CN107167590A - The board-like chemoluminescence method detection kit and preparation method of carbohydrate antigen 242 - Google Patents
The board-like chemoluminescence method detection kit and preparation method of carbohydrate antigen 242 Download PDFInfo
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- CN107167590A CN107167590A CN201710243922.9A CN201710243922A CN107167590A CN 107167590 A CN107167590 A CN 107167590A CN 201710243922 A CN201710243922 A CN 201710243922A CN 107167590 A CN107167590 A CN 107167590A
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Abstract
The present invention relates to a kind of board-like chemoluminescence method detection kit of carbohydrate antigen 242, including reagent have the CA242 antibody-solutions of biotin labeling, the coated ELISA Plate of anti-biotin antibodies, the CA242 antibody-solutions of horseradish peroxidase-labeled, CA242 calibration object A F, concentration washing lotion, substrate solution A, substrate solution B.The main design thought of the present invention is that on the basis of chemiluminescence immune assay, have anti-biotin antibodies biotin system by introducing, and the detection of stable reagent box is specific simultaneously, is added significantly to detection sensitivity and detection time.High-temperature oscillation formula anti-biotin antibodies coating technique is improved, so as to while reducing antibody/antigen usage amount, simplify reagent production procedure, shorten the production cycle.
Description
Technical field
The invention belongs to biotinylation kit Material Field, and in particular to a kind of quantitative detection for carbohydrate antigen 242 is tried
Agent box and detection method, can quickly, it is easy, efficiently, delicately the qualitative, quantitative of carbohydrate antigen 242 in human serum sample is examined
Survey.
Background technology
Carbohydrate antigen 242（Carbohydrate Antigen 242,CA242）It is a kind of sialylated mucin type sugar
Class antigen, content is very low in healthy population and the serum of benign disease patient, and malignant tumour occurs for alimentary canal etc.（Such as pancreas
Cancer, colon cancer, stomach cancer, oophoroma, uterine cancer, lung cancer etc.）When its content substantially rise, it is particularly bright when cancer of pancreas and colorectal cancer
It is aobvious.As a kind of tumor markers, rise is obvious when the advantage of carbohydrate antigen 242 essentially consists in malignant tumour, and benign disease
When do not raise typically.Carbohydrate antigen 242 and carcinomebryonic antigen（CEA）Joint-detection, can increase the early diagnosis to knot, the carcinoma of the rectum quick
Perception, is also a good diagnosis index to monitoring postoperative recurrence.Carbohydrate antigen 242 is that occur first in Patients with Pancreatic Cancer
A kind of antigen related to tumour.It is more more special than CA19-9, CA50 in diagnosis of pancreatic cancer.
CA242 is a kind of sialylated sugar antigen, through what hybridoma technology was obtained one can be by colon cancer cell line
One of list clonal antibody Ca242 is recognized that it is a kind of sugared egg being present in multiple organ malignant tumour in mucin types
CanAg is in vain, i.e., can not can not be reacted with LewisA type antigen-reactives with sialylated galactoside.Immunochemistry is ground
Study carefully and have shown that it is different from other known tumor-associated mucin such as：Ca199, Ca50, Ca125, Ca153 etc., Healthy People and
Content is relatively low in benign disease serum.Ca242 is to be applied to a kind of clinical newer tumor markers, cancer of pancreas and knot in recent years
The preferable tumor markers of intestinal cancer.
CA242 is a kind of sialylated glycosyl sphingolipid class antigen, is almost always expressed together with CA50, but both are by difference
Monoclonal antibody identification.Clinically it is used for the diagnosis of malignant tumor of digestive tract especially cancer of pancreas, colorectal cancer, with
CA19-9, CA50 are compared, and sensitivity in cancer of pancreas, gallbladder cancer and digestive system cancer of CA242 of new generation, specificity are higher
(CA50, CA19-9 easily by liver function and it is cholestatic influence, benign obstructive jaundice and liver parenchyma infringement note disease
There is false positive often in disease).
In order to improve diagnosing tumor level and improve therapeutic effect, from detection angles for, at present clinically be used for survey
Determine the method for CA242 antigens mainly have radioimmunoassay technology, Enzyme-multiplied immune technique, time-resolved fluorescence immunoassay method and
Chemiluminescence etc..Past, people's CA242 antigen measuring kits using radioimmunoassay technology as representative were due to methodology
Limitation, its sensitivity and antijamming capability wretched insufficiency, the drawbacks of having very big, substantially withdraw from the market；At present application compared with
It is many for Enzyme-multiplied immune technique, time resolved fluoro-immunoassay and chemiluminescence, wherein chemiluminescence rise in
In the 80's of eighties of last century, be the emerging technology grown up after Enzyme-multiplied immune technique and radioimmunoassay technology, due to
Its high sensitivity, high specific, while method is easy, quick, mark conjugate is stable, relative time resolved fluorometric immunization point
Analysis is with low cost, easy to operate, the features such as "dead" isotope is damaged and polluted, and is developed rapidly.
