CN101398429A - Tumor-associated antigen 242 magnetic microparticle chemiluminescence immune assay determination kit and method for making same - Google Patents
Tumor-associated antigen 242 magnetic microparticle chemiluminescence immune assay determination kit and method for making same Download PDFInfo
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Abstract
The invention relates to the medical field of immunoassay, particularly provides a measuring kit of the chemiluminescence immunoassay of a CA242 magnetic particle, and a preparation method thereof. The kit of the invention comprises: 1) a CA242 working calibrator; 2) the mixed solution of the magnetic particle coated with a CA242 monoclonal antibody and the CA242 monoclonal antibody marked by biotin; 3) streptavidin marked by horse radish peroxidase; 4) a chemiluminescence substrate; and 5) a reaction tube. Further, the method for preparing the kit according to the invention comprises the following steps: 1) the working calibrator is prepared by CA242 pure products; 2) the mixed solution of the magnetic particle coated with the monoclonal antibody and the monoclonal antibody marked by the biotin is prepared; 3) the horse radish peroxidase is used for marking the streptavidin; 4) the chemiluminescence substrate is prepared; 5) the working calibrator, the mixed solution, enzyme markers, the chemiluminescence substrate and the reaction tube are sub-assembled; and 6) assembly is carried out to obtain a finished product. The kit of the invention has the advantages of simpleness and convenience, fastness, sensitivity and stability and the like.
Description
Technical field
The present invention relates to the immunoassay medical domain, particularly, the invention provides a kind of tumor associated antigen 242 magnetic microparticle chemiluminescence immune assay determination kits and preparation method thereof.Kit of the present invention combines biotin one Avidin immunity amplifying technique, immune magnetic particle technology and chemiluminescence immunoassay technology.
Background technology
Cancer has just constituted serious threat to human existence health from being born.The hospital diagnosis infantile tumour mainly is by iconography and cell pathology diagnostic techniques at present, is just made a definite diagnosis after having tangible occupying lesion and clinical symptoms.Along with early diagnosis of tumor enters the protein epoch, be that the body fluid protein of representative becomes the focus in the tumor-marker research field with blood, thereby promoted the setting-up and development of the novel detection technique of quick, highly sensitive, high special tumor markers.
Tumor associated antigen 242 (carbonhydrate antigen 242, CA242) as a kind of tumor associated antigen, be a kind of glycoprotein of mucin type, content in healthy people and benign disease people's serum is very low, when alimentary canal generation malignant tumour, CA242 occur to raise in the serum, so CA242 has higher specificity than other tumor marker, and false positive rate is very low in benign disease.CA242 also can be used for the diagnosis and the monitoring of cancer of pancreas, colorectal cancer simultaneously, and is for clinical detection such as the judgement of the curative effect behind the treating malignant tumor, tracing studys, also significant.
The immunization method that is used to detect CA242 at present mainly contains that enzyme immunoassay (EIA), radiommunoassay, immunofluorescence assay, radio-immunity are developed, antibody chip technology and chemiluminescence immune assay.According to a large amount of test findings and clinical practice data,, be followed successively by: chemiluminescence immune assay, fluoroimmunoassay, radiommunoassay and EIA enzyme immunoassay from practicality, stability, accuracy and development prospect.
Therefore radiating immuning analysis technology has certain contaminative to environment, and exists instrument cost expensive because of it uses radioelement thing that serves as a mark, and sensitivity is not high, complicated operation, measurement result instability, shortcoming such as the reagent holding time is short.Enzyme immunoassay (EIA) sensitivity is low, and influence factor is more, easily causes false negative and false positive.Chemiluminescence immunoassay is a kind of than elder generation and then effective method, can make detection sensitivity reach 10
-18Mol level, and sensing range can reach 6 orders of magnitude, and its enzyme labeling thing is stable, can use for a long time.Characteristics such as chemiluminescence immunoassay technology is highly sensitive because of it, height is special, quick, high flux obtain application more and more widely.
