CN114076824A - Multiplex ELISA kit for bladder cancer diagnosis - Google Patents
Multiplex ELISA kit for bladder cancer diagnosis Download PDFInfo
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- CN114076824A CN114076824A CN202111225655.5A CN202111225655A CN114076824A CN 114076824 A CN114076824 A CN 114076824A CN 202111225655 A CN202111225655 A CN 202111225655A CN 114076824 A CN114076824 A CN 114076824A
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Abstract
A multiplex ELISA kit for bladder cancer diagnosis belongs to the field of immunological detection. The kit for diagnosing the bladder cancer comprises a solid phase carrier and a primary antibody corresponding to a tumor-associated antigen coated on the solid phase carrier, wherein the tumor-associated antigen is Bladder Tumor Antigen (BTA), CD44 and Survivin (Survivin). The invention provides a multiplex ELISA kit for diagnosing bladder cancer, which has the characteristics of high sensitivity, strong specificity, simple operation and stable performance.
Description
Technical Field
The invention relates to a multiplex ELISA kit for bladder cancer diagnosis, which adopts a double-antibody sandwich enzyme-linked immunosorbent assay and a biotin-avidin biological reaction amplification system, and belongs to the field of immunological detection.
Background
Bladder cancer refers to malignant tumor occurring on the mucous membrane of the bladder, is the most common malignant tumor of the urinary system, is also a common malignant tumor worldwide, and has the 7 th incidence rate in the male systemic tumor. Generally, bladder cancer can be diagnosed only in the middle and later stages, about 75% of bladder cancer is non-muscle-layer-infiltrating bladder cancer, the 5-year survival rate is 88% -98%, while the 5-year survival rate of the muscle-layer-infiltrating bladder cancer is only 46% -63%, and more than 70% of patients still relapse after being treated, so that huge physical and psychological damage is brought to the patients. Therefore, it is important for early diagnosis of bladder cancer patients.
The traditional diagnosis means of bladder cancer are many, and the current early diagnosis methods mainly comprise clinical manifestations, urine cast-off cytology examination, optical imaging, tumor marker detection, imaging examination, cystoscopy biopsy, pathological examination after diagnostic electrostomy and the like. In clinic, cystoscopy is the gold standard for bladder cancer diagnosis, but the method is invasive, complex to operate, low in sensitivity and specificity and easy to have a high false positive rate in early diagnosis of bladder cancer. The cystoscope is an invasive operation, belongs to invasive examination, is painful for patients, is not suitable for the operation when the patients have urinary system infection, and even has the possibility of causing the urinary system infection in the cystoscope examination.
The occurrence of bladder cancer has a direct influence on the composition of blood and urine, so that a non-invasive liquid biopsy will bring a new detection method for early diagnosis of bladder cancer. The liquid biopsy is a bladder cancer biomarker detection method with good development prospect, can perform combined analysis and detection on a plurality of biomarkers, and further improves the sensitivity and specificity of a detection result. The liquid biopsy has higher efficiency, the detection time is generally within a few minutes, and the rapid detection of the tumor marker can be realized. In addition, the liquid biopsy is easy to carry, is widely applied to disease diagnosis and has good application prospect. With the development of molecular biology in recent years, the molecular biological diagnosis of bladder cancer has made great progress, and the method provides powerful support for the bladder cancer liquid biopsy technology.
Bladder tumor antigen is one of the most studied bladder cancer tumor markers in the clinic in recent years. Bladder tumor cells can degrade Bladder basement membrane to form basement membrane complexes, and these degradation products are excreted into the Bladder to form Bladder Tumor Antigen (BTA), also known as complement factor H related protein (HCFHrp). Two BTA detection reagents, BTA-Stat and BTA-Trak, are currently approved by the FDA, and studies have shown that BTA-Stat has a sensitivity and specificity of 64% and 77% respectively, and BTA-Trak has a sensitivity and specificity of 65% and 74% respectively. BTA is therefore a very potential tumor marker.
