CN107490695B - The board-like chemoluminescence method detection kit and preparation method of Carbohydrate Antigen 50 - Google Patents
The board-like chemoluminescence method detection kit and preparation method of Carbohydrate Antigen 50 Download PDFInfo
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Abstract
The present invention relates to a kind of board-like chemoluminescence method detection kit of Carbohydrate Antigen 50, including reagent have the CA50 antibody-solutions of biotin labeling, the coated ELISA Plate of anti-biotin antibodies, the CA50 antibody-solutions of horseradish peroxidase-labeled, CA50 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 detecting reagent for Carbohydrate Antigen 50
Box and detection method, can quickly, it is easy, efficiently, delicately to the quantitative detection of Carbohydrate Antigen 50 in human serum sample.
Background technique
CA50(Carbohydrate Antigen 50, abbreviation CA50) refer to tumor markers (Tumor Marker), be
Reflect chemicals existing for tumour.They are not present in normal adult tissue and are detected in embryonic tissue, or in tumour
Content in tissue substantially exceeds the content in normal tissue, their presence or quantitative change can prompt the property of tumour, borrows
To understand tissue generation, the cell differentiation, cell function of tumour, to help diagnosis, classification, Index for diagnosis and the treatment of tumour
Guidance.
CA50 is a kind of saliva acid esters and sialoglycoprotein, is generally not present in normal tissue, when malignant change of cell, sugar
Base enzyme is activated, and cell surface glycosyl structure is caused to change and become CA50 marker.The μ of normal blood < 20 g/L is many pernicious
It can all be increased in Blood of Tumor Patients, such as 66.6% lung cancer, 88.2% liver cancer, 68.9% gastric cancer, 88.5% ovary or uterus
Neck cancer, 94.4% pancreas or cholangiocarcinoma, such as the dirty cancer of the carcinoma of the rectum, wing all has 70% or more is raised for other.
Glycoprotein antigen CA50 is a kind of nonspecific broad-spectrum tumor marker.It is a kind of saliva acid esters and sialic acid sugar
Albumen is generally not present in normal tissue, and when malignant change of cell, glycosylase is activated, and cell surface glycosyl structure is caused to change
Become and becomes CA50 marker.Normal blood < 20 μ g/L can all be increased in many malignant tumor patient blood, such as 66.6% lung cancer,
88.2% liver cancer, 68.9% gastric cancer, 88.5% ovary or cervix cancer, 94.4% pancreas or cholangiocarcinoma, other are such as the carcinoma of the rectum, wing
It is raised that dirty cancer etc., which all has 70% or more,.
Cancer antigen 50 (CA50) is used as nonspecific broad-spectrum tumor marker, has certain intersect with CA 19-9
Antigenicity is mainly used for the auxiliary diagnosis of cancer of pancreas, the colon/carcinoma of the rectum, gastric cancer, and wherein Patients with Pancreatic Cancer increases most
Obviously.Increase: seeing cancer of pancreas (positive rate is up to 87%), the colon/carcinoma of the rectum, gastric cancer, lung cancer.Liver cancer.Oophoroma, breast cancer
Etc. malignant tumours；Ulcerative colitis, cirrhosis, melanoma, lymthoma, autoimmune disease etc. also increase.
Clinically detection CA50 in order to improve diagnosing tumor level and improve therapeutic effect, from detection angles for, mesh
The preceding method clinically for measuring CA50 antigen mainly has Enzyme-multiplied immune technique and chemiluminescence etc..Past is exempted from enzyme-linked
Epidemic disease technology is limitation of people's CA50 antigen measuring kit due to methodology of representative, and sensitivity and anti-interference ability are seriously not
, there is very big drawback, the requirement of hospital or inspection center to sensitivity can not have been adapted in foot；At present, more applications are enzyme-linked
Immunological technique, time-resolved fluoroimmunoassay and chemiluminescence, wherein chemiluminescence rises in eighties of last century 80
Age is the emerging technology to grow up after Enzyme-multiplied immune technique and radioimmunoassay technology, since it is highly sensitive, high
Specificity, while method is easy, quick, label conjugate is stablized, and low in cost, easy to operate, "dead" isotope damage
It the features such as wound 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 CA50 antigen is researched and developed and is optimized and is relevant various to serology immune response principle
Key technology develops sensitivity to improve for the intracorporal CA50 antigen detection sensitivity of clinical patients and accuracy
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
Body-biotin system, so that detection sensitivity be significantly increased while stable reagent box detection specificity.
