CN102818892B - Detection kit for prostate specific antigen and preparation method thereof - Google Patents
Detection kit for prostate specific antigen and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the field of immunoassay medicine, and specifically provides a chemiluminescence immunoassay diagnostic kit for a free prostate specific antigen (fPSA) and a preparation method thereof. The kit provided by the invention comprises an anti-FITC antibody coated solid phase vector, an FITC marked anti-PSA antibody, an enzyme marked anti-fPSA antibody, a chemiluminescent substrate acted by the above enzyme, a negative control solution and a positive control solution. The preparation method of the above kit comprises the following steps: 1) coating a solid-phase vector with the anti-FITC antibody; 2) marking the anti-PSA antibody with FITC to obtain the FITC marked anti-PSA antibody; 3) marking the anti-PSA antibody with an enzyme to obtain the enzyme marked anti-fPSA antibody; and 4) subpackaging and assembling. The kit provided by the invention has low cost and high sensitivity, and can improve sensitivity and reproducibility of the assay and reduce the usage amount of antibodies compared with an existing chemiluminescence immunoassay detection method.
Description
Technical field
The present invention relates to field of immunoassay medicine, particularly, the invention provides chemical luminescence immune analysis diagnosis reagent kit of a kind of prostate specific antigen and preparation method thereof.
Background technology
Along with the change of China's aging population, dietary structure and environmental factor, and the raising of health care level, the incidence of disease of prostate cancer in increasing trend year by year, leapt to male urinary, the 3rd of reproductive system malignant tumour.The early stage general non-evident sympton of prostate cancer, once find often to appear at late period, has missed best occasion for the treatment, therefore, has needed responsive method of early diagnosis.
Prostate specific antigen (PSA, prostate specific antigen) is the tumor markers that diagnosing prostate cancer is the most responsive clinically at present, has been acknowledged as the important indicator that prostate cancer detects and assesses, has had very high sensitivity.PSA is a kind of containing 237 amino acid whose single chain polypeptides, belongs to and has the tissue-specific serine protease having the effect of chymotrypsin sample.Mainly exist with PSA with in conjunction with state PSA in serum, free state is called fPSA, is called (tPSA) in conjunction with state; PSA has tissue specificity, but it there is no tumour-specific.Have in the prostate cancer of clinical meaning at great majority, PSA can raise, but benign prostatic hyperplasis and prostatitis also there will be the result of the PSA positive.At present, in serum, PSA level is the Grey Diagnosis district of prostate cancer between 4-10 μ g/L.In diagnosis grey area, prostate cancer (PCa) has larger overlapping region with benign prostatic hyperplasis (BPH) and prostatitis.Along with deepening continuously of studying prostatic disorders and PSA, find that in PCa patients serum, most PSA is bonding state, and f-PSA concentration level is compared with patient BPH and normal person, obviously on the low side.Therefore, the method solving this problem detects free prostate gland specificity antigen exactly, and introduces new clinical indices-Free to total PSA ratio.This is for improving the specificity of examination with the PCa of diagnosis gray area, and the false positive rate reducing clinical examination examination has great importance.
The method of current detection fPSA has a lot, labelling immunoassay technology wherein based on antibody-antigene specific reaction, as radiommunoassay, EIA enzyme immunoassay, time resolved fluoro-immunoassay and chemiluminescence immune assay etc., owing to having the advantages such as screening simple to operate, quick, to be applicable to a large amount of sample and detection, it is primary dcreening operation detection method general clinically at present.The basic theories of these ultramicron detection techniques is substantially identical, and their common feature is that sensitivity is good, high specificity; Difference is that tracer agent used and the signal that sends are different.According to a large amount of experimental results and clinical practice data, from practicality, stability, accuracy and development prospect, be followed successively by: chemiluminescence immune assay, time resolved fluoro-immunoassay, radiommunoassay and EIA enzyme immunoassay.There is shortcomings in radio immunoassay, as complicated operation, measurement result is unstable, the reagent holding time is short, radioactive contamination, expensive equipment etc., although fluoroimmunoassay and EIA enzyme immunoassay overcome alpha-contamination problem, its sensitivity is restricted.The highly sensitive chemiluminescence immune assay grown up thus is more and more subject to the favor of people.
