CN103789271A - Hybridoma for resisting generation of CA242 monoclonal antibody and preparation of chemiluminescence immune assay kit - Google Patents
Hybridoma for resisting generation of CA242 monoclonal antibody and preparation of chemiluminescence immune assay kit Download PDFInfo
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- CN103789271A CN103789271A CN201410035829.5A CN201410035829A CN103789271A CN 103789271 A CN103789271 A CN 103789271A CN 201410035829 A CN201410035829 A CN 201410035829A CN 103789271 A CN103789271 A CN 103789271A
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Abstract
The invention discloses a hybridoma for resisting generation of CA242 monoclonal antibody and preparation of a chemiluminescence immune assay kit and belongs to the field of immunoassay medicine. The invention provides a monoclonal antibody for specifically identifying the CA242, a hybridoma for generating the monoclonal antibody, and a high-sensitivity method for detecting the CA242 by using the monoclonal antibody. The invention further provides a CA242 chemiluminescence immune assay kit and a preparation method thereof. The chemiluminescence immune assay kit comprises a CA242 detection reaction plate, an enzyme conjugate, a luminescent substrate, a standard substance, a quality control substance and a concentrated cleaning solution. According to the preparation, the antibody with high sensitivity and good specificity is developed by using a cellular fusion and hybridoma technology; meanwhile, immunology and chemiluminescence are combined by using an independently researched and developed antibody so that the kit with the advantages of wide detection range, high sensitivity, convenience in operation, and low production cost is researched and developed. The kit is used for auxiliary diagnosis of tumors and dynamic monitoring of definite malignant tumor patients so that the course of the disease is judged in an assisting manner, and the like through in vitro measuring the concentration of the CA242 in serum.
Description
[technical field]
The present invention relates to the hybridoma that CA242 monoclonal antibody produces and the test kit that detects CA242 content in serum, can be widely used in medical science and and technological field of biochemistry.
[background knowledge]
CA242 antigen is a kind of new carbohydrate tumor markers, and in sera of patients with malignant tumors, CA242 has higher positive rate.In normal human's bile duct cell, pancreatic ductal cell, contain a small amount of CA242.But CA242 is mainly present in the malignant cell of pancreas and colon, pancreatic tumor cell CA242 immunofluorescence is obviously better than contiguous Normal Pancreas cell.CA242 is a kind of valuable new tumor markers to carcinoma of the pancreas and the rectum cancer.But CA242 is lower to the diagnosis of liver cancer and cancer of the stomach.So far do not have a kind of mark can be completely special to certain tumour yet, so only lower by the accuracy rate of a certain individual event tumor markers detection diagnosing tumour, can not be widely used in the early detection of tumour, but there are some researches show, the joint-detection that adopts Diagnostic Value of Several Serum Tumor Markers, can make up above-mentioned deficiency.As combined utilization such as other tumor markerses CEA, CA199, CA50, CA125, contribute to improve positive diagnosing rate, reduce clinical rate of missed diagnosis.
Clinical study shows, in carcinoma of the pancreas and advanced colorectal cancer patients serum, CA242 generally has significant rising, and for colorectal cancer, CA242 demonstrates than CA50 and CA199 has higher susceptibility; In the comparative study of CA50, show all there is higher susceptibility for the detection of the each phase tumour of Dukes, and significantly increases with the progress of clinical disease period.With the CA242 in the CA242 monoclonal antibody detection serum of high-affinity, its principle based on C242 can with the antigens c A242 specific binding on Saliva Orthana surface, after mark, utilize immunoserology method to detect, this detects CA199 than in the past and CA50 specificity is higher.
CA242 is as the carcinoma of the pancreas tumor markers relevant with colorectal cancer, and the effect aspect clinical diagnosis, monitoring and operation prognosis is very outstanding.Its detection method is simple and feasible, therefore has clinically good using value.Meanwhile, CA242 can be used as a kind of diagnosis, guiding treatment and judging prognosis of tumor markers assistance Several Kinds of Malignancy.
[summary of the invention]
The object of the present invention is to provide the hybridoma that a kind of anti-CA242 monoclonal antibody produces and set up the method for the simple and highly sensitive detection of tool CA242 a kind of, and by the early screening of the method application carcinoma of the pancreas and colorectal cancer.Detect the insufficient sensitivity of CA242 or expensive to solve existing monoclonal antibody, be not suitable for the shortcoming of clinical large-scale application.
Another object of the present invention is to provide the preparation method of a kind of CA242 chemical luminescence immune assay determination reagent kit and this test kit.Utilize that this test kit analyzing and testing is highly sensitive, high specificity.