Survivin (Survivin) protein is a cytoplasmic protein with the molecular weight of 16.5kD, is the protein with the strongest anti-apoptosis activity in an apoptosis inhibiting protein family, has high expression in various malignant tumor tissues of human, such as gastric cancer, prostate cancer and the like, is more obviously expressed in poorly differentiated tissues, is not expressed in normal cells and tissues, can directly act on Caspase, and can block apoptosis mainly by inhibiting Caspase-3, -7. Survivin is expressed at higher levels in bladder cancer tissues than in paracarcinoma tissues, with a corresponding increase in the level of expression as the tumor grade and stage increases. Research shows that the survivin expression rate of the muscle-layer invasive bladder cancer is higher than that of the non-muscle-layer invasive bladder cancer, and the survivin expression rate of the low-differentiation tumor is higher than that of the medium-differentiation tumor, which indicates that the survivin expression is closely related to the staging and grading of the tumor, and is an important reference index for early diagnosis of the bladder tumor.
CD44 is a glycoprotein that plays a role in cell-to-cell, cell adhesion and migration, is involved in a variety of cellular functions, and is often accompanied by abnormal expression of CD44 during the development, progression and metastasis of many tumors. The research finds that the level of CD44 in urine of bladder tumor patients is obviously higher than that of healthy volunteers, the expression of CD44 in the urothelium of normal people is low, when the urothelium is subjected to early catastrophe, the expression of CD44 is gradually increased, and then the expression of CD44 is gradually reduced along with the tumor development and invasion, the sensitivity can reach 81.1 percent, and the specificity is close to 100 percent, so the CD44 is an important index for early diagnosis of bladder cancer.
BTA, CD44 and Survivin have been reported in literature to be related to the diagnosis of bladder cancer, but 3 combined tests for bladder cancer have not been reported. The currently disclosed kit related to bladder cancer diagnosis mostly uses blood or serum as a detection object, and the detection of bladder cancer by a tumor marker in urine is not reported.
Disclosure of Invention
Aiming at the problems and the defects in the prior art, the invention provides a multiplex ELISA kit for diagnosing bladder cancer, which has the characteristics of high sensitivity, strong specificity, simple operation and stable performance.
The technical scheme adopted by the invention is as follows: the combination of BTA, Survivin and CD44 protein joint diagnosis in urine sample is used in the multiple ELISA kit for bladder cancer diagnosis.
The multiplex ELISA kit for bladder cancer diagnosis comprises an ELISA plate, a standard substance, a dilution bottle, a washing solution, streptavidin-coupled horseradish peroxidase working solution, a biotin-labeled secondary antibody, a developing solution and a stop solution. The sample diluent is PBST (phosphate Tween) buffer solution containing 1% (W/V) BSA; the color developing liquid consists of a color developing liquid A and a color developing liquid B, wherein the color developing liquid A is 0.02% (W/V) TMB (3,3 ', 5, 5' -tetramethylbenzidine), and the color developing liquid B is 0.006% (W/V) carbamide peroxide; the stop solution is 2mol/L sulfuric acid; the washing solution is PBST (phosphate-Tween) buffer solution containing 0.2% Tween-20.
The second antibody is coupled with biotin and has the function of amplifying reaction.
The multiplex ELISA kit for diagnosing the bladder cancer is characterized in that the marker is horseradish peroxidase coupled with streptavidin.
A multiplex ELISA kit for bladder cancer diagnosis contains three coated 96-well ELISA plates, which are respectively coated with a first antihuman BTA, a first antihuman CD44 and a first antihuman Survivin, and the well plates are detachable. When in use, three plates must be used simultaneously, and the number of the plates can be selected according to the number of samples.
A multiplex ELISA kit for diagnosing bladder cancer is used for detecting human urine samples.
Compared with the prior art, the technical scheme provided by the invention has the following advantages:
(1) the kit on the market at present mostly uses an index as a detection object, a single marker is difficult to simultaneously achieve higher sensitivity and specificity to detect the occurrence of bladder cancer, but the multiplex ELISA kit for diagnosing bladder cancer prepared by the invention can simultaneously detect the expression levels of 3 proteins of Survivin, CD44 and BTA in a urine sample, compared with the detection of a single marker, the combined detection of the three has high detection success rate and good technical reproducibility, the sensitivity of bladder cancer diagnosis is 96.8%, the specificity is 80.0%, and clinical reference values can be provided for early diagnosis and treatment of bladder cancer patients.