What chemiluminescence immune analysis method was mostly applied in the principle detecting f-PSA is double antibody sandwich method, it be by PSA antibody by the method direct coated of physisorption at solid phase carrier, then removed the analysis thing or sample substrate that do not combine up by washing.This direct coated method can have some to affect on analytical parameters: (1) antigen in solid phase, can cause the inhomogeneity that antigen combines in solid phase by physisorption direct coated, therefore, poor reproducibility, incubative time is long; (2) different from the association reaction of Ag-Ab in the liquid phase, be coated on the albumen (antibody or antigen) in solid phase, its binding site is more close, therefore can cause the immunoactive portions inactivation of albumen, and activity even only reaches about 10% of coating protein amount sometimes; (3) amount being attached to antigen in solid phase is not only relevant with character such as hydrophobicity, the isoelectric point etc. of antigen self, also by the impact of the external conditions such as the preparation of antigenic solution and preservation.And HIV antigen is attached on solid phase carrier by the HIV antigen that the present invention adopts anti-FITC antibody and FITC to mark, thus overcome many shortcomings of physisorphtion direct coated HIV antigen.
In addition, chemical luminescence immune analysis reagent box major part of the prior art is closed full automatic chemiluminescence measuring system, need expensive Full-automatic chemiluminescence measuring instrument, thus limit and promote the use of, cannot effectively be widely used in clinical diagnosis and research work.
Summary of the invention
Propose to solve the problem and complete the present invention.Particularly, the invention solves the problems such as diagnostic kit homogeneity of the prior art, poor reproducibility.
The object of this invention is to provide a kind of free prostate gland specificity antigen (fPSA) chemical luminescence immune analysis diagnosis reagent kit.
Another object of the present invention is to provide a kind of method preparing mentioned reagent box.
FPSA diagnostic kit according to the present invention comprises following component:
1) solid phase carrier of anti-FITC antibody bag quilt;
2) the anti-PSA antibody of FITC mark;
3) the anti-fPSA antibody of enzyme labeling;
4) chemical luminous substrate that acts on of above-mentioned enzyme; And
5) standard items of series concentration.
Wherein, described solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle.Described enzyme is alkaline phosphatase or horseradish peroxidase.The chemical luminous substrate that above-mentioned enzyme acts on is (diamantane)-1,2-dichloroethane, 3-(2
'-spiral diamantane) and-4-methoxyl-4-(3 "-phosphorus acyloxy) phenyl-1,2-dichloroethane, CSPD or CDP-Star, luminol or different luminol.
Present invention also offers a kind of method preparing mentioned reagent box, the method comprises the following steps:
1) with the solid phase carrier of anti-FITC antibody bag quilt;
2) mark anti-PSA antibody with FITC, obtain the anti-PSA antibody of FITC mark;
3) with enzyme labeling anti-fPSA antibody, the anti-fPSA antibody of enzyme labeling is obtained;
4) standard items of the anti-PSA antibody of the FITC mark of the above-mentioned acquisition of packing, the anti-fPSA antibody of enzyme labeling, chemical luminous substrate that this enzyme acts on, series concentration; And
5) finished product is assembled into.
Wherein, the solid phase carrier of anti-FITC antibody bag quilt is used by the following method:
1) by bag by be added to anti-FITC antibody pH=4.4 ~ 5.0, preferably 4.8 citrate buffer solution in mix, with gained mixed liquor bag by solid phase carrier;
2) washing step 1) in the solid phase carrier of bag quilt; And
3) use bovine serum albumin(BSA) closes the solid phase carrier through washing.
