KR100783768B1 - Trichomonas vaginalis and chlamydia trachomatis simultaneous diagnosis kit - Google Patents

Abstract

A kit for simultaneously diagnosing infection of Trichomonas vaginalis and Chlamydia trachomatis by using vaginal excretion is provided to test the Trichomonas vaginalis and Chlamydia trachomatis with high accuracy, high sensitivity and specificity within significantly shortened diagnosis time. A kit for simultaneously diagnosing Trichomonas vaginalis and Chlamydia trachomatis(100) is characterized in that a sample pad(110) in which a buffer for correcting a chemical state of a testing body is absorbed and dried, a conjugate pad(120) in which a colloid gold conjugate is absorbed and dried, a nitrocellulose membrane pad(130) which includes a first detecting portion(132) on which an anti-chlamydia antibody is fixed, a second detection portion(134) on which an anti-Trichomonas vaginalis antibody is fixed, and a third detection portion(136) on which an anti-mouse immunoglobulin G antibody is fixed, and an absorption pad(140) are attached to a plastic card in sequence. The anti-Trichomonas vaginalis antibody is prepared from an antibody obtained by immunizing an excretory-secretory antigen of an animal to be tested and the colloid gold conjugate is characterized in that the anti-Trichomonas vaginalis antibody and the anti-chlamydia antibody are bound to the colloid gold and an average diameter of the colloid gold is 20-40 nanometers.

Description

Trichomonas vaginalis and Chlamydia trachomatis simultaneous diagnosis kit

The present invention relates to a simultaneous diagnosis of vaginal larvae and chlamydia, and more specifically, specificity and sensitivity, short diagnostic time, reduced cost, chromosome and chlamydia can be diagnosed simultaneously It is about the simultaneous diagnosis kit.

Schizophrenia is a disease caused by Trichomonas vaginalis.

A vaginal caterpillar is a protozoa discovered by Donne (1837) and is responsible for Trichomoniasis, one of the sexually transmitted disorders (STDs), and reports that around 170 million people worldwide are infected each year. It is becoming.

Spondyloidosis is one of the most common involuntary infectious diseases caused by sexual contact with vaginal larvae. In women, it is associated with vaginal burning, pruritus, redness and swelling of the vaginal mucosa. Rupture, isopregnancy, infertility, 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). In addition, in men, the symptoms do not appear well, but it is important as a propagator.

Diagnosis of vaginal pterygium includes direct observation of vaginal fluid samples under a microscope, polymerase chain reaction (PCR), and immunological methods.

Chlamydiasis is caused by chlamydia.

In addition, chlamydiasis is currently diagnosed using a culture method, a fluorescence method, etc., but it has a problem in that it takes time and cost or low diagnostic accuracy. In recent years, immunological diagnostic kits such as ELISA or rapid kit have been commercially available as chlamydial diagnostic specific antigens have been developed.

Schizophrenia and chlamydia are known as obstructive infections in the obstetrics and gynecology field, so they may be transmitted to sexual partners without their knowledge.

Currently, vaginal larvae are tested using methods such as smearing or anaerobic culture, but the test takes a long time and requires the skills of a skilled clinician, but the diagnosis rate is low.

Therefore, the most effective method currently used for diagnosing vaginal pneumoconiosis is the PCR method, but expensive equipment is required and the inspection procedure performed until the determination is difficult. Accordingly, most of the clinical practice is performed by smearing or anaerobic culture, but the skills of skilled clinical experts are required.

Therefore, there is a need for the development of a rapid kit method, which is the most sensitive method so far, which can be used easily and quickly in a clinic. Chlamydia diagnosis has many kinds of rapid kits on the market, but in the case of vaginal larvae, only one kit developed by an American company is also difficult to purchase in Korea.

SUMMARY OF THE INVENTION An object of the present invention devised to solve the above-mentioned problems is to provide a vaginal worm and chlamydia co-diagnosis kit that can simultaneously diagnose vaginal worm and chlamydial infection using vaginal secretions.