In the immune detection of reality, from the more background of impure composition, separate, be purified into the purpose determinand fast, be the difficult problem that the clinical examination worker faces always.Immunity magnetic particle technology is to utilize macromolecular material synthesizing micron-grade magnetic solid phase particle to make carrier, carboxyl reactive group and the protein amino covalent bond of utilizing surface organic matter to provide, bag by on have the specificity affinity various immunologic active materials (antigen or antibody), making its sensitization is immune magnetic particle, can carry out antigen, antibody response.Magnetic particle has that velocity of separation is fast, efficient is high, favorable repeatability; Instrument and equipment simple to operate, as not need costliness; Do not influence characteristics such as the biological character of separated cell or other biomaterial and function, adding directed movement under the action of a magnetic field, make some special composition be separated, concentrate or purifying.As a kind of general solid phase isolation methods that is used for the chemiluminescence immune assay detection technique, thereby magnetic particle can improve effective package amount conservation greatly, can widen the range of linearity of detection simultaneously, thereby effectively avoid the generation of crotch effect.
Biotin-avidin system has multistage signal amplification, and does not increase non-specific interference, and has highly sensitive, characteristics such as specificity good, stability is high, applicability is strong and experimental cost is low.Reaction system to be measured can be utilized biotinylated antibody, make Avidin-biotin system and the coupling of immunoassay detection architecture, amplify end reaction: will react simultaneously at the insolubilized antibodies of different antigenic determinants and biotinylated antibody and antigen (standard antigen and determined antigen) earlier, form the double-antibody sandwich immune complex at surface of solid phase carriers, adding Avidin again combines with biotin in the compound, reaction signal is amplified, thus the sensitivity that has improved immunoassay.
Chemiluminescence immune assay is a kind of elder generation and then effective method that detects tumor associated antigen 242 in conjunction with biotin-avidin system and immune magnetic particle solid phase separation system, helps lend some impetus to application and the development of chemiluminescence immunoassay technology in clinical examination, detection.
Summary of the invention
The present invention has solved the problems referred to above simultaneously, be about to biotin-Avidin immunity amplifying technique and immune magnetic particle technology in conjunction with chemiluminescence immunoassay technology, detect tumor associated antigen 242, a kind of mensuration kit that can easy, quick, sensitive, stably detect tumor associated antigen 242 is provided, and this kit is suitable for applying effectively on industry.
The purpose of this invention is to provide a kind of biotin-Avidin immunity amplifying technique and immune magnetic particle technology magnetic microparticle chemiluminescence immune assay determination kit in conjunction with chemiluminscence immunoassay tumor associated antigen 242.
A further object of the present invention provides a kind of method for preparing the mentioned reagent box.
Kit according to the present invention comprises: 1) tumor associated antigen 242 calibration objects; 2) mixed liquor of the magnetic particle of tumor associated antigen 242 monoclonal antibody bag quilts and biotin labeled tumor associated antigen 242 monoclonal antibodies; 3) streptavidin of horseradish peroxidase-labeled; 4) chemical luminous substrate luminol that above-mentioned horseradish peroxidase acted on or different luminol; And 5) reaction tube.
According to kit of the present invention, wherein, the magnetic particle of described tumor associated antigen 242 monoclonal antibody bag quilts and the blending ratio of biotin labeled tumor associated antigen 242 monoclonal antibodies are 1:2.
According to kit of the present invention, wherein, described chemical luminous substrate comprises A liquid and B liquid, wherein,
A liquid is to add Tris and dense HCl in distilled water to be made into 0.1M pH value be 8.5 Tris-HCl damping fluid, comprise the luminol of 4.0mg/mL or different luminol and 0.3mg/mL to iodophenol;
B liquid is to add trisodium citrate and citric acid in distilled water, is mixed with 0.1M pH value and is 4.6 citrate buffer solution, comprises the superoxol of 200mg/mL.
According to kit of the present invention, wherein, the reaction tube material that described kit uses is transparent polystyrene, tygon, polypropylene or glass.