The present invention has obtained hybridoma cell strain 4-1 and the hybridoma cell strain 4-5 of the monoclonal antibody (mAb) that can produce specific recognition CA242, this 2 strain cell strain is preserved in respectively Chinese Typical Representative culture collection center (CCTCC) on January 19th, 2014, preservation address is, China. Wuhan. Wuhan University, deposit number is CCTCC NO:C201419 and CCTCC NO:C201420.In addition, through identifying, two strain monoclonal antibodies are identified respectively two different epi-positions of CA242, have set up DASELISA immune response method by the combination of 4-1 and 4-5, and it is a kind of highly sensitive and high-throughout detection system.
Therefore, the invention provides 1 to 7 content described below:
1. the hybridoma cell strain of anti-CA242, its preserving number is respectively CCTCC NO:C201419 and CCTCC NO:C201420.
2. the monoclonal antibody of anti-CA242, is that the hybridoma cell line of CCTCC NO:C201419 and CCTCC NO:C201420 is secreted by preserving number respectively, described monoclonal antibody called after 4-1 and 4-5.
3. the application of anti-CA242 monoclonal antibody as claimed in claim 2, utilize the DAS-ELISA of described monoclonal antibody to detect CA242, antibody 4-1 and preserving number that the antibody sources that sandwich assay matches is between two the secretion of CCTCC NO:C201419 hybridoma in preserving number are the antibody 4-5 of CCTCC NO:C201420 hybridoma secretion, and its step comprises:
(1) coated with the monoclonal antibody 4-1 of described pairing between two;
(2) add testing sample to hatch;
(3) using another strain monoclonal antibody 4-5 of the described HRP mark of pairing between two as two anti-, add reaction system;
(4) after washing, add enzyme reaction substrate, read OD value with 450nm;
(5) result shows that the sensitivity detecting is very high.
4. a test kit that detects CA242 content in serum, comprising: the opaque polystyrene board that is coated with anti-CA242 monoclonal antibody 4-1; CA242 antigen series standard product; The CA242 monoclonal antibody 4-5 of enzyme labelling; The chemical luminous substrate A liquid of above-mentioned enzyme effect and B liquid and washings.
5. test kit as claimed in claim 4, is characterized in that: opaque polystyrene board adopts direct physical absorption method Sheet clonal antibody 4-1, and coating buffer is phosphate buffered saline buffer; Another strain CA242 monoclonal antibody 4-5 and horseradish peroxidase coupling form enzymic-labelled antibody, employing be that the sodium periodate method of improvement is carried out mark; CA242 antigen series standard product, take calf serum as matrix, add the configuration of CA242 antigen sterling to form; Luminous substrate A, B are HRP-Luminol luminescence system.
6. test kit as claimed in claim 4, is characterized in that: traget antibody marker enzyme used is horseradish peroxidase, use be sodium periodate oxidation, the working concentration of gained enzymic-labelled antibody the best is 1:3000; What be coated with that the opaque polystyrene board of anti-CA242 antibody adopts is that direct physical absorption method is coated; Luminescent solution A liquid borax 7.99g, boric acid 3.46g, luminol,3-aminophthalic acid cyclic hydrazide 0.28g, to iodophenol 0.07g, adds process water and is settled to 700mL, keeps in Dark Place; Luminous substrate B liquid is made up of following ingredients: borax 7.99g, and boric acid 3.46g, urea peroxide 0.07g adds process water and is settled to 700mL.
7. the preparation method of test kit as described in claim 4-6, comprises the following steps:
(1) preparation of standard substance and the correction of concentration
By CA242 antigen sterling, add standard substance diluent, preparation one high density standard substance dope, adopts and is diluted to each concentration by the method for low dilution, is mixed with 0,10,25,50,100, the series standard product of 200U/mL, and standard substance are proofreaied and correct with national standard.
(2) coated
Adopt 0.5mol/L, it is 3 μ g/mL coating buffers that the carbonate buffer solution that pH value is 9.5 and CA242 monoclonal antibody are mixed into antibody concentration, is coated in opaque polystyrene board 4 ℃ of overnight incubation with 100 μ L/ holes;
(3) wash plate
Wash plate hole 2 times by PBS-T washing lotion;
(4) sealing
Confining liquid comprises NaCl8.00g, KCl0.20g, Na
2hPO
4.12H
2o2.90g, KH
2pO
40.20g, sucrose 20.00g, proclin3001.00mL, BSA20.00g, the pH value of confining liquid is 7.3-7.5,150 μ L/ hole sealings, wet box is placed and is hatched 3h;
(5) dry
Remove confining liquid, dried overnight.