According to method of the present invention, wherein, described solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle.Described enzyme is horseradish peroxidase or alkaline phosphatase.The chemical luminous substrate that this enzyme acts on can be (diamantane)-1,2-dichloroethane, 3-(2
'-spiral diamantane) and-4-methoxyl-4-(3 "-phosphorus acyloxy) phenyl-1,2-dichloroethane, CSPD or CDP-Star, luminol or different luminol.
Kit of the present invention can adopt following concrete form, the calf serum that it comprises the opaque microwell plate in 96 holes or 48 holes of anti-FITC antibody bag quilt, the anti-PSA antibody of FITC mark, the anti-fPSA antibody of horseradish peroxidase-labeled, series concentration standard items, calibration object matrix serum are deactivation, 20 times of concentrated cleaning solutions are the 0.02M phosphate buffer (PBS containing 0.05% Tween-20, pH=7.4), chemical luminous substrate A, B liquid is respectively luminol solution, hydrogen peroxide and special efficacy reinforcing agent (to iodophenol) solution thereof, each 1 bottle.
According to the present invention, when use two antibody system is as FITC and anti-FITC Antibody System ankyrin, the immunocompetence of immobilized antigen albumen can be preserved to a great extent.Anti-FITC antibody molecule also can in conjunction with 3 ~ 4 FITC molecules, and specificity and affinity are all very strong.Both are just very stable once combination.When FITC-anti-FITC antibody forming system is as solid phase, first pre-coated anti-FITC antibody in solid phase, PSA antibody immobilization FITC being marked by FITC-anti-FITC antibody response.This method for coating not only can increase the antigen amount on solid phase carrier, but also the binding site on antigen can be made fully to expose.Therefore, use two antibody systems as solid phase, not only can improve the sensitivity of analysis, homogeneity and reappearance, the use amount of PSA antibody can also be reduced.And the bag of anti-FITC antibody can be applied to many analysis detected objects by microwell plate as unified solid phase carrier.
Kit according to the present invention adopts two antibody systems as solid phase carrier, double-antibody sandwich pattern principle, the chemical luminous system of highly sensitive luminol-hydrogen peroxide-horseradish peroxidase and reinforcing agent thereof is as input system, establish a kind of two anti-solid state chemistry chemiluminescence immunoassay methods of highly sensitive, low cost, and be prepared into the diagnostic kit measuring blood or blood plasma Free Prostate-specific Antigen (fPSA), compared with ELISA detection kit, sensitivity improves about 100 times; Compared with existing chemiluminescence immune assay detection method, the sensitivity of analysis can be improved, homogeneity and reappearance, reduce the use amount of PSA antibody.This detection method has very high sensitivity, detects and can shift to an earlier date a couple of days diagnosis, to the early diagnosis of prostate cancer, carry out therapeutic intervention ahead of time and be of great significance than traditional enzyme-linked method.Use kit of the present invention to reduce cost, the health investment also making people limited is used more effectively.
Embodiment
Following embodiment is to explain the present invention in more detail, but should not be construed as the present invention and be confined to this.
embodiment 1 prepares fPSA chemiluminescence diagnostic kit of the present invention
FPSA diagnostic kit of the present invention comprises:
1) the luminous microwell plate in 96 holes that anti-FITC antibody is pre-coated or 48 holes;
2) the anti-PSA antibody of FITC mark;
3) the anti-fPSA antibody of horseradish peroxidase-labeled;
4) standard items of series concentration;
5) sample diluting liquid is the calf serum of deactivation;
6) 20 times of concentrated cleaning solutions are the 0.02M phosphate buffer (PBS, pH=7.4) containing 0.05% Tween-20;
7) chemical luminous substrate A, B liquid is respectively luminol solution, hydrogen peroxide and special efficacy reinforcing agent (to iodophenol) solution.
