CN113791214B - Enhancing solution for detecting chlamydia trachomatis antigen and application thereof - Google Patents
Enhancing solution for detecting chlamydia trachomatis antigen and application thereof Download PDFInfo
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- CN113791214B CN113791214B CN202111344974.8A CN202111344974A CN113791214B CN 113791214 B CN113791214 B CN 113791214B CN 202111344974 A CN202111344974 A CN 202111344974A CN 113791214 B CN113791214 B CN 113791214B
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Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56927—Chlamydia
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
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- G01N2469/10—Detection of antigens from microorganism in sample from host
Abstract
The application belongs to the technical field of immunodetection, and particularly relates to an enhancement solution for detecting a chlamydia trachomatis antigen and an application thereof, wherein the enhancement solution for detecting the chlamydia trachomatis antigen comprises an enhancement solution A and an enhancement solution B, and the enhancement solution A is a mixed solution of titanium potassium oxalate and a surfactant; the enhancement solution B is silver ion solution. The surfactant in the enhancement solution A has the effects of solubilization, wetting, emulsification, scale removal and the like, the condition that the background of the reagent strip is not clean can be eliminated, the observation of a detection result is prevented from being influenced, meanwhile, silver ions in the enhancement solution B are reduced into silver particles through the reducibility of the enhancement solution A, a sandwich compound of colloidal gold antibody-antigen-antibody (fixed on a membrane) is changed into a sandwich compound of silver particles-colloidal gold antibody-antigen-antibody (fixed on the membrane), the sensitivity of the reagent strip is increased, the operation is simple, the traditional production process is not required to be changed, and the sensitivity of the reagent strip can be still improved without an instrument.
Description
Technical Field
The application belongs to the technical field of immunodetection, and particularly relates to an enhancement solution for detecting a chlamydia trachomatis antigen and application thereof.
Background
In vitro diagnostic reagents are widely applied to clinical detection, and tracers thereof are various: colloidal gold, latex particles, fluorescent microspheres, fluorescein, time-resolved microspheres, magnetic particles, and the like. Wherein the colloidal gold has wide application.
The main principle of the colloidal gold reagent is as follows: fixing a specific antibody on a detection area on a nitrocellulose membrane, dropwise adding a sample in the sample area, allowing the sample to migrate to a glass fiber membrane and a colloidal gold combination pad by virtue of capillary action, dissolving a gold-labeled compound in the combination pad by sample liquid, performing antigen-antibody reaction with the sample to form a compound of antigen-labeled antibody-colloidal gold particles, continuously migrating to the detection area of the nitrocellulose membrane, capturing the compound with the gold label by an antigen in the detection area, forming a compound of captured antibody-antigen-labeled antibody-colloidal gold particles, and presenting a red strip. If the sample does not contain the antigen to be detected, no binding occurs, i.e. no color develops. An antibody corresponding to the gold-labeled conjugate is generally fixed near the detection area of the nitrocellulose membrane to serve as a quality control band, and a quality control line is displayed no matter whether the sample contains the substance to be detected or not, and if the sample does not contain the substance to be detected, the detection fails. The whole process is generally completed within 10-30 minutes, the operation is simple and quick, and no instrument is needed.
In order to improve the sensitivity of the gold colloid method, the japanese fuji group performs photographic development based on the gold colloid method by a technique of increasing sensitivity and specificity by a silver amplification technique, in short, by aggregating silver particles in the vicinity of gold particles on the basis of "capturing a complex of antibody-antigen-labeled antibody-gold colloid particles", and amplifying the size of the complex to make the detection line band clearer, thereby improving the gold colloid detection sensitivity. However, the technology changes the traditional structure of the reagent strip, newly adds sample adding holes of the reinforcing liquid and absorbent paper, improves the production complexity and the reagent cost, and cannot adopt the traditional production line for production. Moreover, although the technology improves the detection sensitivity, the detection operation needs to be carried out by a machine, the requirement on detection conditions is high, the detection cost is increased, and the technology is inconvenient for large-scale clinical detection. Moreover, the technology has the phenomenon of unclean background after silver amplification, which affects the observation of detection results.
In addition to the Japanese Fuji group, the English toming Chlamydia rapid detection kit (immunochromatography) is simple to operate, maintains the traditional production process, has low production cost, and needs to improve the sensitivity.