(6) be assembled into finished product
Polystyrene plank is put into aluminium foil bag and put into siccative, and sealing is preserved.
The monoclonal antibody of above-mentioned anti-CA242, is prepared by following method, and step comprises:
(1) the CA242 antigen that is 2.5KU with protein content mixes with Freund's complete adjuvant; After 15 days, carry out same dose and Freund's incomplete adjuvant mixed immunity for the second time; After 15 days, carry out again same dose and Freund's complete adjuvant mixed immunity for the third time; After 10 days, afterbody is got blood indirect ELISA method and is surveyed serum titer, does not add adjuvant booster immunization with the pure antigen of same dose; After 3 days, extracting spleen cell merges;
(2) by immune small white mouse splenocyte and small white mouse myeloma cell (SP2/0) in the ratio of 5:1-10:1, merge as fusogen with 50%PEG;
(3) with containing HAT(xanthoglobulin, aminopterin, thymus pyrimidine) serum free medium, at 37 ℃, 5%CO
2cell culture incubator in cultivate 10 days, conventional indirect ELISA method screens positive hole;
(4) the positive hole of the high specificity limiting dilution assay filtering out is cloned, and obtains the further enlarged culturing of monoclonal antibody cell strain;
(5) collect culture supernatant, affinity chromatography monoclonal antibody purification, is anti-CA242 monoclonal antibody;
(6) detect tiring of monoclonal antibody, choose the high strain of tiring and carry out HRP mark, and the good monoclonal antibody of mark is carried out to titer determination;
(7) monoclonal antibody that mark is good and other unlabelled monoclonal antibodies ELISA that is at war with reacts, and chooses and does not have a competitive strain to carry out sandwich ELISA reaction, determines top condition.
The advantages such as monoclonal antibody of the present invention can be directly used in the CA242 content detecting in sample, by a large amount of cultivation monoclonal antibody cell strains, can obtain a large amount of monoclonal antibodies, compared with polyclonal antibody, has purity high, and specificity is strong, reproducible.
The present invention is directed to clinical labororatory set up one both can manual operations, again can be for the detection means of the full-automatic detecting instrument of standard, set up the quantitative detecting method that detects human serum CA242.CA242 immue quantitative detection reagent box of the present invention (chemoluminescence method) can be single-minded the content that detects the CA242 in human serum, thereby according to how many judgement carcinoma of the pancreas of its content or variation and the result for the treatment of of the colorectal cancer state of an illness.It has advantages of easy, quick, sensitive, stable, test kit of the present invention, CA242 in coated antibody 4-1 and the antibody 4-5 of enzyme labelling and sample forms the sandwich complex structure of " coated antibody-Ag-Ab-enzyme ", so the present invention's employing is the reaction pattern of " double antibodies sandwich single stage method ".Utilize horseradish peroxidase catalytic luminescence substrate, luminous substrate generation chemical reaction discharges a large amount of energy, produce the intermediate of excited state, in the time that it gets back to stable ground state, can launch photon, utilize illumination instrument to measure the yield of photon, the amount of the detection material in yield and the sample of this photon is directly proportional, thus Criterion curve calculate the content of test substance in sample.Kit test method has highly sensitive, and sensing range is wide, easy to operate, to experimenter without injury, simultaneously production cost is low, has alleviated the burden of doctor and patient, is therefore more conducive to the early screening of clinical carcinoma of the pancreas and colorectal cancer.
[accompanying drawing explanation]
What Fig. 1 showed is anti-CA242 monoclonal antibody titration result in ELISA method that the hybridoma in the present invention produces;
What Fig. 2 showed is the anti-CA242 monoclonal antibody titre measuring result of mark HRP;
What Fig. 3 showed is the detection sensitivity of sandwich method ELISA system;
What Fig. 4 showed is the standard substance linear graph of prepared test kit.
[embodiment]
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.In addition, should be understood that after having set forth content of the present invention, those skilled in the art can do various changes and modification to the present invention, and these equivalent form of values are the scope defined in claims in the application equally.
Embodiment 1: animal immune
Select with 8 weeks age of myeloma cell's homology used about male Balb/C healthy mice, antigen is that protein content is that the CA242 antigen of 15KU mixes with Freund's complete adjuvant, PBS, after complete emulsification, takes back multiple spot, and oxter, inguinal region immunity.Immune programme for children: carried out same dose and Freund's incomplete adjuvant mixed immunity for the second time after 15 days; After 15 days, carry out again same dose and Freund's complete adjuvant mixed immunity for the third time; After 10 days, afterbody is got blood indirect ELISA method and is surveyed serum titer, does not add adjuvant booster immunization with the pure antigen of same dose; After 3 days, extracting spleen cell merges.
