CN111024947A

CN111024947A - Candida albicans fluorescence immunochromatography assay kit and preparation method thereof

Info

Publication number: CN111024947A
Application number: CN201911133741.6A
Authority: CN
Inventors: 董常慧
Original assignee: Jiangsu Mics Medical Technology Co ltd
Current assignee: Jiangsu Mics Medical Technology Co ltd
Priority date: 2019-11-19
Filing date: 2019-11-19
Publication date: 2020-04-17

Abstract

The invention provides a candida albicans fluorescence immunochromatography assay kit and a preparation method thereof, wherein the candida albicans fluorescence immunochromatography assay kit comprises a detection card, an SD card and a sample diluent; the detection card consists of a shell and an immunochromatographic test strip, the immunochromatographic test strip is positioned in the shell and comprises a bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and a sample absorption pad are sequentially arranged on the bottom plate along the length direction of the bottom plate, a fluorescent microsphere marked anti-candida albicans monoclonal antibody is fixed on the combination pad, a detection T line and a quality control C line are arranged on the nitrocellulose membrane, the T line is coated with a monoclonal antibody matched with the fluorescent microsphere marked monoclonal antibody, and the C line is coated with a secondary antibody matched with the fluorescent microsphere marked monoclonal antibody. The kit disclosed by the invention has higher detection sensitivity, accuracy and precision, and is of great significance for dynamically monitoring the patient's condition, performing prognosis evaluation and detecting relapse, and guiding clinicians to make important medical decisions in time.

Description

Candida albicans fluorescence immunochromatography assay kit and preparation method thereof

Technical Field

The invention belongs to the technical field of microbial immunodetection, and particularly relates to a candida albicans fluorescence immunochromatography assay kit.

Background

Candida albicans is a putrefactive parasitic bacterium, a unicellular fungus, is a normal flora in the vagina and lives on the skin and mucous membrane of healthy people, and is in a symbiotic state with the body under normal conditions, so that inflammation is avoided. However, when a certain factor breaks the balance state, the local Candida albicans is transformed from the yeast phase to the hyphal phase, and grows and multiplies a lot in the local area, causing skin, mucous membrane and even systemic candidiasis, so the pathogenic bacteria are called as conditional pathogenic bacteria.

Candidal vulvovaginitis (VVC) is one of the most common female vulvovaginitis and is easy to relapse, wherein 80-90% of candida albicans vaginitis is common from adolescence to the time of menopause. VVC is suffered at least once in the life of 75 percent of women. Because the candidal vaginitis has the characteristics of difficult radical cure and easy recurrence, the candidal vaginitis has higher danger to pregnant women, can increase complications such as premature rupture of fetal membranes, amniotic fluid pollution, premature delivery, labor production, postpartum infection and the like, causes premature delivery, fetal infection and malformation, and threatens the health of mothers and babies, so the candidal vaginitis is timely and accurately diagnosed and identified, effective treatment measures are taken, and the candidal vaginitis has important reality and profound significance for improving the health level of the nation, particularly the female reproductive health level.

The laboratory method for checking candida includes: smear microscopy (wet slide); a culture method; antigen detection methods such as latex agglutination, immunochromatography, immunofluorescence; nucleic acid detection methods, such as PCR. The wet plate method is a clinical routine detection method, the culture method is the most sensitive and specific method for detecting candida, but the method is not usually used as a routine detection method due to the troublesome operation and long time consumption, and the direct immunofluorescence staining and nucleic acid detection method needs corresponding instruments and equipment and technical requirements on operators so as to limit the use of the method. At present, existing companies develop kits for detecting candida albicans, but the kits are all qualitative detection, and the used sample diluent cannot well disperse a detection target object, so that the detection error is large, and the repeatability is poor.

