CN105067599A - Chemiluminescence immune detection kit for detecting pro-gastrin-releasing peptide - Google Patents
Chemiluminescence immune detection kit for detecting pro-gastrin-releasing peptide Download PDFInfo
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- CN105067599A CN105067599A CN201510474660.8A CN201510474660A CN105067599A CN 105067599 A CN105067599 A CN 105067599A CN 201510474660 A CN201510474660 A CN 201510474660A CN 105067599 A CN105067599 A CN 105067599A
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Abstract
The invention provides a chemiluminescence immune detection kit for detecting a pro-gastrin-releasing peptide; the chemiluminescence immune detection kit is composed of a non-transparent white elisa plate coated with the pro-gastrin-releasing peptide, a pro-gastrin-releasing peptide standard, a pro-gastrin-releasing peptide-peroxidase labelled antibody working liquid, a luminous substrate liquid, a concentrated sample diluent and a concentrated detergent. A pro-gastrin-releasing peptide protein conjugate is obtained by coupling the pro-gastrin-releasing peptide with a carrier protein by a mixed anhydride method or a carbodiimide method, and the concentrated detergent contains 0.05% of Twain-20. Compared with a conventional enzyme-linked immunosorbent assay, the kit has higher sensitivity, is short in detection time and low in cost, and can be used for detection of the pro-gastrin-releasing peptide in human blood or serum.
Description
Technical field
The present invention relates to immunoassay field, relate to a kind of chemiluminescence immune detection reagent kit detecting gastrin-releasing peptide precursor particularly.
Background technology
Lung cancer is the neoplastic disease that a kind of grade malignancy is higher, and its incidence of disease growth rate is also in first of each malignant tumour.Its small cell lung cancer (smallcelllungcancer, SCLC) accounts for 15% ~ 20% of lung cancer, is the special lung neoplasm that a kind of grade malignancy is high, growth rapidly, is easily shifted in early days.SCLC has the advantages that growth fraction is high, the doubling time is short, and most of patient belongs to late period when medical, and five year survival rate is low, poor prognosis.Therefore, early diagnosis is carried out to SCLC and early treatment is particularly important.Along with chemistry, molecular biological development, tumor markers (tumormarker, TM) is one of important means becoming diagnosing malignant tumor.TM refers to and occurs in tumour and in breeding, produced or secrete and be discharged in blood, cell, body fluid by tumour cell, the class material that reflection tumour exists and grows.The advantage such as TM has efficiently, facilitate, sample easily obtains and wound is little, its auxiliary diagnosis to lung cancer, histological classification, clinical stages, Index for diagnosis and curative effect monitoring have significant application value.
Gastrin-releasing peptide precursor (ProGRP) is newfound a kind of new steroids tumor markers for SCLC in recent years, and it not only can be used for the early diagnosis of SCLC, also contributes to judging curative effect and early detection tumor recurrence.The critical value of proGRP in human serum is 50pg/mL, it is 100% to the specificity of SCLC early diagnosis, detection sensitivity is about 75%, the specificity of its diagnosis SCLC can reach more than 95%, and the detection method of its routine is double antibody sandwich method (ELISA), radioimmunoassays (RIA) and chemoluminescence method.ELISA and RIA detection method detection speed is relatively slow, and the sensitivity of detection and accuracy are also poor.
Chemiluminescence immunoassay detection technique is the product that chemoluminescence method and immunoassay combine, and therefore has the high sensitivity of chemiluminescence detection technology and the high specific of immuno analytical method simultaneously.
Summary of the invention
Goal of the invention: for problems of the prior art, the present invention proposes a kind of chemiluminescence immune detection reagent kit detecting gastrin-releasing peptide precursor, utilize the ultimate principle of the specific immune response of antigen and antibody to realize, there is the high specific of high sensitivity and immunoassay.
Technical scheme: for realizing above-mentioned technical purpose, the chemiluminescence immune detection reagent kit of detection gastrin-releasing peptide precursor of the present invention comprises following component: the opaque white color ELISA Plate being coated with proGRP conjugate; The proGRP standard items of a series of concentration; The peroxidase labeled antibodies of proGRP: luminous substrate liquid; Concentrating sample dilution; 20 times of concentrated cleaning solutions.
