CN102925413A - Hybridoma producing anti-t-PSA monoclonal antibody, and preparation method of t-PSA detection kit - Google Patents
Hybridoma producing anti-t-PSA monoclonal antibody, and preparation method of t-PSA detection kit Download PDFInfo
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Abstract
The invention provides a hybridoma producing anti-t-PSA monoclonal antibody and a preparation method of a t-PSA detection kit. The invention belongs to the field of immunoassay medical science. The invention provides a monoclonal antibody specifically identifying t-PSA, a hybridoma producing the monoclonal antibody, and a high-sensitivity method for detecting t-PSA by using the monoclonal antibody. The invention also provides a prostate-specific antigen t-PSA chemiluminescence immunoassay kit and a preparation method thereof. The kit comprises: a t-PSA detection reaction plate, an enzyme conjugate, a chemiluminescent substrate, a standard sample, a control, and a concentrated washing liquid. According to the invention, the antibody with high sensitivity and good specificity are developed through cell fusion and hybridoma technologies. Also, with the independently developed antibody, immunology and a chemiluminescent technology are combined, and the kit with wide detection range, high sensitivity, convenient operation, and low production cost is developed. The kit is used for clinically detecting t-PSA content in human serum, and provides important references for prostate cancer early screening, treatment program development, and prognosis evaluation.
Description
[technical field]
The present invention relates to the hybridoma that T-PSA (t-PSA) monoclonal antibody produces and the test kit that detects t-PSA content in the serum, can be widely used in medical science and and technological field of biochemistry.
[background knowledge]
Total prostate specific antigen (Prostate specific antigen, t-PSA) be a kind of antigen relevant with prostate cancer, a kind of strand glycoprotein that synthesizes and secrete by prostate epithelial cell, molecular weight 34KD, formed by 237 amino-acid residues, having serine protease, is preferably tumor marker of diagnosing prostate cancer, and its specificity reaches 85%-90%.Prostate cancer is the common cancer of the male sex more than 50 years old, and slower development increased with the age, and in China, along with the change of the serious and dietary structure of day by day the improving of resident living, environmental pollution, its sickness rate rises gradually; In the U.S., with the detection of the t-PSA generaI investigation index as the male sex.The detection of t-PSA is conducive to the early discovery of prostate cancer, the progress that monitors the state of an illness, result for the treatment of detection, and t-PSA plays an important role in the differential diagnosis of primary prostate cancer and Secondary cases prostate cancer simultaneously.
Prostate specific antigen is that prostatosis are worth very high tumor-marker, is widely used clinically, and the diagnosis that t-PSA mainly is used in prostate cancer reaches by stages, can also follow the trail of result for the treatment of.Prostate specific antigen is not that prostate cancer is peculiar, and many prostate hyperplasias or prostate gland inflammation also can make t-PSA rise, therefore, judge whether to suffer from very important of prostate cancer, need to do further inspection, such as doing the prostate gland section, to confirm to suffer from prostate cancer.Also do not find at present the prostate cancer methods for the treatment of in late period, reduce the mortality ratio of prostate cancer, have only rely on that early stage diagnosis is found and to suitable treatment, thereby reduce the mortality ratio of prostate cancer.
The method that purpose detects PSA in clinical labororatory mainly is radioimmunoassay (RIA), immunoturbidimetry and Enzyme Linked Immunoadsorbent Assay (ELISA), wherein RIA must use the radioelement mark, the test set complex and expensive, the transformation period of its radioelement is short, can not prolonged preservation, detected result is unstable, exists simultaneously radiocontamination also to bring injury to experiment operator; Enzyme immunoassay sensitivity is low, and influence factor is more, easily causes false negative and false positive.Chemiluminescence immune analysis method (CLIA) remolding sensitivity that the present invention adopts is higher, can reach 10-18mol/L, fast, luminous signal just can produce in several seconds, easy to operate, and do not use harmful reagent, compare with domestic similar reagent, it is very high to have sensitivity, easy and simple to handle, the more important thing is that its serum amount that needs is few, sensing range wide, the advantage of luminescent solution longer duration, more can satisfy clinical needs, reagent of the present invention is compared with external similar reagent, reaches the same kind of products at abroad level.