Chinese patent CN107132353A discloses a group B streptococcus detection kit and a preparation method thereof, although quantitative detection results can also be given, the ABS system adopted by the kit has a complex preparation process, and requires two antibodies to be labeled respectively, and two conjugate pads, two sample treatment solutions need to be mixed in proportion when in use, and the system error is large, which is not favorable for CV control.

Disclosure of Invention

The invention aims to provide a candida albicans fluorescence immunochromatography assay kit which is small in detection error and high in accuracy.

The second purpose of the invention is to provide a preparation method of the candida albicans fluorescence immunochromatography assay kit, which has small detection error and high accuracy.

In order to achieve the aim, the invention provides a candida albicans fluorescence immunochromatographic assay kit which is characterized by comprising a detection card, an SD card and a sample diluent;

the detection card consists of a shell and an immunochromatographic test strip, the immunochromatographic test strip is positioned in the shell and comprises a bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and a sample absorption pad are sequentially arranged on the bottom plate along the length direction of the bottom plate, a fluorescent microsphere marked anti-candida albicans monoclonal antibody is fixed on the combination pad, a detection T line and a quality control C line are arranged on the nitrocellulose membrane, the T line is coated with a monoclonal antibody matched with the fluorescent microsphere marked monoclonal antibody, and the C line is coated with a secondary antibody matched with the fluorescent microsphere marked monoclonal antibody;

the SD card stores a standard curve, a calibration mode is established by adopting a relation curve of a flat plate counting method and a densitometry, and the standard curve is established by using a relation curve of calibration concentration and an instrument signal value, so that quantitative detection is realized;

the sample diluent comprises a phosphate buffer and glass beads.

As a preferable scheme, the mass ratio of the Candida albicans monoclonal antibody to the fluorescent microspheres is 0.3-0.7: 10 when the combined pad is prepared.

As a preferable scheme, the combination pad is provided with a blocking 1 and a blocking 2, wherein the blocking 1 is blocking by adding glycine and ethanolamine; and the blocking 2 is blocking by adding BSA and rabbit IgG, the mass ratio of the BSA to the fluorescent microspheres is 4-2: 1, and the mass ratio of the rabbit IgG to the fluorescent microspheres is 0.5-2: 1.

As a preferred embodiment, the sample diluent further comprises a hydrophilic surfactant, and the hydrophilic surfactant is preferably tween 20 or triton 100.

As a preferable scheme, the sample diluent comprises 1001-5 parts of Tween 20 or Triton, 1-5 parts of glass beads and the balance of phosphate buffer solution by 1000 parts by weight.

In order to achieve the second object, the invention provides a preparation method of a candida albicans fluorescence immunochromatography assay kit, which comprises a detection card, an SD card and a sample diluent,

the preparation method of the detection card comprises the steps of sequentially fixing a sample sucking pad, a combination pad and a sample pad on a nitrocellulose membrane base plate coated with an anti-candida albicans monoclonal antibody, cutting the nitrocellulose membrane base plate into test strips, and adding a drying agent for sealing and storing at normal temperature;

the conjugate pad was prepared by the following method:

(1) buffer replacement: adding the particles into MES buffer solution for centrifugation, removing supernatant, adding a small amount of MES for ultrasonic resuspension for later use;

(2) and (3) activation: adding NHS and EDC into the microspheres, and uniformly mixing;

(3) cleaning: after activation, adding MES buffer solution for centrifugation, removing supernatant, and adding a small amount of MES for ultrasonic resuspension; adding HEPES buffer solution for ultrasonic resuspension, filling HEPES for centrifugation, and adding HEPES for resuspension for later use;

(4) antibody reaction: adding a corresponding amount of HEPES pre-dialyzed antibody, wherein the mass ratio of the Candida albicans monoclonal antibody to the fluorescent microspheres is 0.3-0.7: 10, and uniformly mixing and reacting;

(5) and (2) sealing 1: adding glycine and ethanolamine for sealing;

