CN101377500A - Free prostate gland specificity antigen chemiluminescence immune analysis determination reagent kit and preparing method thereof - Google Patents
Free prostate gland specificity antigen chemiluminescence immune analysis determination reagent kit and preparing method thereof Download PDFInfo
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Abstract
The invention relates to the medical field of immunoassay, more specially, the invention provides a chemiluminescent immunoassay detection kit for free prostate specific antigens and a preparation method thereof. The kit of the invention comprises: 1) free prostate specific antigen calibrators, 2) solid-phase vectors which are coated by prostate specific antigen monoclonal antibodies, 3) anti-free prostate specific antigen antibodies which are marked by enzyme and 4) chemiluminescent substrate solutions. Further, the preparation method of the kit according to the invention comprises the following steps: 1) preparing the calibrators, 2) coating the solid-phase vectors with the free prostate specific antigen monoclonal antibodies, 3) marking the anti-free prostate specific antigen antibodies with the enzyme, 4) packaging the calibrators, the enzyme markers and chemiluminescent substrate solutions, and 5) assembling finished products. The kit of the invention has the advantages of convenience, rapidness, sensitivity, stability, and the like.
Description
Technical Field
The invention relates to the medical field of immunoassay, and particularly provides a free prostate specific antigen chemiluminescence immunoassay quantitative determination kit and a preparation method thereof.
Background
Prostate Specific Antigen (PSA) is a single-chain glycoprotein molecule with the molecular weight of 30-33 kDa secreted by human prostate epithelial tissue cells. PSA enters seminal fluid after being secreted from prostate, becomes an important protein component of seminal plasma, and has the concentration of about 0.5-5 mg/mL. PSA is usually released at low concentrations into the blood of healthy men, and is found mainly in the prostate tissue, semen and serum of men in humans, and studies have shown that low concentrations of PSA can also be detected in the serum of women.
PSA exists in semen in the form of free PSA (f-PSA), and after entering blood, it will combine with other protein molecules in blood to form PSA in combined state (c-PSA). PSA levels in normal male blood are typically below 4ng/mL, and f-PSA levels are typically below 2 ng/mL. In recent years, the concentration level of PSA in serum of patients with prostate cancer (PCa) is higher than that of normal persons, and as an organ-specific tumor marker, PAP (prostate acid phosphatase) is replaced by PAP (prostate acid phosphatase) as a main index for screening PCa. Relevant studies show that the PSA level in the serum of benign patients with prostatic hyperplasia (BPH) is increased correspondingly, and the PSA level in the human serum is increased to different degrees with the increase of age, so that a 'diagnosis gray zone' is formed in the PSA concentration range of 4-10 ng/mL. Successive researchers have proposed different diagnostic indicators to ameliorate the deficiencies of PSA alone, such as: the prostate growth rate (PSAvelasticity, PSAV), the prostate density (PSAdensity, PSAD), the age-specific reference range (ASR-RS), etc., which have limitations in the use of these indexes in the evaluation of prostate cancer, have been controversial. With the continuous and intensive research on prostate diseases and PSA, the concentration level of f-PSA in the serum of PCa patients is obviously lower than that of BPH patients and normal people, so that a new clinical index, namely the free prostate specific antigen percentage (free/total PSA ratio), is introduced. This parameter is more specific for PCa, while also reducing the false positive rate of clinical trials.
For quantitative analysis of f-PSA, Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) are currently used, wherein RIA must be used125The radioactive elements such as I and the like are labeled, detection equipment is complex, a special instrument is required for detection, the half-life period of the radioactive elements is short, the radioactive elements cannot be stored for a long time, the detection result is unstable, and radioactive damage is brought to experimental operators. The ELISA detection sensitivity is not high enough, the detection range is narrow, the influence factors are more, and the defects of easy false negative and false positive and the like can not meet the clinical requirements. Chemiluminescence immunoassay (CLIA) has been developed.