Embodiment 2: the structure of hybridoma
1, the cultivation of myeloma cell strain and preparation
(1) that the present invention adopts is SP2/0 myeloma cell, and this cell strain growth and fusion efficiencies are all good, and the doubling time is 10-12h.When fusion, select in logarithmic phase, cellular form and active good cell.Myeloma cell should first do to adapt to cultivate before fusion on substratum, makes Growth of Cells to best state (being logarithmic phase);
(2) SP2/0 of cultivation is drawn in the pipe of 50mL, centrifugal, abandon supernatant, hang, add the substratum of 10mL, draw after a small amount of 10 times of dilutions counting.
2, the preparation of splenocyte
(1) mouse is placed in sealing bag, fills CO
2make its death by suffocation;
(2) mouse sterilization is fixed on and is dissected on plate, in Bechtop, get spleen, be placed in the culture dish of 12mL substratum, peel adhesion organization off, grind spleen, until surplus white tissue, suction pipe all picks up, more slowly gets on the tube wall that makes tissue block adhesion, centrifugal, abandon supernatant, add the erythrocyte cracked liquid cracking 10min of 10mL, then the substratum that adds 20-25mL stops its reaction, after centrifugal, abandon supernatant, add the substratum of 10mL, draw after a small amount of 10 times of dilutions counting.
3, cytogamy
Cytogamy is the key link of hybridoma technology, basic step be get in the Sp2/0 of logarithmic phase cell and splenocyte 1:10 mixed, by polyoxyethylene glycol (PEG) method to obtain hybridoma, called after 4-1 and 4-5.The hybridoma obtaining is suspended in the HAT substratum that contains feeder cell, then joins in 96 orifice plates, and at 37 ℃, 5%CO
2incubator in sealing cultivate 12 days.
Embodiment 3: the preparation of monoclonal antibody and screening
1, the preparation of monoclonal antibody
The Kong Gezhong of the hybridoma obtaining from embodiment 2 reclaims the supernatant liquor of substratum, is chosen at the monoclonal antibody of reacting with antigenic peptide in ELISA method.
2, the screening of monoclonal antibody
(1) CA242 that is 10U/ml by 100uL concentration is to each Kong Gezhong of 96 orifice plates, makes it be fixed on solid phase in 4 ℃ after spending the night;
(2) seal 2h with the bovine serum albumin that 150uL concentration is 1%;
(3) medium supernatant of 100uL hybridoma is joined to each Kong Gezhong, in 37 ℃ of reaction 2h, then add the sheep anti-mouse antibody of the horseradish peroxidase of 10000 times of dilutions to react 1h in 37 ℃;
(4) use tetramethyl benzidine micropore peroxidase substrate (TMB) as the substrate 20min that develops the color;
(5) add after the sulfuric acid termination reaction that 50uL concentration is 0.2N, measure the absorbancy of 450nm;
(6) select absorbancy and be approximately 3 4-1 and 4-5, and carry out subclone by limiting dilution assay.
3, a large amount of preparations of monoclonal antibody and and purifying
Cell after subclone is carried out to enlarged culturing with cell-culturing rotating bottle, after approximately 20 days, collect supernatant, carry out affinitive layer purification with staphylococcal protein A,SPA (Protein A).The monoclonal antibody obtaining is called after 4-1 and 4-5 respectively.
4, the mensuration of antibody titer
Tiring of the 2 strain monoclonal antibodies that filter out measured by ELISA method.Add respectively 4-1,4-5(10ug/mL), after reaction, use anti-mouse antibodies and the TMB of horseradish peroxidase to develop the color, result is as shown in Figure 1 more than the tiring and reach 10-9 of two strain monoclonal antibodies.
Embodiment 4: the mark of monoclonal antibody and the mensuration of titre
The antibody being purified into is carried out to HRP mark according to a conventional method, and the titre of the monoclonal antibody that mark is good is measured by method below, and the CA242 that is 10U/mL by concentration is fixed on (100uL/ hole) on 96 hole microplates.The bovine serum albumin of use 1% seals 2h, and tagged monoclonal antibody (the first 100 times, hole dilution) since the dilution of 4 times, the second hole, is reacted 1h under room temperature.Add after TMB, 20min is at room temperature carried out in reaction, by the sulfuric acid stopped reaction of 0.2N.Measure the absorbancy at 450nm, the titre result of the monoclonal antibody of HRP mark as shown in Figure 2.