(6) and (2) sealing: adding BSA and rabbit IgG, wherein the mass ratio of the BSA to the fluorescent microspheres is 2:1, and the mass ratio of the rabbit IgG to the fluorescent microspheres is 0.5-2: 1, uniformly mixing, and sealing;

(7) cleaning: adding PBST for cleaning twice, adding a microsphere preservation solution for cleaning once, adding a proper amount of microsphere preservation solution, and preserving at 2-8 ℃ for later use;

(8) paving a bonding pad: diluting the marked microsphere conjugate to a proper concentration, paving the microsphere conjugate on glass fiber, and drying the glass fiber in a blast drying oven;

the cellulose nitrate membrane coated with the anti-candida albicans monoclonal antibody is prepared by the following method:

(1) preparing a T line marking liquid: diluting the Candida albicans monoclonal antibody to 1.0-2.0 mg/mL by using a buffer solution;

(2) c, preparing a line marking liquid: diluting the goat anti-rabbit IgG antibody to 1.5-2.0 mg/mL by using a buffer solution;

(3) coating the C-line antibody and the T-line antibody on a nitrocellulose membrane fixed on a bottom plate respectively by adopting a gold spraying and membrane scratching instrument;

(4) placing the marked large board in a forced air drying box for drying;

the SD card is prepared by the following method:

(1) establishing a relation curve between a candida albicans plate counting method and a densitometry method: preparing Candida albicans suspension with different concentration gradients, respectively measuring OD values at 600nm wavelength, simultaneously diluting the bacterial suspension samples with different gradients, plating the samples on a flat plate for counting, and measuring OD₆₀₀Performing linear regression with the corresponding thallus concentration to obtain a regression equation;

(2) preparing a reference substance: inoculating candida albicans into a liquid culture medium, carrying out shake culture on a shaking table, centrifuging to remove a supernatant, adding physiological saline for washing, adding a small amount of cell frozen stock solution for resuspending thalli, measuring an OD value at a wavelength of 600nm, counting the concentration of a prepared reference product according to a regression equation of a relation between a candida albicans plate counting method and a densitometry, and subpackaging for freezing for later use;

(3) preparing a calibration product: diluting the reference substance to a plurality of concentration points covering a linear range by using a sample diluent, and subpackaging and freezing for later use;

(4) and (3) testing a calibrator: dropwise adding calibrators with different concentrations into a sample adding hole of an immunochromatographic test strip, and reading a detection signal value through a fluorescence immunoassay analyzer;

(5) and (3) generating a calibration curve: a calibration curve for the batch of reagents is generated by a suitable fitting method according to the prepared concentration and fluorescence value of the calibrator, and the curve is introduced into the SD card by instrument software.

Buffers for preparing nitrocellulose membranes coated with anti-candida albicans monoclonal antibodies are generally used: tris (Tris) buffer, boric acid-borax buffer, preferably 0.01mol/LPBS (containing 10g/L sucrose), pH 7.0-7.4.

MES as present in this text means (2-morpholinoethanesulfonic acid) buffer, NHS means (N-hydroxysuccinimide), EDC means ((1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride)), HEPES means (4-hydroxyethylpiperazineethanesulfonic acid) buffer.

The phosphate buffer solution is 0.01-0.02 mol/L PBS, and the pH value is 7.0-7.4.

The principle of quantitative detection is to link the plate counting method for detecting viable bacteria and the optical density method for representing the final concentration of viable bacteria and dead bacteria, thereby solving the problem that the test strip immunological detection cannot distinguish the dead bacteria and the viable bacteria.

The determination method of the kit comprises the following steps:

(1) and opening the fluorescence immunoassay analyzer, and inserting the SD card into a corresponding socket of the fluorescence immunoassay analyzer.

(2) And (3) placing the sampling swab in a diluent, uniformly mixing, and dropwise adding 100-120 mu L of the sampling swab into a sample adding hole of an immunochromatography test strip.

(3) And incubating for 15-30 min at room temperature, and putting the detection card into a fluorescence immunoassay analyzer to read the detection result.

