WO2008143406A1

WO2008143406A1 - Trichomonas vaginalis diagnostic kit

Info

Publication number: WO2008143406A1
Application number: PCT/KR2008/002292
Authority: WO
Inventors: Kyu Jae Lee
Original assignee: Hdr Co., Ltd.
Priority date: 2007-05-22
Filing date: 2008-04-23
Publication date: 2008-11-27
Also published as: KR100818779B1

Abstract

The present invention relates to a Trichomonas vaginalis diagnostic kit, more specifically, a Trichomonas vaginalis diagnostic kit which is produced by using ant i -Tr i chomonas vaginalis antibodies produced from animals immunized with Trichomonas vaginalis excretory-secretory products, and which detects Trichomonas vaginalis with high sensitivity and specificity, and which increases diagnosis rate by using Trichomonas vaginalis excretory- secretory products in detecting Trichomonas vaginalis.

Description

[DESCRIPTION] [Invention Tit Ie]

TRICHOMONAS VAGINALIS DIAGNOSTIC KIT [Technical Field]

<i> The present invention relates to a Trichomonas vaginalis diagnostic kit, more specifically, a Trichomonas vaginalis diagnostic kit which can detect Trichomonas vaginalis by using ant i-Trichomonas vaginalis antibodies produced from animals immunized with Trichomonas vaginalis excretory- secretory products, and 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 including a first detection zone in which the ant i-Trichomonas vaginalis antibodies are fixed and a second detection zone in which anti-mouse immunoglobulin G antibodies are fixed, and an absorbent pad are sequentially attached onto a plastic card. [Background Art]

<2> The present invention relates to a Trichomonas vaginalis diagnostic kit.

<3> Trichomoniasis is caused by Trichomonas vaginalis infection.

<4> 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.

<5> 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; Muller, 1988; Zhang and Begg, 1994). For men, symptoms of tricnomoniasis are usually not apparent, however, the male is the most important transmitter of the infection. <6> Trichomoniasis can be diagnosed via direct visualization through microscopy, polymerase chain reaction (PCR), immunological approaches, etc. <7> The infection of Trichomonas vaginalis may be inapparent, so people can carry and transmit Trichomonas vaginalis to their sexual partners without knowing it.

<8> Currently, the most widely used methods for diagnosis of trichomoniasis are vaginal smear and anaerobic culture, however, they show low accuracy and are time-consuming. And these methods even require experienced clinical expert for diagnosis. The most effective method for diagnosis of trichomoniasis is PCR, while it requires complex procedures taking more than 5 hours to produce diagnosis results and expensive tools and reagents. <9> Therefore, it is required to develop a rapid kit method for fast and easy clinical diagnosis of trichomoniasis with high sensitivity and specificity. [Disclosure] [Technical Problem]

<io> 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 Trichomonas vaginalis diagnostic kit which can detect Trichomonas vaginalis by using anti-Trichomonas vaginalis antibodies produced from animals immunized with Trichomonas vaginalis excretory- secretory products, and which can detect Trichomonas vaginalis with higher accuracy within a significantly shorter time.

<ii> And, it is another object of the present invention to provide a Trichomonas vaginalis diagnostic kit which can detect Trichomonas vaginalis with high sensitivity and specificity.

<12> And, it is another object of the present invention to provide a Trichomonas vaginalis diagnostic kit which can be used for diagnosis of trichomoniasis in 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]

<i3> In order to accomplish above objects, the present invention provides a Trichomonas vaginalis diagnostic kit which is produced by using anti- Trichomonas vaginalis antibodies produced from animals immunized with Trichomonas vaginalis excretory-secretory products.

<i4> And. the ant i-Trichomonas vaginalis antibodies are characterized as polyclonal.

