CN103293313A - Carbohydrate antigen 15-3 time-resolved immunofluorescence assay and kit - Google Patents
Carbohydrate antigen 15-3 time-resolved immunofluorescence assay and kit Download PDFInfo
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Abstract
The invention discloses a carbohydrate antigen 15-3 time-resolved immunofluorescence assay and a kit. The assay comprises the following steps of: (1) preparing a solid phase antibody; (2) preparing lanthanide ion labeled antibody; (3) adding a carbohydrate antigen 15-3 standard substance or a to-be-tested sample and a reaction buffer solution into the solid phase antibody-containing solid phase material prepared in the step (1), after incubating and washing the solid phase material, adding the diluted labeled antibody, and performing fluorescence detection after incubating again. The invention also provides a corresponding kit. The carbohydrate antigen 15-3 time-resolved immunofluorescence assay and the kit have high sensitivity, specificity and stability, and need short detection time.
Description
Technical field
The present invention relates to a kind of time-resolved fluoroimmunoassay and kit, particularly relate to a kind of sugar antigen 15-3 time-resolved fluoroimmunoassay and kit.
Background technology
Sugar antigen 15-3 (CA 15-3) is the mucin glycoprotein of molecular weight about 400,000, by tumor cell secretion and be released in the blood circulation.Because it by two strain monoclonal antibody DF3 and 115D8 identification, therefore names the 15-3 into CA at first.Result of study shows that CA 15-3 follows up a case by regular visits to one of best mark that monitoring and curative effect judge for breast cancer, to differentiation degree, the tumor prognosis of assisting judgement breast cancer, to lapse to, estimate curative effect etc. all significant.CA15-3 is for the monitoring of patient's postoperative recurrence situation, and particularly cancer metastasis patient's monitoring after operation is very useful.In addition, oophoroma, adenocarcinoma of lung, the cancer of the uterus, colorectal cancer, cancer of pancreas etc. and some optimum mammary gland disease CA15-3 also raise.CA 15-3 also is used for other tumor markers simultaneous determination to improve the recall rate of multiple cancer diagnosis.
Change of serum C A 15-3 detects euzymelinked immunosorbent assay (ELISA) (ELISA), the immune radiating method (IRMA) of mainly containing at present.Because be subjected to the restriction of tracer agent proterties, the sensitivity of ELISA is not high, sensing range is limited, and the IRMA kit is subjected to the influence of isotope half life period, and the term of validity is short, also has certain radioactive contamination in addition.
Time resolution immunofluorescence analysis (time-resolved fluoroimmunoassay, TRFIA) utilize the lanthanide series with unique fluorescent characteristic, replace mould, isotope etc., as a kind of important method in the luminescence immunoassay, have highly sensitive, tracer stable, be not subjected to many advantages such as the interference of sample natural fluorescence, "dead" pollution.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of sugar antigen 15-3 time-resolved fluoroimmunoassay and kit.By method of the present invention, can solve in the prior art about CA 15-3 detect that the sensitivity that exists is low, technical matterss such as poor stability, complex operation and contaminated environment.
For solving the problems of the technologies described above, sugar antigen 15-3 time-resolved fluoroimmunoassay of the present invention comprises step:
1. insolubilized antibody preparation
After will resisting the dilution of sugar antigen 15-3 monoclonal antibody, as coating buffer, bag is formed the solid phase material with insolubilized antibody by solid phase material;
2. lanthanide ion labelled antibody preparation
Select the anti-sugar antigen 15-3 monoclonal antibody of pairing for use, carry out the lanthanide ion mark with conventional method, form labelled antibody; Wherein this labelled antibody and step 1. antibody respectively in conjunction with the different antigenic determinant of sugar antigen 15-3, thereby form sandwich complex.
3. in the solid phase material with insolubilized antibody that 1. step makes, add sugar antigen 15-3 standard items or testing sample, and reaction buffer, after hatching, washing solid phase material, add the labelled antibody of dilution, carry out fluoroscopic examination after hatching again.