Preferably, described opaque white color ELISA Plate is the detachable or non-removable opaque white color ELISA Plate in 36 holes or 96 holes.
Described proGRP conjugate is obtained by mixed anhydride method or carbodlimide method coupling proGRP and carrier protein, and described carrier protein is ovoserum albumin.
The concentration of described proGRP standard items is 0ng/mL, 0.05ng/mL, 0.15ng/mL, 0.45ng/mL, 1.35ng/mL, 4.05ng/mL.
Described proGRP-peroxidase labeled antibodies is the proGRP antibody with peroxidase labelling, wherein, described proGRP antibody is monoclonal antibody or polyclonal antibody, and the working fluid of described proGRP-peroxidase labeled antibodies is diluted to 1:20000 ratio with antibody diluent.
Wherein, described antibody diluent is the PBS damping fluid of 0.01MpH7.2, wherein comprises 1%BSA, 0.1% cold fish glue (coldfishskingelatin) and 0.05% thimerosal (Thimerosal).
The Chemoluminescent substrate that described luminous substrate liquid is is luminous agent with luminol or different luminol.
Particularly, described luminous substrate liquid is divided into A liquid and B liquid to preserve, and press 1:1 before use used in combination, wherein A liquid is the potpourri of luminescence enhancer and luminol, or the potpourri of luminescence enhancer and different luminol; B liquid is superoxol or urea hydrogen peroxide solution.
Preferably, described luminescence enhancer is to iodophenol Huo phenoxazine salt compounds.
Above-mentioned luminous substrate liquid also can be any one Chemoluminescent substrate being luminous agent with luminol or different luminol commercial.
Preferably, described concentrating sample dilution is 2 times of concentrating sample dilutions, and its composition is any one in phosphate buffer, Glycine-HCl buffer or Tris-HCl damping fluid, and concentration is 0.01mol/L, pH is 7.4, with deionized water 1:1 dilution by volume before using.
Preferably, described concentrated cleaning solution is 20 times of concentrated cleaning solutions, and it comprises 0.05% Tween-20, the PBST of 0.01mol/L, between pH value range 7.0 ~ 7.5, uses front deionized water according to concentration and dilution liquid: the volume ratio of deionized water is 1:19 dilution.
When the chemiluminescence immune detection reagent kit of proGRP of the present invention is applied to the detection of proGRP, detecting step is:
(1) pre-service testing sample is fluid sample by sample preparation to be tested;
(2) required reagent is taken out from cold storage environment, be placed in room temperature (20 ~ 25 DEG C) balance more than 30min, notice that often kind of liquid reagent must shake up before using;
(3) get bag by the ELISA Plate of proGRP antigen, add standard items or testing sample 50 μ L/ hole in the micropore of correspondence, standard items and each concentration of sample do two parallel laboratory tests;
(4) add proGRP antibody working fluid, 50 μ L/ holes, mixing of vibrating gently, react 45min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate;
(5) carefully open cover plate film, liquid in hole is dried, with wash operating solution 250 μ L/ hole, fully washs 4 ~ 5 times, every minor tick 10s, pat dry (bubble be not eliminated after patting dry can be poked with original rifle head) with thieving paper;
(6) add luminous substrate liquid mixed liquor (A liquid mixes by 1:1 before use with B liquid) 100 μ L/ hole, mixing of vibrating gently, detects luminous intensity (RLU) in chemiluminescence detector after mixing;
(7) calculating of testing result: calculate with the ratio of the standard solution obtained and sample solution luminous value and blank solution.See following formula:
In relative luminous intensity=RLU/RULmax formula:
The luminous intensity values of RLU=standard (or sample) solution;
The luminous intensity values of RLUmax=blank (concentration is the standard solution of 0);
The natural logarithm of corresponding for the relative luminous intensity value calculated proGRP (μ g/L) is made semilog coordinate system curve figure.
The proGRP concentration of each testing sample is found on typical curve according to its RLU value, or is calculated by the corresponding equation of typical curve.As having dilution in sample preparation, the sample concentration that should draw according to typical curve will be multiplied by its extension rate again.Be the actual concentrations of proGRP in sample.