<i5> And, the ant i-Trichomonas vaginalis antibodies are produced by steps comprising-' removing excessive cervical mucus by using one of the two swabs contained in plastic transport tube, and inserting the other swab into endocervical canal and rotating it for 15-30 seconds to obtain cervical epithelial cells infected with Trichomonas vaginalis; adding 300μβ of the first extraction solution into an extraction tube, and inserting the swab specimen into the extraction tube and rotating it clockwise 10 times for 5 minutes for extraction; and adding 300_/z£ of the second extraction solution immediately into the extraction tube, rotating the swab clockwise 10 times and discarding it, and dropping 100_/iH of the extracted solution onto sample port of a kit by using a dropper and waiting for 20 minutes.

<16> And, the first extraction solution, which is used for obtaining and preserving the ant i-Trichomonas vaginalis antibodies, is characterized by comprising NaOH, NaCl, CHAPS and distilled water.

<i7> And the second extraction solution, which is used for obtaining and preserving the anti-Trichomonas vaginalis antibodies, is characterized by comprising Tricine, NaCl, NaN3, CHAPS, ethylenediamine tetra-acetic acid (EDTA), Tris, casein, Tween 20 and distilled water.

<i8> And, the Trichomonas vaginalis excretory-secretory products are characterized by that maximum amount of Trichomonas vaginalis excretory-

7 secretory products can be produced when 5X10 trichomonads are cultured at 37 °C in an atmosphere containing 5% of CO₂ in CO₂ incubator for 2 hours. <i9> And, 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 including a first detection zone in which the ant i-Trichomonas vaginalis antibodies are fixed and a second detection zone in which anti-mouse immunoglobulin G antibodies are fixed, and an absorbent pad are sequentially attached onto a plastic card.

<20> And, the ant i-Trichomonas vaginalis antibodies and anti-mouse immunoglobulin G antibodies are characterized as detection antibodies.

<21>

[Advantageous Effects]

<22> Accordingly, the present invention provides a Trichomonas vaginalis diagnostic kit which can detect Trichomonas vaginalis by using anti- Trichomonas vaginalis antibodies produced from animals immunized with Trichomonas vaginalis excretory-secretory products, and which can detect Trichomonas vaginalis with higher accuracy within a significantly shorter time.

<23> And, the present invention provides a Trichomonas vaginalis diagnostic kit which can detect Trichomonas vaginalis with high sensitivity and specificity.

<24> And, the present invention provides a Trichomonas vaginalis diagnostic kit which can be used for diagnosis of trichomoniasis in 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]

<25> 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 ^:

<26> FIG. 1 is a perspective view of a Trichomonas vaginalis diagnostic kit according to an embodiment of the present invention; <27> FIG. 2 is a cross-sectional view of the Trichomonas vaginalis diagnostic kit according to an embodiment of the present invention; <28> FIG. 3 is a reference drawing which is showing the detection principle employed in each detection zone of the Trichomonas vaginalis diagnostic kit according to an embodiment of the present invention; <29> FIG. 4 is a reference drawing which is showing electrophoresis result of Trichomonas vaginalis according to an embodiment of the present invention; and, <30> FIG. 5 is the protein band patterns of purified Trichomonas vaginalis excretory-secretory products according to an embodiment of the present invention.

<3i> description of the elements in the drawings> <32> 100: Trichomonas vaginalis diagnostic kit according to an embodiment of the present invention

<33> 110: sample padl20: conjugate pad

<34> 130: nitrocellulose membrane padl32: first detection zone <35> 134: second detection zonel40: absorbent pad

[Best Mode] <36> Detailed description is made hereinafter for a preferred embodiment of a Trichomonas vaginalis diagnostic kit according to the present invention with reference to the attached drawings. <37> FIG.l is a perspective view of the Trichomonas vaginalis diagnostic kit according to an embodiment of the present invention, and FIG. 2 is a cross- sectional view of the Trichomonas vaginalis diagnostic kit according to an embodiment of the present invention. <38> Referring to FIG. 1 and FIG. 2, the Trichomonas vaginalis diagnostic kit (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). <39> The Trichomonas vaginalis diagnostic kit (10C) is produced by using ant i-Trichomonas vaginalis antibodies produced from animals immunized with O

Trichomonas vaginalis excretory-secretory products. The anti-Trichomonas vaginalis antibodies are produced from animals immunized with excretory- secretory products of trichomonads .