Chemiluminescence Immunoassay (chemiluminiscence Immunoassay, CLIA) was published in 1977, and the first generation Chemiluminescence Immunoassay kit was successfully developed and marketed in 1985. In the nineties, the development of chemiluminescence immunoassay kits and the production of automated measuring instruments have made breakthrough progress, and thus have entered a high-speed development stage. The chemiluminescence immune analysis is a new immune analysis technology developed after fluorescence, radioactive isotope and enzyme immune analysis, according to a large amount of experimental results and clinical application data, from the aspects of practicability, stability, accuracy and development prospect, the chemiluminescence immune analysis is in the leading position in the non-radioactive labeling analysis technology, represents the direction and trend of the current world development, not only has the specificity of immune reaction, but also has high sensitivity of chemiluminescence reaction (the detection limit can reach 10)-15～10-18mol/L). The chemiluminescence immunoassay technology has the advantages of high sensitivity, rapidness, accuracy, good repeatability, long effective period, safety, no toxicity, no pollution and the like, and becomes the first choice for replacing the radio immunoassay and enzyme immunoassay technology.
The chemiluminescence immunoassay kit in the prior art is a closed full-automatic chemiluminescence immunoassay system, and needs an expensive full-automatic chemiluminescence immunoassay instrument, so that the popularization and the use are limited, and the chemiluminescence immunoassay kit cannot be widely applied to clinical diagnosis and scientific research work. The invention applies enzyme catalysis luminescence substrate on the basis of enzyme-linked immunoassay, and replaces chromogenic substrate in enzyme-linked immunoassay by detecting light signal generated by the luminescence substrate, thereby greatly improving the sensitivity, having simple and convenient operation and wide applicability, not only being applied to an open semi-automatic chemiluminescence measuring instrument, but also being applied to a full-automatic measuring system, realizing rapid detection in large batch, having low use cost and being easier to popularize and apply.
Disclosure of Invention
The inventor of the present invention has proposed and completed the present invention to solve the above problems, that is, to effectively combine the chemiluminescence technology with the immunoassay of f-PSA, and to use a self-made chemiluminescence substrate solution with strong luminescence signal, long duration and higher signal-to-noise ratio, and to provide a kit for simply, rapidly, sensitively and stably detecting f-PSA. The method adopted by the kit is higher in sensitivity than an enzyme immunoassay method, and the detection window period can be shortened, so that the kit is suitable for effective popularization and application in industry.
The invention aims to provide a free prostate specific antigen chemiluminescence immunoassay quantitative determination kit.
It is a further object of the present invention to provide a method for preparing the above kit.
The free prostate specific antigen chemiluminescence immune analysis quantitative determination reagent kit according to the invention comprises:
1) a free prostate specific antigen calibrator;
2) a solid phase carrier coated by a free prostate specific antigen monoclonal antibody;
3) enzyme-labeled anti-free prostate specific antigen antibody; and
4) a chemiluminescent substrate solution acted by the enzyme.
The kit according to the present invention, wherein the solid phase carrier is a microplate, plastic beads, plastic tubes or magnetic particles.
The kit according to the invention, wherein the labeled enzyme is horseradish peroxidase or alkaline phosphatase.
According to the kit, the chemiluminescence substrate solution is luminol, isoluminol or a1, 2-dioxane derivative.
Preferably, in the kit, the 1, 2-dioxyethane derivative is (adamantane) -1, 2-dioxyethane, 3- (2 '-spiroadamantane) -4-methoxy-4- (3' -phosphoryloxy) phenyl-1, 2-dioxyethane, CSPD or CDP-Star.
Preferably, in the kit, the chemiluminescent substrate solution comprises solution A and solution B, wherein,
the solution A is prepared by adding Tris and concentrated HCl into double distilled water to prepare 0.1M Tris-HCl buffer solution with the pH value of 8.5, and the buffer solution contains 4.0mg/mL Luminol and 0.3mg/mL p-iodophenol;
the solution B is prepared by adding trisodium citrate and citric acid into double distilled water to prepare 0.1M citric acid buffer solution with pH value of 4.6, which contains 200mg/mL hydrogen peroxide solution;
or,
the chemiluminescent substrate solution contained 24g Tris, 160g NaCl, 4g KCl, 15mL HCl, 200mL3- (2 '-spiroadamantane) -4-methoxy-4- (3' -phosphoryloxy) phenyl-1, 2-dioxyethane, 1mL Proclin300, 1000mL double distilled water.
The invention provides a method for preparing the kit, which comprises the following steps:
1) preparing a free prostate specific antigen calibrator;
2) coating a solid phase carrier with a free prostate specific antigen monoclonal antibody;
3) labeling an anti-free prostate specific antigen antibody with an enzyme;
4) subpackaging the free prostate specific antigen calibrator, the enzyme-labeled anti-free prostate specific antigen antibody and the chemiluminescent substrate solution acted by the enzyme; and
5) and assembling to obtain a finished product.
