CN108169486A - A kind of kit and its test method for measuring squamous cell carcinoma-related antigen content - Google Patents
A kind of kit and its test method for measuring squamous cell carcinoma-related antigen content Download PDFInfo
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- CN108169486A CN108169486A CN201711288526.4A CN201711288526A CN108169486A CN 108169486 A CN108169486 A CN 108169486A CN 201711288526 A CN201711288526 A CN 201711288526A CN 108169486 A CN108169486 A CN 108169486A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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Abstract
The invention discloses a kind of kits that squamous cell carcinoma-related antigen (SCC) content in human serum is measured using Magnetism particulate immuno chemistry luminescence method.Kit includes calibration object, quality-control product, anti-reagent, magnetic particle reagent, luminous substrate;Anti- reagent is the squamous cell carcinoma-related antigen coated antibody of marked by fluorescein isothiocyanate and the squamous cell carcinoma-related antigen labelled antibody of alkali phosphatase enzyme mark;Magnetic particle reagent is magnetic particle and goat-anti FITC attachments.Solid phase of the present invention using magnetic microparticles as immune response, coordinated using chemiluminescence enzyme linked immune analytic method and chemiluminescence class determining instrument, for measuring the SCC contents in human serum, and multiple samples can be measured simultaneously on Full-automatic chemiluminescence apparatus, realize the rapid measure of high throughput of SCC, accuracy is high, and high specificity, accuracy and detection efficiency are enhanced.
Description
Technical field
The present invention relates to technological field of biochemistry, and in particular to a kind of to be measured in human body using Magnetism particulate immuno chemistry luminescence method
The kit of squamous cell carcinoma-related antigen (SCC) content.
Background technology
Squamous cell carcinoma-related antigen is one group of glycoprotein for belonging to serine/cysteine proteinase mortifier family,
Molecular weight is 45,000 dalton.SCC is initially separated by Kato et al. in Squamous Cell Carcinoma, at least by 10 not
Subunit with isoelectric point forms.
Tumor markers of the squamous cell carcinoma-related antigen as squamous cell carcinoma, can be to cervical carcinoma, carcinoma of vulva, lung
The diseases such as cancer, cancer of the esophagus are detected.Early prognosis index can be used as, and right to SCC levels in serum before treatment of human cervical cancer
The high risk patient filtered out carries out auxiliary treatment.Importantly, treatment before SCC levels height, reflect patient to putting
The sensitivity for the treatment of, while curative effect can be also assessed, whether early monitoring tumour recurs, but cannot function as diagnosing early malignant tumor
Or the foundation made a definite diagnosis, it should not be used in the tumor screening of general population.
The squamous cell carcinoma-related antigen assay method being currently known has radioimmunology (RIA), enzyme linked immunosorbent assay
(ELISA), latex-enhanced turbidimetry etc..Radioimmunology complex steps, reagent price is expensive, need to use mating instrument and deposit
In radioactive pollution.Enzyme linked immunosorbent assay there are detection time it is long, complicated for operation, poor repeatability, be unsuitable for emergency treatment and clinic
The needs that patient diagnoses in time.Latex enhancing immune turbidimetry is easy to operate, quick, but sensitivity is low, low value poor repeatability.
Invention content
The technical problem to be solved by the present invention is to overcome existing squamous cell carcinoma-related antigen assay method determination steps
It is cumbersome, the defects of reagent price is expensive, provide a kind of accuracy it is high squama in human serum is measured using Magnetism particulate immuno chemistry luminescence method
The kit of columnar epithelium cell carcinoma antigen (SCC) content.