Kit of the present invention can be used for the residues detection of proGRP in blood of human body sample.
Chemiluminescence immune assay is the product that chemoluminescence method and immunoassay combine, and therefore has the high sensitivity of chemoluminescence method and the high specific of immunoassay simultaneously.In whole course of reaction, in sample, proGRP content is higher, and in reaction system, luminous intensity is more weak; Otherwise in sample, proGRP content is fewer, and luminous intensity is higher.The present invention has the proGRP antibody of high-affinity, high specific by distinctive immune animal preparation, and adopts enzymic-labelled antibody, sets up a kind of chemiluminescence immunoassay kit that can detect proGRP in serum.The features such as this method has easy and simple to handle, quick, and highly sensitive, specificity is good.
Beneficial effect: detect the comparison of proGRP residual quantity with existing other, kit of the present invention has following advantage:
(1) kit of the present invention of Chemiluminescence immunoassay is adopted, more more fast and convenient than chromatographic process (efficient liquid phase, LC-MS, gas chromatography mass spectrometry), capillary electrophoresis method, required instrument is more simple, and testing cost is more cheap, has high-throughout feature simultaneously;
(2) adopt the kit of the present invention of Chemiluminescence immunoassay, more sensitiveer than ELISA method, can detect that the proGRP of lower concentration and content remains, the range of linearity is wider simultaneously;
(3) adopt the kit of the present invention of Chemiluminescence immunoassay, decrease two anti-use links, thus shorten detection time; Opaque white color ELISA Plate adds chemiluminescence detector sensitivity.In addition, with proGRP-carrier protein couplet thing but not proGRP antibody wrap by opaque white color ELISA Plate, decrease the instability of proGRP antibody, ensure that the long-term effectiveness of kit.
Accompanying drawing explanation
Fig. 1 is proGRP canonical plotting.
Embodiment
Below by way of specific embodiment, the invention will be further described.These embodiments only for illustration of the present invention, and are not used for limiting the scope of the invention.
Embodiment 1
1, the preparation of each component of kit
(1) the haptenic preparation of proGRP: by proGRP acidifying, with sodium nitrite effect (both amount ratios are 1:1) in 4 DEG C of unglazed low temperature environments, generates the intermediate containing diazo positive ion.Diazotizing proGRP, as haptens, uses and synthesizes immunizing antigen and envelope antigen later;
(2) proGRP-bovine serum albumin(BSA) (BSA) immunogenic preparation: adopt diazotising method to carry out coupling proGRP and bovine serum albumin(BSA) (BSA) and obtain proGRP-BSA, as immunizing antigen;
(3) preparation of proGRP-ovoserum albumin (OVA) envelope antigen: adopt diazotising method to carry out coupling proGRP and ovoserum albumin (OVA) and obtain proGRP-ovoserum albumin (OVA) conjugate, as envelope antigen;
(4) preparation of proGRP-peroxidase labeled antibodies: to the female BAl BIc/c mouse (body weight 18 ~ 20g) in 6 ~ 8 week age, heavy dose of immunization protocol is, first immunisation 160 μ gproGRP-BSA and equivalent Freund's complete adjuvant mix, hypodermic injection.After 3 weeks, then mix with 80 μ gproGRP-BSA and equivalent Freund's complete adjuvant, hypodermic injection.After this mixed with 80 μ gproGRP-BSA and equivalent Freund's complete adjuvant every 3 weeks, lumbar injection.Last immunity in the spleen 80 μ gproGRP-BSA is as booster immunization.Put to death mouse after three days, get its spleen, with myeloma cell fusion.Screen positive hybridoma cell with indirect ELISA method, obtain the monoclonal antibody that can produce pro-GRP.Prepare mouse ascites in a large number by mouse peritoneal injection hybridoma, ascites, after filtration, centrifugal preliminary purification, adopts sad method and affinity chromatography purifying ascites, then obtains the proGRP monoclonal antibody of purifying through dialysis.ProGRP monoclonal antibody and horseradish peroxidase, thus obtain proGRP-peroxidase labeled antibodies;
(5) the opaque white color ELISA Plate preparation of proGRP-OVA conjugate is coated with: be used for wrapping the detect aperture by opaque white color ELISA Plate after being diluted by proGRP-OVA conjugate with damping fluid, 4 DEG C spend the night after use PBST buffer solution, then 180 μ L confining liquids (5% skim milk powder solution) are added, 37 DEG C of incubation 1.5h, incline liquid in hole, and thieving paper pats dry rear sealing and preserves;
(6) luminous substrate liquid preparation: the Chemoluminescent substrate being luminous agent with luminol or different luminol, is divided into A liquid and B liquid to preserve; Wherein A liquid is for adding luminol to iodophenol or adding different luminol to iodophenol, and B liquid is superoxol or urea hydrogen peroxide solution;
(7) 2 times of concentrating sample dilutions: its composition is the phosphate buffer of 0.01mol/L, pH7.4;
(8) 20 times of concentrated cleaning solutions: its composition is for containing 0.05% Tween-20, and the PBST of 0.01mol/L, between pH value range 7.0-7.5.