(2) The multiple ELISA kit for early bladder cancer diagnosis provided by the invention takes urine as a detection object, most of the kits for bladder cancer diagnosis in the market take serum, plasma or tissue homogenate as the detection object, and the content of protein in the plasma or the serum is high, so that interference can be generated in the detection process, false positive or false negative can be caused, and the detection result is inaccurate. Compared with a serum marker, the urine marker has more advantages, the urine is easy to collect and can be repeatedly detected, the urine is directly contacted with the bladder, protein pollution from other organs is reduced, the urine detection belongs to non-invasive operation, and the patient compliance is higher.
(3) The enzyme label plate provided by the invention is detachable, and the number of the plate strips can be selected automatically according to the requirement and can be assembled freely.
(4) The kit provided by the invention has better sensitivity and specificity, simple used equipment and easy operation, and can obtain results within half a day.
Drawings
FIG. 1 is a BTA protein standard curve.
FIG. 2 is a standard curve of CD44 protein.
Fig. 3 is a Survivin protein standard curve.
FIG. 4 shows the ELISA detection of OD in patients and normal human urine by BTA, Survivin and CD44450Test characteristic curve of values.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the scope of the present invention is not limited thereto. In addition, the specific technical operation steps or instruments are not indicated by manufacturers in the examples, and all the conventional products are commercially available.
Example 1 preparation of an Elisa plate coated with a Primary antibody
1. Test materials and reagents
(1) Coating buffer solution: 50mM carbonate buffer, pH 9.6 was used.
(2) Washing liquid: PBST (phosphate-Tween) buffer containing 0.2% (v/v) Tween-20 was selected.
(3) The blocking solution is PBST (phosphate-Tween) buffer containing 2% (W/V) BSA.
2. Coating:
enzyme label plate 1: coating buffer solution and 5.0 mu g/mL first anti-human BTA antibody are prepared into coating solution, the coating solution is gently mixed, 100 mu L/well is added into a 96-well plate, a sealing covering film is placed into a 37 ℃ incubator to be incubated for 1h, and then the coating solution is coated in a 4 ℃ refrigerator overnight. Carefully uncovering the sealing plate membrane, discarding liquid, spin-drying, filling diluted washing liquid into each hole, standing for 1 minute, discarding, repeating the steps for 5 times, and buckling and drying on the filter paper each time. Then adding 100. mu.L/well of blocking solution, blocking at 37 ℃ for 2h, discarding the supernatant, and then washing the plate 3 times with PBST, each time for 3 min. And finally, placing the 96-hole enzyme label plate subjected to sealing treatment in a 37 ℃ drying box for drying, and packaging to obtain a coated 96-hole enzyme label plate, and storing at 4 ℃ for later use.
Enzyme label plate 2: the coating buffer and 5.0 mu g/mL of first anti-human CD44 antibody are prepared into a coating solution, the coating solution is gently mixed, 100 mu L/well is added into a 96-well plate, a sealing covering film is placed into a 37 ℃ incubator to be incubated for 1h, and then the coating solution is coated in a 4 ℃ refrigerator overnight. Carefully uncovering the sealing plate membrane, discarding liquid, spin-drying, filling diluted washing liquid into each hole, standing for 1 minute, discarding, repeating the steps for 5 times, and buckling and drying on the filter paper each time. Then adding 100. mu.L of blocking solution per well, incubating at 37 ℃ for 2h, discarding the supernatant, and then washing the plate 3 times with washing solution, each time for 3 min. And finally, placing the 96-hole enzyme label plate subjected to sealing treatment in a 37 ℃ drying box for drying, and packaging to obtain a coated 96-hole enzyme label plate, and storing at 4 ℃ for later use.