Disclosure of Invention
In order to solve the problems that the current detection kit of the Japanese Fuji has unclean background and complicated operation and the detection kit of the English Ming has insufficient sensitivity, the application discloses an enhancing solution for detecting the chlamydia trachomatis antigen and application thereof, wherein the enhancing solution A is a mixed solution of titanium potassium oxalate and a surfactant, the surfactant has the effects of solubilization, wetting, emulsification, scaling and the like, can help to eliminate the unclean background of a reagent strip and avoid influencing the observation of a detection result, simultaneously silver ions in the enhancing solution B are reduced into silver particles through the reducibility of the enhancing solution A, a sandwich compound of colloidal gold antibody-antigen-antibody (fixed on a membrane) is changed into a sandwich compound of silver particles-colloidal gold antibody-antigen-antibody (fixed on the membrane), and the sensitivity of the reagent strip is increased, the enhancement solution is added on the basis of the original reagent strip, the operation is simple, the traditional production process is not required to be changed, and the sensitivity of the reagent strip can still be improved without an instrument.
In a first aspect, the present application provides an enhancing solution for detecting chlamydia trachomatis antigen, which adopts the following technical scheme:
an enhancement solution for detecting chlamydia trachomatis antigen, which comprises an enhancement solution A and an enhancement solution B, wherein the enhancement solution A is a mixed solution of titanium potassium oxalate and a surfactant; the enhancement solution B is silver ion solution.
The reducing enhancement solution A reduces the free silver ions in the enhancement solution B into silver particles, and the sensitivity of the reagent strip is effectively improved by changing the sandwich compound of the colloidal gold antibody-antigen-antibody (fixed on the membrane) into the sandwich compound of the silver particles-colloidal gold antibody-antigen-antibody (fixed on the membrane).
Preferably, the mass percent concentration of the titanium potassium oxalate in the enhancing liquid A is 2-3%, and the mass percent concentration of the surfactant is 2-10%; the enhancing solution B is 5-15 mu mol/L silver nitrate solution.
Preferably, the surfactant is one or more of triton X-100, tween-20 and NP 40.
Preferably, the surfactant is a mixture of triton X-100 and Tween-20.
Preferably, the mass ratio of the triton X-100 to the Tween-20 is 1: 0.5-1.5.
Preferably, the enhancing solution a is: 2-3% of titanium potassium oxalate with mass percentage concentration, 4-6% of triton X-100 with mass percentage concentration and 4-6% of tween-20 with mass percentage concentration.
Preferably, the concentration of the silver nitrate solution is 15. mu. mol/L.
In a second aspect, the present application provides a method for detecting chlamydia trachomatis antigen, which adopts the following technical scheme:
a method for detecting Chlamydia trachomatis antigen, comprising the following steps: firstly, dropwise adding a sample to be detected to the reagent strip, dropwise adding the enhancement liquid A to the reagent strip after a period of time, dropwise adding the enhancement liquid B to the reagent strip after a period of time, and judging a detection result.
Preferably, the detection method specifically comprises the steps of firstly dripping a sample to be detected on the reagent strip, dripping the enhancement solution A from the sample adding hole after 10min, dripping the enhancement solution B from the sample adding hole after 5min, and judging negative and positive results after 5 min.
Preferably, the amount of enhancing solution A to be added is 0.1 to 0.15 mL, and the amount of enhancing solution B to be added is 0.1 mL.
The application has the following beneficial effects:
(1) the enhancement solution for detecting the chlamydia trachomatis antigen is characterized in that the enhancement solution A is a mixed solution of titanium potassium oxalate and a surfactant, the surfactant has the effects of solubilization, wetting, emulsification, scaling and the like, can help eliminate the condition that the background of the reagent strip is not clean, avoids influencing the observation of a detection result, meanwhile, the silver ions in the enhancing solution B are reduced into silver particles through the reducibility of the enhancing solution A, the sandwich compound of the colloidal gold antibody-antigen-antibody (fixed on the membrane) is changed into the sandwich compound of the silver particles-colloidal gold antibody-antigen-antibody (fixed on the membrane), the sensitivity of the reagent strip is increased, the enhancement solution is added on the basis of the original reagent strip, the operation is simple, the traditional production process is not required to be changed, and the sensitivity of the reagent strip can still be improved without an instrument.