This kit is mainly used in carrying out dynamic monitoring to associated malignancies patient with auxiliary judgment disease process or controlled
Therapeutic effect, it is impossible to the foundation for early diagnosing or making a definite diagnosis as associated malignancies, should not be used in the tumor screening of general population.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of board-like chemoluminescence method detection for above-mentioned prior art
Kit of human serum CA242 antigens and preparation method thereof, researches and develops and optimizes and be related to serology immune response principle each
Key technology is planted, so as to improve for the CA242 antigen detection sensitivities and accuracy in clinical patients body, is developed sensitive
Degree is high, stability is good, production is convenient, easy to operate board-like chemiluminescence diagnostic reagent.
The main design thought of the present invention is on the basis of chemiluminescence immune assay, to be resisted by introducing with antibiotin
Body-biotin system, so that, the detection of stability kit is specific simultaneously, is added significantly to detection sensitivity.
The present invention the used technical scheme that solves the above problems is：
A kind of board-like chemoluminescence method detection kit of carbohydrate antigen 242, it is characterised in that：Including reagent have biotin mark
The coated ELISA Plate of the CA242 antibody-solutions of note, anti-biotin antibodies, the CA242 antibody-solutions of horseradish peroxidase-labeled,
CA242 calibration objects A-F, concentration washing lotion, substrate solution A, substrate solution B.
The concentration of the CA242 antibody-solutions of above-mentioned biotin labeling is 0.02~8.0 μ g/ml, the anti-biotin antibodies
The coating concentration of the anti-biotin antibodies of coated ELISA Plate is 0.2~8.0 μ g/ml, the horseradish peroxidase-labeled
The concentration of CA242 antibody-solutions is 0.03~5.0 μ g/ml, and positive control thinner ratio is 1：500~1：10000.
Above-mentioned CA242 calibration objects A-F points are CA242 antigens（The peak of Beijing nine profit reaches）Tris-HCl using pH value as 7.4
What buffer was obtained, concentration is respectively 0,4,16,48,144,288U/mL.
The preparation process of the CA242 antibody-solutions of above-mentioned biotin labeling：The CA242 antibody of biotin labeling is added to pH
It is worth in the buffer solution for 7.4, convenient multiple is arrived in regulation, is uniformly mixed so as to obtain.
The preparation process of the above-mentioned CA242 antibody-solutions for stating horseradish peroxidase-labeled：Horseradish peroxidase-labeled
CA242 antibody-solutions be added to pH value be 7.4 buffer solution in, regulation arrive convenient multiple, be 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 serums, 1 wt % fish-skins
The 0.1M pH7.4 TBS buffer solutions of glue, 2 wt % trehaloses and 10 wt % sucrose.For the anti-biotin antibodies bag of the present invention
By system, ELISA Plate largely uncombined position is shielded with a small amount of BSA using 0.01M carbonic acid buffer, and in confining liquid
Point, under the synergy of buffer system, nonspecific binding site is eliminated with animal blood serum, it is ensured that the precision of coating plate
And Low background, using sucrose, trehalose and fishskin gelatin as stabilising system, coating plate is enhanced to high temperature, the resistance of drying, is protected
The performance that anti-biotin antibodies are coated with plate is demonstrate,proved.
The preparation process of the coated ELISA Plate of anti-biotin antibodies is：ELISA Plate is coated with using high-temperature oscillation formula, coating temperature
Spend for 37 ± 1 DEG C, the coating time is 1~3 hour, form is coated with using low-speed oscillation, the shaker of selection is Ai Bensen science
Instrument Ltd.'s micropore plate oscillator, oscillation amplitude：3mm, using horizontal rotation, rotating speed is 200 ~ 500rpm, is closed and dry
Dry temperature is 37 ± 1 DEG C, and off-period is 1.5~3 hours, and drying time is 2~4 hours；Work is coated with using high-temperature oscillation formula
Skill, coating plate preparation time is can be controlled in 5~7 hours, and being coated with 3~4 days universal preparation times with room temperature compares, and possesses substantially
Odds for effectiveness, while improve the sensitivity of reaction, while reduction antibody/antigen usage amount, simplify reagent production stream
Journey, shortens the production time.State's endoperidium plate technique using Streptavidin be coated with it is more, using anti-biotin antibodies there is not yet
Patent document is reported.In addition, when low-speed oscillation coating method substantially shortens the production of coating plate compared to conventional room temperature coating method
Between, improve reagent production efficiency.