Further, the method for preparing the mentioned reagent box according to the present invention may further comprise the steps:
1) with tumor associated antigen 242 pure product preparation calibration object;
2) preparation tumor associated antigen 242 monoclonal antibody bags are by the mixed liquor of magnetic particle and biotin labeling tumor associated antigen 242 monoclonal antibodies;
3) with the horseradish peroxidase-labeled streptavidin;
4) preparation chemical luminous substrate luminol or different luminol that horseradish peroxidase acted on;
5) mixed liquor of the above-mentioned calibration object of packing, antibody sandwich magnetic particle and biotin labeling antibody, enzyme labeling thing, chemical luminous substrate luminol or different luminol and reaction tube; And
6) be assembled into finished product.
The method according to this invention is in the step 2 of the mixed liquor of described preparation antibody sandwich magnetic particle and biotin labeling antibody) in:
Described mixed liquor is to be mixing in 1: 2 by the magnetic particle of tumor associated antigen 242 monoclonal antibody bag quilts and biotin labeled tumor associated antigen 242 monoclonal antibodies with volume ratio, and is formulated with 20～40% calf serum;
Described magnetic particle is that 1～2 μ m particle diameter, tri-iron tetroxide kernel, surface parcel have reactive group as amino (NH
2-), (polymkeric substance COOH), its working concentration are 5～10mg/mL to carboxyl;
Described antibody sandwich magnetic particle be by the glutaraldehyde two-step approach with tumor associated antigen 242 monoclonal antibody bags by on magnetic particle;
With the magnetic particle of confining liquid sealing bag quilt, described confining liquid is that to contain 0.2%～1.0% bovine serum albumin(BSA), pH value be 7.2 0.02mol/L phosphate buffer;
Described biotin labeling tumor associated antigen 242 monoclonal antibodies realize with antibody free lysine generation coupling reaction under the alkalescence condition by the biotin succinimide ester.
The method according to this invention in described step 3) with the horseradish peroxidase-labeled streptavidin, adopts improvement sodium periodate method with the horseradish peroxidase-labeled streptavidin.
The method according to this invention, described chemical luminous substrate comprise A liquid and B liquid, wherein,
A liquid is to add Tris and dense HCl in distilled water to be made into 0.1M pH value be 8.5 Tris-HCl damping fluid, comprise the luminol of 4.0mg/mL or different luminol and 0.3mg/mL to iodophenol;
B liquid is to add trisodium citrate and citric acid in distilled water, is mixed with 0.1M pH value and is 4.6 citrate buffer solution, comprises the superoxol of 200mg/mL.
In the method for mentioned reagent box produced according to the present invention, use transparent polystyrene, tygon, polypropylene or glass as the reaction tube material.
Concrete mentioned reagent box can comprise mixed liquor, enzyme labeling thing, chemical luminous substrate and the lavation buffer solution etc. of calibration object, antibody sandwich magnetic particle and biotin labeling antibody.Wherein, the raw material of described calibration object is a standard level, and purity is not less than 90%; Antibody sandwich forms mixed liquor with biotin labeled antibody on magnetic particle; The enzyme of mark streptavidin is a horseradish peroxidase; Chemical luminous substrate is a luminol; Lavation buffer solution is a phosphate buffer.
The present invention's " tumor associated antigen 242 magnetic microparticle chemiluminescence immune assay determination kits " can detect the content of the tumor associated antigen 242 in malignant tumor of digestive tract (as cancer of pancreas, colorectal cancer etc.) the patient body very effectively, can be according to the variation of how much judging the result of treatment and the state of an illness thereof of tumor associated antigen 242 content.Kit of the present invention (chemoluminescence method, biotin-avidin immunity amplifying technique combine with immune magnetic particle) has advantages such as easy, quick, sensitive, stable, and every index all reaches the analytic approach level of similar import reagent box.And, according to detection system of the present invention is open-sky technique, easy to be quick, does not need the expensive luminous measuring instrument of full-automatic chemical, be particularly suitable for vast middle and small hospital and promote the use of, for clinical diagnosis and research work provide a kind of very valuable detection means.