In addition, an object of the present invention is to provide a vaginal larvae and chlamydia co-diagnosis kits that can significantly shorten the diagnostic time and can be tested with a higher accuracy in a shorter time.

In addition, another object of the present invention is to provide a vaginal flagetworm and chlamydia co-diagnosis kit which detects sexually transmitted diseases of vaginal wormworm and chlamydia with high sensitivity and specificity.

In addition, another object of the present invention, can be used as a diagnosis of vaginal larvae and chlamydial infection of obstetrics and gynecology patients, can be used as one of the test items when performing a comprehensive examination in a public examination center, such as public health centers and general hospitals To provide a simultaneous diagnosis of vaginal larvae and chlamydia.

In order to achieve the above object, the vaginal caterpillar and chlamydia co-diagnosis kit according to the present invention, a sample pad dried by absorbing a buffer capable of correcting a chemical state of a sample, and a conjugate pad dried by absorbing a colloidal gold conjugate A nitrocellulose membrane pad and an absorption pad comprising a first detection portion immobilized with an anti-chlamydia antibody, a second detection portion immobilized with an anti-plasmid antibody and a third detection portion immobilized with an anti-mouse immunoglobulin G antibody, on a plastic card Attached.

In addition, the anti-plasmid caterpillar antibody, 5ml TYM broth in a glass tube with a stopper of 15ml size and inoculated with a filler so that the final concentration is 2 × 10 ⁵ cell / ml, 37 ℃, 5% carbon dioxide gas incubator Chlomococcus culturing step of culturing parasites for 24 hours, centrifugation of the cultured vaginal larvae for 15 minutes at 1500rpm, discard the supernatant and wash twice with sterile PBS (phosphate buffer, pH 7.2), washing After resuspending the precipitate with 1 ml of RPMI 1640 free serum medium, 1 × 10 ⁷ vaginal caterpillars were placed in an eppendorf tube, and the RPMI 1640 medium was added to about 1 ml. Incubating in a% carbon dioxide gas incubator, extracting excretory antigen (ESP), 500 μl of 1 mg / mL secretion excretory antigen mixed with the same volume of complete Freund's adjuvant (CFA) female 2 weeks after injection into rabbit skin 500 μl of about 1 mg / ml rTvAP33 was mixed with the same volume of incomplete Freund's adjuvant (IFA) and injected again into the skin, followed by 500 μl of about 1 mg / ml rTvAP33 in the same volume. Intraperitoneal injection mixed with IFA, blood secretion from the ear vessels before injection, secretion excretion antigen phase to separate the serum, rabbit serum transformed with the excretion antigen is dialyzed 3 times 3 hours with 1L loading buffer After centrifugation at 12,000g for 20 minutes, the supernatant is collected and removed by floating particles with a filter of about 0.45 µm.

In addition, the colloidal gold conjugate is characterized in that the anti-plasmid caterpillar antibody and the anti chlamydia antibody are bound to the colloidal gold.

In addition, the colloidal gold is characterized in that the average particle diameter of 20 ~ 40nm.

In addition, the anti-chlamydia antibody, anti-pyroplasmic antibody and anti-mouse immunoglobulin G antibody are characterized in that the detection antibody.

In addition, the anti-chlamydia antibody of the first detection part is developed by reaction with the antibody portion of the sample and the anti-chlamydia colloidal gold conjugate, and the anti-plasmid antibody of the second detection part is developed by the reaction with the antibody part of the sample and the anti-plasmid colloidal gold conjugate. The anti-mouse immunoglobulin G antibody of the third detection portion is characterized in that the color is produced by directly reacting with the antibody portion of the anti-chlamydia colloidal gold conjugate.

In addition, the antibody is characterized in that the monoclonal antibody or polyclonal antibody.

Hereinafter, with reference to the accompanying drawings will be described in detail a preferred embodiment of the vaginal caterpillar and Chlamydia co-diagnosis kit according to the present invention.

Figure 1 is a perspective view of the simultaneous diagnosis of vaginal larvae and chlamydia kit according to an embodiment of the present invention, Figure 2 is a cross-sectional view of the simultaneous diagnosis of the vaginal larvae and chlamydia kit according to an embodiment of the present invention.

