was collected by Bradford assay, dialyzed against Tris (pH 7.5, 1OmM), and finally concentrated by a CentriPreρ-10 device (Amicon, USA).

[STEP 6] Producing a kit using anti-ESP antibody

An absorbent pad, a nitrocellulose membrane pad, a conjugate pad and a sample pad were attached onto a plastic card sequentially. Each pad is characterized by the following. A sample pad was soaked in buffer solution for calibrating chemical state of a specimen and then dried. A conjugate pad was soaked in colloidal gold conjugate comprising 30 nm colloidal gold particles conjugated with anti-Trichomonas vaginalis antibodies developed as above, and soaked in colloidal gold conjugate comprising 30 nm colloidal gold particles conjugated with commercially purchased anti-Chlamydia trachomatis antibodies, and then dried. A nitrocellulose membrane pad include the third detection zone (136) wherein anti -mouse immunoglobulin G antibody was fixed, the first detection zone (132) wherein commercially purchased anti-Chlamydia trachomatis antibody was fixed and the second detection zone (134) wherein anti-Trichomonas vaginalis antibody developed as above was fixed. An absorbent pad underwent no modification or treatment. A kit assembled as above was completed by being inserted in a plastic housing having a sample port for loading a specimen.

[EXAMPLE 1]

In order to examine specificity of the kit produced by the above STEP 6, a specificity test was conducted by dropping a negative specimen (vaginal fluid) 100/d onto the sample port of the kit and then by setting the kit aside for 20 minutes. 361 randomly selected specimens were used in the test. From the result of the test, only 14 specimens were false negative, showing 96.4% of sensitivity. [Table 3]

[EXAMPLE 2]

In order to examine sensitivity of the kit produced by the above STEP 6, a sensitivity test was conducted. [Table 41

FIG. 4 is a reference drawing which is showing a sensitivity test result of the simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia trachomatis in EXAMPLE 2 produced according to an embodiment of the present invention.

Referring to FIG. 4, each detection zone comprises a test line. If a positive reaction occurs in a detection zone, the test line in the detection zone turns red. If the reaction is negative, the test line remains unchanged. If the test line in the third detection zone turns red, the result can be

determined as positive or negative. In FIG. 4, Tl, T2 and C means respectively the first detection zone, the second detection zone and the third detection zone of the simultaneous diagnostic kit for Trichomonas vaginalis and Chlamydia trachomatis.

More specifically, in A-I the third detection zone displayed a red line, but the second detection zone and the first detection zone remained unchanged, showing negative result. In A-2 the third detection zone and the first detection zone turned red, thus anti-Chlamydia trachomatis antibody in the first detection zone was positive and anti-Trichomonas vaginalis antibody in the second detection zone was negative. In A-3 red lines appearing in the third detection zone and the second detection zone means that anti- Trichomonas vaginalis antibody was positive and anti-Chlamydia trachomatis antibody was negative. In A-4 red lines appeared in every detection zone, thus both anti-Chlamydia trachomatis antibody and anti-Trichomonas vaginalis antibody were positive.

And from B-I to B-4, the third detection zone remained unchanged, meaning that the result cannot be determined.

The result of the test is shown in table 4. For anti-Trichomonas vaginalis antibody, 21 out of 27 positive specimens were diagnosed as positive, showing 78% of sensitivity, and for anti-Chlamydia trachomatis antibody, 20 out of 30 positive specimens were diagnosed as positive, showing 66% of sensitivity. For negative specimens, 96% of specificity was shown.

And, judging from the result of the cross-reaction test on the present invention, there was no cross-reaction between anti-Trichomonas vaginalis antibody and anti-Chlamydia trachomatis antibody.

And, judging from the result of the cross-reaction test between various bacteria commonly found in human vagina, namely Candida albicans, Streptococcus pneumonia, Streptococcus viridans, Shigella dysenteria, Shigella sonnei, Salmonella typhi, E.coli and Staphylococcus aureus using strains of the bacteria, all strains except the Staphylococcus aureus showed o no cross-reaction even at more than 1X10 organisms/ml. The Staphylococcus

8 8 aureus showed cross-reaction at IX10 organisms/ml. However, IX10 organisms/ml of Staphylococcus aureus concentration is significantly more than the concentration in a specimen from normal patient, thus it was confirmed that there is no practical problem for trichomoniasis diagnosis.

Although the preferred embodiment of the present invention has been disclosed for illustrative purposes, the present invention is not limited to the preferred embodiment. Furthermore, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.