According to kit of the present invention, the magnetic particle of tumor associated antigen 242 monoclonal antibody bag quilts, the tumor associated antigen in the testing sample 242 combine with biotin labeled tumor associated antigen 242 monoclonal antibodies, form " double-antibody sandwich " composite structure, the streptavidin of enzyme labeling combines with this composite structure more afterwards, form " magnetic particle-antibody-determined antigen-antibody-biotin-avidin-enzyme " compound, the last also amplifying optical signals that produces with the chemical luminous substrate effect carries out highly sensitive, high special detection to tumor associated antigen 242 in the tubular type light-emitting appearance.The present invention adopts " the direct sandwich two-step approach of double antibody " reaction pattern, effectively utilized chemiluminescence in conjunction with magnetic particle and biotin-avidin immunity amplifying technique principle, tumor associated antigen 242 content in detection by quantitative human serum, the plasma sample have been guaranteed the sensitivity that detects.The magnetic particle that uses has super paramagnetic, high dispersive, characteristics that surface area is big.The pre-treatment of sample is required low, sample pretreatment process is simple and reliable, can be fast, the high throughput testing gross sample, be convenient to operation and production.Kit of the present invention is simple in structure, and is easy to use, low price, carrying convenience is compared with the enzyme linked immunological kit on the market, board-like chemical luminescence reagent kit, and the range of linearity is wide, effectively avoid the crotch effect, do not need diluted sample, be applicable to the mass detection sample.
Description of drawings
Fig. 1 is the calibration object linear graph (double-log typical curve) in the prepared kit of embodiment 1.
Fig. 2 is the correlation curve of kit of the present invention with the clinical blood sample measured value comparison of external kit (ELISA of CanAg company kit).
Embodiment
Embodiment 1 preparation tumor associated antigen 242 magnetic microparticle chemiluminescence immune assay determination kits of the present invention
One, the preparation of tumor associated antigen 242 calibration objects
With horse serum tumor associated antigen 242 pure product are diluted to calibration object, concentration is respectively 0U/mL, 10U/mL, 30U/mL, 100U/mL, 300U/mL, 600U/mL, totally 6 bottles.
Two, tumor associated antigen 242 monoclonal antibody bags are by the preparation of the mixed liquor of magnetic particle and biotin labeling tumor associated antigen 242 monoclonal antibodies
1, the preparation of the magnetic particle of tumor associated antigen 242 monoclonal antibody bag quilts
With particle diameter is that the magnetic particle of 1～2 μ m activates with glutaraldehyde, stirring at room, and mixing is after 3 hours, add magnetic field, leave standstill 20～25min, pour out supernatant, with the pH value is that 7.4 0.01mol/L phosphate buffer cleans three times, and suspends with this solution, and concentration is 50～100mg/mL; Add tumor associated antigen 242 monoclonal antibodies 20～50 μ g in every milliliter of suspending liquid, after 4 ℃ stirring is spent the night down, add magnetic field, leave standstill 10～15min, pour out supernatant, sealed 3～4 hours in room temperature with the phosphate buffer (pH is 7.2) that contains 0.2%～1.0% bovine serum albumin(BSA), 0.02mol/L; At last with the pH value be 7.4, the phosphate lavation buffer solution that contains Tween-20 and sodium azide antiseptic cleans 3～5 times, and is mixed with the working fluid of 5～10mg/mL with this solution.Magnetic particle is 4 ℃ of preservations.
2, biotin labeled tumor associated antigen 242 MONOCLONAL ANTIBODIES SPECIFIC FOR
Biotin labeling tumor associated antigen 242 monoclonal antibodies with the biotin succinimide ester under the alkalescence condition with monoclonal antibody generation coupling reaction, PBS is fully dialysed, the biotin labeling thing dilutes with calf serum, adds proclin and preserves below 300 ,-20 ℃.
3, the preparation of mixed liquor
Is that 1:2 mixes with the magnetic particle of tumor associated antigen 242 monoclonal antibody bag quilts with the volume ratio with biotin labeled tumor associated antigen 242 monoclonal antibodies, formulated with 20～40% calf serums.