The preparation method of the board-like chemoluminescence method detection kit of carbohydrate antigen 242 of the present invention, comprises the following steps
First, the preparation of washing lotion is concentrated, step is as follows：
1st, KCl 60g, NaCl 300g are weighed in 1L containers；
2nd, 20.0g Tween-20 are weighed in 100ml containers plus 50ml water makes after it is completely dissolved, to pour into above-mentioned 1L and hold
In device；
3rd, Proclin-300 is measured into 2ml with pipettor, poured into above-mentioned 1L containers；
4th, appropriate purified water is measured in above-mentioned 1L containers with graduated cylinder, be sufficiently stirred for until being completely dissolved；
5th, pH is adjusted, its scope is controlled between 7.35~7.45；
6th, 1000ml is finally settled to, is produced after being completely dissolved with 0.2 μm of filter filtering；
2nd, the preparation of substrate solution
（One）Substrate solution A preparation
1st, weigh borax 11.44g, boric acid 4.948g, luminol 2.0g and to iodophenol 0.2mg in 1L beakers；
2nd, purified water is measured in 1L beakers with graduated cylinder, be sufficiently stirred for until being completely dissolved, tune pH controls its scope in 7.95-
8.05 between；
3rd, filtrate is collected by filtration with 0.2 μm of filter, is produced after 1000ml, mixing are settled to purified water；
（Two）Substrate solution B preparation
1st, borax 11.44g, boric acid 4.948g, the μ l of urea peroxide 0.2g and PC300 500 are weighed in 1L beakers；
2nd, purified water is measured in 1L beakers with graduated cylinder, be sufficiently stirred for until being completely dissolved, tune pH controls its scope to exist
7.95-8.05 between；
3rd, filtrate is collected by filtration with 0.2 μm of filter, is produced after 1000ml, mixing are settled to purified water；
3rd, the compound method of calibration object dilution, step is as follows：
1st, the ml of Tris 12.11g and HCl 7 is weighed in 1L container, is adjusted pH value of solution, is controlled its scope in 7.35-
Between 7.45,1000ml is settled to；
2nd, calf serum 300ml is measured with graduated cylinder, is added in above-mentioned solution, it is standby as calibration object dilution；
4th, the compound method of calibration object, step is as follows：
1st, the preparation of calibration object A points：Calibration object dilution is taken, appropriate gauge is dispensed into；
2nd, the preparation of calibration object B points：Calibration object dilution is taken, CA242 antigenic solutions are diluted into convenient multiple, controlled concentration is
4U/mL, is dispensed into appropriate gauge；
3rd, the preparation of calibration object C points：Take；Calibration object dilution, convenient multiple is diluted by CA242 antigenic solutions, and controlled concentration is
16U/mL, is dispensed into appropriate gauge；
4th, the preparation of calibration object D points：Calibration object dilution is taken, CA242 antigenic solutions are diluted into convenient multiple, controlled concentration is
48U/mL, is dispensed into appropriate gauge；
5th, the preparation of calibration object E points：Calibration object dilution is taken, CA242 antigenic solutions are diluted into convenient multiple, controlled concentration is
144U/mL, is dispensed into appropriate gauge；
6th, the preparation of calibration object F points：Calibration object dilution is taken, CA242 antigenic solutions are diluted into convenient multiple, controlled concentration is
288U/mL, is dispensed into appropriate gauge；
5th, the coated ELISA Plate of anti-biotin antibodies
1st, 0.01M carbonic acid buffer is prepared, pH is to pH9.6 ± 0.1 for regulation, standby；
2nd, anti-biotin antibodies solution is added in above-mentioned 0.01M carbonic acid buffer to stir to final concentration of 0.1~5 μ g/ml
30 minutes to well mixed；
3rd, it is that 100 μ L are added in ELISA Plate per hole by package amount by the solution after above-mentioned mixing, is coated with using high-temperature oscillation formula,
Coating temperature is 37 ± 1 DEG C, and the coating time is 1~3 hour, and mode is coated with using low-speed oscillation, and the shaker of selection is to end this
Gloomy scientific instrument Co., Ltd micropore plate oscillator, oscillation amplitude：3mm, using horizontal rotation, rotating speed is 200 ~ 500rpm；
4th, 0.1M pH7.4TBS buffer solutions are prepared, containing 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 every holes of 150 μ L, envelope
Close as 37 ± 1 DEG C, off-period is 2~5 hours；
5th, confining liquid is sopped up, is placed in drying box, drying temperature control is at 37 ± 1 DEG C, and drying time is 2~4 hours, then
It is vacuum-packed with aluminium foil bag, labeling is standby；
6th, the preparation of horseradish peroxidase-labeled CA242 antibody-solutions
（One）The preparation of enzyme reaction thing dilution
1st, take Tris 4.846g, HCl 2900 μ l in beaker, purified water is then added in flask, is sufficiently stirred for making reagent
It is completely dissolved；
2nd, pH is adjusted, pH is in 7.35-7.45 for control；
3rd, BSA 4g are weighed to pour into above-mentioned beaker；
4th, last beaker is settled to 400ml, is produced with the filtering of 0.2um filters；
（Two）Horseradish peroxidase（HRP）With the coupling of CA242 antibody