<40> It is preferred that the conjugate pad (120) is soaked in colloidal gold conjugate, which comprises colloidal gold particles conjugated with the ant i-Trichomonas vaginalis antibodies and then dried.

<4i> The nitrocellulose membrane pad (130) includes a first detection zone (132) and a second detection zone (134).

<42> It is preferred that the first detection zone (132) is wherein the anti-Trichomonas vaginalis antibodies are fixed.

<43> It is preferred that the second detection zone (134) is wherein anti- mouse immunoglobulin G antiDodies are fixed.

<44> FIG. 3 is a reference drawing which is showing the detection principle employed in each detection zone of the Trichomonas vaginalis diagnostic kit according to an embodiment of the present invention.

<45> Referring to FIG. 3, the detection principle of each detection zone is explained

<46> Tl means the first detection zone (132) and C means the second detection zone (134). Conjugate of anti-Trichomonas vaginalis antibody and colloidal gold is namely ant i-Trichomonas vaginalis antibody-colloidal gold conjugate.

<47> In line Tl, which is the first detection zone (132), fixed anti- Trichomonas vaginalis antibodies develop a color when reacting with a specimen and antibody part of the ant i-Trichomonas vaginalis antibody- colloidal gold conjugate.

<48> In line C, which is the second detection zone (134), fixed anti-mouse immunoglobulin G antibodies develop a color when directly reacting with antibody part of the ant i-Trichomonas vaginalis antibody-colloidal gold conjugate, without a specimen.

<49> A detailed description is given for a Trichomonas vaginalis diagnostic kit according to the present invention. It snould be noted that the present invention is not limited to the embodiments described below.

<50> <51> [STEP 1] Culturing of Trichomonas vaginalis

<52> 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 xiθ^" cell/ml, and cultured at 37^°C in an atmosphere containing 5% of CO₂ for

24 hours in CO₂ incubator. And, Trichomonas vaginalis protozoa were inoculated into a capped 50ml glass tube culture containing 20ml of TYM broth to final cellular concentration of 2XX10 cell/ml, and cultured under the same condition for 24 hours in CO₂ incubator. And, 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 diluted in a ratio of 1:1 with trypan blue.

<53> [Table 1]

Recipe for TYM broth

<54> <55> [STEP 2] Extracting Trichomonas vaginalis excretory-secretory products <56> 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, lxxiO of the trichomonads and ImI of RPMI 1640 were added into 1.5ml tubes and cultured at 37^°C in an atmosphere containing 5% of CO₂ in

COgincubator , for respectively 60 minutes. 90 minutes, 120 minutes, 150 minutes and 180 minutes.

<57> To asses the amount of excretory-secretory products secretion by Trichomonads concentration, by the same method mentioned above, trichomonads were added into tubes and mixed with RPMI 1640 in the ratio of, respectively,

IXlO⁶ cells, 5xiθ' cells, IXlO⁷ cells, 5X1O^? cells and lxiΛells to 1 ml, and cultured for 120 minutes.

<58> And, after the cultured trichomonads were centrifuged at 10,000 rpm for 30 minutes, each supernatant liquid was collected and moved into another tubes, mixed with lOOμl solution made of 0.15% deoxycholic acid in ethanol, and then vortexed and set aside for 10 minutes at room temperature. And after 50μl of 100% tricholoroacetic acid was added, the tubes were centrifuged at 10,000 rpm for 5 minutes and the supernatant liquid from each tube was disposed of, the sediment from the tubes were dissolved by adding 40μl of 0.1N NaOH. To examine them by SDS-PAGE electrophoresis method, they were mixed thoroughly with 10 μ I 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.

<59>

<60> [STEP 3] Purifying excretory-secretory products by centrifugation

<6i> 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 resuspended in RPMI 1640 serum-free media. Then, 2XX10 of the trichomonads and ImI of RPMI 1640 were added into an 1.5m-£ 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 tube were centrifuged at 10,000 rpm for 30 minutes, the supernatant liquid was collected and moved into an Amicon Centriprep-10, and then centrifuged at 3,00Og for about an hour at 4^°C . And after the liquid transferred to a supporting membrane was disposed, the rest was centrifuged at 3,00Og for about 30 minutes each at 4^°C . Finally, the concentration of remaining proteins was measured by Bradford assay.