Three, the preparation of the streptavidin of horseradish peroxidase-labeled
Adopt improvement sodium periodate method with the horseradish peroxidase-labeled streptavidin, concrete labeling process is as follows: dissolving 4.4mg HRP is in 1mL distilled water, add 0.4mL sodium periodate (50mmol/L) stirring at room 20min, through the 1mmol/L sodium-acetate buffer, pH4.4 dialysis back adds the 8mg streptavidin, stir 2h, use 200mmol/L NaBH at last
4Reduce, after the dialysis of 0.02M PBS damping fluid, add equal-volume glycerine, preserve below-20 ℃.
Four, the concentration of enzyme labeling thing is selected
Adopting the square formation method to select the working concentration scope of enzyme labeling thing is 1:3000～5000.
Five, enzyme labeling thing dilution
Tris 12.120g
BSA 5g
Proclin 1mL
Distilled water 1000mL
Six, chemical luminous substrate
The compound method of the chemical luminous substrate liquid of horseradish peroxidase used in the present invention (HRP):
A liquid: adding Tris and dense HCl in distilled water, to be made into 0.1M pH value be 8.5 Tris-HCl damping fluid.In this damping fluid, add the Luminol of 4.0mg/mL and 0.3mg/mL to iodophenol.
B liquid: in distilled water, add trisodium citrate and citric acid, be mixed with 0.1M pH value and be 4.6 citrate buffer solution, the superoxol of adding 200mg/mL in this solution.
Using method: A, B liquid bi-component reagent mix according to the use amount equal-volume before use.
Seven, lavation buffer solution
Lavation buffer solution is the phosphate buffer that contains 0.1～0.5% Tween-20,0.1% sodium azide antiseptic, and the pH value is 7.4.Use 20 times of distilled water dilutings during use.
Eight, semi-manufacture and finished product are formed
The packing of above-mentioned steps products obtained therefrom is semi-manufacture.Extract three parts out and just can be assembled into tumor associated antigen 242 magnetic microparticle chemiluminescence immune assay determination kits, be assembled into also need inspect by random samples behind the kit and just can dispatch from the factory after qualified through specificity, accuracy, sensitivity and stable assay approvals.
The using method of embodiment 2 kits of the present invention
One, sample pre-treatments
Get people's empty stomach blood serum sample in morning, the centrifugal 5min of 3000rpm gets upper strata liquid 50 μ L and analyzes.
Two, detection method
Before using this kit to experimentize, the Avidin of the mixed liquor of taking-up antibody sandwich magnetic particle and biotin labeling antibody, calibration object/testing sample, enzyme labeling was placed 15～30 minutes in room temperature earlier, made them equilibrate to room temperature; Afterwards, constant temperature incubator or water-bath are transferred to 37 ℃; Again, be ready to suitable micro sample adding appliance and corresponding suction nozzle and check whether operate as normal of Chemiluminescence Apparatus.
Use this kit as follows according to the concrete operations step that the method for embodiment 1 experimentizes:
After round bottom polystyrene test tube numbering, in test tube, add 50 μ L blood serum samples or serial calibration object solution, the every pipe of calibration object adds 0U/mL, 10U/mL, 30U/mL, 100U/mL, each 50 μ L of 300U/mL, 600U/mL, the mixed liquor 50 μ L that add antibody sandwich magnetic particle and biotin labeling antibody again, 37 ℃ of oscillating reactions 30min.After cleansing solution cleaning three times, add the streptavidin 50 μ L of horseradish peroxidase-labeled, 37 ℃ of oscillating reactions 15min.Afterwards, test tube rack placed separate 5min on the magnetic separator, the separation vessel that reverses is then poured out supernatant, the test tube of reversing is placed on the filter paper blots, and bounces separation vessel to remove wall built-up liquid.Every pipe adds cleansing solution 500 μ L, and fully mixing places and separates 5min on the magnetic separator, pours out supernatant, the test tube that reverses is placed on the filter paper blots, and bounces separation vessel to remove wall built-up liquid, repeats 3 times.Each pipe adds chemical luminous substrate 200～400 μ L, and fully mixing places in the magnetic separator, treat that magnetic particle is enriched in the bottom after, 10min is placed in the dark place, then measures the luminous intensity (RLU) of each pipe on tubular type chemiluminescence measuring instrument in regular turn.Log value with calibration object concentration is a horizontal ordinate, the Log value of RLU is drawn typical curve (double logarithmic curve) for ordinate, on typical curve, find the concentration of the CA242 of this serum with each test serum RLU value, see accompanying drawing 1, wherein, I is a luminous intensity, and C is CA242 concentration (unit is U/mL), correlation coefficient r=0.9998, Y=4.6896+0.7283X.