2, the establishment of the chemiluminescence immune detection reagent kit of proGRP is detected
The chemiluminescence immune detection reagent kit of the detection proGRP set up, contains following ingredient:
(1) 96 hole opaque white color ELISA Plate (8 hole × 12) is coated with proGRP-OVA conjugate, uses aluminium foil bag vacuum sealed package;
(2) proGRP standard solution 6 bottles, concentration is respectively: 0ng/mL, 0.05ng/mL, 0.15ng/mL, 0.45ng/mL, 1.35ng/mL, 4.05ng/mL;
(3) proGRP-Horseradish Peroxidase Conjugates solution;
(4) luminous substrate A liquid (luminol and to iodophenol), luminous substrate B liquid (urea hydrogen peroxide);
(5) 2 times of concentrating sample dilutions.Please dilute (1 part of concentration and dilution liquid+1 part of deionized water) by 1:1 before using and become working prototype dilution, the working prototype dilution after its dilution is the PBST damping fluid of 0.05mol/L, pH7.4;
(6) 20 times of concentrated cleaning solutions.Please dilute (1 part of concentration and dilution liquid+19 parts of deionized waters) by 1:19 before using and become work cleansing solution, the work cleansing solution after its dilution is between pH value range 7.0-7.5, containing 0.05% Tween-20, and the PBST damping fluid of 0.01mol/L.
3, chemiluminescence immune detection reagent kit on probation of proGRP
(1) pre-treatment of sample
Get water sample to be checked, if when sample is more muddy, can more than 5000r/min, centrifugal 5min or filtration;
(2) chemiluminescence immune detection reagent kit operation steps
The hole bar of standard and sample requirement is inserted in microwell plate framework, the position of record standard and sample.50 μ L/ hole proGRP standard solution and testing samples are added respectively in suitable micropore.Add 50 μ L/ hole proGRP-Horseradish Peroxidase Conjugates in each micropore, fully after mixing, under room temperature, lucifuge leaves standstill incubation 45min.Liquid in hole is dried, fully washs 4 ~ 5 times with wash operating solution.Remove the liquid in hole completely, pat dry with thieving paper, add luminous substrate liquid mixed liquor A liquid and mix by 1:1 before use with B liquid) 100 μ L/ holes.In chemiluminescence detector, luminous intensity (RLU) is detected immediately after mixing;
(3) computational analysis of testing result
Calculate with the ratio of obtained standard solution and sample solution luminous value and blank solution.See following formula:
Relative luminous intensity=RLU/RULmax, in its Chinese style:
The luminous intensity values of RLU=standard (or sample) solution;
The luminous intensity values of RLUmax=blank (concentration is the standard solution of 0);
The natural logarithm of corresponding for the relative luminous intensity value calculated proGRP (μ g/L) is made semilog coordinate system curve figure, as shown in Figure 1.The proGRP concentration of each testing sample is found on typical curve according to its RLU value, or is calculated by the corresponding equation of typical curve.As sample have passed through beforehand dilution, the sample concentration that should draw according to typical curve will be multiplied by its extension rate again.