(2) The surfactant in the enhancement solution A is preferably a non-ionic surfactant such as triton X-100, tween-20, NP40 and the like, so that the stability of the enhancement solution A can be effectively improved, and the enhancement solution A is not easily influenced by inorganic salt and acid-base solution.
(3) The surfactant in the enhancing solution A is preferably a mixture of triton X-100 and Tween-20, wherein the mass ratio of triton X-100 to Tween-20 is 1:0.5-1.5, and the inventor finds through a large amount of experiments that the selection of the two surfactants in the enhancing solution A in the above dosage ratio is helpful for obtaining the background of a clean reagent strip and does not influence the sensitivity of the reagent strip.
Drawings
The present application is further described below with reference to the drawings and examples.
Fig. 1 is a detection schematic diagram of the present application.
Detailed Description
The present application will now be described in further detail with reference to examples.
Preparing a reagent strip:
(1) preparation of colloidal gold
Colloidal gold is prepared by a sodium citrate reduction method. 100ml of a 0.01% aqueous chloroauric acid solution were placed on a magnetic stirrer and heated to boiling. 2mL of a 1% aqueous solution of trisodium citrate was added with stirring, and heating and boiling were continued. Boiling for 15min until the solution appears transparent wine red in color. Then, cooled at room temperature, made up to the original volume with deionized water, and stored at 4 ℃ until use. And scanning an absorption peak at 200nm-800nm by using an ultraviolet spectrophotometer to determine the maximum absorption peak and the absorbance of the fired colloidal gold.
(2) Preparation of colloidal gold marker
Determining the optimal pH value and the minimum labeling concentration of the colloidal gold labeled anti-chlamydia trachomatis antibody: with 0.2M K2CO3Adjusting the pH value of the colloidal gold solution to 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, respectively taking 1mL, respectively adding 100mL (1 mg/mL) of the chlamydia trachomatis antibody, shaking, mixing uniformly, and standing at room temperature for 15 min. 20 mL of 10% NaC1 was added to each tube and mixed well, and the mixture was allowed to stand at room temperature for 15min to stabilize the color of the gold colloid solution.
Adjusting the pH value of the colloidal gold solution to an optimal value (pH 7.0), respectively taking 1mL of the colloidal gold solution into a series of clean centrifuge tubes, respectively adding 0, 0.5, 1, 2, 4, 6, 8, 10, 12 and 14 mg of chlamydia trachomatis antibodies, uniformly mixing, standing for 5min, respectively adding 100mL of 10% NaCI, uniformly mixing, standing for 2h at room temperature, and taking the amount of the chlamydia trachomatis antibody with stable and unchanged colloidal gold color as the minimum labeled protein amount. The optimal amount of marker protein was determined by increasing the amount by 20% from the minimum amount of marker.
Preparation of colloidal gold-chlamydia antibody conjugates: 1mL of the colloidal gold solution prepared above was first added to a beaker, the pH of the colloidal gold was adjusted to the optimum pH (i.e., pH = 8.4) using 200mM boric acid buffer, and the Chlamydia trachomatis antibody (CT, concentration 2.0 mg/mL) diluted with 50mM PBS was added dropwise with stirring, and stirring was continued for 15min, and then 40ul of 10% BSA blocking solution was added for blocking for 3 h. After the blocking is finished, the mixed solution is placed in a high-speed centrifuge for centrifugation under the conditions of 4 ℃ of rotation speed, 8000r/min and 10min, the supernatant is discarded, the precipitate is washed by 1mL of PBST with 20mM, the centrifugation and washing are repeated for 3 times to remove the redundant antibody not combined with the colloidal gold, and finally the mixed solution is resuspended to 100mL by 100mM of PBS containing 0.5 percent of casein and 0.03 percent of sodium azide, and the mixed solution is vortexed and is stored at 4 ℃ for standby.
(3) Preparation of sample pad
The sample pad was soaked with 50mM phosphate buffer (0.5% CaseinA, 0.5% PEG 8000) for 2h and placed at 37 ℃ for vacuum drying for 6 h.
(4) Preparation of gold-labeled bonding pad
The conjugate pad was soaked with 50mM Tris-HCI (1% BSA, 0.5% Tween-20) buffer for 1 h, drained and placed under vacuum at 37 ℃ for 6 h.