③ the ELISA plate 3: coating buffer solution and 2.5 mu g/mL first antihuman Survivin antibody are prepared into coating solution, the coating solution is gently mixed, 100 mu L/well is added into a 96-well plate, a sealing covering film is placed into a 37 ℃ incubator to be incubated for 1h, and then the coating solution is coated in a 4 ℃ refrigerator overnight. Carefully uncovering the sealing plate membrane, discarding liquid, spin-drying, filling diluted washing liquid into each hole, standing for 1 minute, discarding, repeating the steps for 5 times, and buckling and drying on the filter paper each time. Then adding 100. mu.L/well of blocking solution, blocking at 37 ℃ for 2h, discarding the supernatant, and then washing the plate 3 times with PBST, each time for 3 min. And finally, placing the 96-hole enzyme label plate subjected to sealing treatment in a 37 ℃ drying box for drying, and packaging to obtain a coated 96-hole enzyme label plate, and storing at 4 ℃ for later use.
Example 2 preparation of working solution of Biotin-labeled Secondary antibody
1. Preparation of sample diluent:
sample dilutions contained 1% (W/V) BSA in PBST buffer.
2. Preparation of biotin-labeled secondary antibody working solution:
diluting a biotin-labeled second anti-BTA antibody with a sample diluent according to the proportion of 1:10000(v/v), and uniformly mixing for later use;
secondly, diluting a biotin-labeled second anti-CD 44 antibody with a sample diluent according to the proportion of 1:10000(v/v), and uniformly mixing for later use;
③ diluting the biotin-labeled secondary antibody Survivin antibody with sample diluent according to the proportion of 1:500(v/v), and mixing uniformly for later use.
EXAMPLE 3 composition of the kit of the invention
(1) Coated 96-well microplate prepared in example 1;
(2) sample diluent: PBST buffer containing 1% (W/V) BSA;
(3) washing liquid: PBST (phosphate-tween) buffer containing 0.2% tween 20;
(4) a biotin-labeled secondary antibody working solution prepared in example 2;
(5) horse radish peroxidase-labeled streptavidin solution;
(6) color development liquid: the developing solution consists of a developing solution A and a developing solution B, wherein the developing solution A is 0.02% (W/V) TMB, and the developing solution B is 0.006% (W/V) carbamide peroxide; when in use, the color development liquid A and the color development liquid B are uniformly mixed in equal volume according to the ratio of 1: 1; (7) stopping liquid: 2mol/L sulfuric acid.
Second, using method of kit
(A) Using step
1. Preparation before experiment: the kit was removed from the 4 ℃ freezer and allowed to equilibrate to room temperature (18-25 ℃). The reagents in the kit are placed in an ice box, and attention is paid to fully and uniformly mixing all the reagents before use, so that a large amount of bubbles are not generated in the liquid to avoid error of the result.
2. Sample adding and incubation:
the kit comprises three coated 96-well enzyme-labeled plates which are respectively coated with a first anti-BTA, a first anti-CD 44 and a first anti-Survivin protein, the pore plates are detachable, three enzyme-labeled plates are required to be used simultaneously when the kit is used, and the number of the plates can be selected according to the number of samples.