In order to solve the above technical problem, the present invention provides following technical solutions:
A kind of kit for measuring squamous cell carcinoma-related antigen content, it is micro- including calibration object, quality-control product, anti-reagent, magnetic
Grain reagent, luminous substrate;
The magnetic particle reagent is that anti-fluorescein isothiocynate antibody and the coupling of carboxyl magnetic bead are made, the luminous substrate
It is that ALPS is dissolved in luminous substrate buffer solution to be made;
Calibration object, quality-control product, anti-reagent, magnetic particle reagent, luminous substrate are prepared respectively;By calibration object, quality-control product, anti-examination
Agent, magnetic particle reagent, luminous substrate, are separately applied in packing container, obtain the quantitative determination of squamous cell carcinoma-related antigen
Kit;
(1), the preparation of the calibration object, quality-control product, includes the following steps:
SCC is dissolved with calibration object buffer solution, prepares the calibration object and quality-control product of anti-reagent;Wherein, the calibration object delays
It is newly mould by tetracycline and the sulfuric acid of 0.1g~0.5g that 0.01g~0.05g is added in the newborn bovine serum of 1L to rush solution
Element is prepared after being completely dissolved by 0.22 μm of filter membrane processing;
(2), the preparation of the anti-reagent:
1) preparation of anti-reagent buffer:
By the NaCl of Tris, 5g of 10g~20g~6g, the sheep serum of NaN3,1g~5g of 0.5~1.0g, 3g~
The newborn bovine serum of 10g, the horse serum of 1g~5g are added in 1L purified waters, are stirred well to and are completely dissolved, with MgCl and ZnCl
PH to 8~9 is adjusted, anti-reagent buffer is made;
2) fluorescein isothiocynate and SCC antibody couplings obtain the dermoid cancer of marked by fluorescein isothiocyanate
Antigen coat antibody:
Fluorescein isothiocynate is configured to the fluorescein isothiocynate of a concentration of 1.0~5.0mg/mL with buffer solution first
Solution, then according to SCC antibody:Fluorescein isothiocynate solution=1:1.1~1:1.5 mass ratio the two is transferred to brown
In vial, abundant mixing;It is fully balanced after reaction using the carbonate buffer solution that pH is 8~9, then using gel chromatography point
The squamous cell carcinoma-related antigen coated antibody of marked by fluorescein isothiocyanate is obtained from purifying;
3) alkaline phosphatase and SCC antibody couplings obtain the SCC antibody of alkali phosphatase enzyme mark:
Alkaline phosphatase is configured to the alkaline phosphatase enzyme solutions of a concentration of 1.0~5.0mg/mL, SCC with buffer solution first
Antibody and alkaline phosphatase reactive group is activated respectively respectively after according to molar ratio be alkaline phosphatase:SCC antibody=1:1~
1:3 reaction carries out coupling reaction than mixing abundant under the catalysis of catalyst, fully after reaction, uses the tris that pH is 8~9
Buffer solution balances, and gel column carries out isolating and purifying for different molecular big or small slice degree, obtains the scaly epithelium of alkali phosphatase enzyme mark
Cell carcinoma antigen labelled antibody;
The alkaline phosphorus that will be obtained in the SCC coated antibodies of the marked by fluorescein isothiocyanate obtained in step 2) and step 3)
The SCC labelled antibodies of sour enzyme label are added in the phosphate buffer containing surfactant, are obtained after being sufficiently stirred described anti-
Reagent;Preferably, the surfactant in the step 3) is one kind in Tween 20, Triton X-100, Bronidox
Or it is a variety of, the additive amount of surfactant is 0.01%~0.5%.