Spraying the prepared colloidal gold-chlamydia antibody solution onto the treated bonding pad by using an automatic spraying machine under the conditions of temperature and humidity of 37 ℃ and 40% respectively, wherein the spraying amount is 2.0 mL/cm, and thus obtaining the gold-labeled bonding pad.
(5) Production of reagent strip
Under the conditions that the temperature and the humidity are respectively 37 ℃ and 40%, an automatic spraying machine is used for spraying the chlamydia trachomatis antibody and the rabbit anti-mouse secondary antibody onto a nitrocellulose NC membrane to respectively serve as the mass concentration of a detection line (T) and the mass concentration of a quality control line (C line) which are both 1.2 mg/mL and 1.0 mL/cm, and then the membrane is placed at 37 ℃ for vacuum overnight drying; and adhering the processed sample pad, the gold-labeled combination pad, the NC membrane and the absorbent paper on a PVC (polyvinyl chloride) bottom plate, then cutting the sample pad, the gold-labeled combination pad, the NC membrane and the absorbent paper into 4mm wide by an automatic slitter, and packaging the cut sample pad, the gold-labeled combination pad and the NC membrane into cards to obtain the prepared immunochromatographic test strip for detecting the chlamydia trachomatis.
Preparing a reinforcing liquid (the components in the reinforcing liquid A are in mass percentage concentration):
enhancing solution A1: 2% of titanium potassium oxalate and 10% of triton X-100.
Enhancing solution A2: 2.5 percent of titanium potassium oxalate, 5 percent of triton X-100 and 5 percent of Tween-20.
Enhancing solution A3: 3% of titanium potassium oxalate, 3% of triton X-100, 3% of NP40 and 3% of Tween-20.
Enhancing solution A4: 2.5 percent of titanium potassium oxalate, 5 percent of NP40 and 5 percent of Tween-20.
Enhancing solution A5: 2.5% of titanium potassium oxalate, 5% of triton X-100 and 5% of NP 40.
Enhancing solution A6: 2.5% of titanium potassium oxalate, 4% of triton X-100, 3% of Tween-20 and 3% of NP 40.
Enhancing solution B1: 5 μmol/L silver nitrate solution.
Enhancing solution B2: 10. mu. mol/L silver nitrate solution.
Enhancing solution B3: 15 μmol/L silver nitrate solution.
Enhancing solution B4: 20 μmol/L silver nitrate solution.
The enhancement solution is respectively applied to the detection of the chlamydia trachomatis antigen, and the influence of the selection of different enhancement solutions on the detection result is tested, wherein the specific method comprises the following steps: the Chlamydia trachomatis antigen is first diluted to 5X 107 IFU/mL、1×106IFU/mL, using a Chlamydia trachomatis reagent strip to detect, dripping 0.1 mL of the enhancing solution A to a sample adding hole 10min after all the test strips are added into a sample to be detected, dripping 0.1 mL of the enhancing solution B to the sample adding hole 5min after all the test strips are added, and judging negative and positive results after 5 min. The results of the experiments are reported in tables 1 and 2, respectively:
TABLE 1 Chlamydia trachomatis antigen concentration of 5X 107Experimental results at IFU/mL
TABLE 2 Chlamydia trachomatis antigen concentration of 1X 106 IFUResults of the experiment at/mL
As can be seen by combining table 1 and table 2: when the silver ion concentration of the enhancement solution B is 5-15 mu mol/L, a cleaner background can be obtained by adopting the enhancement solutions A1-A6 (except the situation that the enhancement solution A5 is matched with the enhancement solution B3 in the table 1), which indicates that the silver ion concentration of 5-15 mu mol/L is helpful for obtaining the cleaner background. In addition, as can be seen from Table 2, when the concentration of Chlamydia trachomatis antigen is 1X 106 In IFU/mL, only the case of enhancement solution A2 with enhancement solution B3 has higher sensitivity, and combining the case of enhancement solution A2 with enhancement solution B3 in Table 1 has clean background and strong positive result of "+++++", it can be known that enhancement solution A2 using 5% triton X-100 and 5% Tween-20 as surfactant and enhancement solution B3 using 15 μmol/L silver nitrate solution can be matched to obtain higher sensitivity while ensuring clean background.
Therefore, experimental comparison shows that the enhancement solution A prepared by taking triton X-100 and Tween-20 as surfactants and compounding with potassium titanium oxalate is more favorable for improving the detection sensitivity, and the higher silver ion concentration in the enhancement solution B is not favorable for obtaining a clean background, and the silver ion concentration is preferably controlled below 15 mu mol/L.