The methodology calibrating of embodiment 3 kits of the present invention
According to manufacturing conventional in this area and vertification regulation the kit of preparation among the embodiment 1 is examined and determine, the result is as follows:
1, kit precision is measured
(1) calibration object precision experiment
The kit of preparation among the embodiment 1 is got three batches respectively carry out the precision experiment, every batch is extracted 10 kits.With the CA242 calibration object of the kit measurement 30U/mL that extracted among the embodiment 15 times.Calculate the coefficient of variation of measuring concentration.The measurement result of three batches of kits among the embodiment 1 is as shown in table 1, and the result shows that the coefficient of variation is between 3.2%～7.6%.
The repeatable experiment of table 1 CA242 calibration
(2) the repeatable experiment of sample
Get two portions of normal human serums, add the CA242 calibration object respectively to 30U/mL and 100U/mL.Get each 3 of the kits of three different batches among the embodiment 1,, calculate the coefficient of variation respectively sample replication 5 times.Measurement result is shown in table 2, table 3, and the coefficient of variation that shows the CA242 magnetic microparticle chemiluminescence immune assay determination kit mensuration serum sample that the present invention is prepared is less than 6.1%.
The repeatable experiment of table 2 30U/mL CA242 blood serum sample
The repeatable experiment of table 3 100U/mL CA242 blood serum sample
2, kit accuracy determination
Get two clinical definite value serum of CA242, concentration is respectively 34.8U/mL and 25.6U/mL, respectively to calibration object solution 10U/mL, the 30U/mL and the 100U/mL that wherein add CA242, according to the method for embodiment 1 to each concentration do 3 parallel, calculate recovery rate.The result is as shown in table 4, and the interpolation recovery that shows CA242 is between 85.3%～98.7%.
The recovery of the kit of table 4 embodiment 1
3, kit stability experiment
After the kit of embodiment 1 carried out 37 ℃ of 7 days accelerated tests, measure the recovery of standard addition of the maximum of kit, minimum luminous intensity, determinand, the kit index that shows embodiment 1 is all within normal range.Kit key component (the streptavidin solution of the mixed liquor of antibody sandwich magnetic particle and biotin labeling antibody, horseradish peroxidase-labeled, calibration object, luminous substrate liquid and lavation buffer solution) to embodiment 1 is carried out 2～8 ℃ of 8 months tracking tests, and the result shows that every index meets clinical requirement fully.Consider the generation of the freezing situation of kit, with the kit of embodiment 1 put into-20 ℃ freezing 7 days, measurement result shows that also every index of kit is normal fully.Kit can be preserved more than 6 months at least at 2～8 ℃ as can be seen from the above results.
As follows through a large amount of methodology indexs that experiment showed, kit of the present invention:
Sensing range: 0～700U/mL;
Sensitivity: minimum detects and is limited to 0.5U/mL;
Precision: less than 10% (n=30);
Accuracy: average recovery rate is between 85.0%～100.0%;
Specificity: with the cross reacting rate of common tumor markers such as CA19-9, CEA, AFP, CA50, CA125, CA153 less than 0.1%;
Stability: each reagent set splits 37 ℃, investigates after 7 days, and each component is still stable.
Clinical detection scope, sensitivity, precision, accuracy, specificity and stability that " tumor associated antigen 242 magnetic microparticle chemiluminescence immune assay determination kits " are described are to meet clinical requirement fully.