Described step 1. in, concrete preparation process comprises:
(1) will resisting sugar antigen 15-3 monoclonal anti body and function bag to be cushioned liquid, to be diluted to antibody concentration be 1.0～10.0 μ g/ml, as coating buffer, contacts and leave standstill 5～24 hours with solid phase material;
Wherein, bag is cushioned liquid and comprises: the 50mM phosphate buffered solution of pH 7.2-7.6;
Solid phase material comprises: micropore reaction bar or the micro reaction plate that is formed by micropore reaction bar;
(2) with the damping fluid that contains surfactant, behind the solid phase material of the antibody sandwich of washing step (1) preparation, add the damping fluid that contains closed protein, left standstill 3～12 hours;
Wherein, contain the damping fluid of surfactant, comprising: contain the 50mM pH7.8TSA damping fluid of 0.1%Tween-20, and 50mM pH 7.8TSA damping fluid is 50mM Tris, contains 150mM NaCl, 1%BSA, 16% sucrose;
Contain the damping fluid of closed protein, comprising: the 50mM pH7.8TSA damping fluid that contains 0.5% caseic 0.1%Tween-20;
(3) discard the damping fluid that contains closed protein, dry solid phase material is finished the insolubilized antibody preparation.
Described step 2. in, lanthanide ion comprises: Eu
3+, Sm
3+, De
3+, Te
3+Deng, be preferably Eu
3+The mass ratio of lanthanide ion and anti-sugar antigen 15-3 monoclonal antibody is 1: 2-1: 1.
Described step 3. in, the preparation method of sugar antigen 15-3 standard items is: earlier respectively sugar antigen 15-3 is diluted to required maximum concentration, adopts coubling dilution to be diluted to desired concn again, obtain sugar antigen 15-3 standard items; Wherein, these standard items are preferably with containing 20% calf serum, 0.1%NaN
3Physiological saline sugar antigen 15-3 is mixed with standard items;
Reaction buffer comprises: the 50mmol/L Tris-HCl of pH 7.6-8.0 includes 0.9%NaCl, 2%BSA, 0.05% N of IgG, 0.1%Tween-20 and 0.1%NaN
3
Hatch, wash in the solid phase material, after 20～25 ℃ (room temperatures) hatch 1～2h, employing contains the dilution of damping fluid of surfactant as cleansing solution, the washing solid phase material, wherein, contain the damping fluid of surfactant, comprising: the 50mM pH7.8TSA damping fluid that contains 0.1%Tween-20, and 50mM pH 7.8TSA damping fluid is 50mM Tris, contains 150mM NaCl, 1%BSA, 16% sucrose.
The dilution that contains the damping fluid of surfactant is to contain the damping fluid of surfactant and the dilution proportion that water is 1: 24～1: 25 with volume ratio;
In the labelled antibody of dilution, labelled antibody is dilution in 1: 20 with reaction buffer with volume ratio; The labelled antibody concentration of dilution is 1～5 μ g/ml.
In hatching again, hatch 1～2h 20～25 ℃ (room temperatures).
In addition, after hatching again, after washing solid phase material (containing the dilution washing of the damping fluid of surfactant) again, can add enhancing liquid and carry out fluoroscopic examination, wherein, strengthening liquid is the damping fluid that contains 1.0% glacial acetic acid and 2.0% Potassium Hydrogen Phthalate, pH 3.0-4.5, contain 0.4% betanaphthyl formyl trifluoroacetone (β-NTA), 2% 3 n-octyl phosphine oxide (TOPO), 0.1%Triton X-100 in this damping fluid simultaneously.
According to said method, the present invention also provides a kind of sugar antigen 15-3 quantitative detection kit, comprising: bag is by the solid phase material of anti-sugar antigen 15-3 monoclonal antibody, reaction buffer, the anti-sugar antigen monoclonal antibody of lanthanide ion mark, sugar antigen 15-3 standard items.