The detection principle of the enhancement liquid of the present application is adopted:
during detection, the processed sample is dripped into a sample adding hole of the detection card, and the sample liquid is mixed with the chlamydia trachomatis specific antibody marked by colloidal gold particles and the avidin marked by blue latex, which are coated in advance in the colloidal gold combined pad. The mixture is then subsequently chromatographed under capillary effect towards the other end. In the case of positive specimen, the chlamydia trachomatis antibody labeled with the colloidal gold particles is firstly combined with the chlamydia trachomatis antigen in the specimen to form a colloidal gold antibody-antigen complex, and when the colloidal gold antibody-antigen complex passes through a test region in the chromatography process, the complex is captured by another chlamydia trachomatis antibody fixed in the test region to form a colloidal gold antibody-antigen-antibody (fixed on a membrane) sandwich complex. Under the action of the enhancing solution A and the enhancing solution B, the enhancing solution A has reducibility, free silver ions in the enhancing solution B are reduced into silver particles, a large amount of free colloidal gold antibody-antigen-antibody (fixed on a membrane) sandwich complexes are gathered near a gold core, a silver shell is formed on the periphery of the colloidal gold at the test area (T) to finally form the silver particle-colloidal gold antibody-antigen-antibody (fixed on the membrane) sandwich complexes, and a black strip appears in the test area (T). If the specimen is negative, the sandwich complex cannot be formed in the test zone (T) because the specimen does not contain the Chlamydia trachomatis antigen, and no black band will appear. biotin-BSA conjugate is fixed in the quality control region (C) on the membrane, and the blue latex particles marked avidin after chromatography in the capture mixture forms blue (green) latex-avidin-BSA (fixed on the membrane) complex in the quality control region (C). Thus, a blue band appears in the quality control region (C) regardless of the presence of Chlamydia trachomatis antigen in the clinical specimen. The blue band appearing in the quality control region (C) is a standard for judging whether the sample is enough or not and whether the chromatography process is normal or not, and is also used as an internal control standard of the reagent.
And (3) detecting the performance of the chlamydia trachomatis colloidal gold immune test strip:
according to the detection principle, in the following detection process, after all test strips are added into a sample to be detected, the enhancement liquid A is dripped into the sample adding hole 10min, after 5min, the enhancement liquid B is dripped into the sample adding hole, and after 5min, the detection result is judged. (remark: according to the requirement of performance test verification, the experimental data in tables 3, 6 and 7, when judging the result of the reagent strip, the reagent strip is placed on an immunochromatography detector for detection, and the obtained experimental data)
Firstly, sensitivity detection: chlamydia trachomatis antigen was first diluted to 5X 107IFU/mL, then diluted 10 times, using Chlamydia trachomatis reagent strips for detection, recording the results of the experiment, repeating the detection three times for each concentration, calculating the average value of the colloidal gold signal values of the three detection T-lines, see Table 3. Chlamydia trachomatis detection reagent can realize 105And (5) detecting IFU/mL. And produced by British corporationThe British Chlamydia Rapid test kit (immunochromatography) of (U.K.) performs a comparative experiment, and the results are shown in Table 4.
TABLE 3
As can be seen from table 3: the application adopts the chlamydia trachomatis detection reagent of the enhancement liquid to realize 105IFU/mL, and the higher the sample concentration, the more stable the signal value and the more stable the detection result of the product.
TABLE 4
As can be seen from tables 3 and 4: the chlamydia trachomatis detection reagent can realize 1.95 star 105 IFU/mL detection, while British could achieve 3.13 × 106IFU/mL detection, namely compared with British Chlamydia rapid detection kit (immunochromatography) produced by British corporation, the Chlamydia trachomatis detection reagent has 10-fold enhanced sensitivity.