To sum up, in research process of the present invention, the present inventor has at first carried out screening experiment and quality arbitration to used starting material, comprises the luminous intensity of activity, chemical luminous substrate of the absorption property of activity, magnetic particle of coated antibody and variation size, HRP and luminous duration etc.Then method for coating is studied, be cushioned liquid and confining liquid experimentizes, select optimal coating buffer and confining liquid, found best concentration conditions by the concentration experiment by the different bags of antibody with different bags.Mark for HRP can have diverse ways, by explore repeatedly and the contrast experiment finally found easy, productive rate is high, cost is low, the reliable quality labeling method, and different enzyme dilutions has been carried out long-term investigation tested, made and can make the activity stabilized dilution of the long-term maintenance of enzyme labeling thing.
The present inventor has also carried out experimental study to the reaction pattern and the reaction conditions of kit, finally determined double-antibody sandwich two-step approach reaction pattern, and the influence of experimental result is tested with regard to the different reaction time, determine the optimal reaction time.
Utilize the inventive method to detect, highly sensitive, high specificity, sensing range is wide, and is simple to operate, "dead" pollution, the kit cost is low, and clinical applicability is strong, more is applicable to China's clinical detection and examination laboratory, can diagnose malignant tumor of digestive tract (as cancer of pancreas, colorectal cancer etc.) effectively, and can be used for the observation of curative effect after malignant tumor of digestive tract operation and the chemotherapy.
Embodiment 4 kits of the present invention are with the clinical blood sample measured value comparison of external kit
Detect simultaneously with the CA242 ELISA kit of kit of the present invention and the CanAg company clinical blood serum sample to 80 parts of tumour patients, 80 parts of clinical blood samples that adopted are from the attached Friendship Hospital in the attached Tiantan Hospital of the Capital University of Medical Sciences and the Capital University of Medical Sciences.Its testing result is seen accompanying drawing 2, and the blood sample CA242 result who measures with the inventive method is a horizontal ordinate, and the result who measures with CanAg is that ordinate is done regretional analysis, and dependent equation is: Y=1.0951+0.9520X, correlation coefficient r=0.9872.Learn result by statistics and show, the inventive method is good with the external clinical blood sample measured value of kit correlativity.
Claims (10)
1, a kind of tumor associated antigen 242 magnetic microparticle chemiluminescence immune assay determination kits is characterized in that described kit comprises:
1) tumor associated antigen 242 calibration objects;
2) mixed liquor of the magnetic particle of tumor associated antigen 242 monoclonal antibody bag quilts and biotin labeled tumor associated antigen 242 monoclonal antibodies;
3) streptavidin of horseradish peroxidase-labeled;
4) chemical luminous substrate luminol that above-mentioned horseradish peroxidase acted on or different luminol; And
5) reaction tube.
2, kit as claimed in claim 1 is characterized in that, the magnetic particle of described tumor associated antigen 242 monoclonal antibody bag quilts and the blending ratio of biotin labeled tumor associated antigen 242 monoclonal antibodies are 1:2.
3, kit as claimed in claim 1 is characterized in that, described chemical luminous substrate comprises A liquid and B liquid, wherein,
A liquid is that 0.1M pH value is 8.5 Tris-HCl damping fluid, comprise the luminol of 4.0mg/mL or different luminol and 0.3mg/mL to iodophenol;
B liquid is that 0.1M pH value is 4.6 citrate buffer solution, comprises the superoxol of 200mg/mL.
4, kit as claimed in claim 1 is characterized in that, the reaction tube material that described kit uses is transparent polystyrene, tygon, polypropylene or glass.
5, a kind of method for preparing the described kit of claim 1 is characterized in that may further comprise the steps:
1) with tumor associated antigen 242 pure product preparation calibration object;
2) preparation tumor associated antigen 242 monoclonal antibody bags are by the mixed liquor of magnetic particle and biotin labeling tumor associated antigen 242 monoclonal antibodies;
3) with the horseradish peroxidase-labeled streptavidin;
4) preparation chemical luminous substrate luminol or different luminol that horseradish peroxidase acted on;
5) mixed liquor of the above-mentioned calibration object of packing, antibody sandwich magnetic particle and biotin labeling antibody, enzyme labeling thing, chemical luminous substrate luminol or different luminol and reaction tube; And
6) be assembled into finished product.