Described kit also comprises: cleansing solution (dilution that contains the damping fluid of surfactant as above-mentioned employing), enhancing liquid.
Among the present invention, the solid phase material of the bag quilt that adopts is the solid phase material of specific antibody bag quilt, and the use double antibody sandwich method, pat dry, add steps such as strengthening liquid, survey fluorescent value by reagent preparation, application of sample reaction, washing and detect, have following beneficial effect:
1) have stability preferably, and the sensitivity for analysis height (＜1U/ml);
2) detection time short (185min);
3) specificity is good, and is little with the cross reaction of interfering material.
Description of drawings
The present invention is further detailed explanation below in conjunction with accompanying drawing and embodiment:
Fig. 1 is the reaction principle figure of employing double-antibody sandwich two step method of the present invention, and wherein, A is anti-people CA 15-3 monoclonal antibody bag by in 96 hole fluorescence microwell plates, with the CA 15-3 antigen in standard items or the sample; B is that CA 15-3 antigen and the Sheet in standard items or the sample resists after micropore inside surface generation immune response, and washing; C is for adding europium ion (Eu
3+) the anti-CA15-3 monoclonal antibody of mark, carry out immune response after, the sandwich immunoassay compound of micropore surface separates by washing with the anti-CA 15-3 monoclonal antibody of free label; D is the Eu in the micropore surface immune complex
3+After being dissociated by fluorescence enhancement solution, form the stable fluorescence complex, the proportional example of CA 15-3 antigen concentration in fluorescence intensity and standard items or the sample can draw CA 15-3 antigen concentration in the sample by dose-response curve;
Fig. 2 is operational flowchart of the present invention, and wherein, a is the dilution testing sample, and b is for adding standard items and testing sample, c is for adding reaction buffer, and d is for hatching, and e is for washing plate, and f is for adding the europium label after diluting, g is for hatching, and h is for washing plate, and i strengthens liquid for adding, and j is for detecting;
Fig. 3 is the sugar antigen 15-3 value that records of the present invention of 36 parts of blood samples among the embodiment and the correlation figure of Luo Shi measured value.
Embodiment
Embodiment: sugar antigen 15-3 (CA 15-3) measures
1, standard items preparation
With containing 20% (v/v) calf serum, 0.1%NaN
3(w/v) physiological saline will purchase in the sugar antigen 15-3 of Hytest company be mixed with 0,15,30,60,117, the 256U/ml titer.Packing is in 2～8 ℃ of preservations.
2, bag is by the preparation of micropore reaction bar
(1) the anti-sugar antigen 15-3 monoclonal antibody (purchasing the company in Hystest) of purifying is diluted to 4 μ g/ml with the 50mM phosphate buffer of pH 7.2-7.6, adds in each hole of micropore reaction bar by 100 μ l/ holes amount then, left standstill 5～24 hours;
(2) with the damping fluid that contains surfactant, behind the micropore reaction bar of the antibody sandwich of washing step (1) preparation, add the damping fluid that contains closed protein, left standstill 3～12 hours, seal;
Wherein, the damping fluid that contains surfactant, be the 50mM pH7.8TSA damping fluid that contains 0.1% (V/V) Tween-20, and 50mM pH 7.8TSA damping fluid is 50mM Tris, contains the sucrose of 150mM NaCl, 1% (w/v) BSA (bovine serum albumin(BSA)), 16% (w/v);
The damping fluid that contains closed protein is the 50mM pH7.8TSA damping fluid that contains 0.5% (w/v) casein, 0.1% (v/v) Tween-20;
(3) discard the damping fluid that contains closed protein, dry micropore reaction bar, the micropore that obtains anti-sugar antigen 15-3 monoclonal antibody bag quilt reacts bar;
(4) will resist the micropore of sugar antigen 15-3 monoclonal antibody bag quilt to react the Aluminium Foil Package pack that bar is packed into special-purpose, seal and refrigerate standby.