[summary of the invention]
The method that the object of the present invention is to provide a kind of anti-prostate specific antigen monoclonal antibody and set up a kind of simple and highly sensitive detection of tool t-PSA, and the method is applied to the detection of prostate cancer.Detect the insufficient sensitivity of t-PSA 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 prostate gland specificity antigen chemiluminescence immune analysis determination reagent kit and this test kit.Utilize highly sensitive, the high specificity of this test kit analyzing and testing.
The present invention has obtained to produce hybridoma cell strain 3-20-1 and the hybridoma cell strain 3-26-1 of the monoclonal antibody (mAb) of specific recognition t-PSA, this cell strain has been preserved in Chinese Typical Representative culture collection center (CCTCC) on March 6th, 2012, the preservation address is, China. Wuhan. Wuhan University, deposit number is CCTCC C201202 and CCTCC C201208.In addition, through identifying that two strain monoclonal antibodies are identified respectively two different epi-positions of t-PSA, have set up DASELISA immune response method by the combination of 3-20-1 and 3-26-1, it is a kind of highly sensitive and high-throughout detection system.
Therefore, the invention provides the content of (1) described below to (8):
1. the hybridoma cell strain of anti-prostate specific antigen, its preserving number is respectively CCTCC C201202 and CCTCCC201208.
2. the monoclonal antibody of anti-prostate specific antigen is secreted by preserving number CCTCC C201202 and CCTCCC201208 hybridoma cell line respectively, described monoclonal antibody called after 3-20-1 and 3-26-1.
3. the preparation method of hybridoma cell strain as claimed in claim 1, it is characterized in that: behind Balb/c mouse immune 3 times, 3 heaven-made last booster immunizations before cytogamy, adopt the ELISA method to divide 2 stepping row filter fused cells: the first step adopts indirect elisa method to filter out the positive hole of anti-prostate specific antigen PSA; Second step adopts the indirect competitive ELISA method that the positive hole nutrient solution that the first step filters out is detected, select absorbancy and the higher positive hole of sensitivity, adopt limiting dilution assay to clone, clone and adopted same two step screening method to detect in rear about 10 days, after so repeating 3 times, filter out at last 2 strain of hybridoma system.
4. the application of anti-prostate specific antigen monoclonal antibody as claimed in claim 2, namely utilize the DAS-ELISA of described monoclonal antibody to detect t-PSA, the antibody sources that sandwich assay matches in twos is in cell strain 3-20-1 and 3-26-1, and its step comprises:
(1) coated with one of described monoclonal antibody of matching in twos;
(2) adding testing sample hatches;
(3) anti-as two with another strain monoclonal antibody of the described in twos HRP mark of pairing, add reaction system;
(4) add enzyme reaction substrate after the washing, read the OD value with 450nm;
(5) result shows that the sensitivity of detection is very high.
5. a test kit that detects t-PSA content in the serum comprises: the opaque polystyrene board that is coated with anti-prostate specific antigen antibody; Prostate specific antigen series standard product; Another strain monoclonal antibody of the prostate specific antigen of enzyme labelling; The chemical luminous substrate A liquid of above-mentioned enzyme effect and B liquid and washings.
6, test kit as claimed in claim 5 is characterized in that: opaque polystyrene board adopts direct physical absorption method Sheet clonal antibody, and coating buffer is in the phosphate buffered saline buffer; Another strain prostate specific antigen monoclonal antibody and horseradish peroxidase coupling form enzymic-labelled antibody, employing be that the sodium periodate method of improvement is carried out mark; Prostate specific antigen series standard product add the configuration of prostate specific antigen sterling and form take calf serum as matrix; Luminous substrate A, B are the HRP-Luminol luminescence system.