Referring to FIGS. 1 and 2, the vaginal caterpillar and chlamydia co-diagnosis kit 100 according to the present invention includes a sample pad 110, a conjugate pad 120, a nitrocellulose membrane 130 pad, and an absorbent pad 140. It includes. In addition, the nitrocellulose film 130 pad includes a first detector 132, a second detector 134, and a third detector 136.

The conjugate pad 120 is preferably dried by absorbing the colloidal gold conjugate in which the anti-plasmid caterpillar antibody and the anti-chlamydia antibody are bound to the colloidal gold.

The first detection unit 132 is preferably an anti-chlamydia antibody is fixed.

The second detection unit 134 is preferably an anti-plasmid caterpillar antibody is fixed.

The third detection unit 136 is preferably fixed to the anti-mouse immunoglobulin G antibody.

In addition, the anti-plasmid caterpillar antibody is prepared from an antibody obtained by immunizing a secretory excretory antigen to a test animal as a raw material. Rabbits, sheep and goats can be used as experimental animals.

3 is a reference diagram illustrating a detection principle of each detection unit of a manufactured simultaneous diagnosis kit according to an embodiment of the present invention.

Referring to Figure 3, it can be seen the detection principle of each detector. The first detector 132 denotes T1, the second detector 134 denotes T2, and the third detector 136 denotes C. The conjugate in which the anti-chlamydia antibody is bound to the colloidal gold is referred to as the anti-chlamydia colloidal gold conjugate, and the conjugate in which the anti-plasmid antibody is bound to the colloidal gold is referred to as the anti-plasmid colloidal gold conjugate. Anti trichomonas antibodies refer to anti-pyromolar antibodies.

In the T1 line, which is the first detection unit 132, the immobilized anti-chlamydia antibody is reacted with the antibody portion of the sample and the anti-chlamydia colloidal gold conjugate.

In the T2 line, which is the second detection unit 134, the immobilized anti-pneumococcal antibody is colored by reacting with the antibody portion of the sample and the anti-pneumococcal colloidal gold conjugate.

Referring to line C of the third detection unit 136, the immobilized anti mouse immunoglobulin G antibody is colored by directly reacting with the antibody portion of the anti chlamydia colloidal gold conjugate except for the specimen, unlike other detection units.

Simultaneous diagnosis of vaginal worms and chlamydia kit of the present invention is a vaginal secretion of the subject, so it can be confirmed whether or not the infection of the vaginal worms and chlamydia at the same time, it will be widely used in the early diagnosis of vaginal worms and chlamydia.

Hereinafter, a preferred embodiment of the vaginal caterpillar and chlamydia co-diagnosis kit according to the present invention will be described in detail.

However, these examples are only for illustrating the present invention specifically, the scope of the present invention is not limited to these examples.

[Step 1] Cultivation of Vernal Chronicles

Parasite culture was first inoculated with a 15 ml stoppered glass tube containing about 5 ml of TYM broth [Table 1] to give a final concentration of about 2 × 10 ⁵ cells / ml. It was confirmed by the light, inoculated in a glass tube with a 50 ml stopper containing about 20 ml of medium of about 2 × 10 ⁵ cells / ml and incubated for 24 hours under the same culture conditions. Then, about 400 ml of medium was placed in a 1 L glass Erlenmeyer flask for mass culturing of the vaginal larvae, and the inoculated vaginal larvae at the same concentration and incubated for 24 hours under the same conditions. The number of parasites was calculated under a 100-fold microscope after dilution with trypan blue and the bacterial solution 1: 1.