Further, the magnetic particle reagent is prepared according to following steps:
1) the carboxyl magnetic bead concentrate after abundant mixing is put into reaction bulb, which is placed in magnetic field to 15~
20min, supernatant is sucked after carboxyl magnetic bead all sedimentation, is added in into reaction bulb and is equivalent to carboxyl magnetic bead volume 2 in reaction bulb
~5 times of magnetic particle buffer solution, 20~30min of concussion cleaning;Reaction bulb is placed in magnetic field after 15~20min again and is sucked
Clearly;Repeated washing carboxyl magnetic bead 3 times;Finally by carboxyl magnetic bead solution constant volume to 10~50mg/mL, mixing is for use;
2) coupled reaction according to quality than carboxyl magnetic bead solution:Anti- fluorescein isothiocynate antibody=100:1 ratio exists
Anti- fluorescein isothiocynate antibody is added in carboxyl magnetic bead solution obtained by step 1), keeps mixing state anti-in 2~8 DEG C
It answers 18 hours;
3) reaction bulb places 15min in magnetic field, is washed 3 times with magnetic particle buffer solution after the sedimentation of carboxyl magnetic bead, then fixed
Hold to 10mg/mL, 2~8 DEG C of preservations, required for use magnetic particle reagent is made.Preferably, the configuration side of the magnetic particle buffer solution
Method is by the Tris of 12.12~15.26mg, the sodium chloride of 5.82~8.58mg, and it is pure that the Methyl cellulose ether of 50~60g is added to 1L
Change in water, be stirred well to and be completely dissolved to obtain the final product.
Using the method for the kit measurement squamous cell carcinoma-related antigen content, include the following steps:
1) three test tubes is taken to add 15 μ L calibration objects, 15 μ L quality-control products, 15 μ L samples to be tested respectively;
2) the 60 anti-reagents of μ L are added in every test tube, with covered rearing with plastic film test tube, test tube 30s is gently vibrated, is placed in 37
Water-bath 15min at DEG C;
3) 30 μ L magnetic particle reagents are added in every test tube, with covered rearing with plastic film test tube, test tube 30s is gently vibrated, puts
Water-bath 5min at 37 DEG C;
4) test tube on magnetic separator is precipitated to 3min, test tube and magnetic separator is slowly reversed, pours out supernatant;Falling
The test tube turned is placed on filter paper together with magnetic separator, flops magnetic separator bottom to remove all liquid being sticked on tube wall
Drop;
5) 300 μ L cleaning solutions are added in every test tube, with covered rearing with plastic film test tube, gently vibrate test tube 30s, after mixing
Test tube and magnetic separator are slowly reversed, pours out supernatant, the test tube of reversing together with magnetic separator, is placed on filter paper,
Separator bottom is firmly flopped to remove all drops being sticked on tube wall;
6) it is primary to repeat step 5);
7) 200 μ L luminous substrates are added in every test tube, vibrate mixing 3s, luminous intensity is detected with Chemiluminescence Apparatus.
The magnetic particle reagent of the present invention is magnetic particle and goat-anti FITC attachments, the buffer solution containing BSA.
The calibration object of the present invention is the buffering containing BSA for being added to not same amount squamous cell carcinoma-related antigen antigen respectively
Liquid.
The present invention technical principle be:This kit uses direct sandwich method principle, using magnetic microparticles as immune response
Solid phase, coordinated using chemiluminescence enzyme linked immune analytic method and chemiluminescence class analyzer, for measuring in human serum
SCC contents.Technical principle is:The SCC antibody of fluorescein isothiocynate (FITC) label and alkaline phosphatase (AP) label
The SCC that SCC is matched in antibody and sample, calibration object or quality-control product, which is combined, forms " sandwich " compound.Then addition is connected with anti-
The magnetic particle of FITC antibody makes antigenantibody complex be incorporated in magnetic by the specific binding of anti-FITC antibody and FITC
On particle.Under the action of externally-applied magnetic field, the compound that immune response is formed is detached with other unbonded substances, cleaning is multiple
After closing object, enzyme-catalyzed chemical luminescence substrate is added in.Substrate, by catalytic pyrolysis, is formed among unstable excitation state under enzyme effect
Body sends out photon when excitation state intermediate returns to ground state, forms luminescence-producing reaction, you can uses Chemiluminescence Apparatus detection reaction
Luminous intensity.In detection range, luminous intensity is directly proportional to the content of the SCC in sample, uses four parameters of improvement
Logistic equation models can calculate SCC concentration in sample.