Embodiment 5: double-antibody sandwich elisa detects the foundation of CA242 method
(1) coated with the monoclonal antibody 4-1 of described pairing between two, the monoclonal antibody of 3ug/ml is added on microwell plate with 100uL/ hole, hatches 24h be fixed on solid phase in 4 ℃;
(2) microwell plate uses the 20mM PBS that the pH value that contains 0.1%Tween-20 is 7.4 to wash 2 times with the amount in 200uL/ hole.Add 1% bovine serum albumin to seal 2h with the amount in 150uL/ hole.
(3) microwell plate is washed 4 times with the amount in 200uL/ hole with PBST, adds continuous 4 times of dilution CA242 by initial concentration 10ug/ml, in room temperature incubation 2h, then adds another strain antibody 4-5(1:3000 of mark HRP; 100uL/ hole) and in room temperature incubation 2h.
(4) add after TMB, 20min is at room temperature carried out in reaction, adds the sulfuric acid stopped reaction of 0.2N and measures the absorbancy of 450nm.
(5) detected result is shown in shown in accompanying drawing 3.
Embodiment 6: prepare CA242 quantitative determination reagent kit of the present invention (chemoluminescence method)
(1) sodium periodate oxidation mark horseradish peroxidase
(1) oxidation of enzyme (whole process lucifuge)
Take 3-5mg HRP and be dissolved in 600 μ L ddH
2o
2in;
B, add freshly prepared 150uL0.1M sodium periodate (NaIO
4) (MW:213.89g/mol gets 0.22g and is dissolved in 10mL10mM pH7.0 sodium phosphate buffer);
C, mix, room temperature, lucifuge is hatched 20min;
D, solution is dialysed with dialysis tubing, 3000rpm/min, 4 ℃, 20min, solution is 1.0mM pH4.0 sodium-acetate buffer, changes 3-4 time;
E, finally dialyse to 800 μ L, to EP pipe;
(2) preparation of antibody and mark (lucifuge)
A, get ready 3mg monoclonal antibody, be condensed into 500 μ L-1000 μ L volumes with centrifuge tube, sucking-off is in new 15mL centrifuge tube;
B, add 500 μ L0.2M pH9.5 carbonate buffer solutions, mix, detect pH value 9.0～9.5;
C, immediately the HRP solution of having dialysed is mixed with monoclonal antibody solution, room temperature is rocked 2h, lucifuge;
D, add the freshly prepared NaBH4(4.0mg/mL of 80 μ L, get 40mg and be dissolved in 10mL ddH
2o
2in);
E, mix (lucifuge), hatch 1.5h for 4 ℃;
F, with PBS dialyse to volume be 2mL;
G, add equal-volume glycerine, 1mL/ pipe ,-20 ℃ of preservations
(2) preparation of enzyme labelled antibody working fluid
(1) preparation of enzyme mark diluent
Accurately take Tutofusin tris 7.27g according to standard recipe, add process water 800.0mL, after stirring makes fully to dissolve, add concentrated hydrochloric acid appropriate, stir, the pH value that uses digital display acidometer to measure liquid is 7.1～7.3, again load weighted other recipe ingredients are added in above-mentioned solution successively, fully after stirring and dissolving, operation water is settled to 1200.0mL, carry out Sterile Filtration by " 316L SGP150K type stainless steel cymbals formula liquid precise filter criteria working specification ", filter 2～8 ℃ of rear liquid and save backup.
(2) preparation of enzyme labelled antibody working fluid
Through evidence, the best effort concentration of enzyme labelled antibody is 1:3000, uses (1) described diluent that enzyme labelled antibody is diluted to needed working concentration.
The enzyme conjugates damping fluid being up to the standards adds CA242-HRP according to the Dilution ratio of antibody used, and after stirring clockwise 30min and fully mixing, 2～8 ℃ save backup, when packing by 96 person-portions: 11.0mL/ bottle, validity period 18 months.
(3) preparation of CA242 standard substance
Accurately take above-mentioned each component according to standard recipe content, add process water 800.0mL to stir after fully dissolving, the pH value that uses digital display acidometer to measure liquid should be 7.1～7.3, is settled to 1000.0mL with process water, mixes, by CA242 antigen sterling, add standard substance diluent, preparation one high density standard substance dope, adopts and is diluted to each concentration by the method for low dilution, be mixed with 0,10,25,50,100, the series standard product of 200U/mL, 2～8 ℃ save backup.