Secondly, specific detection: detecting the influence of the following concentrations of interferents and cross-members on the performance of the Chlamydia trachomatis colloidal gold immune test strip: 50 μ L/mL whole blood, 10mg/mL mucin, 50 μ L/mL urine, 5mg/mL nystatin (suppository), 5mg/mL miconazole (suppository), 5mg/mL tinidazole (gel), 5mg/mL metronidazole (gel), 20 μ L/mL jie' er yin (lotion), 20 μ L/mL skinpro (lotion); proteus, shigella dysenteriae, alpha hemolytic streptococcus, beta hemolytic streptococcus, alpha streptococcus, mycoplasma hominis, ureaplasma urealyticum, human papilloma virus, staphylococcus epidermidis, staphylococcus aureus, escherichia coli, candida albicans, trichomonas vaginalis (all at a microbial concentration of 1 × 10)7IFU/mL). The results are shown in Table 5:
TABLE 5
As can be seen from Table 5, the above interferents and cross-members have no effect on the performance of the Chlamydia detection reagent.
Thirdly, repeatability: the reagent strip was subjected to a repetitive detection test using three concentration samples of high value (2.5E 7), median value (3.13E 6) and low value (7.81E 5), each concentration was measured 10 times, and the average value, standard deviation and coefficient of variation of T-line colloidal gold signal values measured 10 times were calculated. The repeatability is within 20%. The results are shown in Table 6:
TABLE 6
From table 6 it can be seen that: the chlamydia trachomatis detection reagent using the enhancement solution has the variation coefficient within 20% in high, medium and low concentration samples, and the product has good repeatability, thereby meeting the requirements of clinical detection.
Fourthly, stability: placing the test strip at 37 ℃ for one month, taking out the test strip on days 1, 3, 5, 7, 14, 21 and 30 respectively, carrying out repeated detection tests on the test strip by using three concentration samples with a high value (2.5E 7), a medium value (3.13E 6) and a low value (7.81E 5), detecting each concentration for 5 times, recording the average value of T-line colloidal gold signal values measured for 5 times, recording the average value into a table, calculating the statistical result of 7 times of detection, and calculating the average value, the standard deviation and the abnormal coefficient of the T-line colloidal gold signal values. The results are shown in Table 7:
TABLE 7
As can be seen from Table 7, the stability of the test strips was good.
Based on the traditional colloidal gold immunochromatography kit, the application reduces silver ions around gold particles into silver particles under the action of a reducing agent, and the sandwich compound of colloidal gold antibody-antigen-antibody (fixed on a membrane) is prepared "The method is changed into a sandwich compound of silver particles, colloidal gold antibodies, antigens and antibodies (fixed on a membrane), the sensitivity of the reagent strip is effectively increased, and 10 can be realized5 And (5) detecting IFU/mL. Compared with the British Chlamydia immediately testing kit (immunochromatography) produced by British corporation, the sensitivity is enhanced by 10 times. Provides greater help for extensive basic clinical examination.
The present embodiment is merely illustrative and not restrictive, and various changes and modifications may be made by persons skilled in the art without departing from the scope of the present invention as defined in the appended claims. The technical scope of the present application is not limited to the contents of the specification, and must be determined according to the scope of the claims.
Claims (5)
1. An enhancement solution for detecting a chlamydia trachomatis antigen, comprising: the composition comprises an enhancement solution A and an enhancement solution B, wherein the enhancement solution A is 2-3% of titanium potassium oxalate with mass percentage concentration, 4-6% of triton X-100 with mass percentage concentration and 4-6% of tween-20 with mass percentage concentration; the enhancing solution B is 5-15 mu mol/L silver nitrate solution.
2. The enhancing solution for detecting a chlamydia trachomatis antigen according to claim 1, wherein: the concentration of the silver nitrate solution is 15 mu mol/L.
3. Use of an enhancing solution according to claim 1 for the preparation of a kit for the detection of chlamydia trachomatis antigens, wherein: the method comprises the following application steps: firstly, dropwise adding a sample to be detected to the reagent strip, dropwise adding the enhancement liquid A to the reagent strip after a period of time, dropwise adding the enhancement liquid B to the reagent strip after a period of time, and judging a detection result.
4. Use of the enhancing solution of claim 3 for the preparation of a kit for the detection of chlamydia trachomatis antigen, wherein: the application steps are specifically that a sample to be detected is firstly dripped into the reagent strip, the enhancement liquid A is dripped from the sample adding hole after 10min, the enhancement liquid B is dripped from the sample adding hole after 5min, and negative and positive results are judged after 5 min.
5. Use of the enhancing solution of claim 3 for the preparation of a kit for the detection of chlamydia trachomatis antigen, wherein: the dripping amount of the enhancing solution A is 0.1-0.15 mL, and the dripping amount of the enhancing solution B is 0.1 mL.
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