Enzyme label plate 1: and respectively taking enough coating battens according to the number of the samples to be detected and the number of the standard products, fixing the coating battens on the frame, and recording the positions of the holes. Each standard, sample and blank well is recommended to be 2 replicate wells, 100. mu.L/well. BTA standard was diluted to 0.5ng/ml, 1.0ng/ml, 2.0ng/ml, 4.0ng/ml, 8.0ng/ml, 16.0ng/ml and added to the standard. Adding the sample to the bottom of the enzyme label plate, keeping the sample from touching the hole wall as much as possible, slightly shaking and uniformly mixing, covering a sealing plate film, and incubating for 60 minutes at room temperature. (setting the concentration of the standard substance as 0. mu.g/mL as a negative control, and finally, only adding the chromogenic substrate and the stop solution without other antigen-antibody components as blank wells for zero setting)
Enzyme label plate 2: and respectively taking enough coating battens according to the number of the samples to be detected and the number of the standard products, fixing the coating battens on the frame, and recording the positions of the holes. Each standard, sample and blank well is recommended to be 2 replicate wells, 100. mu.L/well. The CD44 standard was diluted to 0.31ng/ml, 0.62ng/ml, 1.25ng/ml, 2.5ng/ml, 5ng/ml, 10ng/ml and added to the standard. Adding the sample to the bottom of the enzyme label plate, keeping the sample from touching the hole wall as much as possible, slightly shaking and uniformly mixing, covering a sealing plate film, and incubating for 60 minutes at room temperature. (setting the concentration of the standard substance as 0. mu.g/mL as a negative control, and finally, only adding the chromogenic substrate and the stop solution without other antigen-antibody components as blank wells for zero setting)
③ the ELISA plate 3: and respectively taking enough coating battens according to the number of the samples to be detected and the number of the standard products, fixing the coating battens on the frame, and recording the positions of the holes. Each standard, sample and blank well is recommended to be 2 replicate wells, 100. mu.L/well. Survivin standards were diluted to 0.625ng/ml, 1.25ng/ml, 2.5ng/ml, 5ng/ml, 10ng/ml,20ng/ml and added to the standards. Adding the sample to the bottom of the well of the ELISA plate, keeping the sample from touching the wall of the well as far as possible, slightly shaking and uniformly mixing, covering a sealing plate film, and incubating for 60 minutes at room temperature. (setting the concentration of the standard substance as 0. mu.g/mL as a negative control, and finally, only adding the chromogenic substrate and the stop solution without other antigen-antibody components as blank wells for zero setting)
3. Washing the plate: carefully uncovering the sealing plate membrane, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 1 minute, then discarding, repeating the steps for 5 times, and buckling and drying on the filter paper each time. (Manual plate washing or plate washing machine)
4. Biotin-labeled secondary antibody:
taking the enzyme label plate dried in the step 3 for the following operation
Enzyme label plate 1: diluting a biotin-labeled second anti-BTA antibody with a sample diluent according to the ratio of 1:10000(v/v), carrying out 100 mu L/hole dilution, shaking and uniformly mixing, covering a membrane sealing plate, and incubating for 90 minutes at 37 ℃;
enzyme label plate 2: diluting a biotin-labeled second anti-CD 44 antibody with a sample diluent according to the proportion of 1:10000(v/v), carrying out 100 mu L/hole dilution, shaking and mixing uniformly, covering a membrane sealing plate, and incubating for 90 minutes at 37 ℃;
③ the ELISA plate 3: the biotin-labeled secondary antibody to Survivin was diluted with a sample diluent at a ratio of 1:500(v/v) at 100. mu.L/well, mixed well with shaking, covered with a sealing plate, and incubated at 37 ℃ for 90 minutes.
The kit has three bottles of biotin-labeled second antibody bottles, namely a biotin-labeled second anti-BTA antibody, a biotin-labeled second anti-CD 44 antibody and a biotin-labeled second anti-Survivin antibody, and when the kit is used, an enzyme label plate is matched with the corresponding biotin-labeled second antibody bottle to use, so that the biotin-labeled second antibody bottle is not crossed.
5. Washing the plate: carefully uncovering the sealing plate membrane, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 1 minute, then discarding, repeating the steps for 5 times, and buckling and drying on the filter paper each time.
6. Streptavidin-labeled HRP working solution: and adding 100 mu l of streptavidin HRP reagent into each hole, shaking and uniformly mixing, covering a sealing membrane plate, and incubating for 30 minutes at 37 ℃.
7. Washing the plate: carefully uncovering the sealing plate membrane, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 1 minute, then discarding, repeating the steps for 5 times, and buckling and drying on the filter paper each time.
8. Adding a color development liquid: the color development liquid A and the color development liquid B are uniformly mixed in equal volume according to the ratio of 1:1, then the mixed color development liquid is quickly added into the reaction hole of the 96-hole enzyme label plate, 100 mu l/hole, and the mixture is placed at 37 ℃ for light-shielding reaction for 15 min.
9. Adding a stop solution: the ELISA plates were removed, 100. mu.L of stop buffer was added quickly, and the results were immediately determined.