1st, anti-CA242 antibody 1mg is taken to be positioned in 1ml glass tubes；
2nd, take 200 μ l DMSO lytic antibodies the final concentration of antibody is reached 5mg/ml, then fully mix；
3rd, the molar ratio that 10mol disuccinimidyl suberate is added according to 1mol antibody adds the succinyl of suberic acid two
Imines ester reacts 1.5 hours into above-mentioned 2 solution in 37 DEG C of insulating boxs；
4th, 1mol HRP mol ratio is added according to 3mol antibody toward HRP is added in above-mentioned 3 solution, then adds 1ml's
PH is the PB buffer solutions that 7.4 concentration are 0.1M, is placed in 37 DEG C of insulating boxs and reacts 3 hours；
5th, the solution prepared above-mentioned 4 PD-10 posts are purified, and refined solution are collected, according to 1:3000 volume addition is homemade
Enzyme reaction thing dilution, controls final concentration in 0.03-5.0 μ g/ml, well mixed to produce enzyme reaction thing；
Seven：The preparation of the CA242 antibody-solutions of biotin labeling
（One）The preparation of biotin reaction thing dilution
1st, take Tris 4.846g, HCl 2847 μ l in flask, purified water is then added in flask, is sufficiently stirred for making reagent
It is completely dissolved；
2nd, pH is adjusted, pH is in 7.35-7.45 for control；
3rd, BSA 4g are weighed to pour into above-mentioned beaker；
4th, last beaker is settled to 400ml, is produced with 0.2 μm of filter filtering；
（Two）Biotinylated antibody is taken, with biotin reaction thing diluted, controls final concentration left in 0.5 μ g/ml
It is right（0.01~5.0 μ g/ml）, mix, you can obtain biotin conjugate.
The detection method of kit of the present invention is that the CA242 antibody of sample and biotin labeling is added into antibiotin
In the coated ELISA Plate of antibody, CA242 antigens and the antibody binding in sample form immune complex, meanwhile, this is immune multiple
Compound is fixed on ELISA Plate by the combination between anti-biotin antibodies and biotin.Use washing lotion cleaning of enzyme mark
Plate, removes uncombined free composition.The CA242 antibody-solutions of horseradish peroxidase-labeled are added, the enzyme labelled antibody passes through
Immune response is combined with by fixed immune complex.Again after cleaning ELISA Plate, add substrate solution and excite chemiluminescence, determine
Relative light intensity RLU, in the range of finite concentration, RLU values and the linear proportional relation of CA242 antigenic contents in sample.Pass through
Calibration object measured value, fitted calibration curve calculates CA242 antigen concentrations in sample according to curve, thus assess in human serum whether
Content containing CA242 antigens and CA242 antigens.
Compared with prior art, the kit and its system of board-like chemoluminescence method of the invention detection change of serum C A242 antigens
Preparation Method, on the basis of chemiluminescence immune assay, has the coated ELISA Plate of anti-biotin antibodies, and improve by introducing
High-temperature oscillation formula anti-biotin antibodies coating technique, by anti-biotin antibodies-Biotin method system, improves the sensitive of reaction
Degree, saves production cost, so that while reducing antibody/antigen usage amount, reagent production procedure is simplified, when shortening production
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, the degree of accuracy is high for market
Detection kit of new generation.
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, help 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 conventional method unless otherwise specified；The material, unless otherwise specified, is commercially obtained.
The board-like chemoluminescence method detection kit main agents of the carbohydrate antigen 242 of the present embodiment include：Including examination
Agent has the CA242 antibody-solutions of biotin labeling, anti-biotin antibodies coated ELISA Plates, horseradish peroxidase-labeled
CA242 antibody-solutions, CA242 calibration objects A-F, concentration washing lotion, substrate solution A, substrate solution B.
The preparation method of kit
First, the preparation of washing lotion is concentrated, step is as follows：
1st, KCl 60g, NaCl 300g are weighed in 1L containers；
2nd, 20.0g Tween-20 are weighed in 100ml containers plus 50ml water makes after it is completely dissolved, to pour into above-mentioned 1L and hold
In device；
3rd, Proclin-300 is measured into 2ml with pipettor, poured into above-mentioned 1L containers；
4th, appropriate purified water is measured in above-mentioned 1L containers with graduated cylinder, be sufficiently stirred for until being completely dissolved；
5th, pH is adjusted, its scope is controlled between 7.35~7.45；
6th, 1000ml is finally settled to, is produced after being completely dissolved with 0.2 μm of filter filtering.
2nd, the preparation of substrate solution
（One）Substrate solution A preparation steps
1st, weigh borax 11.44g, boric acid 4.948g, luminol 2.0g and to iodophenol 0.2mg in 1L beakers；
2nd, measure purified water in 1L beakers with graduated cylinder, be sufficiently stirred for until be completely dissolved, adjust pH, control its scope 8 ±
Between 0.05；
3rd, filtrate is collected by filtration with 0.2 μm of filter, is produced after 1000ml, mixing are settled to purified water.