 TYM broth composition Ingredient content Trypticase 20.0 g Yeastextract 10.0 g Maltose 5.0 g L-cyteine 1.0 g Ascorbic Acid (Vit.C) 1.0 g KH2PO4 1.0 g K2HPO4 1.0 g Penicillin 0.06 g Streptomycin 0.5g Horse serum 100 ml D.W Up to 1L

[Step 2] Extraction of Excretory-secretory Product (ESP)

After centrifugation of the vaginal larvae cultured for about 24 hours in [Step 1] at about 1500 rpm for about 15 minutes, the supernatant was discarded and washed twice with sterile phosphate buffer (PBS, pH 7.2). The precipitate was then resuspended in about 1 ml of RPMI 1640 free serum medium. Place about 1 × 10 ⁷ vaginal larvae in about 1.5 ml Eppendorf tubes, add about 1 ml of RPMI 1640 medium, about 60 minutes, about 90 minutes, about 120 minutes at 37 ° C., about 5% CO ₂ , respectively, Incubated for about 150 minutes and about 180 minutes. In order to observe the secretion of ESP by the concentration of vaginal larvae, the number of vaginal larvae in the 1 ml RPMI 1640 medium was 1 × 10 ⁶ cells, 5 × 10 ⁷ cells, and 1 × 10 ⁷ cells, respectively. 5 × 10 ⁷ cells and 1 × 10 ⁸ cells were placed and incubated for about 120 minutes. The cultured larvae were centrifuged at about 10,000 rpm for about 30 minutes, the supernatant was transferred to another tube, vortexed with about 100 μl of about 0.15% Deoxycholic acid in ethanol, and left at room temperature for about 10 minutes. Then, 50 μl of 100% Trichloroacetic acid was added, vortexed, and centrifuged at about 10,000 rpm for about 5 minutes. The supernatant was discarded and the precipitate dissolved in 40 μl of approximately 0.1 N NaOH. In order to confirm by SDS-PAGE electrophoresis method, 10 μl of 5 × loading buffer was added, mixed well, and boiled at about 100 ° C. for about 5 minutes, and then SDS-PAGE was performed on about 12% polyacrylamide gel. [Step 2] is a preliminary experiment to find the proper experimental conditions of [Step 3].

[Step 3] Purification of secretory excreted antigen by centrifugation

The vaginal larvae incubated for about 24 hours in a glass flask of about 1 L containing about 400 ml TYM medium were centrifuged at about 1500 rpm for about 15 minutes, and then the supernatant was discarded and washed twice with PBS. After resuspending with RPMI 1640 free serum medium, 2 × 10 ⁷ vaginal caterpillars each were placed in a 1.5 ml Eppendorf tube and RPMI 1640 medium was added to about 1 ml, then at about 37 ° C. and about 5% CO ₂ . Incubated for about 2 hours. Centrifuge the cultured vaginal larvae at about 10,000 rpm for about 30 minutes, collect the supernatant, transfer to centriprep (10,000 kDa,), and add a complete protease inhibitor cocktail (Roche, Germeny). After centrifugation at 4 ° C. and about 3,000 g for about 1 hour, the solution that passed through the cell membrane was discarded and centrifuged twice at about 4 ° C. and about 3,000 g for about 30 minutes. The concentration of the last remaining protein was measured by bredford assay.

Amounts of ESP Protein Produced by Squamous Caterpillars Cultured in 400ml TYM Media for 24 Hours Degree T.viginalis number (10 ⁸ cell) Final Protein Concentrate (μl) Protein amount (μl) One 3.2 730 1474.6 2 4.0 580 899.0 3 4.6 580 823.6 4 4.4 650 962.0 5 2.4 540 1209.6 Average 3.72 616 1073.76

[Step 4] Immunize the excretory antigen to rabbit

About 500 μl of secretory excretory antigen at a concentration of about 1 mg / ml was mixed with the same volume of the complete Freund's adjuvant and then injected into the skin of female rabbits. After about two weeks, about 500 μl of about 1 mg / ml TvESP (plasmid worm secretory antigen) was mixed with the same volume of incomplete Freund's adjuvant and injected again into the skin. After another two weeks, about 500 μl of about 1 mg / ml vaginal larvae excretory antigen was injected intraperitoneally with the same volume of incomplete Freund's adjuvant. Blood was collected from the ear vessels before each injection and serum was isolated from it.