The advantageous effect that is reached of the present invention is:
1st, chemiluminescence is combined by the kit with immune magnetic particle, is provided a kind of close to homogeneous reactant
System, and employs one-step method reaction pattern so that detection sensitivity, accuracy greatly improve, detection range expands, during reaction
Between greatly shorten, from starting to be loaded onto testing result, the time is less than 45min, hence it is evident that is faster than similar kit;
2nd, a kind of new FITC antibody and magnetic particle coupling method have been invented, this method coupling efficiency is high, is firmly combined with, and
Process stabilizing while enhancing product performance, greatly reduces product cost.
3rd, kit moderate resistance reagent, magnetic particle reagent, calibration object, quality-control product, luminous substrate liquid and concentration washing lotion are these
Optimization formula under reaction system imitates the phase to the use of the kit and detection performance provides powerful guarantee.
4th, the present invention can measure multiple samples simultaneously on Full-automatic chemiluminescence apparatus, realize that dermoid cancer resists
The former rapid measure of high throughput, accuracy is high, and high specificity, accuracy and detection efficiency are enhanced.
Description of the drawings
Attached drawing is used to provide further understanding of the present invention, and a part for constitution instruction, the reality with the present invention
Example is applied together for explaining the present invention, is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is the correlation of the kit and other commercial reagent box detection clinical serums of the present invention;Wherein abscissa is
The testing result of other commercial reagent boxes, ordinate are the testing result of kit of the present invention.
Specific embodiment
The preferred embodiment of the present invention is illustrated below, it should be understood that preferred embodiment described herein is only used
In the description and interpretation present invention, it is not intended to limit the present invention.
The operation sequence of kit test sample using the present invention is as follows:
1st, sample collection
Serum (sample of significant hemolysis or piarhemia cannot be used for measuring), the sample after collection are collected using correct medical technology
This may not exceed 8 hours being placed at room temperature for;Sample need to be positioned in 2-8 DEG C of refrigerator if detected not in 8 hours;If it needs
It preserves or transports within 72 hours or more, then should freeze in -20 DEG C hereinafter, avoiding multigelation.Room temperature is returned to before use, is gently shaken
Dynamic mixing.
2nd, prepare before experiment
1. one bottle of washing lotion is taken to carry out 15 times of dilutions with distilled water;
2. insulating box or water-bath pot temperature are adjusted to 37 DEG C, used after temperature stabilization;
3. by the abundant mixing of magnetic particle suspension to be visible by naked eyes precipitation.
3 experimental methods
1. taking out a certain amount of reaction vessel (flat based tubes), number.15ul calibration objects/Quality Control is added according to requirement of experiment
Product/clinical sample;
2. it is separately added into anti-reagent 60ul per hole;
3. solution in reaction vessel is uniformly mixed, 37 DEG C incubate 30 minutes;
4. shaking up magnetic particle suspension, 30ul is separately added into per hole;
5. solution in reaction vessel is uniformly mixed, 37 DEG C incubate 5 minutes;
6. reaction vessel is taken out, using Magneto separate and washing facility, by the wash liquid 3 times of magnetic particle in reaction vessel;
7. going washing lotion after the completion of washing, add luminous substrate liquid 200ul per hole, shake;
8. chemiluminescence detector device detects luminous intensity;
9. using four parameter fitting modes, using calibration object concentration value as X-axis, using calibration object luminous intensity values as Y-axis, establish
Calibration curve.Corresponding concentration value is back-calculated according to the luminous intensity values of sample to be tested.
Kit of the present invention according to methodology is identified, can reach following index:
Standard curve is linear:R > 0.9900.
Minimum detection limit:≤0.3ng/ml.
Accuracy:TIANZHU XINGNAO Capsul 85%-115%.
Repeatability:Coefficient of variation CV≤8%.
Difference between batch:The coefficient of variation≤15%.
Linear dilution:R is more than 0.9900.