(4) kit precision is determined
Examine and determine kit of the present invention according to manufacture conventional in this area and vertification regulation, result is as follows:
Kit provided by the invention is got two batches, measure the serum of basic, normal, high variable concentrations (proGRP concentration is respectively 30ng/ml, 135ng/ml, 180ng/ml) respectively, 10 hole replicate determinations, show that variation within batch coefficient is 1.94%, 2.31% and 4.24%, as shown in table 1.
Table 1 kit precision and accuracy are tested
(5) stabilization of kit test:
Kit preservation condition is 2 ~ 8 DEG C, preserves after 6 months, measures the indices of kit, finds all within normal range.Consider in transport and use procedure, have improper preservation condition and occur, placed 6 days under 37 DEG C of conditions of preserving by kit, carry out accelerated aging tests, result shows that this kit indices meets the requirements completely.Consider that the freezing situation of kit occurs, kit is put into-20 DEG C of refrigerator freezings 5 days, measurement result also shows that kit indices is completely normal.Can show that kit at least can preserve more than 6 months at 2 ~ 8 DEG C from above result.
The above is only the preferred embodiment of the present invention; be noted that for those skilled in the art; under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. detect a chemiluminescence immune detection reagent kit for gastrin-releasing peptide precursor, it is characterized in that, comprise following component: the opaque white color ELISA Plate being coated with proGRP conjugate; The proGRP standard items of a series of concentration; The peroxidase labeled antibodies of proGRP: luminous substrate liquid; Concentrating sample dilution; 20 times of concentrated cleaning solutions.
2. chemiluminescence immune detection reagent kit according to claim 1, is characterized in that, described opaque white color ELISA Plate is the detachable or non-removable opaque white color ELISA Plate in 36 holes or 96 holes.
3. chemiluminescence immune detection reagent kit according to claim 1, is characterized in that, described proGRP conjugate is obtained by mixed anhydride method or carbodlimide method coupling proGRP and carrier protein, and described carrier protein is ovoserum albumin.
4. chemiluminescence immune detection reagent kit according to claim 1, is characterized in that, the concentration of described proGRP standard items is 0ng/mL, 0.05ng/mL, 0.15ng/mL, 0.45ng/mL, 1.35ng/mL, 4.05ng/mL.
5. chemiluminescence immune detection reagent kit according to claim 1, it is characterized in that, described proGRP-peroxidase labeled antibodies is the proGRP antibody with peroxidase labelling, wherein, described proGRP antibody is monoclonal antibody or polyclonal antibody, and the working fluid of described proGRP-peroxidase labeled antibodies is diluted to 1:20000 ratio with antibody diluent.
6. chemiluminescence immune detection reagent kit according to claim 5, is characterized in that, described antibody diluent is the PBS damping fluid of 0.01MpH7.2, wherein comprises 1%BSA, 0.1% cold fish glue and 0.05% thimerosal.
7. chemiluminescence immune detection reagent kit according to claim 1, it is characterized in that, described luminous substrate liquid is divided into A liquid and B liquid to preserve, and presses 1:1 before use used in combination, wherein A liquid is the potpourri of luminescence enhancer and luminol, or the potpourri of luminescence enhancer and different luminol; B liquid is superoxol or urea hydrogen peroxide solution.
8. chemiluminescence immune detection reagent kit according to claim 7, is characterized in that, described luminescence enhancer is to iodophenol Huo phenoxazine salt compounds.
9. chemiluminescence immune detection reagent kit according to claim 1, it is characterized in that, described concentrating sample dilution is 2 times of concentrating sample dilutions, its composition is any one in phosphate buffer, Glycine-HCl buffer or Tris-HCl damping fluid, concentration is 0.01mol/L, pH is 7.4, with deionized water 1:1 dilution by volume before using.
10. chemiluminescence immune detection reagent kit according to claim 1, it is characterized in that, described concentrated cleaning solution is 20 times of concentrated cleaning solutions, it comprises 0.05% Tween-20, the PBST of 0.01mol/L, between pH value range 7.0 ~ 7.5, use front deionized water according to concentration and dilution liquid: the volume ratio of deionized water is 1:19 dilution.
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Application publication date: 20151118 |