Prepare mentioned reagent box by the following method:
1, bag is added to mixing in citrate buffer solution (pH=4.8) with anti-FITC antibody, is added in luminous micropore plate hole, every hole 100 μ L, placed 15 h bag quilts for 4 DEG C.Wash after five times with cleansing solution, use ox
Seralbumin (BSA) solution room temperature closes 2 hours.Discard liquid in hole, dry ELISA Plate, sealing, namely completes the preparation of pre-coated elisa plate.
2, with direct labelling method, FITC and PSA antibody is marked, obtain the PSA antibody of FITC mark.
Direct labelling method concrete steps are as follows:
(I) A liquid: get the Na that PSA antibody 1 mg puts 50 mM pH 9.3
2cO
3-NaHCO
34 DEG C of dialysis 24 h in damping fluid, last fixing fabric structure is within 200 μ l;
(II) B liquid: get the Na that FITC (Beijing Xin Jingke biotech company) 2 mg are dissolved in 1 ml 50 mM pH 9.3
2cO
3-NaHCO
3in damping fluid;
(III) A liquid is placed in magnetic stirring apparatus, gets B liquid 25 μ l and slowly drop in A liquid, added between 5-8 minute, prevent bubble, then put 4 DEG C and stir 12-16 hour;
(IV) reactant liquor is taken out the centrifugation 20 minutes in 10000 revs/min.Removing microprecipitation thing, supernatant dress bag filter, puts in the PBS solution of 10 mM pH=7.4 and dialyses 4 days, change dislysate every day 2 times;
(V) centrifugally remove sediment, label is diluted to 1 ml, adds appropriate NaN
3, preserve at packing ， – 20 DEG C.
3, utilize the Over-voltage protection of improvement, mark fPSA antibody and horseradish peroxidase are combined, obtains the f-PSA antibody labeling thing of horseradish peroxidase-labeled.
The concrete steps of the Over-voltage protection of improvement are as follows:
(Ι) taking 2mgHRP is dissolved in 1ml distilled water;
(II) NaIO of the 0.1mol/L that 200 μ l newly configure is added
4solution, stirs 20 minutes in room temperature gently.Solution is brown-green;
(III) by sodium acetate buffer dialysed overnight at 4 DEG C of the 0.001mol/L of pH=4.4;
(IV) 20 μ l 0.2mol/L Na are added
2cO
3damping fluid, makes pH value of solution be promoted to about 9.0-9.5;
(V) with the carbonate buffer solution of 0.01mol/L, pH=9.5, fPSA antibody is deployed into the concentration of 8mg/ml.And antibody-solutions is slowly added in isopyknic above-mentioned HRP solution, stirring at room temperature 2 hours;
(VI) add the dobell's solution of the 4mg/ml of fresh configuration, be placed in 4 DEG C 2 hours;
(VII) in 4 DEG C, dialyse 24 hours in the sodium borate buffer liquid of 0.1 mol/L, exchange buffering liquid 3 times therebetween;
(VIII) add equivalent 60% neutral glycerine solution, preserve at packing ， – 20 DEG C after mixing.
4, serial standards concentration is selected to be 0,0.15 by experiment, 0.6,1.7,3.3,8.3,20 μ g/L.
5, other components of reagent preparation box: 20 times of concentrated cleaning solutions (the 0.02M phosphate buffer containing 0.05% Tween-20, pH=7.4), sample diluting liquid (calf serum of deactivation), chemical luminous substrate A, B liquid are respectively luminol solution, hydrogen peroxide and special efficacy reinforcing agent (to iodophenol) solution thereof.
6, the accuracy of the method generate a reagent box, sensitivity, precision and qualified stability is identified.
7, according to the requirement of kit by these component packing in the vial, finished product is assembled into.
the using method of embodiment 2 kit of the present invention:
1, in 4 DEG C of refrigerators, take out kit, equilibrium at room temperature 20 minutes, get one bottle of concentrated washing lotion, adding distil water (1ml+19ml) is for subsequent use.
2, luminous plaque is taken out from sealing bag, the anti-fPSA antibody-solutions (1:2000) that 100 μ L FITC mark is joined in the ELISA Plate being coated with anti-FITC antibody, incubation 1 hour at 37 DEG C.