3, the anti-sugar antigen 15-3 MONOCLONAL ANTIBODIES SPECIFIC FOR of europium mark
(1) gets antibody to be marked (anti-sugar antigen 15-3 monoclonal antibody is purchased the company in Fitzgerald) and add in the bag filter, put into the 50mM carbonate buffer solution of the pH 9.4-9.6 for preparing, the room temperature dialysed overnight;
(2) next day, take out antibody, be diluted to 0.2～5mg/ml, be 1: 1 ratio (DTTA-Eu: antibody) add DTTA-Eu while vibrating, on the vibration plate machine, vibrated at a slow speed 10 minutes under the room temperature, left standstill in the dark then 24 hours in mass ratio;
(3) (1 * 50cm) separates the antibody that mark is good, and detachment process is monitored with the nucleic acid-protein instrument by Sephadex G50 chromatographic column with free DTTA-Eu;
(4) accept effluent with small test tube segmentation successively, use spectrophotometer, survey absorbance at the 280nm place, survey fluorescence intensity simultaneously, calculate Eu labeling antibody concentration; Labelled antibody concentration is generally 0.1mg/ml;
(5) filtrator with 0.2 μ m filters;
(6) add 0.3% (w/v) BSA, 2～8 ℃ of preservations.
4, operation steps
1) preparation of reagent
A) cleansing solution: the damping fluid and the 960mL deionized water that 40mL are contained surfactant mix in clean bottle for handling liquid toilet or cosmetic substance, and be standby as the work cleansing solution.
Wherein, the damping fluid that contains surfactant is the 50mM pH7.8TSA damping fluid that contains 0.1% (V/V) Tween-20, and 50mM pH 7.8TSA damping fluid is 50mM Tris, contains the sucrose of 150mM NaCl, 1% (w/v) BSA, 16% (w/v);
B) europium label: using last hour, is the anti-sugar antigen 15-3 monoclonal antibody of the europium mark of the above-mentioned preparation of dilution in 1: 20 by volume with reaction buffer.
Wherein, reaction buffer is the 50mmol/L Tris-HCl of pH 7.8, includes 0.9% (w/v) NaCl, 2% (w/v) BSA, 0.05% (w/v) ox IgG, 0.1% (v/v) Tween-20 and 0.1% (w/v) NaN
3
C) micropore that is coated with antibody with reaction buffer, testing sample, standard items and requirement reacts the bar balance to room temperature (20～25 ℃).
2) be 1: 41 dilute sample by volume with physiological saline.
3) draw the standard items of 25 μ l or the sample that has diluted, be sequentially added into corresponding micropore bottom.
4) in each micropore, add 100 μ l reaction buffers, vibrate 120 minutes in slow shelves under the room temperature.
5) use the above-mentioned a) cleansing solution of preparation to wash plate 4 times, the thieving paper of lath in cleaning is patted dry.
6) in each micropore, add the europium label solution that 100 μ l have diluted [above-mentioned b) preparation], under the room temperature in slow shelves vibration 60 minutes.
7) use the cleansing solution that a) prepares to wash plate 6 times, the thieving paper of lath in cleaning is patted dry.
8) enhancing liquid [1.0% (v/v) glacial acetic acid and 2.0% (w/v) Potassium Hydrogen Phthalate damping fluid of adding 100 μ l in each micropore, pH 3.0-4.5, and contain 0.4% (w/v) betanaphthyl formyl trifluoroacetone (β-NTA), 2% (w/v) three n-octyl phosphine oxides (TOPO), 0.1% (v/v) Triton X-100], under the room temperature in slow shelves vibration 5 minutes.
9) differentiating fluor tester (the Anytest2000 fluorescence detector that the biological company limited of new ripple produces) with the time detects.
Above sugar antigen 15-3 (CA 15-3) measures flow process and sees Table 1 or Fig. 2.