According to the method of the present invention, preferably, the step 2) of coating the solid phase carrier comprises the steps of:
I) coating quilt
Mixing 0.05M carbonate buffer solution with pH value of 9.6 or 0.046M citric acid buffer solution with pH value of 4.6 with free prostate specific antigen monoclonal antibody with proper concentration to prepare coating solution, and loading the coating solution on a solid phase carrier;
II) washing the solid phase carrier with physiological saline; and
III) sealing
Preparing a confining liquid containing 0.2g NaH based on 1000mL of the confining liquid2PO4·2H2O、2.9gNaH2PO4·12H2O, 10g BSA and 1mL biological preservative, wherein the pH value of the blocking solution is 7.0-7.6, and then the obtained blocking solution is loaded on the washed solid phase carrier.
Specifically, the coating method may include:
I) coating quilt
Preparing a buffer solution by using 1000mL of a 0.05M carbonate buffer solution with the pH value of 9.6, containing 1.59g of anhydrous sodium carbonate and 2.94g of sodium bicarbonate deionized water solution, or 1000mL of a 0.046M citric acid buffer solution with the pH value of 4.6, containing 4.44g of citric acid and 7.3g of trisodium citrate deionized water solution, mixing the buffer solution with a free prostate specific antigen monoclonal antibody with a proper concentration to prepare a coating solution, and loading the coating solution on a solid phase carrier;
II) washing the solid phase carrier with physiological saline; and
III) sealing
Preparing a confining liquid containing 0.2g NaH based on 1000mL of the confining liquid2PO4·2H2O、2.9gNaH2PO4·12H2O, 10g BSA and 1mL biological preservative, wherein the pH value of the blocking solution is 7.0-7.6, and then the obtained blocking solution is loaded on the washed solid phase carrier.
In the above method, preferably, the solid phase carrier is a microplate, a plastic bead, a plastic tube or a magnetic particle.
In the above method, preferably, the enzyme is horseradish peroxidase or alkaline phosphatase.
In the above method, preferably, the chemiluminescent substrate solution is luminol, isoluminol or a1, 2-dioxane derivative.
In the above process, preferably, the 1, 2-dioxane derivative is (adamantane) -1, 2-dioxane, 3- (2' -spiroadamantane) -4-methoxy-4- (3 "-phosphoryloxy) phenyl-1, 2-dioxane, CSPD or CDP-Star.
In the above method, preferably, the chemiluminescent substrate solution comprises solution A and solution B, wherein,
the solution A is prepared by adding Tris and concentrated HCl into double distilled water to prepare 0.1M Tris-HCl buffer solution with the pH value of 8.5, and the buffer solution contains 4.0mg/mL Luminol and 0.3mg/mL p-iodophenol;
the solution B is prepared by adding trisodium citrate and citric acid into double distilled water to prepare 0.1M citric acid buffer solution with pH value of 4.6, which contains 200mg/mL hydrogen peroxide solution;
or,
the chemiluminescent substrate solution contained 24g Tris, 160g NaCl, 4g KCl, 15mL HCl, 200mL3- (2 '-spiroadamantane) -4-methoxy-4- (3' -phosphoryloxy) phenyl-1, 2-dioxyethane, 1mL Proclin300, 1000mL double distilled water.
The invention provides a detection means which can be manually operated and is also suitable for some standard semi-automatic detection instruments aiming at a clinical examination laboratory, and establishes a method for quantitatively analyzing the free prostate specific antigen (f-PSA) in human serum. An enzymatic chemiluminescence detection system of a luminescent substrate which adopts an 96/48-hole polystyrene micropore plate or a coated test tube or magnetic microparticles as a solid phase carrier, horseradish peroxidase (HRP) or alkaline phosphatase (ALP) as a labeling enzyme and Luminol or AMPPD as a main component is adopted to carry out quantitative analysis on free prostate specific antigen (f-PSA) in human serum.
All indexes of the free prostate specific antigen chemiluminescence immunoassay quantitative determination kit reach the level of the analytical method of the similar imported kit. The method for detecting the tumor has the advantages of high sensitivity, strong specificity, wide detection range, simple operation, no radioactive pollution, low cost of the kit and strong clinical applicability, and is more suitable for clinical detection screening laboratories in China.