10. And (3) determination: OD values per well were determined within 5 minutes using a microplate reader set at 450 nm.
11. And (4) judging and analyzing results: recording related experimental data, respectively drawing standard curves by using Curve Expert 1.3 software, wherein an X axis is an OD value of a standard substance, a Y axis is a concentration corresponding to each OD value, and a concentration value obtained according to the curves is multiplied by a corresponding dilution multiple to obtain the actual concentration of the sample.
(II) Performance test of the kit
1. Sensitivity of the probe: performing gradient dilution on the antigen, detecting the antigen with PBS as negative control, detecting the lowest detected concentration (LOQ) of the antigen according to the determined critical value of yin and yang,
TABLE 1 sensitivity test measurement data
| ELISA plate 1 | ELISA plate 2 | |
|
| Sensitivity ng/mL | 0.32 | 0.113 | 0.21 |
| Detection range ng/mL | 0.5-16 | 0.31-10 | 0.625-20 |
2. Precision: the batch repeatability is that the same batch of well-assembled ELISA kits stored at 4 ℃ are selected for continuous detection for 3 days, 3 antigen standard substances with different concentrations and larger concentration difference are used as samples, 5 times of the detection are set, the operation is carried out according to the instruction of the kit, and the average value of the detection is calculated
Standard Deviation (SD) and Coefficient of Variation (CV),
x is 100%; the batch-to-batch repeatability is that 3 batches of ELISA kits stored at 4 ℃ are selected, 3 antigen standard substances with different concentrations and larger concentration difference are used as samples, 5 repeats are set, and the average value is calculated according to the operation of the instruction of the kit
Standard Deviation (SD) and Coefficient of Variation (CV).
TABLE 2 precision determination data (mean)
3. The accuracy is as follows: adopting a standard addition recovery test, selecting 2 cases of urine of bladder cancer patients, adding 2.0ng/ml and 5.0ng/ml of standard substance solutions with equal volumes respectively, analyzing and determining the concentration of the added samples, performing 3 parallel determinations on each sample, and finally calculating the average recovery rate.
Recovery rate (standard sample measurement value-sample measurement value)/standard addition value × 100%
TABLE 3 results of recovery test
| Recovery (%) | ELISA plate 1 | ELISA plate 2 | |
| Analysis of sample 1 | 89-102 | 96.5-106 | 88-110 |
| Analysis of sample 2 | 98-111 | 95-115 | 99-104.5 |
4. Stability: the results were compared after testing the same product with the kit placed at 37 ℃ for 3 weeks and at 4 ℃ for 3 weeks.
TABLE 4 stability test results of the kit
5. Clinical validation
(1) Sample collection
Tumor group: 40 males and 20 females, mean age of the tumor group was 66.4 ± 9.8 years, with the criteria: all patients have complete case data and do not receive urogenital operation, radiotherapy and chemotherapy and the like recently, and finally the patients are proved to be bladder tumor patients through pathological examination of cystoscopy or TURBT.
Healthy control group: 40 normal people who have undergone health examination at the same time have no tumor-related evidence, 17 men and 23 women, and the average age is 65.8 +/-8.0 years.
(2) Sample processing
All patients are left to take fresh clean midnight urine 50-100mL in the morning before urinary related operations such as catheterization, cystoscopy, digital rectal examination and the like are performed after admission, 1000g of clean midnight urine is numbered and centrifuged for 15 minutes at the temperature of 2-8 ℃ on a centrifuge, supernatant in a sample is left, the test sample is equally divided, and the sample is stored at the temperature of-80 ℃ to be detected.