Be up to the standards the packing of CA242 standard substance time, 0.3mL/ bottle, validity period 18 months.
(4) the coated polystyrene board of CA242 monoclonal antibody
(1) by 0.05mol/L, the dipotassium hydrogen phosphate solution of pH9.6 and CA242 monoclonal antibody 4-1 are mixed and made into coating buffer, are coated in polystyrene board, place in wet box, spend the night coated;
(2) washing: wash plate hole 2 times by PBS-Tween washing lotion;
(3) sealing:
The preparation of confining liquid:
Accurately weigh NaCl8.00g, KCl0.20g, Na
2hPO
412H
2o2.90g, KH
2pO
40.20g, stirs, and the pH value that uses digital display acidometer to measure liquid is 7.3～7.5, load weighted other recipe ingredients are added in above-mentioned solution successively, fully, after stirring and dissolving, operation water is settled to 1000.00mL again, 2～8 ℃ save backup, validity period 15 days.150 μ L/ holes when use, wet box is hatched 2h, gets rid of confining liquid, on clean thieving paper, pats dry;
(4) dry: polystyrene board to be placed on to freeze-drying 2h on Freeze Drying Equipment, vacuum sealing bag, the rearmounted 2-8 ℃ of preservation of labeling.
(5) Chemoluminescent substrate
The compound method of the Chemoluminescent substrate of horseradish peroxidase used in the present invention (HRP) is:
The compound method of chemical luminous substrate A:
Accurately take above-mentioned each component, add purified water 600.0mL, stir fully and be settled to 700.0mL after dissolving, the pH that measures solution should be 9.1～9.4, and 2～8 ℃ keep in Dark Place for subsequent use.
By pressing after the assay was approved 96 person-portions: the packing of 6.0mL/ bottle, validity period 18 months.
The compound method of chemical luminous substrate B:
Accurately take above-mentioned each component, add purified water 600.00mL, stir fully and be settled to 700.00mL after dissolving, the pH that measures solution should be 9.1～9.4, and 2～8 ℃ save backup.
After the assay was approved by 96 person-portions: the packing of 6.0mL/ bottle, validity period 18 months.
Using method: before using, A liquid and B liquid are mixed and used in 1:1 ratio.
(6) 10 × washingss
Accurately take above-mentioned each component, add process water to be settled to 800.00mL, stir after fully dissolving, add process water and be settled to 1000.00mL, the pH that measures solution should be 7.1～7.4, and room temperature preservation is for subsequent use.
After the assay was approved according to 96 person-portions, the packing of 30.00mL/ bottle, validity period 18 months.
(7) composition of work in-process and finished product
The various enclosure group such as various work in-process compositions and product description of above-mentioned steps gained are dressed up CA242 quantitative determination reagent kit (chemoluminescence method).
Insolubilized antibody in test kit is to be coated with in advance, do not need on-the-spot coated, when easy to use and joint; Calibration object is liquid; Enzyme labelling thing is the liquid that has been diluted to working concentration, also can directly use.
Embodiment 7: CA242 quantitative determination reagent kit of the present invention (chemoluminescence method) working method is as follows:
(1) prepare
Will serum sample to be checked and detection kit return to room temperature (18-25 ℃) (approximately 15min), opaque polystyrene board in test kit of the present invention is fixed on CA242 antibody in plate hole, can directly use, need not be coated with now, very easy to use.
(2) working method:
1, the coated lath of requirement is placed on support;
2, in coated hole, add respectively standard substance and the blood sample of 10 μ L;
3, every hole adds the enzyme labelling thing of 100 μ L, and vibration mixes it a little; Put in wet box and hatch 1h;
4, discard liquid in hole, with the washings after dilution, automatic washer or manual wash plate 4 times finally buckle and is done on clean thieving paper;
5, luminous substrate A, B equal proportion are mixed to this volume used, every hole adds mixed luminous substrate 100 μ L, and vibration mixes it a little, lucifuge room temperature reaction 3min;
6, Chemiluminescence Apparatus detects relative light unit (RLU), and Measuring Time is 0.1-1 second/hole;
7,, respectively to standard substance concentration and relative light unit (RLU) value of taking the logarithm, Criterion curve (seeing accompanying drawing 4), finds the concentration value of CA242 in serum with the value of test serum RLU at typical curve, calculates detected result;
8, statistical study detected result.
With mentioned reagent box of the present invention according to above-mentioned steps measure the time used short, only need within more than one hour, just can all complete, fast and easy.