Table 1 sugar antigen 15-3 (CA 15-3) measures flow process
Therefore, can be made into kit of the present invention according to above-mentioned steps, namely comprise: above-mentioned bag is by the micropore of anti-sugar antigen 15-3 monoclonal antibody reaction bar, reaction buffer, the anti-sugar antigen 15-3 monoclonal antibody of europium mark, sugar antigen 15-3 standard items, cleansing solution, enhancing liquid.
In addition, according to above-mentioned testing process, to blood sample (available from the serum of Da An genome company), utilize Luo Shi CA 15-3 kit and method of the present invention (kit of the present invention), compare experiment, testing result sees Table 2, table 4 and Fig. 3.
Wherein, the Luo Shi concentration in the table 2 refers to the sugar antigen 15-3 concentration value that blood sample records at Luo Shi cobas e 411 instruments through Luo Shi CA 15-3 kit; Fluorescent value (CPS) is the value that the inventive method records.
Table 3 is yin and yang attribute criterions of the sugar antigen 15-3 measured value of the blood sample in the his-and-hers watches 2, and according to this standard, his-and-hers watches 2 are added up, and the results are shown in Table 4.
Table 2 sugar antigen 15-3 (CA 15-3) testing result
The yin and yang attribute criterion of the sugar antigen 15-3 measured value of the blood sample in table 3 his-and-hers watches 2
Table 4 is according to the yin and yang attribute statistics of 36 parts of blood samples in the criterion his-and-hers watches 2 of table 3
Therefore, by showing 2-4 and Fig. 3 as can be known, compare with the Luo Shi kit, kit of the present invention is good to the measurement result correlativity of blood sample, and yin and yang attribute coincidence rate height.
5, specific detection
According to the aforesaid operations step, alpha-fetoprotein and neuronspecific enolase are detected as carrying out cross reacting material, the results are shown in Table 5.Wherein, alpha-fetoprotein and neuronspecific enolase are all available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
The testing result of table 5 cross reacting material
Wherein, the apparent CA 15-3 concentration in the table 5 is the cross reacting material of concentration shown in the table 2, adopts the resulting CA 15-3 of detection method of the present invention concentration.As seen have specificity preferably, and the cross reaction of the present invention and interfering material is little.
Claims (11)
1. sugar antigen 15-3 time-resolved fluoroimmunoassay is characterized in that: comprise step:
1. insolubilized antibody preparation
After will resisting the dilution of sugar antigen 15-3 monoclonal antibody, as coating buffer, bag is formed the solid phase material with insolubilized antibody by solid phase material;
2. lanthanide ion labelled antibody preparation
Select the anti-sugar antigen 15-3 monoclonal antibody of pairing for use, carry out the lanthanide ion mark, form labelled antibody;
3. in the solid phase material with insolubilized antibody that 1. step makes, add sugar antigen 15-3 standard items or testing sample, and reaction buffer, after hatching, washing solid phase material, add the labelled antibody of dilution, carry out fluoroscopic examination after hatching again.
2. analytic approach as claimed in claim 1 is characterized in that: described step 1. in, concrete preparation process comprises:
(1) will resisting sugar antigen 15-3 monoclonal anti body and function bag to be cushioned liquid, to be diluted to antibody concentration be 1.0～10.0 μ g/ml, as coating buffer, contacts and leave standstill 5～24 hours with solid phase material;
Wherein, bag is cushioned liquid and comprises: the 50mM phosphate buffered solution of pH 7.2-7.6;
Solid phase material comprises: micropore reaction bar or the micro reaction plate that is formed by micropore reaction bar;
(2) with the damping fluid that contains surfactant, behind the solid phase material of the antibody sandwich of washing step (1) preparation, add the damping fluid that contains closed protein, left standstill 3～12 hours;
Wherein, contain the damping fluid of surfactant, comprising: contain the 50mM pH7.8TSA damping fluid of 0.1%Tween-20, and 50mM pH 7.8TSA damping fluid is 50mM Tris, contains 150mM NaCl, 1%BSA, 16% sucrose;
Contain the damping fluid of closed protein, comprising: the 50mM pH7.8TSA damping fluid that contains 0.5% caseic 0.1%Tween-20;
(3) discard the damping fluid that contains closed protein, dry solid phase material is finished the insolubilized antibody preparation.
3. analytic approach as claimed in claim 1 is characterized in that: described step 2. in, lanthanide ion comprises: Eu
3+, Sm
3+, De
3+, Te
3+
The mass ratio of lanthanide ion and anti-sugar antigen 15-3 monoclonal antibody is 1: 2～1: 1.
4. analytic approach as claimed in claim 3 is characterized in that: described step 2. in, lanthanide ion is Eu
3+
5. analytic approach as claimed in claim 1, it is characterized in that: described step 3. in, the preparation method of sugar antigen 15-3 standard items is: respectively sugar antigen 15-3 is diluted to required maximum concentration earlier, adopts coubling dilution to be diluted to desired concn again, obtain sugar antigen 15-3 standard items.
6. analytic approach as claimed in claim 5 is characterized in that: described standard items are for containing 20% calf serum, 0.1%NaN
3Physiological saline sugar antigen 15-3 is mixed with standard items.
7. analytic approach as claimed in claim 1 is characterized in that: described step 3. in, reaction buffer comprises: the 50mmol/L Tris-HCl of pH 7.6-8.0 includes 0.9%NaCl, 2%BSA, 0.05% N of IgG, 0.1%Tween-20 and 0.1%NaN
3
8. analytic approach as claimed in claim 1, it is characterized in that: described step 3. in, 20～25 ℃ hatch 1～2h after, employing contains the dilution of damping fluid of surfactant as cleansing solution, the washing solid phase material, wherein, the damping fluid that contains surfactant, comprise: contain the 50mM pH7.8TSA damping fluid of 0.1%Tween-20, and 50mM pH 7.8TSA damping fluid is 50mM Tris, contains 150mM NaCl, 1%BSA, 16% sucrose;
The dilution that contains the damping fluid of surfactant is to contain the damping fluid of surfactant and the dilution proportion that water is 1: 24～1: 25 with volume ratio;
The labelled antibody of dilution is: labelled antibody is dilution in 1: 20 with reaction buffer with volume ratio; The labelled antibody concentration of dilution is 1～5 μ g/ml;
In hatching again, hatch 1～2h at 20～25 ℃.
9. analytic approach as claimed in claim 8 is characterized in that: described step 3. in, after hatching again, after washing solid phase material again, add and strengthen liquid and carry out fluoroscopic examination;
Wherein, strengthening liquid is the damping fluid that contains 1.0% glacial acetic acid and 2.0% Potassium Hydrogen Phthalate, and pH 3.0-4.5 contains 0.4% betanaphthyl formyl trifluoroacetone, 2% 3 n-octyl phosphine oxide, 0.1%Triton X-100 simultaneously in this damping fluid.
10. the quantitative detection kit of a kind of sugar antigen 15-3 as claimed in claim 1, it is characterized in that: comprising: bag is by the solid phase material of anti-sugar antigen 15-3 monoclonal antibody, reaction buffer, the anti-sugar antigen monoclonal antibody of lanthanide ion mark, sugar antigen 15-3 standard items.
11. kit as claimed in claim 10 is characterized in that: also comprise: cleansing solution, enhancing liquid.
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| CN107121548A (en) * | 2016-02-25 | 2017-09-01 | 深圳市迈科龙生物技术有限公司 | Quantitatively detect test paper, preparation method and the detection method of tumor markers |
| CN108982843A (en) * | 2018-06-11 | 2018-12-11 | 广东轻工职业技术学院 | A kind of test strips and preparation method thereof of effective cypermethrin pesticide quantitative detection |
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