The invention has the advantages that (1) the invention adopts the fluorescence immunochromatography technology, not only retains the advantages of on-site rapid detection of the traditional colloidal gold test strip, but also increases the advantages of high sensitivity, good stability, low interference of natural fluorescence and the like, has higher detection sensitivity, and the linear range of the detection is 10⁴cfu/mL～10⁷cfu/mL, Linear correlation coefficient>0.99, and simultaneously, the matched equipment is exquisite, does not need large-scale instrument equipment, is suitable for primary hospitals and large hospital clinical laboratories. (2) The sample diluent contained in the invention can disperse the Candida albicans which is easy to gather into a single thallus, so that the sample is more uniform, and the accuracy and precision of detection are improved. The kit isAverage recovery rate is 90% -110%, and average precision<15 percent, and the clinical sample coincidence rate is more than 99 percent.

Drawings

FIG. 1 is a schematic structural diagram of a Candida albicans fluorescence immunochromatographic test strip;

FIG. 2 is a microscopic image of Candida albicans in PBS dilution;

FIG. 3-1 is a microscopic view of Candida albicans in PBS +1 part Tween 20 dilution;

FIG. 3-2 is a microscopic image of Candida albicans in PBS +3 parts Tween 20 dilution;

FIGS. 3-3 are microscopic images of Candida albicans in PBS +5 parts Tween 20 dilution;

FIG. 4-1 is a microscopic image of Candida albicans in PBS +1 part of Triton 100 dilution;

FIG. 4-2 is a microscopic image of Candida albicans in PBS +3 parts of Triton 100 dilution;

FIGS. 4-3 are microscopic images of Candida albicans in PBS +5 parts of Triton 100 dilution;

FIG. 5 is a graph showing the relationship between the plate count method and the densitometry method for Candida albicans;

FIG. 6 is a four parameter fit plot of calibrator concentration versus signal value.

Icon: 101-a base plate; 102-a sample pad; 103-a conjugate pad; 104-nitrocellulose membrane; 105-a draw pad; 106-detection line; 107-quality control line.

Detailed Description

In order to further understand the technical features of the present invention, the technical solutions of the present invention will be described in detail below with reference to the specific embodiments and the accompanying drawings. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. The examples are given for illustrative purposes only and are not intended to be limiting. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The reagents or instruments used, not specifically noted by the manufacturer, are conventional products available commercially.

Example 1: bond pad preparation

1. Buffer solution replacement: 1mg of 200nm fluorescent microspheres was added with 1mL of 100mM MES (hereinafter, MES was 100mM) and centrifuged (14000g 30 min). Add 500. mu.L MES and resuspend by ultrasound for further use.

2. And (3) activation: adding 0.06mg NHS, mixing, adding 0.1mg EDC, adding 500 μ L MES, and rotating at 37 deg.C for 1hr (rotation speed 40-60).

3. Cleaning: after the activation, 1mL MES was added, centrifuged (14000g 30min), the supernatant was removed, 500. mu.L of HEPES was added and the mixture was ultrasonically resuspended, and the procedure was repeated once, 50mM HEPES was added, 500. mu.L of pH8.0 (hereinafter all HEPES are 50mM, pH8.0), ultrasonically resuspended, 1mL HEPES was added and centrifuged, and 500. mu.L of HEPES was added and ultrasonically resuspended for use.

4. Antibody reaction: adding 0.03mg antibody (antibody HEPES pre-dialyzed), mixing, and reacting at 37 deg.C for 3 hr.

5. And (2) sealing 1: add 2M Gly7 μ L and 1M EA (ethanolamine) 14 μ L, and rotate at 37 deg.C for 1 hr.

6. And (2) sealing: respectively closed as follows: adding 2mg BSA and 0.5mg rabbit IgG, and performing rotary reaction at 2-8 ℃ for 12-18 h.