100 parts of serum sample measured values and commercially available squamous cell carcinoma-related antigen detection kit A are compared, it is more of the invention
Kit and the correlation of commercially available squamous cell carcinoma-related antigen kit testing result, the results are shown in Figure 1.
Finally it should be noted that:The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention,
Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used
To modify to the technical solution recorded in foregoing embodiments or carry out equivalent replacement to which part technical characteristic.
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in the present invention's
Within protection domain.
Claims (5)
1. a kind of kit for measuring squamous cell carcinoma-related antigen content, which is characterized in that including calibration object, quality-control product, resist
Reagent, magnetic particle reagent, luminous substrate;
The magnetic particle reagent be by anti-fluorescein isothiocynate antibody and carboxyl magnetic bead coupling be made, the luminous substrate be by
ALPS is dissolved in luminous substrate buffer solution and is made;
Calibration object, quality-control product, anti-reagent, magnetic particle reagent, luminous substrate are prepared respectively;By calibration object, quality-control product, anti-reagent,
Magnetic particle reagent, luminous substrate, are separately applied in packing container, obtain the quantitative determination reagent of squamous cell carcinoma-related antigen
Box;
(1), the preparation of the calibration object, quality-control product, includes the following steps:
SCC is dissolved with calibration object buffer solution, is prepared after being completely dissolved by 0.22 μm of filter membrane processing;
(2), the preparation of the anti-reagent:
1) preparation of anti-reagent buffer:
By the NaCl of Tris, 5g of 10g~20g~6g, the NaN of 0.5~1.0g3, the sheep serum of 1g~5g, 3g~10g it is new
Raw cow's serum, 1g~5g horse serum add in 1L purified waters, be stirred well to and be completely dissolved, with MgCl and ZnCl adjust pH to
8~9, the anti-reagent buffer is made;
2) fluorescein isothiocynate and SCC antibody couplings obtain the SCC coated antibodies of marked by fluorescein isothiocyanate:
Fluorescein isothiocynate is configured to the isosulfocyanic acid fluorescence of a concentration of 1.0~5.0mg/mL with anti-reagent buffer first
Plain solution, then according to SCC antibody:Fluorescein isothiocynate solution=1:1.1~1:1.5 mass ratio the two is transferred to palm fibre
In color vial, abundant mixing;It is fully balanced after reaction using the carbonate buffer solution that pH is 8~9, then using gel chromatography
It isolates and purifies to obtain the squamous cell carcinoma-related antigen coated antibody of marked by fluorescein isothiocyanate;
3) alkaline phosphatase and SCC antibody couplings obtain the SCC antibody of alkali phosphatase enzyme mark:
Alkaline phosphatase is configured to the alkaline phosphatase enzyme solutions of a concentration of 1.0~5.0mg/mL, SCC antibody with buffer solution first
After reactive group is activated respectively respectively with alkaline phosphatase according to molar ratio be alkaline phosphatase:SCC antibody=1:1~1:3
Reaction than under the catalysis of catalyst abundant mixing carry out coupling reaction, fully after reaction, delayed using pH for 8~9 tris
Fliud flushing balances, and gel column carries out isolating and purifying for different molecular big or small slice degree, and the scaly epithelium for obtaining alkali phosphatase enzyme mark is thin
Born of the same parents' cancer antigen labelled antibody;
It will be in the squamous cell carcinoma-related antigen coated antibody and step 3) of the marked by fluorescein isothiocyanate that obtained in step 2)
The SCC antibody of the alkali phosphatase enzyme mark of acquisition is added in the phosphate buffer containing surfactant, is obtained after being sufficiently stirred
Obtain the anti-reagent.
2. the kit according to claim 1 for measuring squamous cell carcinoma-related antigen content, which is characterized in that the step
It is rapid 3) in surfactant be Tween 20, it is Triton X-100, one or more in Bronidox, surfactant
Additive amount is 0.01%~0.5%.