3, discard liquid in each hole, cleansing solution is filled each hole, leave standstill 10-20 second, get rid of cleansing solution; Repeat to wash plate, finally pat dry on clean thieving paper.
4, establish each 2 holes of serial standards concentration, each sample well 2 hole, get standard items or each 50 μ l of sample solution in corresponding plate hole, add enzyme marker 50 μ l again, micro oscillator fully vibrates mixing, with adhesive sticker sheet capping reaction plate, then puts reaction plate 37 DEG C of incubations 30 minutes.
5, carefully get rid of dereaction liquid, then wash five subsynchronous rapid 3.
6, after using first 5 minutes and being mixed with B liquid 100:1 by luminous substrate A liquid, in every hole, add 50 μ L chemical luminous substrates successively, room temperature lucifuge is measured luminous intensity after reacting 10 minutes.
7, with RLU value, fPSA concentration is mapped with log-log coordinate, by typical curve, quantitative test is realized to fPSA in Sample serum.
The advantage that the present invention has:
Result high specificity, highly sensitive, stable, convenient etc.The present invention is easy and simple to handle, and required detecting instrument (Chemiluminescence Apparatus) generally uses at various big hospital and inspection center.
Claims (1)
1. prepare a method for free prostate gland specificity antigen (fPSA) chemical luminescence immune analysis diagnosis reagent kit, comprise the following steps:
1) with anti-FITC antibody bag by solid phase carrier, obtain the solid phase carrier of anti-FITC antibody bag quilt;
2) mark anti-PSA antibody by following step FITC, obtain the anti-PSA antibody of FITC mark:
(I) Na that PSA antibody 1 mg puts 50 mM pH 9.3 is got
2cO
3-NaHCO
34 DEG C of dialysis 24 h in damping fluid, last fixing fabric structure is within 200 μ l;
(II) Na that FITC 2 mg is dissolved in 1 ml 50 mM pH 9.3 is got
2cO
3-NaHCO
3in damping fluid;
(III) liquid obtained in above-mentioned steps (I) is placed in magnetic stirring apparatus, getting the liquid 25 μ l obtained in above-mentioned steps (II) slowly drops in A liquid, added between 5-8 minute, prevent bubble, then put 4 DEG C and stir 12-16 hour;
(IV) reactant liquor is taken out the centrifugation 20 minutes in 10000 revs/min, removing microprecipitation thing, supernatant dress bag filter, puts in the PBS solution of 10 mM pH=7.4 and dialyses 4 days, change dislysate every day 2 times;
(V) centrifugally remove sediment, label is diluted to 1 ml, adds appropriate NaN
3, packing, preserves at-20 DEG C;
3) by following step enzyme labeling anti-fPSA antibody, the anti-fPSA antibody of enzyme labeling is obtained:
I () takes 2mgHRP and is dissolved in 1ml distilled water;
(ii) NaIO of the 0.1mol/L that 200 μ l newly configure is added
4solution, stirs 20 minutes in room temperature gently, and solution is brown-green;
(iii) by sodium acetate buffer dialysed overnight at 4 DEG C of the 0.001mol/L of pH=4.4;
(iv) 20 μ l 0.2mol/L Na are added
2cO
3damping fluid, makes pH value of solution be promoted to 9.0-9.5;
V fPSA antibody is deployed into the concentration of 8mg/ml with the carbonate buffer solution of 0.01mol/L, pH=9.5 by (), and slowly added by antibody-solutions in isopyknic above-mentioned HRP solution, stirring at room temperature 2 hours;
(vi) add the dobell's solution of the 4mg/ml of fresh configuration, be placed in 4 DEG C 2 hours;
(vii) in 4 DEG C, dialyse 24 hours in the sodium borate buffer liquid of 0.1 mol/L, exchange buffering liquid 3 times therebetween;
(viii) add equivalent 60% neutral glycerine solution, preserve at packing ， – 20 DEG C after mixing;
4) the anti-PSA antibody of the FITC mark of the above-mentioned acquisition of packing, the anti-fPSA antibody of enzyme labeling and this enzyme act on chemical luminous substrate, series concentration standard items;
5) finished product is assembled into.