[Step 5] Purification of Antibody Against Secretory Excretory Antigen

Rabbit serum immunized with secretory excreted antigen was dialyzed about 3 hours with about 1 L of loading buffer. After centrifugation at about 12,000 g for about 20 minutes, the supernatant was recovered to remove suspended particles with a 0.45 μm filter. Dialysed serum was loaded onto a column packed with Affi-Gel resin conjugated with secretory excretory antigens, allowing the sample to pass through the column under natural pressure. After washing the resin with about 10 ml of loading buffer, the resin was washed twice with about 10 ml of wash buffer (10 mM Tris pH 7.5). After addition of elution buffer (100 mM glycine pH 2.5), each tube was received in about 1 mL fraction tubes, and each fraction tube was neutralized with elution buffer to which 1 M Tris was added so that the acidity could be pH 7.5. Anti-rTvAP33 antibody elution fractions were collected by Bradford assay, dialyzed at 10 mM Tris pH7.5, and concentrated using CentriPrep-10 (Amicon).

[Step 6] Preparation of kit using antibody against secretory excretory antigen

Four pads of an absorbent pad, a nitrocellulose membrane pad, a conjugate pad, and a sample pad were attached on the plastic card in this order. The characteristics of each pad were as follows. The sample pad was dried after absorbing the buffer material to correct the chemical state of the sample. The conjugate pad was dried after the anti-plasmid antibody developed according to the above description was conjugated to a 30 nm colloidal gold, and the anti-Chlamydia antibody purchased commercially was conjugated to the 30 nm colloidal gold. In the nitrocellulose membrane pad, anti-mouse immunoglobulin G antibody was developed in the third detection unit 136, anti-chlamydia antibody purchased commercially in the first detection unit 132, and in the second detection unit 134 according to the above method. Anti-pyromolar antibodies are immobilized. The absorbent pad was not treated. The kit was assembled in a plastic housing with a sample window.

Experimental Example 1

To determine the specificity of the kit prepared in [Step 6], 100 μl of a negative sample (vaginal secretion) was added to the sample window of the kit, and a specificity test was performed for 20 minutes. The sample used a randomly selected 28 negative samples. As a result, only one sample was false positive for the negative sample, which showed a specificity of 96.4%.

Item unit Specificity rate Sample used Specificity % 96.4% after 20 minutes 100 μl negative sample (vaginal discharge)

Experimental Example 2

A sensitivity test was performed to find out the sensitivity of the kit prepared in [Step 6].

Positive specimen Voice sample Sample count Positive diagnosis responsiveness Sample count Voice Diagnosis Specificity  Vaginal caterpillar 27 21 78% 361 347 96% Chlamydia 30 20 66%

Figure 4 is a reference diagram showing the sensitivity test results of Experimental Example 2 of the prepared simultaneous diagnostic kit according to an embodiment of the present invention.

Referring to FIG. 4, when a red line appears in a line corresponding to each detection unit of the simultaneous diagnosis kit, a positive response is displayed, and when there is no change, a negative response is indicated. If a red line appears in the third detection unit, a positive diagnosis and a negative diagnosis can be determined. In the diagnostic kit, the first detector T1, the second detector T2, and the third detector C are shown.

That is, A-1 is a diagnostic kit indicating a negative diagnosis due to no change in the second detection unit and the first detection unit while the third detection unit has a red line, and A-2 is the red detection unit in the third detection unit and the first detection unit. A line appears so that the anti-chlamydia antibody corresponding to the first detection part shows a positive reaction, and the anti-plasmid antibody shows a negative reaction. A-3 shows a red line in the third detection part and the second detection part, so that the anti-plasmid caterpillar antibody corresponding to the second detection part shows a positive reaction, the antichlamydia antibody shows a negative reaction, and A-4 shows the first detection part to the first detection part. All three detection areas show red lines, indicating that both the anti-chlamydia and anti-plasmid antibodies are positive.

In addition, in B-1 to B-4, no change occurs in all of the third detection unit, and if there is no change in the third detection unit, the positive diagnosis and the negative diagnosis cannot be determined.