<62> [Table 2]

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

<63> <64> [STEP 4] Immunizing a rabbit with excretory-secretory products <65> 500μ£ of Trichomonas vaginalis excretory-secretory products with concentration of lmg/m-β was mixed with the same volume of complete Freund's adjuvant (CFA) and injected into the skin of a female rabbit. After two weeks, 500μi of Trichomonas vaginalis excretory-secretory products with concentration of lmg/itt£ 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, 500μl of Trichomonas vaginalis excretory-secretory products with concentration of lmg/m-C 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 .

<66>

<67> [STEP 5] Purifying anti-excretory-secretory antibodies <68> The immune rabbit serum was dialyzed against Ii of loading buffer 3 times for 3 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 excretory-secretory products, then flown through the column by gravity. After washing resin with 10ml of loading buffer, the resin was washed again with 10mA of wash buffer (pH 7.5, 1OmM Tris). After adding elution buffer (pH 2.5, 100 mM glycine), lmteach 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 an Amicon CentriPrep-10.

<69>

<70> [STEP 6] Producing a kit using anti-excretory-secretory antibodies

<7i> A sample pad, a conjugate pad, a nitrocellulose membrane pad and an absorbent pad were attached onto a plastic card sequentially.

<72> Each pad is characterized by the following: the sample pad was soaked in buffer solution for calibrating chemical state of a specimen and then dried the conjugate pad was soaked in colloidal gold conjugate comprising 30 nm colloidal gold particles conjugated with the ant i-Trichomonas vaginalis antibodies developed as above, and then dried the nitrocellulose membrane pad includes the second detection zone (134) wherein anti-mouse immunoglobulin G antibodies were fixed and the second detection zone (134) wherein anti- Trichomonas vaginalis antibodies developed as above were fixed and the absorbent pad underwent no modification or treatment.

<73> The kit assembled as above was completed by being inserted in a plastic housing having a sample port for loading a specimen. <74> <75> [STEP 7] Producing the first and the second extraction solution <76> The produced amount of the first and the second extraction solution was

IH each.

<77> The first extraction solution is visually transparent and colorless.

Table 3 shows the recipe for the first extraction solution. <78> [Table 3]

<79> The second extraction is citron-colored. Table 4 shows the recipe for the second extraction solution.

<80> [Table 4]

<81> <82> [STEP 8] Obtaining and preserving a vaginal fluid specimen <83> Excessive cervical mucus is removed by one of the two swabs contained in plastic transport tube.

<84> The other swab was inserted into endocervical canal until the tip of the swab was no longer visible and reached beyond the squamocolumnar junction. And the swab was rotated carefully for 15-30 seconds to obtain cervical epithelial cells infected with Trichomonas vaginalis. And then the swab was carefully withdrawn without touching any vaginal surface and placed in the transport tube. <85> A swab specimen needs to be examined immediately, or preserved in a transport tube for up to 6 hours at room temperature or for up to 72 hours at

2~8°C. In the case of preserving a swab specimen for more than 72 hours, it needs to be frozen at -70^°C. <86> 300_/z£ of the first extraction solution was added into an extraction tube, and the swab was inserted into the extraction tube and rotated clockwise 10 times for 5 minutes for extraction. The extraction tube is preferred to be made of transparent or white plastic. <87> And then 300μJt of the second extraction solution was added into the extraction tube immediately, and the swab was rotated clockwise 10 times and discarded. <88> And then 100μ£ of the extracted solution was dropped onto sample port of a kit by using a dropper. The dropper is preferred to be made of transparent or white plastic.