（Two）Substrate solution B preparation
1st, borax 11.44g, boric acid 4.948g, the μ l of urea peroxide 0.2g and PC300 500 are weighed in 1L beakers；
2nd, measure purified water in 1L beakers with graduated cylinder, be sufficiently stirred for until be completely dissolved, adjust pH control its scope 8 ±
Between 0.05；
3rd, filtrate is collected by filtration with 0.2 μm of filter, is produced after 1000ml, mixing are settled to purified water.
3rd, the compound method of calibration object dilution, step is as follows：
1st, the ml of Tris 12.11g and HCl 7 is weighed in 1L container, is adjusted pH value of solution, is controlled its scope in 7.35-
7.45 between, it is settled to 1000ml；
2nd, calf serum 300ml is measured with graduated cylinder, is added in above-mentioned solution, it is standby as calibration object dilution；
4th, the compound method of calibration object, step is as follows：
1st, the preparation of calibration object A points：Calibration object dilution is taken, appropriate gauge is dispensed into；
2nd, the preparation of calibration object B points：Calibration object dilution is taken, by CA242 antigens（The peak of Beijing nine profit reaches）Suitable times of solution dilution
Number, controlled concentration is 4U/mL, is dispensed into appropriate gauge；
3rd, the preparation of calibration object C points：Take；Calibration object dilution, convenient multiple is diluted by CA242 antigenic solutions, and controlled concentration is
16U/mL, is dispensed into appropriate gauge；
4th, the preparation of calibration object D points：Calibration object dilution is taken, CA242 antigenic solutions are diluted into convenient multiple, controlled concentration is
48U/mL, is dispensed into appropriate gauge；
5th, the preparation of calibration object E points：Calibration object dilution is taken, CA242 antigenic solutions are diluted into convenient multiple, controlled concentration is
144U/mL, is dispensed into appropriate gauge；
6th, the preparation of calibration object F points：Calibration object dilution is taken, CA242 antigenic solutions are diluted into convenient multiple, controlled concentration is
288U/mL, is dispensed into appropriate gauge.
5th, the coated ELISA Plate of anti-biotin antibodies
6th, 0.01M carbonic acid buffer is prepared, pH is to pH9.6 ± 0.1 for regulation, standby；
7th, anti-biotin antibodies solution is added in above-mentioned 0.01M carbonic acid buffer to final concentration of 0.3 μ g/ml stirrings 30
Minute to well mixed；
8th, it is that 100 μ L are added in ELISA Plate per hole by package amount by the solution after above-mentioned mixing, is coated with using high-temperature oscillation formula,
It is 37 ± 1 DEG C to be coated with temperature, and the coating time is 1~3 hour；
9th, 0.1M pH7.4 TBS buffer solutions are prepared, containing 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 every holes of 150 μ L,
Closure temperature is 37 ± 1 DEG C, and off-period is 2~5 hours；
10th, confining liquid is sopped up, is placed in drying box, drying temperature control is at 37 ± 1 DEG C, and drying time is 2~4 hours, then
It is vacuum-packed with aluminium foil bag, labeling is standby；
6th, the preparation of horseradish peroxidase-labeled CA242 antibody-solutions
（One）The preparation of enzyme reaction thing dilution
1st, take Tris 4.846g, HCl 2900 μ l in beaker, purified water is then added in flask, is sufficiently stirred for making reagent
It is completely dissolved；
2nd, pH is adjusted, pH is 7.4 ± 0.05 for control；
3rd, BSA 4g are weighed to pour into above-mentioned beaker；
4th, last beaker is settled to 400ml, is produced with the filtering of 0.2um filters；
（Two）Horseradish peroxidase（HRP）With the coupling of CA242 antibody
1st, anti-CA242 antibody 1mg is taken to be positioned in 1ml glass tubes；
2nd, take 200 μ l DMSO lytic antibodies the final concentration of antibody is reached 5mg/ml, then fully mix；
3rd, the molar ratio that 10mol disuccinimidyl suberate is added according to 1mol antibody adds the succinyl of suberic acid two
Imines ester reacts 1.5 hours into above-mentioned 2 solution in 37 DEG C of insulating boxs；
4th, 1mol HRP mol ratio is added according to 3mol antibody toward HRP is added in above-mentioned 3 solution, then adds 1ml's
PH is the PB buffer solutions that 7.4 concentration are 0.1M, is placed in 37 DEG C of insulating boxs and reacts 3 hours；
5th, the solution prepared above-mentioned 4 PD-10 posts are purified, and refined solution are collected, according to 1:3000 volume addition is homemade
Enzyme reaction thing dilution, controls final concentration in 0.5 μ g/ml, well mixed to produce enzyme reaction thing；
Seven：The preparation of the CA242 antibody-solutions of biotin labeling
（One）The preparation of biotin reaction thing dilution
1st, take Tris 4.846g, HCl 2847 μ l in flask, purified water is then added in flask, is sufficiently stirred for making reagent
It is completely dissolved；
2nd, pH is adjusted, pH is in 7.35-7.45 for control；
3rd, BSA 4g are weighed to pour into above-mentioned beaker；
4th, last beaker is settled to 400ml, is produced with 0.2 μm of filter filtering.