7. Cleaning: adding 1mL of PBST, centrifuging (14000g for 30min), removing supernatant, adding 500 mu L of PBST, performing ultrasonic resuspension, supplementing 1mLPBST, centrifuging (14000g for 30min), removing supernatant, adding 500 mu L of microsphere storage solution, centrifuging (14000g for 30min), removing supernatant, adding 100 microliter of microsphere fixing solution, performing ultrasonic resuspension, fixing on a bonding pad according to the using concentration of 1/50, placing in a forced air drying oven, and drying at 37 ℃ for 4 hr.

Example 2: bond pad preparation

The addition of the antibody was 0.05mg and the addition of the rabbit IgG was 1mg, and the other steps were the same as in example 1.

Example 3: bond pad preparation

The addition of the antibody was 0.05mg and the addition of the rabbit IgG was 2mg, the other steps were the same as in example 1.

Example 4: bond pad preparation

The addition of the antibody was 0.07mg and the addition of the rabbit IgG was 2mg, and the rest of the procedure was the same as in example 1.

Example 5: preparation of anti-candida albicans antibody coated by nitrocellulose membrane

Preparing a T-line marking liquid: the anti-Candida albicans monoclonal antibody is diluted to 1.0mg/mL by adopting a scratching diluent, and is uniformly mixed, wherein the scratching diluent is 0.01mol/L PBS (containing 10g/L sucrose), and the pH value is 7.0-7.4.

Preparing a C line marking liquid: diluting goat anti-rabbit IgG antibody to 1.5mg/mL by adopting a membrane-scratching diluent, and uniformly mixing, wherein the membrane-scratching diluent is 0.01mol/L PBS (containing 10g/L sucrose) and has a pH value of 7.0-7.4.

3. Adopting a gold spraying membrane scribing instrument, coating the C line antibody and the T line antibody on a nitrocellulose membrane fixed on a bottom plate respectively, and scribing the liquid volume: 1 mu L/cm; film scratching speed: 50 mm/s.

4. Placing the marked large board in a forced air drying oven, and drying for 16h at 37 ℃;

example 6: preparation of anti-candida albicans antibody coated by nitrocellulose membrane

The concentration of the anti-Candida albicans monoclonal antibody in the T-line marking solution is 1.5mg/mL, and the rest steps are the same as the example 5.

Example 7: preparation of anti-candida albicans antibody coated by nitrocellulose membrane

The concentration of the anti-Candida albicans monoclonal antibody in the T-line marking solution is 2.0mg/mL, and the rest steps are the same as the example 5.

Example 8: preparation of anti-candida albicans antibody coated by nitrocellulose membrane

The concentration of goat anti-rabbit IgG antibody in the C-line marking solution is 2.0mg/mL, and the rest steps are the same as example 5.

Example 9: preparation of anti-candida albicans antibody coated by nitrocellulose membrane

The concentration of the anti-candida albicans monoclonal antibody in the T line marking solution is 2.0mg/mL, the concentration of the goat anti-rabbit IgG antibody in the C line marking solution is 1.5mg/mL, and the rest steps are the same as the example 5.

Example 10: preparation of fluorescence immunochromatographic test strip

A fluorescence immunochromatographic test strip (figure 1) comprises a bottom plate 101, wherein a sample pad 102, a combination pad 103, a nitrocellulose membrane 104 and a sample absorbing pad 105 are sequentially arranged on the bottom plate 101 along the length direction; the sample pad 102 is partially overlapped with the combination pad 103, the combination pad 103 is partially overlapped with the nitrocellulose membrane 104, one side edge of the combination pad 103 is positioned below the sample pad 102, and the other opposite side edge of the combination pad 103 is positioned above one side edge of the nitrocellulose membrane 104; the nitrocellulose membrane 104 is partially overlapped with the blotting pad 105, and the opposite side edge of the nitrocellulose membrane 104 is positioned below the blotting pad 105; the nitrocellulose membrane 104 is provided with a detection line 106 and a quality control line 107, and the detection line 106 is close to the binding pad 103.