In other words, as shown in Table 4, 21 of 27 samples showed positive diagnosis, and 78% showed sensitivity, while anti- chlamydia antibody showed positive diagnosis with 20 of 30 samples. 66% and 96% specificity of negative sample.

The present invention is not limited to the above-described preferred embodiments and can be easily modified by anyone of ordinary skill in the art without departing from the gist of the invention claimed in the claims, Such changes are intended to fall within the scope of the claims.

According to the present invention as described above, it is possible to provide a vaginal worm and chlamydia co-diagnosis kit that can simultaneously diagnose vaginal worm and chlamydia infection using vaginal secretions.

In addition, according to the present invention, it is possible to provide a vaginal larvae and chlamydia co-diagnosis kits that can significantly reduce the diagnostic time, which can be examined with higher accuracy in a shorter time.

In addition, according to the present invention, it is possible to provide a vaginal flagetworm and chlamydia co-diagnosis kit which detects the sexually transmitted diseases of vaginal pneumoniae and chlamydia with high sensitivity and specificity.

In addition, according to the present invention, it can be used as a diagnosis of vaginal larvae and chlamydial infection of obstetrics and clinic visits, and can be used as one of the test items when performing a comprehensive examination in a group examination institution such as a public health center and a general hospital and Chlamydia co-diagnosis kits can be provided.

1 is a perspective view of a simultaneous diagnosis of vaginal caterpillar and chlamydia according to an embodiment of the present invention

Figure 2 is a cross-sectional view of the vaginal caterpillar and Chlamydia co-diagnosis kit according to an embodiment of the present invention

3 is a reference diagram illustrating a detection principle of each detection unit of a manufactured simultaneous diagnostic kit according to an embodiment of the present invention.

Figure 4 is a reference diagram showing the sensitivity test results of Experimental Example 2 of the prepared simultaneous diagnostic kit according to an embodiment of the present invention

Figure 5 is a protein band pattern of the purified secretion excretion antigen according to an embodiment of the present invention.

<Explanation of symbols for the main parts of the drawings>

100: simultaneous diagnosis kit 110 according to the invention: sample pad

120: conjugate pad 130: nitrocellulose film pad

132: first detection unit 134: second detection unit

136: third detection unit 140: absorption pad

Claims (7)

A sample pad in which a buffer capable of correcting a chemical state of the sample is absorbed and dried; Conjugate pads in which the colloidal gold conjugate has been absorbed and dried; A nitrocellulose membrane pad comprising a first detection portion immobilized with an anti-chlamydia antibody, a second detection portion immobilized with an anti-plasmid insect antibody, and a third detection portion immobilized with an anti-mouse immunoglobulin G antibody; and Absorption pads; Are attached in sequence on the plastic card, The anti-vaginal caterpillar antibody, It is prepared from the antibody obtained by immunizing the excretory antigen to the experimental animal as a raw material, The colloidal gold conjugate, Anti-cytoplasmic antibodies and anti-chlamydia antibodies are bound to the colloidal gold, Simultaneous diagnosis of vaginal larvae and chlamydia, characterized in that the average particle diameter of the colloidal gold is 20 ~ 40nm. delete delete delete The method of claim 1, An anti-Chlamydia antibody, an anti-Chromeworm antibody, and an anti-mouse immunoglobulin G antibody are detection antibodies. The method of claim 1, The anti-chlamydia antibody of the first detection part is developed by reaction with the antibody portion of the sample and the anti-chlamydia colloidal gold conjugate, and the anti-plasmid antibody of the second detection part is developed by reaction with the antibody part of the sample and the anti-plasmid colloidal gold conjugate. Anti-mouse immunoglobulin G antibody of the third detection part is a vaginal caterpillar and chlamydia co-diagnosis kit, characterized in that the color is produced by directly reacting with the antibody portion of the anti-chlamydia colloidal gold conjugate. The method of claim 1, wherein the antibody is A vaginal caterpillar and chlamydia co-diagnosis kit, characterized in that the monoclonal antibody or polyclonal antibody.