（Two）Biotinylated antibody is taken, with biotin reaction thing diluted, final concentration is controlled in 0.5 μ g/
Ml, is mixed, you can obtain biotin conjugate.
8th, optimization and checking
Main performance index meets luminous detection reagent Technical Review specification.
1st, negative match-rate
The clinical blood sample of 130 Beckman chemical illuminating reagent detections of detection, negative match-rate（-/-）For 130/130, coincidence rate
100%。
2nd, positive coincidence rate
The clinical blood sample of 204 parts of Beckman chemical illuminating reagent detections of detection, positive coincidence rate（+/+）For 201/204, coincidence rate
For 98.53%, it see the table below.
3rd, it is repeated
Detection 10 times is repeated with enterprise's precision reference material through National reference mark, CV is in 3.23- for its coefficient of variation
Between 3.66%, meet no more than 10.0% industry standard（Full-automatic instrument is operated）.
| Accuracy quality-control product | Average value | Standard deviation | CV% |
| L | 4.85 | 0.2 | 3.58% |
| M | 68.95 | 2.5 | 3.66% |
| H | 226.88 | 7.3 | 3.23% |
4th, difference between batch
Three lot number kits, its interassay coefficient of variation are detected with enterprise's precision quality-control product through National reference mark（CV）
Between 2.50~4.24%, meet the requirement that rower is less than 15%（Full-automatic instrument is operated）.
Analysis result is as follows：
| Accuracy quality-control product | Average value | Standard deviation | CV% |
| L | 4.79 | 0.1 | 2.39% |
| M | 68.61 | 2.9 | 4.17% |
| H | 220.96 | 10.1 | 4.59% |
5th, stability
Heat endurance：Kit is detected that compared with the control, amplitude of variation is no more than detection signal after being placed 7 days at 37 DEG C
15%。
A kind of semi-automated instrument detection method of kit, micro- using Beijing Hamamatsu Technology Co., Ltd. in embodiment
Orifice plate luminescence analyzer BHP9504 is detected that detecting step includes
（1）Take out kit and be placed in room temperature, make the equalized temperature of kit to room temperature（18~25 DEG C）.
（2）Sample should be well mixed, if sample is freeze thawing sample, balance to room temperature（18~25 DEG C）Detected again afterwards.
（3）According to concentration washing lotion:Distilled water=1:39 proportions washing lotion, after being well mixed, is transferred in bottle for handling liquid toilet or cosmetic substance.
（4）Detection ELISA Plate lath is taken out, if calibration sample wells, measuring samples hole well.Remaining lath is put back in aluminium foil bag,
Seal up for safekeeping.
（5）By the order of table 1, semi-automatic sample-adding and operation are carried out.
Table 1 is loaded and operated the table of comparisons
Substrate solution A and B can also be used, mix with 100 μ L are added per hole after preceding isometric mixing if substrate solution A and B are mixed
Liquid should be used in 30 minutes.
A kind of half full-automatic instrument detection method of kit in embodiment, using Full-automatic chemiluminescence immunoassay analysis meter ADC
CLIA 200/300/400/500/600 and Smart3000/Smart300/ Smart3000S detected, detecting step bag
Include
（1）Using Full-automatic chemiluminescence immunoassay analysis meter（Hereinafter referred to as " instrument "）When, instrument do be read over
Operation manual, must be configured, examination and maintenance according to corresponding operating instruction to instrument, to realize optimum detection performance.
（2）According to the operation manual explanation configuration reagent information of instrument, and reference substance is set in " microplate layout is set "
Hole and the position of sample aperture.Especially, please detection method is carried out according to parameter shown in table 2 at " method editor " interface of instrument to match somebody with somebody
Put.
（3）According to the full-automatic instrument parameter setting table of table 2, parameter setting and data are carried out
The Full-automatic chemiluminescence immunoassay analysis meter parameter list of table 2
In addition to the implementation, present invention additionally comprises have other embodiment, all use equivalent transformation or equivalent replacement modes
The technical scheme of formation, all should fall within the scope of the hereto appended claims.
Claims (6)
1. a kind of board-like chemoluminescence method detection kit of carbohydrate antigen 242, it is characterised in that：Including reagent have biotin
The coated ELISA Plate of the CA242 antibody-solutions of mark, anti-biotin antibodies, horseradish peroxidase-labeled CA242 antibody it is molten
Liquid, CA242 calibration objects A-F, concentration washing lotion, substrate solution A, substrate solution B.