According to the kit, an enzyme-labeled antibody, an antibody coated on a carrier and a free prostate specific antigen of a detected sample form a sandwich compound structure of coated antibody-antigen-antibody-enzyme, so that a reaction mode of a double-antibody sandwich one-step method adopted by the kit not only effectively utilizes the technical principle of chemiluminescence, but also applies an enzyme-catalyzed luminescent substrate on the basis of enzyme-linked immunoassay, and a chromogenic substrate in the enzyme-linked immunoassay is replaced by a light signal generated by detecting the luminescent substrate, so that the sensitivity of the kit is greatly improved. In addition, this mode is also convenient for operation and production.
Drawings
FIG. 1 is a line graph of a calibrator in the kit prepared in example 1
FIG. 2 is the comparison result of the clinical blood sample measurement value of the kit of the present invention and the foreign kit
Detailed Description
EXAMPLE 1 preparation of free prostate specific antigen chemiluminescence immunoassay quantitative determination kit of the invention
Preparation of enzyme-labeled free prostate specific antigen monoclonal antibody
(1) Preparation of free prostate specific antigen monoclonal antibody labeled by horseradish peroxidase
Dissolving 4.4mg HRP in 1mL distilled water, adding 0.4mL sodium periodate (50mmol/L), stirring at room temperature for 20min, dialyzing with 1mmol/L sodium acetate buffer solution at pH4.4, adding 8mg free prostate specific antigen monoclonal antibody, stirring for 2h, and adding 200mmol/L NaBH4Reducing, dialyzing with 0.02MPB buffer solution, adding equal volume of glycerol, and storing at-20 deg.C.
(2) Preparation of monoclonal antibody of alkaline phosphatase-labeled free prostate specific antigen
Free prostate specific antigen monoclonal antibody was coupled to alkaline phosphatase using the glutaraldehyde method, dialyzed extensively against PBS, added with an equal volume of glycerol, and stored at-20 deg.C or below.
Second, enzyme-labeled antibody concentration selection
Experiments prove that the working concentration range of the enzyme-labeled antibody is 1: 500-50000 by adopting a square matrix method.
Preparation of free prostate specific antigen calibrator
The calf serum is taken as a substrate, and the purified concentrated human seminal plasma raw material is diluted in series by taking a national standard product as a standard. The subpackaging concentration ranges are as follows: 0. 6 bottles of calibrators with the concentration of 0.5, 1, 2.5, 5.0 and 10 ng/mL.
Fourthly, coating the solid phase carrier with the free prostate specific antigen monoclonal antibody
(1) Coating quilt
Mixing 0.05M carbonate buffer solution with pH value of 9.6 or 0.046M citric acid buffer solution with pH value of 4.6 with free prostate specific antigen monoclonal antibody with proper concentration to prepare coating solution, and loading the coating solution on a solid phase carrier;
specifically, the coating method may include:
anhydrous sodium carbonate 1.59g
Sodium bicarbonate 2.94g
1000mL of deionized water
Dissolving, mixing, adjusting pH to 9.6, adding 5.0mg of free prostate specific antigen monoclonal antibody, mixing, adding into each well of microporous plate, each well at 110 μ L, and standing at 4 deg.C overnight.
Or,
citric acid 4.44g
Trisodium citrate 7.3g
1000mL of deionized water
Dissolving, mixing, adjusting pH to 4.6, adding 5.0mg of free prostate specific antigen monoclonal antibody, mixing, adding into each well of microporous plate, each well at 110 μ L, and standing at 4 deg.C overnight.
(2) Washing: washed three times with physiological saline.
(3) Sealing of
NaH2PO4·2H2O 0.2g
Na2HPO4·12H2O 2.9g
BSA 10g
Proclin 300 1mL
Double distilled water to 1000mL
Weighing the above reagents, placing into a clean container, adding double distilled water to a constant volume, dissolving, mixing uniformly, and measuring the pH value to be 7.0.
Add 300. mu.L of blocking solution to each well, and then let stand at room temperature for 3 hours. The confining liquid is thrown off and patted dry on absorbent paper. And dehumidifying and drying for 24 hours at room temperature. Vacuum bagging was immediately performed. After the bag is sealed, the bag is placed for 15 minutes to check whether the air leakage exists, and if the air leakage exists, the bag needs to be sealed again. And (5) after labeling, preserving at 2-8 ℃.