<89> And then the kit was examined after 20 minutes. <90> To asses the sensitivity and specificity of the Trichomonas vaginalis diagnostic kit (100), an additional PCR method was performed using lOμi of the extracted solution. <9i> The swab comprises Dacron absorbent tip and a stick to which the Dacron absorbent tip is attached, and is provided and contained in a plastic transport tube. The swab is preferred to be preserved in a transport tube with sealing tape for sterilization.

<92>

<93> [STEP 9] PCR(Polymerase chain Reaction) and electrophoresis

<94> (1) Preparing DNA

<95> The sealed tube with iθμ£ of the extracted solution was boiled in a microwave for 5 minutes. And then it was centrifuged at lO.OOOrpm at 4^°C , and then the supernatant liquid containing Trichomonas vaginalis DNA was moved into a new tube. Trichomonas vaginalis strain KT-4 was used as positive control group. <96> (2) PCR condition

<97> Primer was designed based on repetitive DNA sequence specific for

Trichomonas vaginalis (TV-E650-1), which was originally cloned by Paces et . al. (1992). PCR was carried out using following primer sequences:

<98>

<99> Primer-sense

<ioo> 5 ' - GAGTTAGGGTATAATGTTTGATGTG-3 '

<101>

<iO2> Pr imer-ant i sense

<iO3> 5 ' - AGAATGTGATAGCGAAATGGG-3 '

<104>

<iO5> After PCR, products of 330bp were obtained. The PCR reaction solution comprised 2μJL of 1Ox PCR buffer, 2μi of 2.5mM dNTPs, 1.5//4 of sense primer (lOuM), 1.5μJL of antisense primer (lOuM), 2μi of the supernatant liquid containing Trichomonas vaginalis DNA and 0.3μΛ of Taq. polymerase (5\J/μH), and was made up to final volume of 20μ£ with distilled water. The PCR reaction solution was incubated at 95^°C for 5 minutes followed by 30 cycles of 94^°C 30 seconds, 52°C 10 seconds and 72^°C 30 seconds. And then it was electrophoresized on 2% agarose gel, and pictured under UV (ultraviolet) light.

<iO6> FIG. 4 is a reference drawing which is showing electrophoresis result of Trichomonas vaginalis according to an embodiment of the present invention. Referring to FIG. 4, 'SM^' means a DNA ladder used as a molecular size marker, 'NC means negative control group, 'PC means positive control group and 147 shows the result of the diagnostic test for Trichomonas vaginalis infection of a infected patient.

<107>

<iO8> [EXAMPLE 1]

<iO9> In order to examine specificity of the kit produced by the above STEP 6, a specificity test ^as conducted by dropping lOOμH of PCR-confirmed negative specimen (vaginal fluid) onto the sample port of the kit and then by 4

setting the kit aside for 20 minutes. 496 negative specimens were used in the test. Table 5 is showing the result of the test, in which only 6specimens were false negative, showing 98.8% of specificity.

<110> And, in order to examine sensitivity of the kit produced by the above STEP 6, a sensitivity test was conducted by dropping 100//4 of PCR-confirmed positive specimen (vaginal fluid) onto the sample port of the kit and then by setting the kit aside for 20 minutes. Table 5 is showing the result of the test, in which 29 out of 34 positive specimens were positive, showing 85.3% of sensitivity.

<111> [Table 5]

<112> 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.

Claims

ID

[CLAIMS] [Claim 1]

A Trichomonas vaginalis diagnostic kit, characterized by: being produced by using anti-Trichomonas vaginalis antibodies produced from animals immunized with Trichomonas vaginalis excretory-secretory

7 products produced by culturing 5X10 trichomonads at 37 C in an atmosphere containing 5% of CO2 in CO2 incubator for 2 hours.

[Claim 2]

The kit of claim 1, wherein: a sample pad, soaked in buffer solution for calibrating chemical state of a specimen and then dried; a conjugate pad, soaked in colloidal gold conjugate and then dried; a nitrocellulose membrane pad, including a first detection zone wherein the anti-Trichomonas vaginalis antibodies are fixed and a second detection zone wherein anti-mouse immunoglobulin G antibodies are fixed; and an absorbent pad; are sequentially attached onto a plastic card.