2. the board-like chemoluminescence method detection kit of carbohydrate antigen 242 according to claim 1, it is characterised in that：Institute
The concentration for stating the CA242 antibody-solutions of biotin labeling is 0.02~8.0 μ g/ml, the coated enzyme mark of anti-biotin antibodies
The coating concentration of the anti-biotin antibodies of plate is 0.2~8.0 μ g/ml, and the CA242 antibody of the horseradish peroxidase-labeled is molten
The concentration of liquid is 0.03~5.0 μ g/ml, and positive control thinner ratio is 1：500~1：10000.
3. the board-like chemoluminescence method detection kit of carbohydrate antigen 242 according to claim 1, it is characterised in that：Institute
It is CA242 antigens to state CA242 calibration object A-F points（The peak of Beijing nine profit reaches）Obtained by 7.4 Tris-HCl buffers of pH value
Arrive, concentration is respectively 0,4,16,48,144,288U/mL.
4. the board-like chemoluminescence method detection kit of carbohydrate antigen 242 according to claim 1, it is characterised in that：Institute
State the carbonic acid buffer that coating buffer used in the coated ELISA Plate of anti-biotin antibodies is 0.01M, pH9.6 ± 0.1, closing used
Liquid contain 0.3wt%BSA, 0.1 % Proclin300 (v/v), 1 wt % sheep blood serums, 1 wt % fishskin gelatins, 2 wt % marine algas
The 0.1M pH7.4 TBS buffer solutions of sugar and 10 wt % sucrose.
5. the board-like chemoluminescence method detection kit of carbohydrate antigen 242 according to claim 1, it is characterised in that：Institute
State the coated ELISA Plate of anti-biotin antibodies to be coated with using high-temperature oscillation formula, coating temperature is 37 ± 1 DEG C, the coating time is 1~3
Hour, form, oscillation amplitude are coated with using low-speed oscillation：3mm, using horizontal rotation, rotating speed is 200 ~ 500rpm, is closed and dry
Dry temperature is 37 ± 1 DEG C, and off-period is 1.5~3 hours, and drying time is 2~4 hours；Work is coated with using high-temperature oscillation formula
Skill, coating plate preparation time is can be controlled in 5~7 hours.
6. a kind of method for the board-like chemoluminescence method detection kit for preparing carbohydrate antigen 242 described in claim 1, its feature
It is：Comprise the following steps
First, the preparation of washing lotion is concentrated, step is as follows：
1st, KCl 60g, NaCl 300g are weighed in 1L containers；
2nd, 20.0g Tween-20 are weighed in 100ml containers plus 50ml water makes after it is completely dissolved, to pour into above-mentioned 1L and hold
In device；
3rd, Proclin-300 is measured into 2ml with pipettor, poured into above-mentioned 1L containers；
4th, appropriate purified water is measured in above-mentioned 1L containers with graduated cylinder, be sufficiently stirred for until being completely dissolved；
5th, pH is adjusted, its scope is controlled between 7.35~7.45；
6th, 1000ml is finally settled to, is produced after being completely dissolved with 0.2 μm of filter filtering；
2nd, the preparation of substrate solution
（One）Substrate solution A preparation steps
1st, weigh borax 11.44g, boric acid 4.948g, luminol 2.0g and to iodophenol 0.2mg in 1L beakers；
2nd, purified water is measured in 1L beakers with graduated cylinder, be sufficiently stirred for until being completely dissolved, tune pH controls its scope in 7.95-
8.05 between；
3rd, filtrate is collected by filtration with 0.2 μm of filter, is produced after 1000ml, mixing are settled to purified water；
（Two）Substrate solution B preparation
1st, borax 11.44g, boric acid 4.948g, the μ l of urea peroxide 0.2g and PC300 500 are weighed in 1L beakers；
2nd, purified water is measured in 1L beakers with graduated cylinder, be sufficiently stirred for until being completely dissolved, tune pH controls its scope to exist
7.95-8.05 between；
3rd, filtrate is collected by filtration with 0.2 μm of filter, is produced after 1000ml, mixing are settled to purified water；
3rd, the compound method of calibration object dilution, step is as follows：
1st, the ml of Tris 12.11g and HCl 7 is weighed in 1L container, is adjusted pH value of solution, is controlled its scope in 7.35-
Between 7.45,1000ml is settled to；
2nd, calf serum 300ml is measured with graduated cylinder, is added in above-mentioned solution, it is standby as calibration object dilution；
4th, the compound method of calibration object, step is as follows：
1st, the preparation of calibration object A points：Calibration object dilution is taken, appropriate gauge is dispensed into；
2nd, the preparation of calibration object B points：Calibration object dilution is taken, CA242 antigenic solutions are diluted into convenient multiple, controlled concentration is