The present invention solves the above problems used technical solution are as follows:
A kind of board-like chemoluminescence method detection kit of Carbohydrate Antigen 50, including reagent have the CA50 of biotin labeling
The coated ELISA Plate of antibody-solutions, anti-biotin antibodies, the CA50 antibody-solutions of horseradish peroxidase-labeled, CA50 calibration object
A-F, concentration washing lotion, substrate solution A, substrate solution B.
The concentration of the CA50 antibody-solutions of above-mentioned biotin labeling is 0.02~8.0 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 CA50 antibody-solutions is 0.03~5.0 μ g/ml.
Above-mentioned CA50 calibration object A-F point is that CA50 antigen (nine peak of Beijing profit reaches) is delayed with the Tris-HCl that pH value is 7.4
What fliud flushing was prepared, concentration is respectively 0,5,20,70,150,240U/mL.
The preparation process of the CA50 antibody-solutions of above-mentioned biotin labeling: the CA50 antibody of biotin labeling is added to pH value
To adjust and arriving convenient multiple, be uniformly mixed so as to obtain in 7.4 buffer.
The preparation process of the CA50 antibody-solutions of above-mentioned horseradish peroxidase-labeled: horseradish peroxidase-labeled
CA50 antibody-solutions 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 it is bright containing 0.3wt%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 performance 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 Carbohydrate Antigen 50 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 CA50 antigenic solution is diluted convenient multiple, controlled concentration
For 5U/mL, it is dispensed into appropriate gauge；
3, it the preparation of calibration object C point: takes；CA50 antigenic solution is diluted convenient multiple, controlled concentration by calibration object dilution
For 20U/mL, it is dispensed into appropriate gauge；
4, the preparation of calibration object D point: taking calibration object dilution, and CA50 antigenic solution is diluted convenient multiple, controlled concentration
For 70U/mL, it is dispensed into appropriate gauge；
5, the preparation of calibration object E point: taking calibration object dilution, and CA50 antigenic solution is diluted convenient multiple, controlled concentration
For 150U/mL, it is dispensed into appropriate gauge；
6, the preparation of calibration object F point: taking calibration object dilution, and CA50 antigenic solution is diluted convenient multiple, controlled concentration
For 240U/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 CA50 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 10g 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 CA50 antibody
1, anti-CA50 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 0.03-5.0 μ g/ml up to enzyme reaction object；
Seven: the preparation of the CA50 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 10g 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 or so (0.01~8.0 μ g/ml) mixes, biotin conjugate can be obtained.
The detection method of kit of the present invention is that the CA50 antibody of sample and biotin labeling is added to antibiotin and is resisted
In the coated ELISA Plate of body, CA50 antigen and the antibody in sample, which combine, forms immune complex, meanwhile, the immune complex
It is fixed on ELISA Plate by the combination between anti-biotin antibodies and biotin.ELISA Plate is cleaned using washing lotion, is gone
Except unbonded free ingredient.The CA50 antibody-solutions of horseradish peroxidase-labeled are added, which passes through immune anti-
It should be in conjunction with fixed immune complex.After cleaning ELISA Plate again, substrate solution is added and excites chemiluminescence, measurement light relatively
Intensity RLU, within the scope of a certain concentration, the linear proportional relation of CA50 antigenic content in RLU value and sample.It is surveyed by calibration object
Whether value, fitted calibration curve calculate CA50 antigen concentration in sample according to curve, to assess anti-containing CA50 in human serum
Former and CA50 antigen content.