(3) Experimental methods
The kit prepared in the embodiment 1 of the invention is used for detecting the content of 3 antigens in urine samples of 40 healthy control groups and 60 tumor groups. Analysis was then performed using SPSS19.0 software based on experimental data and comparisons between groups were performed using chi-square test on relevant metrology data with a significant statistical difference of P < 0.05. SPSS plotting is used to analyze the sensitivity and specificity of each tumor marker for bladder cancer detection using a receiver operating curve (ROC curve), and the cutoff value of each index is determined by the Youden index. The area under the ROC curve (AUC) can reflect the accuracy of the tumor marker on bladder cancer diagnosis experiments, and when the AUC is less than or equal to 0.5, the tumor index has no diagnostic value on bladder cancer; when AUC is more than 0.5 and less than or equal to 0.7, the diagnosis accuracy is lower; when AUC is more than 0.7 and less than or equal to 0.9, the accuracy is certain; when AUC > 0.9, higher accuracy is indicated. The accuracy of the combined detection of the 3 marker combinations was compared using logistic regression analysis and ROC analysis.
(4) Analysis of results
As shown in Table 5, the combination of BTA, CD44 and Survivin has high sensitivity and specificity, the specificity is 80%, the sensitivity is 96.8%, when the three indexes are jointly detected, an ROC curve is made, the AUC is 0.905 (95% CI: 0.822-0.958), and the efficacy of the joint diagnosis of the three markers is the best.
TABLE 5 Combined detection results of different tumor markers
| Antigen combination | Sensitivity% | Specific degree of% | AUC |
| BTA | 71.9 | 80 | 0.820 |
| CD44 | 82.5 | 80 | 0.859 |
| Survivin | 80.95 | 82.64 | 0.8552 |
| BTA+ |
80 | 86.54 | 0.89 |
| BTA+CD44+Survivin | 96.8 | 80 | 0.905 |
Claims (7)
1. Use of a combination of cystoma urine markers BTA, CD44 and Survivin in the preparation of a kit for the early diagnosis of bladder cancer.
2. The kit for diagnosing the bladder cancer comprises a solid phase carrier and a primary antibody corresponding to a tumor-associated antigen coated on the solid phase carrier, wherein the tumor-associated antigen is Bladder Tumor Antigen (BTA), CD44 and Survivin (Survivin).
3. The multiplex ELISA kit for bladder cancer diagnosis is characterized by comprising an ELISA plate, a standard substance, a diluent, a washing solution, streptavidin-coupled horseradish peroxidase working solution, a biotin-labeled secondary antibody, a color development solution and a stop solution.
4. The concatemeric ELISA kit for bladder cancer diagnosis according to claim 3, wherein the kit comprises three coated 96-well ELISA plates, each coated with a first anti-human BTA monoclonal antibody, a first anti-human CD44 monoclonal antibody and a first anti-human Survivin monoclonal antibody, wherein the well plates are detachable:
a) a 96-well plate coated with a first anti-human BTA monoclonal antibody;
b) a 96-well plate coated with a first anti-human CD44 monoclonal antibody;
c) a 96-well plate coated with a first anti-human Survivin monoclonal antibody.
5. The multiplex ELISA kit for bladder cancer diagnosis as claimed in claim 3, wherein the standard solution has 3 bottles for drawing a standard curve, and when in use, the ELISA plate is matched with the corresponding standard and biotin-labeled secondary antibody, so as not to cross;
a) the BTA standard was used at concentrations: 0.5ng/ml, 1.0ng/ml, 2.0ng/ml, 4.0ng/ml, 8.0ng/ml, 16.0 ng/ml;
b) the CD44 standard was used at concentrations of: 0.31ng/ml, 0.62ng/ml, 1.25ng/ml, 2.5ng/ml, 5ng/ml, 10 ng/ml;
c) survivin standards were used at concentrations of: 0.625ng/ml, 1.25ng/ml, 2.5ng/ml, 5ng/ml, 10ng/ml,20 ng/ml.
6. The concatemeric ELISA kit for diagnosing bladder cancer as claimed in claim 3, wherein the biotin-labeled secondary antibody has 3 vials, and when in use, the microplate is used in combination with the corresponding standard and the biotin-labeled secondary antibody, so as not to cross;
a) a biotin-labeled second anti-BTA antibody;
b) a biotin-labeled secondary anti-CD 44 antibody;
c) a biotin-labeled secondary anti-Survivin antibody.
7. The multiplex ELISA kit for diagnosis of bladder cancer as claimed in claim 3, wherein the subject is human urine.
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