Fixing a sample sucking pad, a combination pad and a sample pad on a nitrocellulose membrane base plate coated with an antibody in sequence; cutting into test paper strips, adding a drying agent, and sealing and storing at normal temperature.

Example 11: preparation of sample dilutions

The sample dilution is 0.01mol/L PBS, pH7.4, in 1000 portions, which contains 1 portion of Tween 20, 1 portion of glass beads.

Example 12: preparation of sample dilutions

The sample dilution is 0.01mol/L PBS, pH7.4, in 1000 portions, which contains 3 portions of Tween 20, 1 portion of glass beads.

Example 13: preparation of sample dilutions

The sample dilution is 0.01mol/L PBS, pH7.4, in 1000 portions, which contains 5 portions of Tween 20, 1 portion of glass beads.

Example 14: preparation of sample dilutions

The sample dilution was 0.01mol/L PBS, pH7.4, and contained 1 part of

Triton

100 and 1 part of glass beads in 1000 parts.

Example 15: preparation of sample dilutions

The sample dilution was 0.01mol/L PBS, pH7.4, and contained 3 parts of

Triton

100, 1 part of glass beads, in 1000 parts.

Example 16: preparation of sample dilutions

The sample dilution was 0.01mol/L PBS, pH7.4, and contained 5 parts of

Triton

100, 1 part of glass beads, in 1000 parts.

To verify the dispersion effect of Candida albicans in the modified sample diluent, Candida albicans was diluted to 10 with 0.01mol/L PBS and different amounts of Tween 20 and Triton 100, respectively⁷cfu/mL, stained with a double fluorescent staining solution produced by Jiangsu Meike medical technology Co., Ltd, placed under a biological (fluorescent) microscope, and subjected to microscopic examination by selecting blue light with a wavelength of 450nm-500nm as excitation light to observe the dispersibility of Candida albicans. The result of the PBS diluent is shown in figure 2, and the result of the PBS plus Tween 20 diluent is shown in figures 3-1, 3-2 and 3-3. The microscopic examination results of the PBS + Triton 100 diluent are shown in figures 4-1, 4-2 and 4-3, and the microscopic examination results show that the diluents with Tween 20 and Triton 100 can uniformly disperse Candida albicans in the solution, and the Candida albicans can obviously agglomerate in the pure PBS diluent.

Example 17: calibration curve creation

1. Preparation of bacterial suspensions with different optical density values: the candida albicans activated on the SDA solid culture medium is picked by an inoculating needle and evenly mixed in sterile physiological saline to prepare bacterial suspension. And the OD value was measured at a wavelength of 600 nm. Preparing bacterial suspension with OD value of about 0.8 as original bacterial liquid sample, diluting the original bacterial liquid sample in gradient and preparing 5-7 bacterial suspension samples with OD value of 0.2-0.8.

2. Diluting and coating a flat plate: diluting the prepared bacterial suspension samples with different gradients, coating the samples on a flat plate, and culturing the samples on a PDA culture medium at 37 ℃ for 24h for counting.

3. And (3) generating a calibration curve: the cell concentration was calculated from the dilution factor and the amount of applied solution, and the results are shown in Table 1. Will OD₆₀₀And the corresponding cell concentration is linearly regressed to obtain a regression equation y as ax + b (as shown in FIG. 5).

TABLE 1 Candida albicans plate count results

Example 18: reference preparation

1. Inoculation: the Candida albicans activated on the SDA solid medium is inoculated in the SDA liquid medium and shake-cultured for 24h at 37 ℃.

2. Cleaning: centrifuging the cultured candida albicans to remove supernatant, adding physiological saline, centrifuging and washing for 2-3 times, and adding a small amount of cell frozen stock solution to resuspend the bacteria.