Enzyme-labeled monoclonal antibody diluent
Tris 12.120g
BSA 5g
Proclin 1mL
1000mL of double distilled water
Sixthly, chemiluminescent substrate
The preparation method of the chemiluminescent substrate solution of the horseradish peroxidase (HRP) comprises the following steps:
solution A: tris and concentrated HCl are added into double distilled water to prepare 0.1M Tris-HCl buffer solution with the pH value of 8.5. To this buffer were added 4.0mg/mL Luminol and 0.3mg/mL p-iodophenol.
And B, liquid B: trisodium citrate and citric acid were added to double distilled water to prepare a 0.1M citric acid buffer solution having a pH of 4.6, to which 200mg/mL hydrogen peroxide solution was added.
The using method comprises the following steps: before use, the liquid A and the liquid B are mixed according to the proportion of 1: 1 proportion and then used.
The preparation method of the chemiluminescent substrate solution of alkaline phosphatase (ALP) used in the invention comprises the following steps:
Tris 24g
NaCl 160g
KCl 4g
HCl 15mL
1000mL of double distilled water
Proclin 300 1mL
AMPPD 200mL
Seventh, washing buffer solution
Tris 24g
NaCl 160g
KCl 4g
HCl 15mL
1000mL of deionized water
The pH was adjusted to 7.4.
Eighthly, semi-finished product and finished product composition
And subpackaging the products obtained in the steps to obtain semi-finished products. Three parts are extracted and can be assembled into the f-PSA quantitative determination kit after being qualified by the detection of specificity, precision, sensitivity and stability. The kit can be delivered after being assembled and qualified by sampling inspection.
The solid phase antibody in the kit is coated in advance, and does not need to be coated on site; the calibrator is liquid; the labeled antibody is also a working solution that has been diluted to a working concentration, and both can be used directly. The sample adding volumes of the calibrator/sample to be tested, the enzyme-labeled antibody solution and the luminescent substrate are all 50 mu L, and the sample adding volumes do not need to be adjusted in the experimental process, so that the calibrator/sample to be tested is convenient to use to the maximum extent. The measuring method of the invention is used for measuring according to the procedures, and has short time, convenience and quickness.
Examples 2 to 3 preparation of the free prostate specific antigen chemiluminescence immunoassay quantitative determination kit of the present invention
f-PSA chemiluminescence immunoassay quantitative determination kits were prepared in the same manner as in example 1, except that plastic beads and magnetic particles were used as carriers, respectively.
Example 4 methods of Using the kits of the invention
Before the kit is used for an experiment, the solid-phase antibody, the calibrator/detection sample and the labeled antibody solution are taken out and placed at room temperature for 15-30 minutes to balance the solid-phase antibody, the calibrator/detection sample and the labeled antibody solution to the room temperature; then, adjusting the constant temperature oven or the water bath to 37 ℃; then, the appropriate microsyrinths and corresponding tips are prepared and the chemiluminescence apparatus and auxiliary instruments, such as a plate washer, are checked for proper operation.
The specific procedures for carrying out the experiment using the kit according to the method of example 1 are as follows:
1) the plate required for the experiment (coated with antibody, 96/48 wells were split into 12/6 x 8 x 1 wells) was placed on the plate rack;
2) respectively adding a sample to be detected and a calibrator with each concentration into reaction holes, adding 50 mu L of each calibrator with the concentration of 0, 0.5, 1, 2.5, 5.0 and 10ng/mL into each reaction hole, setting a blank hole with 1 hole in each test, and adding 50 mu L of enzyme-labeled antibody solution into each hole except the blank hole;
3) oscillating on a micro oscillator for 30 seconds and mixing uniformly;
4) incubation at 37 ℃ for 60 minutes;
5) discarding the solution in the hole, washing the plate for 5 times by using Tris-HCl washing liquor and an automatic plate washing machine or a manual plate washing machine, and patting the plate dry on absorbent paper;
6) adding 50 mu L of luminescent substrate into each hole, oscillating and mixing uniformly, and reacting for 30-90 minutes at room temperature (22-28 ℃);
7) measuring relative luminescence value (RLU) with chemiluminescence apparatus, measuring time 1 second/hole;
8) respectively taking logarithm of the calibrator concentration and corresponding RLU, establishing a standard curve on the established double-logarithm curve, finding out the concentration of f-PSA of the serum on the standard curve according to the RLU value of each serum to be detected, and calculating the detection result (see figure 1);
9) and printing a detection result report.