3. measuring the kit of squamous cell carcinoma-related antigen content according to claims 1 or 2 any one of them, feature exists
In the magnetic particle reagent be to be prepared according to following steps:
1) the carboxyl magnetic bead concentrate after abundant mixing is put into reaction bulb, which is placed in magnetic field to 15~
20min, supernatant is sucked after carboxyl magnetic bead all sedimentation, is added in into reaction bulb and is equivalent to carboxyl magnetic bead volume 2 in reaction bulb
~5 times of magnetic particle buffer solution, 20~30min of concussion cleaning;Reaction bulb is placed in magnetic field after 15~20min again and is sucked
Clearly;Repeated washing carboxyl magnetic bead 3 times;Finally by carboxyl magnetic bead solution constant volume to 10~50mg/mL, mixing is for use;
2) coupled reaction:According to quality than carboxyl magnetic bead solution:Anti- fluorescein isothiocynate antibody=100:1 ratio is in step
1) anti-fluorescein isothiocynate antibody is added in the carboxyl magnetic bead solution obtained by, mixing state response 18 is kept in 2~8 DEG C
Hour;
3) reaction bulb places 15min in magnetic field, is washed 3 times with magnetic particle buffer solution after the sedimentation of carboxyl magnetic bead, is then settled to
Required for use magnetic particle reagent is made in 10mg/mL, 2~8 DEG C of preservations.
4. the kit of squamous cell carcinoma-related antigen content is measured according to claim 3 any one of them, which is characterized in that
The configuration method of the magnetic particle buffer solution be by the Tris of 12.12~15.26mg, the sodium chloride of 5.82~8.58mg, 50~
The Methyl cellulose ether of 60g is added in 1L purified waters, is stirred well to and is completely dissolved to obtain the final product.
It is 5. special using the method for 2 any one of them kit measurement squamous cell carcinoma-related antigen content of claims 1 or 2
Sign is that this method includes the following steps:
1) three test tubes is taken to add 15 μ L calibration objects, 15 μ L quality-control products, 15 μ L samples to be tested respectively;
2) the 60 anti-reagents of μ L are added in every test tube, with covered rearing with plastic film test tube, test tube 30s is gently vibrated, is placed at 37 DEG C
Water-bath 30min;
3) 30 μ L magnetic particle reagents are added in every test tube, with covered rearing with plastic film test tube, test tube 30s is gently vibrated, puts 37 DEG C
Lower water-bath 5min;
4) test tube on magnetic separator is precipitated to 3min, test tube and magnetic separator is slowly reversed, pours out supernatant;Reversing
Test tube is placed on filter paper together with magnetic separator, flops magnetic separator bottom to remove all drops being sticked on tube wall;
5) 30 μ L cleaning solutions are added in every test tube, with covered rearing with plastic film test tube, gently vibrate test tube 30s, after mixing slowly
Reversing test tube and magnetic separator, pour out supernatant, the test tube of reversing together with magnetic separator, be placed on filter paper, firmly
Separator bottom is flopped to remove all drops being sticked on tube wall;
6) it is primary to repeat step 5);
7) 200 μ L luminous substrates are added in every test tube, vibrate mixing 3s, luminous intensity is detected with Chemiluminescence Apparatus.
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CN110780079A (en) * | 2019-11-28 | 2020-02-11 | 南京迪安医学检验所有限公司 | Squamous cell carcinoma antigen detection reagent |
CN111175492A (en) * | 2020-02-27 | 2020-05-19 | 江苏泽成生物技术有限公司 | Kit for detecting procalcitonin content in serum and use method thereof |
CN112129941A (en) * | 2020-09-22 | 2020-12-25 | 武汉生之源生物科技股份有限公司 | Chemiluminescence kit for detecting squamous cell carcinoma antigen |
CN113092759A (en) * | 2021-02-26 | 2021-07-09 | 北京九强生物技术股份有限公司 | Squamous cell carcinoma antigen detection kit |
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