Compared with prior art, the kit of board-like chemoluminescence method of the invention detection change of serum C A50 antigen and its preparation
Method has the coated ELISA Plate of anti-biotin antibodies by introducing, and improve height on the basis of chemiluminescence immune assay
Warm oscillatory type 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 Carbohydrate Antigen 50 of the present embodiment include: including reagent
Have the CA50 antibody-solutions of biotin labeling, the coated ELISA Plate of anti-biotin antibodies, horseradish peroxidase-labeled CA50
Antibody-solutions, CA50 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；
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 CA50 antigenic solution is diluted convenient multiple, controlled concentration
For 5U/mL, it is dispensed into appropriate gauge；
3, it the preparation of calibration object C point: takes；CA50 antigenic solution is diluted convenient multiple, controlled concentration by calibration object dilution
For 20U/mL, it is dispensed into appropriate gauge；
4, the preparation of calibration object D point: taking calibration object dilution, and CA50 antigenic solution is diluted convenient multiple, controlled concentration
For 70U/mL, it is dispensed into appropriate gauge；
5, the preparation of calibration object E point: taking calibration object dilution, and CA50 antigenic solution is diluted convenient multiple, controlled concentration
For 150U/mL, it is dispensed into appropriate gauge；
6, the preparation of calibration object F point: taking calibration object dilution, and CA50 antigenic solution is diluted convenient multiple, controlled concentration
For 240U/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 stir to final concentration of 0.3 μ g/ml
Mix 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, shake coating form using low speed, are together Ai Bensen science
Instrument Ltd.'s microplate oscillator；
4, 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；
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 CA50 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 10g 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 CA50 antibody
1, anti-CA50 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 the solution of step 2；
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 for preparing above-mentioned steps 4 PD-10 column purification is collected refined solution, is added according to the volume of 1:3000
Add homemade enzyme reaction object dilution, control is uniformly mixed finally using concentration in 0.8 μ g/ml up to enzyme reaction object；
Seven: the preparation of the CA50 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 10g 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 159 Beckman chemical illuminating reagents detection is detected, negative match-rate (-/-) is 159/159, symbol
Conjunction rate 100%.
2, positive coincidence rate
The clinical blood sample of 205 parts of Beckman chemical illuminating reagents detection is detected, positive coincidence rate (+/+) is 201/205, symbol
Conjunction rate is 98.05%, be see the table below.
3, repeated
Detection 10 times is repeated with enterprise's precision reference material through National reference mark, coefficient of variation CV is in 2.02-
Between 4.77%, meet the industry standard (full-automatic instrument operation) no more than 10.0%.