Methodology index when embodiment eight kit measurement of the present invention is as follows:
1, sensing range: 0-200U/mL;
2, sensitivity: minimum detection limit is 2U/mL;
3, precision: in analyzing, precision (detects basic, normal, high three groups of samples (n=10) and is all less than 15%, precision between analysis (detects basic, normal, high three groups of samples (n=10) and is all less than 15%, higher than national standard, illustrate that test kit of the present invention has good repeatability in detection test;
4, linearity: r >=0.99;
5, specificity: and the kinds of tumor mark no cross reaction such as CA153, CA125, CA724, CA50.Test kit of the present invention serum amount used is few, only needs 10 μ L, vitro detection to patient without any side effect; What the present invention simultaneously used is chemiluminescence immune analysis method, indices is also better than ELISA adsorption analysis method (ELISA), therefore for clinical detection CA242 provides, one is easier, method fast and accurately in the present invention, more can meet demand clinically.
The clinical blood sample measured value of embodiment nine test kit of the present invention
Take hospital clinical and collect 60 parts of patients serum samples, carry out clinical detection with test kit of the present invention, its measured value is as follows:
| Mark | Calculating concentration | Theoretical concentration | Luminous value |
| S0 | 0.00 | 0.00 | 7016 |
| S1 | 8.98 | 10.00 | 210657 |
| S2 | 24.11 | 25.00 | 499623 |
| S3 | 49.54 | 50.00 | 1062407 |
| S4 | 94.20 | 100.00 | 2022271 |
| S5 | 193.96 | 200.00 | 3477739 |
First to the standard substance concentration in upper table and relative light unit (RLU) value of taking the logarithm, Criterion curve, typical curve coefficient R value is 0.99, and in typical curve, find the concentration value of CA242 in serum with the value of test serum RLU, statistical study detected result, with the comparison of clinical detection value, coincidence rate reaches 99%.
In sum, test kit of the present invention is simple to operate, low price, to patient have no side effect, fast, high accuracy for examination, be more suitable for promoting the use of.
Claims (7)
1. the hybridoma cell strain of anti-CA242, its preserving number is respectively CCTCC NO:C201419 and CCTCC NO:C201420.
2. the monoclonal antibody of anti-CA242, is that the hybridoma cell line of CCTCC NO:C201419 and CCTCC NO:C201420 is secreted by preserving number respectively, described monoclonal antibody called after 4-1 and 4-5.
3. the application of anti-CA242 monoclonal antibody as claimed in claim 2, utilize the DAS-ELISA of described monoclonal antibody to detect CA242, antibody 4-1 and preserving number that the antibody sources that sandwich assay matches is between two the secretion of CCTCC NO:C201419 hybridoma in preserving number are the antibody 4-5 of CCTCC NO:C201420 hybridoma secretion, and its step comprises:
(1) coated with the monoclonal antibody 4-1 of described pairing between two;
(2) add testing sample to hatch;
(3) using another strain monoclonal antibody 4-5 of the described HRP mark of pairing between two as two anti-, add reaction system;
(4) after washing, add enzyme reaction substrate, read OD value with 450nm;
(5) result shows that the sensitivity detecting is very high.
4. a test kit that detects CA242 content in serum, comprising: the opaque polystyrene board that is coated with anti-CA242 monoclonal antibody 4-1; CA242 antigen series standard product; The CA242 monoclonal antibody 4-5 of enzyme labelling; The chemical luminous substrate A liquid of above-mentioned enzyme effect and B liquid and washings.
5. test kit as claimed in claim 4, is characterized in that: opaque polystyrene board adopts direct physical absorption method Sheet clonal antibody 4-1, and coating buffer is phosphate buffered saline buffer; Another strain CA242 monoclonal antibody 4-5 and horseradish peroxidase coupling form enzymic-labelled antibody, employing be that the sodium periodate method of improvement is carried out mark; CA242 antigen series standard product, take calf serum as matrix, add the configuration of CA242 antigen sterling to form; Luminous substrate A, B are HRP-Luminol luminescence system.
6. test kit as claimed in claim 4, is characterized in that: traget antibody marker enzyme used is horseradish peroxidase, use be sodium periodate oxidation, the working concentration of gained enzymic-labelled antibody the best is 1:3000; What be coated with that the opaque polystyrene board of anti-CA242 antibody adopts is that direct physical absorption method is coated; Luminescent solution A liquid borax 7.99g, boric acid 3.46g, luminol,3-aminophthalic acid cyclic hydrazide 0.28g, to iodophenol 0.07g, adds process water and is settled to 700mL, keeps in Dark Place; Luminous substrate B liquid is made up of following ingredients: borax 7.99g, and boric acid 3.46g, urea peroxide 0.07g adds process water and is settled to 700mL.