2. the method for claim 1, is characterized in that, wraps by solid phase carrier by the following method:
1) mixing wrapping to be added to anti-FITC antibody in the citrate buffer solution of pH=4.4 ~ 5.0, then using gained mixed liquor bag by solid phase carrier;
2) coated solid phase carrier is washed;
3) bovine serum albumin(BSA) is used to close the above-mentioned carrier through washing.
3. method as claimed in claim 2, it is characterized in that, the pH value of described citrate buffer solution is 4.8.
4. method as claimed in claim 1 or 2, it is characterized in that, described solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle.
5. the method for claim 1, is characterized in that, described chemical luminous substrate is 1,2-dichloroethane analog derivative, luminol or different luminol.
6. method as claimed in claim 5, it is characterized in that, described 1,2-dichloroethane analog derivative is (diamantane)-1,2-dichloroethane, 3-(2 '-spiral diamantane)-4-methoxyl-4-(3 "-phosphorus acyloxy) phenyl-1,2-dichloroethane, CSPD or CDP-Star.
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| CN103033611A (en) * | 2012-12-13 | 2013-04-10 | 北京新华联协和药业有限责任公司 | Chemiluminiscence diagnostic kit for sensitization allergens and preparation method thereof |
| CN103087171B (en) * | 2012-12-24 | 2015-01-14 | 中国人民解放军第四军医大学 | Bispecific antibody of resisting PSMA/FITC (prostate specific membrane antigen/fluorescein isothiocyanate) for early diagnosis and treatment of prostatic cancer and preparation method of bispecific antibody |
| EA201691952A1 (en) | 2014-03-28 | 2017-05-31 | Опкоу Дайагностикс, Ллк. | COMPOSITIONS AND METHODS RELEVANT TO DIAGNOSTIC PROSTATE CANCER |
| DK3253800T3 (en) * | 2015-03-27 | 2021-05-31 | Opko Diagnostics Llc | Prostate antigen standards and use thereof |
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| US6180417B1 (en) * | 1999-04-22 | 2001-01-30 | Bayer Corporation | Immunochromatographic assay |
| CN1880958A (en) * | 2005-06-14 | 2006-12-20 | 郑州安图绿科生物工程有限公司 | Enzyme-catalyzed chemiluminescence immune detection system |
| CN101377490B (en) * | 2007-08-30 | 2012-07-04 | 北京科美生物技术有限公司 | Magnetic microparticle separating chemiluminescence immune analysis determination reagent kit for detecting related sign object and preparing method thereof |
| CN101377500A (en) * | 2007-08-31 | 2009-03-04 | 北京科美东雅生物技术有限公司 | Free prostate gland specificity antigen chemiluminescence immune analysis determination reagent kit and preparing method thereof |
| CN101377515A (en) * | 2008-04-16 | 2009-03-04 | 北京科美东雅生物技术有限公司 | Thyrotropin chemiluminescence immune analysis determination reagent kit and preparing method thereof |
| CN101377504A (en) * | 2008-04-16 | 2009-03-04 | 北京科美东雅生物技术有限公司 | Chemiluminescence immune analysis determination reagent kit for detecting Toxoplasma Gondi IgM antibody |
| CN101661036A (en) * | 2009-08-27 | 2010-03-03 | 北京倍爱康生物技术有限公司 | Aflatoxin B1 magnetic particle separation enzyme-linked immunoassay |
| CN101907627A (en) * | 2010-08-05 | 2010-12-08 | 北京倍爱康生物技术有限公司 | Clenbuterol hydrochloride magnetic particle separation enzyme-linked immunosorbent assay method |
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