Example 5 methodological identification of the kits of the invention
The kit prepared in example 1 was characterized according to the manufacturing and assay protocols conventional in the art, and numerous experiments demonstrated that the kit of the present invention, when used in the determination of f-PSA concentration levels, has the following methodological criteria:
detection range: 0-10 ng/mL;
sensitivity: the minimum detection limit is 0.01 ng/mL;
precision: the intra-assay precision (three quality control ranges of high, medium and low) is less than 7% (n is 10), and the inter-assay precision (three quality control ranges of high, medium and low) is less than 7% (n is 10);
the accuracy is as follows: the recovery rate of the added standard is between 90 and 110 percent;
specificity: the cross reaction rate with common tumor markers such as CA199, CEA, AFP and the like is less than 0.1 percent;
stability: the reagent components were left at 37 ℃ and after 6 days of investigation, the components remained stable.
The kit of the invention has small sample amount, only 50 mu L of sample is needed for one-time detection, and the in vitro detection has no toxic or side effect on a test object. Meanwhile, the chemiluminescence enzyme immunoassay method adopted by the invention does not produce radioactive pollution, and each index is superior to the enzyme-linked immunosorbent assay (ELISA) widely applied at present. The method for detecting the tumor has the advantages of high sensitivity, strong specificity, wide detection range, simple operation, no radioactive pollution, low cost of the kit and strong clinical applicability, and is more suitable for clinical detection screening laboratories in China. Therefore, the invention provides a more accurate, specific, convenient and quick method for clinically detecting the free prostate specific antigen (f-PSA), assisting in diagnosing the prostate cancer and observing after the operation.
Example 6 comparison of the clinical blood sample measurement value of the kit of the present invention with that of a foreign kit
The reagent and the Monobind free prostate specific antigen chemiluminescence reagent simultaneously detect 102 clinical serum samples, the detection results are shown in the attached figure 2, the result of the blood sample f-PSA determined by the method is a horizontal coordinate, the result determined by the Monbind is a vertical coordinate to perform regression analysis, and the related equation is as follows: y is 1.1182X-0.0309 and the correlation coefficient r is 0.9918. The method has good correlation with the clinical blood sample measuring value of the foreign kit.
In this example, among 102 clinical blood samples, 21 clinical Prostate Cancer (PCA) samples, 30 benign prostate diseases (bpa) samples, and 51 normal human samples were taken, PSA and f-PSA levels of the samples were measured simultaneously, and when the PSA level was within the range of 4 to 10ng/ml, the percentage of free prostate-specific antigen was calculated, and 20% was used as a reference value for clinical diagnosis, and the measurement results are shown in table 1:
table 1: results of measurements on 102 clinical diagnostic blood samples
If only PSA levels greater than 4.0ng/ml are used as the threshold, false positives would be associated with benign patients with prostate hyperplasia, and Table 2 shows the results of using only PSA levels greater than 4.0ng/ml as the positive criteria:
table 2: results of measurements on 102 clinical diagnostic blood samples
Compared with the two judgment standards, the method and the judgment standards provided by the kit can obviously distinguish prostate cancer patients with PSA in the range of 4-10 ng/ml from benign prostate disease patients, greatly improve the clinical diagnosis rate of the prostate cancer, avoid false positive, and have good conformity rate and correlation with the detection results of foreign similar imported reagents.
According to the data analysis of the detection results of the listed reagents, the sensitivity, specificity and accuracy of the kit have no obvious difference with those of similar imported kits; convenient popularization and application, safety and reliability.
Claims (13)
1. A free prostate specific antigen chemiluminescence immunoassay assay kit, wherein the kit comprises:
1) a free prostate specific antigen calibrator;
2) a solid phase carrier coated by a free prostate specific antigen monoclonal antibody;
3) enzyme-labeled anti-free prostate specific antigen antibody; and
4) a chemiluminescent substrate solution acted by the enzyme.