7. the preparation method of test kit as described in claim 4-6, comprises the following steps:
(1) preparation of standard substance and the correction of concentration
By CA242 antigen sterling, add standard substance diluent, preparation one high density standard substance dope, adopts and is diluted to each concentration by the method for low dilution, is mixed with 0,10,25,50,100, the series standard product of 200U/mL, and standard substance are proofreaied and correct with national standard.
(2) coated
Adopt 0.5mol/L, it is 3 μ g/mL coating buffers that the phosphate buffered saline buffer that pH value is 7.4 and CA242 monoclonal antibody are mixed into antibody concentration, is coated in opaque polystyrene board 4 ℃ of overnight incubation with 100 μ L/ holes;
(3) wash plate
Wash plate hole 2 times by PBS-T washing lotion;
(4) sealing
Confining liquid comprises NaCl8.00g, KCl0.20g, Na
2hPO
4.12H
2o2.90g, KH
2pO
40.20g, sucrose 20.00g, proclin3001.00mL, BSA20.00g, the pH value of confining liquid is 7.3-7.5,150 μ L/ hole sealings, wet box is placed and is hatched 3h;
(5) dry
Remove confining liquid, dried overnight.
(6) be assembled into finished product
Polystyrene plank is put into aluminium foil bag and put into siccative, and sealing is preserved.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108196054A (en) * | 2017-07-27 | 2018-06-22 | 数字本草中医药检测有限公司 | A kind of test strips for detecting glycyrrhizic acid and its preparation method and application |
| CN113156137A (en) * | 2021-02-19 | 2021-07-23 | 山东省大健康精准医疗产业技术研究院 | Chemiluminescence immunoassay kit for detecting CD47, and preparation method and application 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 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101324579A (en) * | 2007-06-13 | 2008-12-17 | 清华大学 | Magnetic microparticle chemiluminescence enzyme immune analytic reagent kit for detecting saccharide antigen and its use method |
| CN102435739A (en) * | 2011-08-31 | 2012-05-02 | 内蒙古科慧生物科技有限责任公司 | Quantitative carbohydrate antigen 242 (CA242) determination kit and assay method thereof |
| CN102925413A (en) * | 2012-03-13 | 2013-02-13 | 大庆麦伯康生物技术有限公司 | Hybridoma producing anti-t-PSA monoclonal antibody, and preparation method of t-PSA detection kit |
| CN103149359A (en) * | 2012-09-20 | 2013-06-12 | 河南生生医疗器械有限公司 | Time resolution fluorescence method comprehensive detection pancreatic cancer kit and application thereof |
| CN203275412U (en) * | 2013-06-04 | 2013-11-06 | 苏州长光华医生物试剂有限公司 | Kit for chemical luminescence immunoassay of CA242 (tumor marker) |
-
2014
- 2014-01-26 CN CN201410035829.5A patent/CN103789271A/en not_active Withdrawn
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101324579A (en) * | 2007-06-13 | 2008-12-17 | 清华大学 | Magnetic microparticle chemiluminescence enzyme immune analytic reagent kit for detecting saccharide antigen and its use method |
| CN102435739A (en) * | 2011-08-31 | 2012-05-02 | 内蒙古科慧生物科技有限责任公司 | Quantitative carbohydrate antigen 242 (CA242) determination kit and assay method thereof |
| CN102925413A (en) * | 2012-03-13 | 2013-02-13 | 大庆麦伯康生物技术有限公司 | Hybridoma producing anti-t-PSA monoclonal antibody, and preparation method of t-PSA detection kit |
| CN103149359A (en) * | 2012-09-20 | 2013-06-12 | 河南生生医疗器械有限公司 | Time resolution fluorescence method comprehensive detection pancreatic cancer kit and application thereof |
| CN203275412U (en) * | 2013-06-04 | 2013-11-06 | 苏州长光华医生物试剂有限公司 | Kit for chemical luminescence immunoassay of CA242 (tumor marker) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108196054A (en) * | 2017-07-27 | 2018-06-22 | 数字本草中医药检测有限公司 | A kind of test strips for detecting glycyrrhizic acid and its preparation method and application |
| 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 |
| CN113156137A (en) * | 2021-02-19 | 2021-07-23 | 山东省大健康精准医疗产业技术研究院 | Chemiluminescence immunoassay kit for detecting CD47, and preparation method and application thereof |
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Application publication date: 20140514 |