3. Calibrating a reference product: diluting to a certain proportion with 0.01mol/LPBS, measuring OD value (OD value should be between 0.2-0.8) at 600nm wavelength, and counting the concentration of the reference product according to the relation regression equation y ═ ax + b between Candida albicans plate counting method and densitometry, and the result is shown in Table 2. Subpackaging and freezing for later use. The cell cryopreservation solution formulation is shown in table 3.

TABLE 2 reference calibration results

TABLE 3 cell cryopreservation solution formula

Example 19: preparation of SD card

1. Preparing a calibration product: the reference was diluted with sample diluent to several concentration points covering the linear range, 1X 10 each⁴cfu/mL、1×10⁵cfu/mL、5×10⁵cfu/mL、1×10⁶cfu/mL、5×10⁶cfu/mL、1×10⁷And (5) subpackaging cfu/mL for freezing and storing for later use. The sample dilutions were as in example 12.

2. And (3) testing a calibrator: and (3) dripping 120 mu L of the calibrator with different concentrations into a sample adding hole of the immunochromatographic test strip, and reading a detection signal value through a fluorescence immunoassay analyzer after 20 min. The results are shown in Table 4. The immunochromatographic test strip used was prepared as in example 3, example 5, and example 10.

Table 4 calibrator signal value test results

3. And (3) generating a calibration curve: and generating a calibration curve of the batch of reagents by adopting a four-parameter fitting mode according to the prepared concentration and the fluorescence value of the calibrator, and introducing the curve into the SD card by using instrument software, wherein the equation of the calibration curve is shown in Table 5, and the curve shape is shown in FIG. 6.

TABLE 5 calibration curve equation

Calibration curve equation of (A-D)/[1+ (x/C) ^ B ] + D

A＝13.51976

B＝-0.69661

C＝1254.20974

D＝-0.05757

r^2＝0.99959

Example 20: blank limit test of kit

And taking the sample diluent as a blank sample, dropwise adding 120 mu L of the sample diluent into a sample adding hole of an immunochromatographic test strip, and reading a detection signal value through a fluorescence immunoassay analyzer after 20 min. And repeating the test for 20 times, calculating an average signal value M and a standard deviation SD, substituting M +2SD into a standard curve, and calculating a corresponding concentration value, namely the blank limit of the kit. The results are shown in Table 6. The immunochromatographic test strip is prepared as in example 3, example 5 and example 10, the sample diluent is shown in example 12, and the SD card is shown in example 19.

TABLE 6 blank limits of the kit

Example 21: in-batch precision testing of kits

Diluting the reference substance to high, medium and low concentrations by using a sample diluent, dropwise adding 120 mu L of the reference substance to a sample adding hole of an immunochromatographic test strip, and reading the detection concentration by a fluorescence immunoassay analyzer after 20 min. The test was repeated 10 times for each concentration, and the mean value M and standard deviation SD were calculated, and CV was calculated, respectively (CV ═ SD/mx 100%). The results are shown in Table 7. The immunochromatographic test strip is prepared as in example 3, example 5 and example 10, the sample diluent is shown in example 12, and the SD card is shown in example 19.

TABLE 7 in-batch precision of kits

Example 22: inter-batch precision testing of kits

And (3) diluting the reference substance to high, medium and low concentrations by using the sample diluent, respectively using three kits of different batches to repeatedly test for 3 times, and calculating the relative range difference. The results are shown in Table 8. The kit was prepared as in example 3, example 5, example 10, example 12, example 19.

TABLE 8 batch-to-batch precision of kits

Example 23: accuracy testing of kits

Adding reference substance with certain concentration into low-value clinical sample to make its final concentration be 10⁵About cfu/mL (medical decision level), the volume of the reference substance added should not be higher than 1/20 of the final volume, and 3 replicates were performed using the kit to test the low sample concentration and the sample concentration after addition of the reference substance. The recovery rates R were calculated according to the following formulas, respectively, and the results are shown in Table 9.