| Accuracy quality-control product | Average value | Standard deviation | CV% |
| L | 2.46 | 0.1 | 4.77% |
| M | 19.3 | 0.6 | 3.22% |
| H | 47.21 | 1.0 | 2.02% |
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 2.58~4.47%, 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 | 2.46 | 0.1 | 4.47% |
| M | 19.49 | 0.6 | 3.07% |
| H | 47.70 | 1.2 | 2.58% |
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 (4)
1. a kind of board-like chemoluminescence method detection kit of Carbohydrate Antigen 50, it is characterised in that: including reagent have biotin
The coated ELISA Plate of the CA50 antibody-solutions of label, anti-biotin antibodies, horseradish peroxidase-labeled CA50 antibody-solutions,
CA50 calibration object A-F, concentration washing lotion, substrate solution A, substrate solution B；
Coating buffer used in the coated ELISA Plate of anti-biotin antibodies is the carbonic acid buffer of 0.01M, pH9.6 ± 0.1, institute
Contain 0.3wt%BSA, 0.1 (v/v) % Proclin300,1 wt % sheep blood serum, 1 wt % fishskin gelatin, 2 wt with confining liquid
The 0.1M pH7.4 TBS buffer of % trehalose and 10 wt % sucrose；
The preparation method of the 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 CA50 antigenic solution is diluted convenient multiple, controlled concentration 5U/
ML is dispensed into appropriate gauge；
3, the preparation of calibration object C: taking calibration object dilution, and CA50 antigenic solution is diluted convenient multiple, controlled concentration 20U/
ML is dispensed into appropriate gauge；
4, the preparation of calibration object D: taking calibration object dilution, and CA50 antigenic solution is diluted convenient multiple, controlled concentration 70U/
ML is dispensed into appropriate gauge；
5, the preparation of calibration object E: taking calibration object dilution, and CA50 antigenic solution is diluted convenient multiple, controlled concentration 150U/
ML is dispensed into appropriate gauge；
6, the preparation of calibration object F: taking calibration object dilution, and CA50 antigenic solution is diluted convenient multiple, controlled concentration 240U/
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 0.1~5 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 using low-speed oscillation；
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 CA50 antibody-solutions
(1) preparation of enzyme reaction object dilution
1, it takes Tris 4.846g, 2900 HCl μ l in flask, purified water is then added in flask, being sufficiently stirred makes reagent
It is completely dissolved；
2, pH is adjusted, controls pH in 7.35-7.45；
3, BSA 10g is weighed to pour into above-mentioned flask；
4, last flask is settled to 400ml, is filtered with 0.2um filter to obtain the final product
(2) coupling of horseradish peroxidase (HRP) and CA50 antibody
1, anti-CA50 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 certainly according to the volume of 1:3000
The enzyme reaction object dilution of system, control are uniformly mixed finally using concentration in 0.03-5.0 μ g/ml up to enzyme reaction object；
Seven: the preparation of the CA50 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
It is completely dissolved；
2, pH is adjusted, controls pH in 7.35-7.45；
3, BSA 10g is weighed to pour into above-mentioned flask；
4, last flask 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 0.02~8.0 μ
G/ml mixes, biotin conjugate can be obtained.
2. the board-like chemoluminescence method detection kit of Carbohydrate Antigen 50 according to claim 1, it is characterised in that: described
The concentration of the CA50 antibody-solutions of biotin labeling is 0.02~8.0 μ g/ml, the coated ELISA Plate of anti-biotin antibodies
The peridium concentration of anti-biotin antibodies is 0.2~8.0 μ g/ml, the CA50 antibody-solutions of the horseradish peroxidase-labeled
Concentration is 0.03~5.0 μ g/ml.
3. the board-like chemoluminescence method detection kit of Carbohydrate Antigen 50 according to claim 1, it is characterised in that: institute
Stating CA50 calibration object A-F point is CA50 antigen, is obtained with the Tris-HCl buffer that pH value is 7.4, concentration is respectively
0,5,20,70,150,240U/mL。
4. the board-like chemoluminescence method detection kit of Carbohydrate Antigen 50 according to claim 1, it is characterised in that: described
The coated ELISA Plate of anti-biotin antibodies is coated with using high-temperature oscillation formula, and coating temperature is 37 ± 1 DEG C, and the coating time is 1~3 small
When, form is coated with using low-speed oscillation, closing and drying temperature are 37 ± 1 DEG C, and off-period is 1.5~3 hours, drying time
It is 2~4 hours；Using high-temperature oscillation formula coating technique, it is coated with plate preparation time and can be controlled in 5~7 hours.
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| CN111018991B (en) * | 2018-10-10 | 2022-11-04 | 东莞市朋志生物科技有限公司 | anti-CA50 antibody and application thereof |
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| 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 |
| CN101178405B (en) * | 2006-11-10 | 2011-05-18 | 北京科美东雅生物技术有限公司 | Tumor-associated antigen 50 chemiluminescence immune analyze quantitative determination reagent box and method of producing the same |
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