2. The kit of claim 1, wherein the solid support is a microplate, plastic beads, plastic tubes, or magnetic particles.
3. The kit of claim 1, wherein the enzyme is horseradish peroxidase or alkaline phosphatase.
4. The kit of claim 1, wherein the chemiluminescent substrate solution is luminol, isoluminol, or a1, 2-dioxane derivative.
5. The kit of claim 4, wherein the 1, 2-dioxane derivative is (adamantane) -1, 2-dioxyethane, 3- (2' -spiroadamantane) -4-methoxy-4- (3 "-phosphoryloxy) phenyl-1, 2-dioxyethane, CSPD or CDP-Star.
6. The kit of claim 1, 4 or 5,
the chemiluminescent substrate solution comprises solution A and solution B, wherein,
the solution A is prepared by adding Tris and concentrated HCl into double distilled water to prepare 0.1M Tris-HCl buffer solution with the pH value of 8.5, and the buffer solution contains 4.0mg/mL Luminol and 0.3mg/mL p-iodophenol;
the solution B is prepared by adding trisodium citrate and citric acid into double distilled water to prepare 0.1M citric acid buffer solution with pH value of 4.6, which contains 200mg/mL hydrogen peroxide solution;
or,
the chemiluminescent substrate solution contained 24g Tris, 160g NaCl, 4g KCl, 15mL HCl, 200mL3- (2 '-spiroadamantane) -4-methoxy-4- (3' -phosphoryloxy) phenyl-1, 2-dioxyethane, 1mL Proclin300, 1000mL double distilled water.
7. A method for preparing the kit of claim 1, comprising the steps of:
1) preparing a free prostate specific antigen calibrator;
2) coating a solid phase carrier with a free prostate specific antigen monoclonal antibody;
3) labeling an anti-free prostate specific antigen antibody with an enzyme;
4) subpackaging the free prostate specific antigen calibrator, the enzyme-labeled anti-free prostate specific antigen antibody and the chemiluminescent substrate solution acted by the enzyme; and
5) and assembling to obtain a finished product.
8. The method of claim 7, wherein step 2) of coating the solid support comprises the following steps:
I) coating quilt
Mixing 0.05M carbonate buffer solution with pH value of 9.6 or 0.046M citric acid buffer solution with pH value of 4.6 with free prostate specific antigen monoclonal antibody with proper concentration to prepare coating solution, and loading the coating solution on a solid phase carrier;
II) washing the solid phase carrier with physiological saline; and
III) sealing
Preparing a confining liquid containing 0.2g NaH based on 1000mL of the confining liquid2PO4·2H2O、2.9gNaH2PO4·12H2O, 10g BSA and 1mL biological preservative, wherein the pH value of the blocking solution is 7.0-7.6, and then the obtained blocking solution is loaded on the washed solid phase carrier.
9. The method of claim 7 or 8, wherein the solid support is a microplate, plastic beads, plastic tubes, or magnetic particles.
10. The method of claim 7, wherein the enzyme is horseradish peroxidase or alkaline phosphatase.
11. The method of claim 7, wherein the chemiluminescent substrate solution is luminol, isoluminol, or a1, 2-dioxane derivative.
12. The method of claim 11, wherein the 1, 2-dioxane derivative is (adamantane) -1, 2-dioxyethane, 3- (2' -spiroadamantane) -4-methoxy-4- (3 "-phosphoryloxy) phenyl-1, 2-dioxyethane, CSPD, or CDP-Star.
13. The method of claim 7, 11 or 12,
the chemiluminescent substrate solution comprises solution A and solution B, wherein,
the solution A is prepared by adding Tris and concentrated HCl into double distilled water to prepare 0.1M Tris-HCl buffer solution with the pH value of 8.5, and the buffer solution contains 4.0mg/mL Luminol and 0.3mg/mL p-iodophenol;
the solution B is prepared by adding trisodium citrate and citric acid into double distilled water to prepare 0.1M citric acid buffer solution with pH value of 4.6, which contains 200mg/mL hydrogen peroxide solution;
or,
the chemiluminescent substrate solution contained 24g Tris, 160g NaCl, 4g KCl, 15mL HCl, 200mL3- (2 '-spiroadamantane) -4-methoxy-4- (3' -phosphoryloxy) phenyl-1, 2-dioxyethane, 1mL Proclin300, 1000mL double distilled water.
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