R-recovery rate;

v-volume of added reference solution;

v0 — volume of human vaginal secretion sample;

c is the concentration of the human vaginal secretion sample added with the reference solution;

c0 — measured concentration of human vaginal secretion sample;

cs — concentration of reference solution.

TABLE 9 recovery test results

Example 24: detecting clinical samples

Collecting the samples of a case group and a control group determined by a gold standard, synchronously measuring by using an examination reagent (the kit) and a reference reagent (Beijing Thailand letter kit), and calculating the statistical index of the coincidence or difference degree of the measurement result of the examination reagent and the measurement result of the reference reagent, wherein the evaluation method comprises the following steps:

positive compliance rate: the proportion of the positive samples detected by the examination reagent to the total number of the positive samples detected by the reference reagent is the positive coincidence rate.

Negative coincidence rate: the proportion of the sample which is negative detected by the examination reagent to the total number of the negative samples detected by the reference reagent is negative coincidence rate.

The total coincidence rate is as follows: and the proportion of the samples with consistent detection results of the examination reagent and the reference reagent in the total samples, namely the total coincidence rate.

The consistency ratio is: and calculating the Kappa coefficient, wherein if the Kappa coefficient is more than or equal to 0.75, the consistency rate is good.

Expressed by the following formula:

positive coincidence rate a/(a + C) × 100% negative coincidence rate D/(B + D) × 100%

The total coincidence rate is (a + D)/(a + B + C + D) × 100%

Kappa coefficient 2 × (a × D-B × C)/[ (a + B) × (B + D) + (a + C) × (C + D) ]

The results are shown in Table 10.

TABLE 10 comparative test results

The positive coincidence rate is 99/(1+99) × 100% is 99%;

the negative coincidence rate is 99/(1+ 99). times.100%. 99%;

the total coincidence rate is (99+99)/(100+100) 99%;

the Kappa coefficient was 2 × (99 × 99-1 × 1)/[ (99+1) × (1+99) + (99+1) × (1+99) ] -0.98.

As can be seen from the comparison results, the reagent has higher consistency with the reference reagent. Two samples with inconsistent comparison are compared with the microscopic examination result, the detection result of the reagent is consistent with the microscopic examination result, and the performance of the reagent is better than that of a reference reagent.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. The candida albicans fluorescence immunochromatography assay kit is characterized by comprising a detection card, an SD card and a sample diluent;

the sample diluent comprises a phosphate buffer and glass beads.

2. The Candida albicans fluorescence immunochromatography assay kit according to claim 1, wherein the mass ratio of the Candida albicans monoclonal antibody to the fluorescent microspheres is 0.3-0.7: 10 when the conjugate pad is prepared.

3. The candida albicans fluorescence immunochromatography assay kit according to claim 1, wherein the binding pad is provided with a block 1 and a block 2, the block 1 is a block added with glycine and ethanolamine; and the blocking 2 is blocking by adding BSA and rabbit IgG, the mass ratio of the BSA to the fluorescent microspheres is 4-2: 1, and the mass ratio of the rabbit IgG to the fluorescent microspheres is 0.5-2: 1.

4. The candida albicans fluorescence immunochromatographic assay kit according to claim 1, wherein the sample diluent further comprises a hydrophilic surfactant, and the hydrophilic surfactant is preferably tween 20 or triton 100.

5. The Candida albicans fluorescence immunochromatography assay kit according to claim 4, wherein the sample diluent comprises, by weight of 1000 parts, 1001-5 parts of Tween 20 or Triton, 1-5 parts of glass beads and the balance of phosphate buffer.

6. The method for preparing Candida albicans fluorescence immunochromatography assay kit according to claim 1, wherein the kit comprises a detection card, an SD card and a sample diluent,

the conjugate pad was prepared by the following method:

(5) and (2) sealing 1: adding glycine and ethanolamine for sealing;

(4) placing the marked large board in a forced air drying box for drying;

the SD card is prepared by the following method: