CN107462721B - The board-like chemoluminescence method detection kit and preparation method of cytokeratin 19 fragment - Google Patents
The board-like chemoluminescence method detection kit and preparation method of cytokeratin 19 fragment Download PDFInfo
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Abstract
The present invention relates to a kind of board-like chemoluminescence method detection kit of cytokeratin 19 fragment, including reagent: the coated ELISA Plate of the CYFRA21-1 antibody-solutions of biotin labeling, anti-biotin antibodies, the CYFRA21-1 antibody-solutions of horseradish peroxidase-labeled, CYFRA21-1 calibration object A-F, concentration washing lotion, substrate solution A, substrate solution B.Main design thought of the invention is that have anti-biotin antibodies-biotin system on the basis of chemiluminescence immune assay by introducing, while the detection of stable reagent box is specific, be added significantly to detection sensitivity and detection time.High-temperature oscillation formula anti-biotin antibodies coating technique is improved, to simplify reagent production procedure while reducing antibody/antigen usage amount, shorten the production cycle.
Description
Technical field
The invention belongs to biotinylation kit Material Fields, and in particular to a kind of quantitative inspection for cytokeratin 19 fragment
Test agent box and preparation method quick, easy, efficiently, delicately can determine cytokeratin 19 fragment in human serum sample
Amount detection.
Background technique
Cytokeratin 19 fragment (Cytokeratin-19-fragment CYFRA21-1) is alveolar epithelial cells apoptosis
When, become solable matter after the fragment degradation of the keratin contained in cell and enter blood, increases content in blood.
CYFRA21-1, which is that the preferred marker of non-small cell lung cancer, especially dermoid cancer are presently preferred, to swell
Tumor markers, sensitivity is up to 60%, and specificity is up to 95%.It supervises the early diagnosis of non-small cell lung cancer (NSCLC), curative effect
It surveys and Index for diagnosis is significant.CYFRA21-1 is commonly used to the course of disease and prognosis of monitoring non-small cell lung cancer, non-small thin
In born of the same parents' lung cancer patient body, the horizontal significant raising of CYFRA21-1 in blood.
Prompt advanced stage or the poor prognosis of tumour；If the therapeutic effect to non-small cell lung cancer is good, under level meeting quickly
Normal level is dropped or be restored to, is such as worth constant or slight attenuating prompt tumour and does not completely remove or with the presence of multiple lump.
In order to improve diagnosing tumor level and improve therapeutic effect, from detection angles for, at present clinically for surveying
The method for determining CYFRA21-1 is less, mainly there is Enzyme-multiplied immune technique and chemiluminescence etc..Wherein, chemiluminescence is emerging
The 80's of eighties of last century are arised from, are the emerging technologies to grow up after Enzyme-multiplied immune technique and radioimmunoassay technology,
Due to its highly sensitive, high specific, while method is easy, quick, and label conjugate is stablized, and relative time resolved fluorometric is immune
The features such as method analysis cost is cheap, easy to operate, "dead" isotope damage and pollution, is developed rapidly.
This kit is mainly used for carrying out dynamic monitoring to associated malignancies patient with auxiliary judgment disease process or control
Therapeutic effect cannot function as associated malignancies early diagnosis or the foundation made a definite diagnosis, should not be used in the tumor screening of general population.
Summary of the invention
The technical problem to be solved by the present invention is to provide a kind of board-like chemoluminescence method detection for the above-mentioned prior art
The kit and preparation method thereof of human serum CYFRA21-1 is researched and developed and is optimized and is relevant to serology immune response principle each
Kind key technology is directed to the intracorporal CYFRA21-1 detection sensitivity of clinical patients and accuracy to improve, develops sensitivity
The board-like chemiluminescence diagnostic reagent high, stability is good, production is convenient, easy to operate.
Main design thought of the invention is resisted by introducing with antibiotin on the basis of chemiluminescence immune assay
Detection sensitivity is significantly increased while the detection of stable reagent box is specific in body-biotin system.
The present invention realizes technical solution used by above-mentioned technical purpose are as follows:
A kind of board-like chemoluminescence method detection kit of cytokeratin 19 fragment, it is characterised in that: including reagent
There are the CYFRA21-1 antibody-solutions of biotin labeling, the coated ELISA Plate of anti-biotin antibodies, horseradish peroxidase-labeled
CYFRA21-1 antibody-solutions, CYFRA21-1 calibration object A-F, concentration washing lotion, substrate solution A, substrate solution B.
The concentration of the CYFRA21-1 antibody-solutions of above-mentioned biotin labeling is 0.01~1.0 μ g/ml, the antibiotin
The peridium concentration of the anti-biotin antibodies of the coated ELISA Plate of antibody is 0.2~8.0 μ g/ml, the horseradish peroxidase mark
The concentration of the CYFRA21-1 antibody-solutions of note is 0.02~3.0 μ g/ml.
Above-mentioned CYFRA21-1 calibration object A-F point is CYFRA21-1(Beijing Hua Xinhang) with pH value be 7.4 Tris-
HCl buffer obtains, and concentration is respectively 0,1,5,20,60,120ng/mL.
The preparation process of the CYFRA21-1 antibody-solutions of above-mentioned biotin labeling: the CYFRA21-1 antibody of biotin labeling
It is added in the buffer that pH value is 7.4, adjusts and arrive convenient multiple, be uniformly mixed so as to obtain.
The preparation process of the above-mentioned CYFRA21-1 antibody-solutions for stating horseradish peroxidase-labeled: horseradish peroxidase
The CYFRA21-1 antibody-solutions of label are added in the buffer that pH value is 7.4, are adjusted and are arrived convenient multiple, are uniformly mixed so as to obtain.
Coating buffer used in the above-mentioned coated ELISA Plate of anti-biotin antibodies be 0.01M carbonic acid buffer, pH9.6 ±
0.1.Confining liquid used is bright containing 0.1wt%BSA, 0.1 % Proclin300 (v/v), 1 wt % sheep blood serum, 1 wt % fish-skin
The 0.1M pH7.4 TBS buffer of glue, 2 wt % trehaloses and 10 wt % sucrose.For anti-biotin antibodies packet of the invention
By system, ELISA Plate largely unbonded position is shielded with a small amount of BSA using the carbonic acid buffer of 0.01M, and in confining liquid
Point eliminates nonspecific binding site with animal blood serum, ensure that the precision of coating plate under the synergistic effect of buffer system
And Low background is enhanced coating plate to the resistance of high temperature, drying, is protected using sucrose, trehalose and fishskin gelatin as stabilising system
The effective active of anti-biotin antibodies coating plate is demonstrate,proved.
The preparation process of the coated ELISA Plate of anti-biotin antibodies are as follows: ELISA Plate is coated with using high-temperature oscillation formula, coating temperature
Degree is 37 ± 1 DEG C, and the coating time is 1~3 hour, is coated with form using low-speed oscillation, the shaker of selection is Ai Bensen science
Instrument Ltd.'s microplate oscillator, oscillation amplitude and mode: 3mm(horizontal rotation), speed low speed (200~500rpm),
Closing and drying temperature are 37 ± 1 DEG C, and off-period is 1.5~3 hours, and drying time is 2~4 hours；Using high-temperature oscillation
Formula coating technique, coating plate preparation time are can be controlled in 5~7 hours, are compared with 3~4 days universal preparation times of room temperature coating,
Have apparent odds for effectiveness, while improving the sensitivity of reaction, while reducing antibody/antigen usage amount, simplifies examination
Agent production procedure, shortens the production time.State's endoperidium plate technique is more using Streptavidin coating, anti-using antibiotin
Body is there is not yet patent document is reported.In addition, low-speed oscillation coating method has been obviously shortened coating plate compared to conventional room temperature coating method
Production time, improve reagent production efficiency.
The preparation method of the board-like chemoluminescence method detection kit of CYFRA21-1 of the present invention, includes the following steps
One, the preparation of washing lotion is concentrated, steps are as follows:
1, KCl 60g, NaCl 300g are weighed in 1L container；
2,20.0g Tween-20 is weighed in 100ml container plus after 50ml water makes it completely dissolved, and pours into above-mentioned 1L
In container；
3, Proclin-300 is measured into 2ml with pipettor, poured into above-mentioned 1L container；
4, appropriate purified water is measured in above-mentioned 1L container with graduated cylinder, be sufficiently stirred until being completely dissolved；
5, pH is adjusted, controls its range between 7.35~7.45；
6, it is finally settled to 1000ml, is filtered after being completely dissolved with 0.2 μm of filter to obtain the final product；
Two, the preparation of substrate solution
(1) preparation of substrate solution A
1, weigh borax 11.44g, boric acid 4.948g, luminol 2.0g and to iodophenol 0.2mg in 1L beaker；
2, purified water is measured in 1L beaker with graduated cylinder, be sufficiently stirred until being completely dissolved, tune pH controls its range and exists
Between 7.95-8.05；
3, filtrate is collected by filtration with 0.2 μm of filter, is settled to 1000ml with purified water, after mixing to obtain the final product；
(2) preparation of substrate solution B
1, borax 11.44g, boric acid 4.948g, 500 μ l of urea peroxide 0.2g and PC300 are weighed in 1L beaker；
2, purified water is measured in 1L beaker with graduated cylinder, be sufficiently stirred until being completely dissolved, tune pH controls its range and exists
Between 7.95-8.05；
3, filtrate is collected by filtration with 0.2 μm of filter, is settled to 1000ml with purified water, after mixing to obtain the final product；
Three, the preparation method of calibration object dilution, steps are as follows:
1,7 ml of Tris 12.11g and HCl is weighed in the container of 1L, is adjusted pH value of solution, is controlled its range and exist
Between 7.35-7.45, it is settled to 1000ml；
2, calf serum 300ml is measured with graduated cylinder, be added in above-mentioned solution, it is spare as calibration object dilution
Four, the preparation method of calibration object, steps are as follows:
1, the preparation of calibration object A point: calibration object dilution is taken, appropriate gauge is dispensed into；
2, the preparation of calibration object B point: taking calibration object dilution, and CYFRA21-1 solution is diluted convenient multiple, controlled concentration
For 1 ng/mL, it is dispensed into appropriate gauge；
3, it the preparation of calibration object C point: takes；CYFRA21-1 solution is diluted convenient multiple, controlled dense by calibration object dilution
Degree is 5 ng/mL, is dispensed into appropriate gauge；
4, the preparation of calibration object D point: taking calibration object dilution, and CYFRA21-1 solution is diluted convenient multiple, controlled concentration
For 20 ng/mL, it is dispensed into appropriate gauge；
5, the preparation of calibration object E point: taking calibration object dilution, and CYFRA21-1 solution is diluted convenient multiple, controlled concentration
60 ng/mL, are dispensed into appropriate gauge；
6, the preparation of calibration object F point: taking calibration object dilution, and CYFRA21-1 solution is diluted convenient multiple, controlled concentration
For 120 ng/mL, it is dispensed into appropriate gauge
Five, the coated ELISA Plate of anti-biotin antibodies
1, the carbonic acid buffer of 0.01M is prepared, pH is to pH9.6 ± 0.1 for adjusting, spare；
2, anti-biotin antibodies solution is added in the carbonic acid buffer of above-mentioned 0.01M to final concentration of 0.1~5 μ g/ml
Stirring 30 minutes to be uniformly mixed；
It 3, is that the every hole 100 μ L is added in ELISA Plate by package amount by the solution after above-mentioned mixing, using high-temperature oscillation formula packet
Quilt, coating temperature are 37 ± 1 DEG C, and the coating time is 1~3 hour, are coated with mode using low-speed oscillation；
4, prepare 0.1M pH7.4TBS buffer, contain 0.3%BSA, 1% sheep blood serum, 0.1% Proclin300 (v/v), 1%
Fishskin gelatin, 2% trehalose and 10% sucrose are added in the coating plate after cleaning as confining liquid, and closing amount is that 150 μ L are every
Hole is closed as 37 ± 1 DEG C, and off-period is 2~5 hours；
5, confining liquid is sopped up, is placed in drying box, at 37 ± 1 DEG C, drying time is 2~4 hours for drying temperature control,
It is vacuum-packed again with aluminium foil bag, labeling is spare；
Six, the preparation of horseradish peroxidase-labeled CYFRA21-1 antibody-solutions
(1) preparation of enzyme reaction object dilution
1, it takes Tris 4.846g, 2900 HCl μ l in beaker, purified water is then added in flask, being sufficiently stirred makes
Reagent is completely dissolved；
2, pH is adjusted, controls pH in 7.35-7.45；
3, BSA 1g is weighed to pour into above-mentioned beaker；
4, last beaker is settled to 400ml, is filtered with 0.2um filter to obtain the final product；
(2) coupling of horseradish peroxidase (HRP) and CYFRA21-1 antibody
1, anti-CYFRA21-1 antibody 1mg is taken to be placed in 1ml glass tube；
2, the final concentration for taking 200 μ l DMSO lytic antibodies to make antibody reaches 5mg/ml, then mixes well；
3, the molar ratio that the disuccinimidyl suberate of 10mol is added according to 1mol antibody adds two amber of suberic acid
Amber imide ester reacts 1.5 hours in 37 DEG C of insulating boxs into above-mentioned 2 solution；
4, HRP is added into above-mentioned 3 solution according to the molar ratio that 1mol HRP is added in 3mol antibody, be then added
The pH of 1ml is the PB buffer that 7.4 concentration are 0.1M, is placed in 37 DEG C of insulating boxs and reacts 3 hours；
5, the solution prepared above-mentioned 4 PD-10 column purification collects refined solution, adds certainly according to the volume of 1:3000
The enzyme reaction object dilution of system, control are uniformly mixed finally using concentration in 0.02-3.0 μ g/ml up to enzyme reaction object；
Seven: the preparation of the CYFRA21-1 antibody-solutions of biotin labeling
(1) preparation of biotin reaction object dilution
1, it takes Tris 4.846g, 2847 HCl μ l in flask, purified water is then added in flask, being sufficiently stirred makes
Reagent is completely dissolved；
2, pH is adjusted, controls pH in 7.35-7.45；
3, BSA 1g is weighed to pour into above-mentioned beaker；
4, last beaker is settled to 400ml, is filtered with 0.2 μm of filter to obtain the final product；
(2) biotinylated antibody is taken, with biotin reaction object diluted, control is finally using concentration in 0.3 μ g/
Ml or so (0.01~1.0 μ g/ml) mixes, biotin conjugate can be obtained.
The detection method of kit of the present invention is that the CYFRA21-1 antibody of sample and biotin labeling is added to antibiosis
In the coated ELISA Plate of object element antibody, CYFRA21-1 and the antibody in sample, which are combined, forms immune complex, meanwhile, this is exempted from
Epidemic disease compound is fixed on ELISA Plate by the combination between anti-biotin antibodies and biotin.Use washing lotion cleaning of enzyme
Target removes unbonded free ingredient.The CYFRA21-1 antibody-solutions of horseradish peroxidase-labeled are added, the enzyme mark is anti-
Body is by immune response in conjunction with fixed immune complex.After cleaning ELISA Plate again, substrate solution excitation chemistry hair is added
Light measures relative light intensity RLU, and within the scope of a certain concentration, the linear direct ratio of CYFRA21-1 content is closed in RLU value and sample
System.By calibration object measured value, fitted calibration curve calculates CYFRA21-1 concentration in sample according to curve, to assess human serum
In whether the content containing CYFRA21-1 and CYFRA21-1.
Compared with prior art, the kit and its system of board-like chemoluminescence method of the invention detection change of serum C YFRA21-1
Preparation Method has the coated ELISA Plate of anti-biotin antibodies by introducing, and improve on the basis of chemiluminescence immune assay
High-temperature oscillation formula anti-biotin antibodies coating technique improves the sensitive of reaction by anti-biotin antibodies-Biotin method system
Degree saves production cost, so that reagent production procedure is simplified, when shortening production while reducing antibody/antigen usage amount
Between, detecting step is simplified, can provide that of high quality and at a reasonable price, reliable and stable, reproducible, difference between batch is small, accuracy is high for market
Detection kit of new generation.
Specific embodiment
Below in conjunction with specific embodiment, the present invention will be described, for embodiment be only to product of the present invention or method
Make generality illustration, helps to more fully understand the present invention, but be not limiting upon the scope of the invention.It is real described in following embodiments
Proved recipe method is unless otherwise specified conventional method；The material commercially obtains unless otherwise specified.
The board-like chemoluminescence method detection kit main agents of the CYFRA21-1 of the present embodiment include: including reagent
There are the CYFRA21-1 antibody-solutions of biotin labeling, the coated ELISA Plate of anti-biotin antibodies, horseradish peroxidase-labeled
CYFRA21-1 antibody-solutions, CYFRA21-1 calibration object A-F, concentration washing lotion, substrate solution A, substrate solution B.
The preparation method of kit
One, the preparation of washing lotion is concentrated, steps are as follows:
1, KCl 60g, NaCl 300g are weighed in 1L container；
2,20.0g Tween-20 is weighed in 100ml container plus after 50ml water makes it completely dissolved, and pours into above-mentioned 1L
In container；
3, Proclin-300 is measured into 2ml with pipettor, poured into above-mentioned 1L container；
4, appropriate purified water is measured in above-mentioned 1L container with graduated cylinder, be sufficiently stirred until being completely dissolved；
5, pH is adjusted, controls its range between 7.35~7.45；
6, it is finally settled to 1000ml, is filtered after being completely dissolved with 0.2 μm of filter to obtain the final product.
Two, the preparation of substrate solution
(1) preparation steps of substrate solution A
1, weigh borax 11.44g, boric acid 4.948g, luminol 2.0g and to iodophenol 0.2mg in 1L beaker；
2, purified water is measured in 1L beaker with graduated cylinder, be sufficiently stirred until being completely dissolved, tune pH controls its range 8
Between ± 0.05；
3, filtrate is collected by filtration with 0.2 μm of filter, is settled to 1000ml with purified water, after mixing to obtain the final product.
(2) preparation of substrate solution B
1, borax 11.44g, boric acid 4.948g, 500 μ l of urea peroxide 0.2g and PC300 are weighed in 1L beaker；
2, purified water is measured in 1L beaker with graduated cylinder, be sufficiently stirred until being completely dissolved, tune pH controls its range and exists
Between 8 ± 0.05；
3, filtrate is collected by filtration with 0.2 μm of filter, is settled to 1000ml with purified water, after mixing to obtain the final product.
Three, the preparation method of calibration object dilution, steps are as follows:
1,7 ml of Tris 12.11g and HCl is weighed in the container of 1L, is adjusted pH value of solution, is controlled its range and exist
Between 7.35-7.45, it is settled to 1000ml；
2, calf serum 300ml is measured with graduated cylinder, be added in above-mentioned solution, it is spare as calibration object dilution；
1,7 ml of Tris 12.11g and HCl is weighed in the container of 1L, is adjusted pH value of solution, is controlled its range and exist
Between 7.35-7.45, it is settled to 1000ml；
2, calf serum 300ml is measured with graduated cylinder, be added in above-mentioned solution, it is spare as calibration object dilution；
Four, the preparation method of calibration object, steps are as follows:
1, the preparation of calibration object A point: calibration object dilution is taken, appropriate gauge is dispensed into；
2, the preparation of calibration object B point: taking calibration object dilution, and CYFRA21-1 solution is diluted convenient multiple, controlled concentration
For 1 ng/mL, it is dispensed into appropriate gauge；
3, it the preparation of calibration object C point: takes；CYFRA21-1 solution is diluted convenient multiple, controlled dense by calibration object dilution
Degree is 5 ng/mL, is dispensed into appropriate gauge；
4, the preparation of calibration object D point: taking calibration object dilution, and CYFRA21-1 solution is diluted convenient multiple, controlled concentration
For 20 ng/mL, it is dispensed into appropriate gauge；
5, the preparation of calibration object E point: taking calibration object dilution, and CYFRA21-1 solution is diluted convenient multiple, controlled concentration
60 ng/mL, are dispensed into appropriate gauge；
6, the preparation of calibration object F point: taking calibration object dilution, and CYFRA21-1 solution is diluted convenient multiple, controlled concentration
For 120 ng/mL, it is dispensed into appropriate gauge
Five, the coated ELISA Plate of anti-biotin antibodies
6, the carbonic acid buffer of 0.01M is prepared, pH is to pH9.6 ± 0.1 for adjusting, spare；
7, anti-biotin antibodies solution is added in the carbonic acid buffer of above-mentioned 0.01M to stir to final concentration of 0.3 μ g/ml
Mix 30 minutes to be uniformly mixed；
It 8, is that the every hole 100 μ L is added in ELISA Plate by package amount by the solution after above-mentioned mixing, using high-temperature oscillation formula packet
Quilt, coating temperature are 37 ± 1 DEG C, and the coating time is 1~3 hour, are coated with form, oscillation amplitude and mode using low-speed oscillation:
3mm horizontal rotation, revolving speed are 200~500rpm；
9, prepare 0.1M pH7.4 TBS buffer, contain 0.3%BSA, 1% sheep blood serum, 0.1% Proclin300 (v/v), 1%
Fishskin gelatin, 2% trehalose and 10% sucrose are added in the coating plate after cleaning as confining liquid, and closing amount is that 150 μ L are every
Hole, closure temperature are 37 ± 1 DEG C, and off-period is 2~5 hours；
10, confining liquid is sopped up, is placed in drying box, at 37 ± 1 DEG C, drying time is 2~4 small for drying temperature control
When, then be vacuum-packed with aluminium foil bag, labeling is spare；
Six, the preparation of horseradish peroxidase-labeled CYFRA21-1 antibody-solutions
(1) preparation of enzyme reaction object dilution
1, it takes Tris 4.846g, 2900 HCl μ l in beaker, purified water is then added in flask, being sufficiently stirred makes
Reagent is completely dissolved；
2, pH is adjusted, controls pH 7.4 ± 0.05；
3, BSA 1g is weighed to pour into above-mentioned beaker；
4, last beaker is settled to 400ml, is filtered with 0.2um filter to obtain the final product；
(2) coupling of horseradish peroxidase (HRP) and CYFRA21-1 antibody
1, anti-CYFRA21-1 antibody 1mg is taken to be placed in 1ml glass tube；
2, the final concentration for taking 200 μ l DMSO lytic antibodies to make antibody reaches 5mg/ml, then mixes well；
3, the molar ratio that the disuccinimidyl suberate of 10mol is added according to 1mol antibody adds two amber of suberic acid
Amber imide ester reacts 1.5 hours in 37 DEG C of insulating boxs into above-mentioned 2 solution；
4, HRP is added into above-mentioned 3 solution according to the molar ratio that 1mol HRP is added in 3mol antibody, be then added
The pH of 1ml is the PB buffer that 7.4 concentration are 0.1M, is placed in 37 DEG C of insulating boxs and reacts 3 hours；
5, the solution prepared above-mentioned 4 PD-10 column purification collects refined solution, adds certainly according to the volume of 1:3000
The enzyme reaction object dilution of system, control are uniformly mixed finally using concentration in 0.5 μ g/ml up to enzyme reaction object；
Seven: the preparation of the CYFRA21-1 antibody-solutions of biotin labeling
(1) preparation of biotin reaction object dilution
1, it takes Tris 4.846g, 2847 HCl μ l in flask, purified water is then added in flask, being sufficiently stirred makes
Reagent is completely dissolved；
2, pH is adjusted, controls pH in 7.35-7.45；
3, BSA 1g is weighed to pour into above-mentioned beaker；
4, last beaker is settled to 400ml, is filtered with 0.2 μm of filter to obtain the final product.
(2) biotinylated antibody is taken, with biotin reaction object diluted, control is finally using concentration in 0.5 μ g/
Ml mixes, biotin conjugate can be obtained.
Eight, optimization and verifying
Main performance index meets luminous detection reagent Technical Review specification.
1, negative match-rate
The clinical blood sample of 103 Roche chemical illuminating reagents detection is detected, negative match-rate (-/-) is 103/103, is met
Rate 100%.
2, positive coincidence rate
The clinical blood sample of 190 parts of Roche chemical illuminating reagents detection is detected, positive coincidence rate (+/+) is 189/190, is met
Rate is 99.47%, be see the table below.
With through markization enterprise's precision reference material repeat detection 10 times, coefficient of variation CV between 2.64-6.92%,
Meet the industry standard (full-automatic instrument operation) no more than 10.0%.
| Accuracy quality-control product | CONC | CONC | CONC | CONC | CONC | CONC | CONC | CONC | CONC | CONC |
| L | 1.9 | 1.9 | 2.2 | 2 | 2.2 | 1.9 | 2.2 | 2.1 | 1.9 | 1.9 |
| M | 20.2 | 20.7 | 19.5 | 20.3 | 21.2 | 21.2 | 19.5 | 21.1 | 20.6 | 19.2 |
| H | 41.7 | 43.5 | 42.7 | 41.1 | 40.9 | 40.2 | 41 | 41.2 | 43.3 | 41.4 |
| Accuracy quality-control product | Average value | Standard deviation | CV% |
| L | 2.02 | 0.1 | 6.92% |
| M | 20.35 | 0.7 | 3.66% |
| H | 41.7 | 1.1 | 2.64% |
4, difference between batch
Three lot number kits, interassay coefficient of variation are detected with enterprise's precision quality-control product through National reference mark
(CV) between 1.95~5.83%, meet requirement (full-automatic instrument operation) of the rower less than 15%.
It is as follows to analyze result:
| Accuracy quality-control product | Average value | Standard deviation | CV% |
| L | 2.06 | 0.1 | 5.83% |
| M | 20.17 | 0.5 | 2.70% |
| H | 41.36 | 0.8 | 1.95% |
5, stability
Thermal stability: kit is detected after placing 7 days at 37 DEG C, and compared with the control, amplitude of variation is or not detection signal
More than 15%.
A kind of semi-automated instrument detection method of kit in embodiment, using the limited public affairs of Beijing shore pine photon technology share
Department microwell plate luminescence analyzer BHP9504 is detected, and detecting step includes
(1) it takes out kit and is placed in room temperature, make the equalized temperature of kit to room temperature (18~25 DEG C).
(2) sample should be uniformly mixed, if sample is freeze thawing sample, be detected again after balance to room temperature (18~25 DEG C).
(3) according to concentration washing lotion: distilled water=1:39 ratio is prepared washing lotion and is transferred in bottle for handling liquid toilet or cosmetic substance after mixing.
(4) detection ELISA Plate lath is taken out, if calibrating sample wells, measuring samples hole well.Remaining lath is put back in aluminium foil bag,
It seals up for safekeeping.
(5) by 1 sequence of table, semi-automatic sample-adding and operation are carried out.
The sample-adding of table 1 and the operation table of comparisons
Substrate solution A and B can also add 100 μ L in every hole after with preceding isometric mixing, use if substrate solution A and B are mixed,
Mixed liquor should be used in 30 minutes.
The half full-automatic instrument detection method of one kind of kit in embodiment, using Full-automatic chemiluminescence immunoassay analysis meter
ADC CLIA 200/300/400/500/600 and Smart3000/Smart300/ Smart3000S are detected, detection step
Suddenly include:
(1) when using Full-automatic chemiluminescence immunoassay analysis meter (hereinafter referred to as " instrument "), instrument do be read over
The operation manual of device must be configured instrument according to corresponding operation explanation, examination and maintenance, to realize optimum detection
Energy.
(2) illustrate to configure reagent information according to the operation manual of instrument, and reference substance is set in " setting of microplate layout "
The position in hole and sample aperture.Particularly, please detection method is carried out according to parameter shown in table 2 at " method editor " interface of instrument to match
It sets.
(3) according to 2 full-automatic instrument parameter setting table of table, parameter setting and data are carried out
2 Full-automatic chemiluminescence immunoassay analysis meter parameter list of table
In addition to the implementation, all to use equivalent transformation or equivalent replacement the invention also includes there is an other embodiments
The technical solution that mode is formed should all be fallen within the scope of the hereto appended claims.
Claims (5)
1. a kind of board-like chemoluminescence method detection kit of cytokeratin 19 fragment, it is characterised in that: including reagent: it is raw
CYFRA21-1 antibody-solutions, the anti-biotin antibodies coated ELISA Plates, horseradish peroxidase-labeled of object element label
CYFRA21-1 antibody-solutions, CYFRA21-1 calibration object A-F, concentration washing lotion, substrate solution A, substrate solution B；
The preparation method of kit, includes the following steps
One, the preparation of washing lotion is concentrated, steps are as follows:
1, KCl 60g, NaCl 300g are weighed in 1L container；
2,20.0g Tween-20 is weighed in 100ml container plus after 50ml water makes it completely dissolved, and is poured into above-mentioned 1L and is held
In device；
3, Proclin-300 is measured into 2ml with pipettor, poured into above-mentioned 1L container；
4, appropriate purified water is measured in above-mentioned 1L container with graduated cylinder, be sufficiently stirred until being completely dissolved；
5, pH is adjusted, controls its range between 7.35~7.45；
6, it is finally settled to 1000ml, is filtered after being completely dissolved with 0.2 μm of filter to obtain the final product；
Two, the preparation of substrate solution
(1) preparation steps of substrate solution A
1, weigh borax 11.44g, boric acid 4.948g, luminol 2.0g and to iodophenol 0.2mg in 1L beaker；
2, purified water is measured in 1L beaker with graduated cylinder, be sufficiently stirred until being completely dissolved, tune pH controls its range in 7.95-
Between 8.05；
3, filtrate is collected by filtration with 0.2 μm of filter, is settled to 1000ml with purified water, after mixing to obtain the final product；
(2) preparation of substrate solution B
1, borax 11.44g, boric acid 4.948g, 500 μ l of urea peroxide 0.2g and PC300 are weighed in 1L beaker；
2, purified water is measured in 1L beaker with graduated cylinder, be sufficiently stirred until being completely dissolved, tune pH controls its range and exists
Between 7.95-8.05；
3, filtrate is collected by filtration with 0.2 μm of filter, is settled to 1000ml with purified water, after mixing to obtain the final product；
Three, the preparation method of calibration object dilution, steps are as follows:
1,7 ml of Tris 12.11g and HCl is weighed in the container of 1L, is adjusted pH value of solution, is controlled its range in 7.35-
Between 7.45, it is settled to 1000ml；
2, calf serum 300ml is measured with graduated cylinder, be added in above-mentioned solution, it is spare as calibration object dilution；
Four, the preparation method of calibration object, steps are as follows:
1, the preparation of calibration object A: calibration object dilution is taken, appropriate gauge is dispensed into；
2, the preparation of calibration object B: taking calibration object dilution, and CYFRA21-1 solution is diluted convenient multiple, controlled concentration 1
Ng/mL is dispensed into appropriate gauge；
3, the preparation of calibration object C: taking calibration object dilution, and CYFRA21-1 solution is diluted convenient multiple, controlled concentration 5
Ng/mL is dispensed into appropriate gauge；
4, the preparation of calibration object D: taking calibration object dilution, and CYFRA21-1 solution is diluted convenient multiple, controlled concentration 20
Ng/mL is dispensed into appropriate gauge；
5, the preparation of calibration object E: taking calibration object dilution, and CYFRA21-1 solution is diluted convenient multiple, 60 ng/ of controlled concentration
ML is dispensed into appropriate gauge；
6, the preparation of calibration object F: taking calibration object dilution, and CYFRA21-1 solution is diluted convenient multiple, controlled concentration 120
Ng/mL is dispensed into appropriate gauge；
Five, the coated ELISA Plate of anti-biotin antibodies
1, the carbonic acid buffer of 0.01M is prepared, pH is to pH9.6 ± 0.1 for adjusting, spare；
2, anti-biotin antibodies solution is added in the carbonic acid buffer of above-mentioned 0.01M to final concentration of 0.2~8 μ g/ml, stirring
30 minutes to be uniformly mixed；
3, it is that the every hole 100 μ L is added in ELISA Plate by package amount by the solution after above-mentioned mixing, is coated with using high-temperature oscillation formula,
Being coated with temperature is 37 ± 1 DEG C, and the coating time is 1~3 hour, is coated with form, oscillation amplitude and mode: 3mm using low-speed oscillation,
Using horizontal rotation, revolving speed is 200~500rpm；
4,0.1M is prepared, pH7.4TBS buffer contains 0.3%BSA, 1% sheep blood serum, 0.1% Proclin300 (v/v), 1% fish-skin
Gelatin, 2% trehalose and 10% sucrose are added in the coating plate after cleaning as confining liquid, and closing amount is the 150 every holes μ L, envelope
Closing temperature is 37 ± 1 DEG C, and off-period is 2~5 hours；
5, confining liquid is sopped up, is placed in drying box, at 37 ± 1 DEG C, drying time is 2~4 hours for drying temperature control, then with
Aluminium foil bag vacuum packaging, labeling are spare；
Six, the preparation of horseradish peroxidase-labeled CYFRA21-1 antibody-solutions
(1) preparation of enzyme reaction object dilution
1, it takes Tris 4.846g, 2900 HCl μ l in beaker, purified water is then added in beaker, being sufficiently stirred makes reagent
It is completely dissolved；
2, pH is adjusted, controls pH in 7.35-7.45；
3, BSA 1g is weighed to pour into above-mentioned beaker；
4, last beaker is settled to 400ml, is filtered with 0.2um filter to obtain the final product；
(2) coupling of horseradish peroxidase (HRP) and CYFRA21-1 antibody
1, anti-CYFRA21-1 antibody 1mg is taken to be placed in 1ml glass tube；
2, the final concentration for taking 200 μ l DMSO lytic antibodies to make antibody reaches 5mg/ml, then mixes well；
3, the molar ratio that the disuccinimidyl suberate of 10mol is added according to 1mol antibody adds two succinyl of suberic acid
In the solution that imines ester is obtained to above-mentioned steps 2, reacted 1.5 hours in 37 DEG C of insulating boxs；
4, according to 3mol antibody be added 1mol HRP molar ratio HRP is added into the solution that above-mentioned steps 3 obtain, then plus
The pH for entering 1ml is PB buffer that 7.4 concentration are 0.1M, is placed in 37 DEG C of insulating boxs and reacts 3 hours；
5, the solution for preparing above-mentioned steps 4 PD-10 column purification collects refined solution, adds enzyme according to the volume of 1:3000
Reactant dilution, control are uniformly mixed finally using concentration in 0.02-3.0 μ g/ml up to enzyme reaction object；
Seven: the preparation of the CYFRA21-1 antibody-solutions of biotin labeling
(1) preparation of biotin reaction object dilution
1, it takes Tris 4.846g, 2847 HCl μ l in beaker, purified water is then added in beaker, being sufficiently stirred makes reagent
It is completely dissolved；
2, pH is adjusted, controls pH in 7.35-7.45；
3, BSA 1g is weighed to pour into above-mentioned beaker；
4, last beaker is settled to 400ml, is filtered with 0.2 μm of filter to obtain the final product；
(2) biotinylated antibody is taken, with biotin reaction object diluted, control is finally using concentration in 0.01~1.0 μ
G/ml mixes, biotin conjugate can be obtained.
2. the board-like chemoluminescence method detection kit of cytokeratin 19 fragment according to claim 1, feature exist
In: the concentration of the CYFRA21-1 antibody-solutions of the biotin labeling is 0.01~1.0 μ g/ml, the anti-biotin antibodies packet
The peridium concentration of the anti-biotin antibodies of the ELISA Plate of quilt is 0.2~8.0 μ g/ml, the horseradish peroxidase-labeled
The concentration of CYFRA21-1 antibody-solutions is 0.02~3.0 μ g/ml.
3. the board-like chemoluminescence method detection kit of cytokeratin 19 fragment according to claim 1, feature exist
It is that CYFRA21-1 is obtained with the Tris-HCl buffer that pH value is 7.4 in: the CYFRA21-1 calibration object A-F point,
Concentration is respectively 0,1,5,20,60,120ng/mL.
4. the board-like chemoluminescence method detection kit of cytokeratin 19 fragment according to claim 1, feature exist
In: coating buffer used in the coated ELISA Plate of anti-biotin antibodies is the carbonic acid buffer of 0.01M, and pH9.6 ± 0.1 is used
Confining liquid is containing 0.3wt%BSA, 0.1 % Proclin300 (v/v), 1 wt % sheep blood serum, 1 wt % fishskin gelatin, 2 seas wt %
The 0.1M pH7.4 TBS buffer of algae sugar and 10 wt % sucrose.
5. the board-like chemoluminescence method detection kit of cytokeratin 19 fragment according to claim 1, feature exist
In: the coated ELISA Plate of anti-biotin antibodies is coated with using high-temperature oscillation formula, and coating temperature is 37 ± 1 DEG C, is coated with the time
It is 1~3 hour, form, oscillation amplitude and mode: 3mm horizontal rotation is coated with using low-speed oscillation, revolving speed is 200~500rpm,
Closing and drying temperature are 37 ± 1 DEG C, and off-period is 1.5~3 hours, and drying time is 2~4 hours；Using high-temperature oscillation
Formula coating technique, coating plate preparation time are can be controlled in 5~7 hours.
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| CN111856040A (en) * | 2020-06-01 | 2020-10-30 | 福建省妇幼保健院 | Chemiluminescence kit for quantitatively detecting human mullerian hormone and preparation method thereof |
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