4U/mL, is dispensed into appropriate gauge；
3rd, the preparation of calibration object C points：Take；Calibration object dilution, convenient multiple is diluted by CA242 antigenic solutions, and controlled concentration is
16U/mL, is dispensed into appropriate gauge；
4th, the preparation of calibration object D points：Calibration object dilution is taken, CA242 antigenic solutions are diluted into convenient multiple, controlled concentration is
48U/mL, is dispensed into appropriate gauge；
5th, the preparation of calibration object E points：Calibration object dilution is taken, CA242 antigenic solutions are diluted into convenient multiple, controlled concentration is
144U/mL, is dispensed into appropriate gauge；
6th, the preparation of calibration object F points：Calibration object dilution is taken, CA242 antigenic solutions are diluted into convenient multiple, controlled concentration is
288U/mL, is dispensed into appropriate gauge；
5th, the coated ELISA Plate of anti-biotin antibodies
0.01M carbonic acid buffer is prepared, pH is to pH9.6 ± 0.1 for regulation, standby；
Anti-biotin antibodies solution is added in above-mentioned 0.01M carbonic acid buffer to final concentration of 0.1~5 μ g/ml stirrings 30
Minute to well mixed；
Solution after above-mentioned mixing is added in ELISA Plate by package amount for 100 μ L per hole, is coated with using high-temperature oscillation formula, bag
It it is 37 ± 1 DEG C by temperature, the coating time is 1~3 hour, and form is coated with using low-speed oscillation；
Preparation 0.1M, pH7.4TBS buffer solutions, containing 0.3%BSA, 1% sheep blood serum, 0.1% Proclin300 (v/v), 1% fish-skin is bright
Glue, 2% trehalose and 10% sucrose, are added in the coating plate after cleaning as confining liquid, and closing amount is the every holes of 150 μ L, closing
Temperature is 37 ± 1 DEG C, and off-period is 2~5 hours；
Confining liquid is sopped up, is placed in drying box, drying temperature control is at 37 ± 1 DEG C, and drying time is 2~4 hours, then with aluminium
Paper tinsel bag vacuum packaging, labeling is standby；
6th, the preparation of horseradish peroxidase-labeled CA242 antibody-solutions
（One）The preparation of enzyme reaction thing dilution
1st, take Tris 4.846g, HCl 2900 μ l in beaker, purified water is then added in flask, is sufficiently stirred for making reagent
It is completely dissolved；
2nd, pH is adjusted, pH is in 7.35-7.45 for control；
3rd, BSA 4g are weighed to pour into above-mentioned beaker；
4th, last beaker is settled to 400ml, is produced with the filtering of 0.2um filters；
（Two）Horseradish peroxidase（HRP）With the coupling of CA242 antibody
1st, anti-CA242 antibody 1mg is taken to be positioned in 1ml glass tubes；
2nd, take 200 μ l DMSO lytic antibodies the final concentration of antibody is reached 5mg/ml, then fully mix；
3rd, the molar ratio that 10mol disuccinimidyl suberate is added according to 1mol antibody adds the succinyl of suberic acid two
Imines ester reacts 1.5 hours into above-mentioned 2 solution in 37 DEG C of insulating boxs；
4th, 1mol HRP mol ratio is added according to 3mol antibody toward HRP is added in above-mentioned 3 solution, then adds 1ml's
PH is the PB buffer solutions that 7.4 concentration are 0.1M, is placed in 37 DEG C of insulating boxs and reacts 3 hours；
5th, the solution prepared above-mentioned 4 PD-10 posts are purified, and refined solution are collected, according to 1:3000 volume addition is homemade
Enzyme reaction thing dilution, controls final concentration in 0.03-5.0 μ g/ml, well mixed to produce enzyme reaction thing；
Seven：The preparation of the CA242 antibody-solutions of biotin labeling
（One）The preparation of biotin reaction thing dilution
1st, take Tris 4.846g, HCl 2847 μ l in flask, purified water is then added in flask, is sufficiently stirred for making reagent
It is completely dissolved；
2nd, pH is adjusted, pH is in 7.35-7.45 for control；
3rd, BSA 4g are weighed to pour into above-mentioned beaker；
4th, last beaker is settled to 400ml, is produced with 0.2 μm of filter filtering；
（Two）Biotinylated antibody is taken, with biotin reaction thing diluted, final concentration is controlled in 0.01~5.0 μ
G/ml, is mixed, you can obtain biotin conjugate.
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| CN109541207A (en) * | 2017-09-21 | 2019-03-29 | 苏州新波生物技术有限公司 | A kind of diagnostic kit and preparation method thereof detecting CA72-4 |
| CN116087503A (en) * | 2023-01-08 | 2023-05-09 | 杭州联科生物技术股份有限公司 | Method for realizing ELISA simple operation |
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