6, method as claimed in claim 5 is characterized in that, in the step 2 of the mixed liquor of described preparation antibody sandwich magnetic particle and biotin labeling antibody) in:
Described mixed liquor is to be the 1:2 mixing by the magnetic particle of tumor associated antigen 242 monoclonal antibody bag quilts and biotin labeled tumor associated antigen 242 monoclonal antibodies with the volume ratio, and is formulated with 20～40% calf serum;
Described magnetic particle is 1～2 μ m particle diameter, tri-iron tetroxide kernel, the surperficial polymkeric substance that has reactive group that wraps up, and its working concentration is 5～10mg/mL;
Described antibody sandwich magnetic particle be by the glutaraldehyde two-step approach with tumor associated antigen 242 monoclonal antibody bags by on magnetic particle;
With the magnetic particle of confining liquid sealing bag quilt, described confining liquid is that to contain 0.2%～1.0% bovine serum albumin(BSA), pH value be 7.2 0.02mol/L phosphate buffer;
Described biotin labeling tumor associated antigen 242 monoclonal antibodies realize with antibody free lysine generation coupling reaction under the alkalescence condition by the biotin succinimide ester.
7, method as claimed in claim 6 is characterized in that, described reactive group is amino or carboxyl.
8, method as claimed in claim 5 is characterized in that, in described step 3) with the horseradish peroxidase-labeled streptavidin, adopts improvement sodium periodate method with the horseradish peroxidase-labeled streptavidin.
9, method as claimed in claim 5 is characterized in that, described chemical luminous substrate comprises A liquid and B liquid, wherein,
A liquid is that 0.1M pH value is 8.5 Tris-HCl damping fluid, comprise the luminol of 4.0mg/mL or different luminol and 0.3mg/mL to iodophenol;
B liquid is that 0.1M pH value is 4.6 citrate buffer solution, comprises the superoxol of 200mg/mL.
10, method as claimed in claim 5 is characterized in that, uses transparent polystyrene, tygon, polypropylene or glass as the reaction tube material.
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| CNA2007101224565A CN101398429A (en) | 2007-09-26 | 2007-09-26 | Tumor-associated antigen 242 magnetic microparticle chemiluminescence immune assay determination kit and method for making same |
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2007101224565A CN101398429A (en) | 2007-09-26 | 2007-09-26 | Tumor-associated antigen 242 magnetic microparticle chemiluminescence immune assay determination kit and method for making same |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102435739A (en) * | 2011-08-31 | 2012-05-02 | 内蒙古科慧生物科技有限责任公司 | Carbohydrate antigen 242(CA242) quantitative determination kit and detection method thereof |
| CN103175954A (en) * | 2013-03-08 | 2013-06-26 | 北京海瑞祥天生物科技有限公司 | Human IgE antibody detection kit, and making method and detection method thereof |
| WO2022121414A1 (en) * | 2020-12-08 | 2022-06-16 | 东莞市朋志生物科技有限公司 | Anti-ca24-2 antibody, reagent and kit for detecting ca24-2 |
| CN115372609A (en) * | 2022-08-24 | 2022-11-22 | 四川沃文特生物技术有限公司 | Kit for determining carbohydrate antigen 242 and application thereof |
-
2007
- 2007-09-26 CN CNA2007101224565A patent/CN101398429A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102435739A (en) * | 2011-08-31 | 2012-05-02 | 内蒙古科慧生物科技有限责任公司 | Carbohydrate antigen 242(CA242) quantitative determination kit and detection method thereof |
| CN103175954A (en) * | 2013-03-08 | 2013-06-26 | 北京海瑞祥天生物科技有限公司 | Human IgE antibody detection kit, and making method and detection method thereof |
| WO2022121414A1 (en) * | 2020-12-08 | 2022-06-16 | 东莞市朋志生物科技有限公司 | Anti-ca24-2 antibody, reagent and kit for detecting ca24-2 |
| CN115372609A (en) * | 2022-08-24 | 2022-11-22 | 四川沃文特生物技术有限公司 | Kit for determining carbohydrate antigen 242 and application thereof |
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