7. test kit as claimed in claim 5, it is characterized in that: the used marker enzyme of traget antibody is horseradish peroxidase, usefulness be sodium periodate oxidation, the working concentration of gained enzymic-labelled antibody the best is 1: 2000; What be coated with that the opaque polystyrene board of anti-prostate antigen antibody adopts is that the 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 the technique water and is settled to 700mL, keeps in Dark Place; Luminous substrate B liquid is comprised of following ingredients: borax 7.99g, boric acid 3.46g, urea peroxide 0.07g add the technique water and are settled to 700mL.
8. such as the preparation method of test kit as described in the claim 5-7, may further comprise the steps:
(1) correction of the preparation of standard substance and concentration
With T-PSA t-PSA sterling, add the standard substance diluent, prepare a high density standard substance dope, adopt the method by low dilution to be diluted to each concentration, be mixed with 0,2.5,5,15,45, the series standard product of 100ng/mL, standard substance are proofreaied and correct with national standard.
(2) coated
Adopt 0.5mol/L, pH value be the prostate specific antigen monoclonal antibody of 9.5 carbonate buffer solution and proper concn to be mixed into antibody concentration be 1 μ g/mL coating buffer, be coated on the opaque polystyrene board 4 ℃ of overnight incubation with 100 μ L/ holes;
(3) wash plate
Wash plate hole 2 times with the PBS-T washing lotion;
(4) sealing
Confining liquid comprises NaCl 8g, KCl 0.2g, and Na2HPO4.12H2O 2.9g, KH2PO4 0.2g, sucrose 20.00g, proclin300 1.00ml, BSA 20.00g, the pH value of confining liquid are 7.3-7.5, the sealing of 150 μ L/ holes, wet box is hatched 2h;
(5) drying
Remove confining liquid, dried overnight.
(6) be assembled into finished product
The polystyrene plank is put into aluminium foil bag and put into siccative, and sealing is preserved.
The monoclonal antibody of above-mentioned anti-prostate specific antigen is prepared by following method, and step comprises:
(1) be that the prostate specific antigen of 50ug is mixed with Freund's complete adjuvant with protein content; After 15 days, carry out same dose and the Freund's incomplete adjuvant mixed immunity second time; Carry out again for the third time same dose and Freund's complete adjuvant mixed immunity after 15 days; Afterbody is got blood and is surveyed serum titer with indirect ELISA method after 10 days, does not add the adjuvant booster immunization with the pure antigen of same dose; Extracting spleen cell merges after 3 days;
(2) with immune small white mouse splenocyte and small white mouse myeloma cell (SP2/0) in 5-10: 1 ratio, merge as fusogen with 50% PEG;
(3) with the serum free medium that contains HAT (xanthoglobulin, aminopterin, thymus pyrimidine), at 37 ℃, to cultivate 10 days in the cell culture incubator of 5% CO2, conventional indirect ELISA method screens positive hole;
(4) the positive hole of the high specificity that filters out obtains the further enlarged culturing of monoclonal antibody cell strain with conventional limiting dilution assay clone;
(5) collect culture supernatant, the affinity chromatography monoclonal antibody purification, but be the prostate specific antigen monoclonal antibody;
(6) titre of detection monoclonal antibody is chosen a preferably strain and is carried out mark (HRP mark), and the good monoclonal antibody of mark is carried out titer determination;
(7) the be at war with ELISA reaction of the monoclonal antibody that mark is good and other unlabelled monoclonal antibodies is chosen and is not had a competitive strain to carry out the sandwich ELISA reaction, determines top condition.
Monoclonal antibody of the present invention can be directly used in the people t-PSA content in the test sample, by a large amount of cultivation monoclonal antibody cell strains, can obtain a large amount of monoclonal antibodies, compares with polyclonal antibody, has the purity height, the advantages such as specificity is strong, good reproducibility.
The present invention is directed to clinical labororatory set up a kind of both can manual operations, can be used for again the detection means of the full-automatic detecting instrument of standard, set up the quantitative detecting method that detects human serum t-PSA.Prostate specific antigen immue quantitative detection reagent box of the present invention (chemoluminescence method) can be single-minded the content that detects the PSA in the human serum, thereby according to the variation of how much judging the prostate cancer state of an illness and the result for the treatment of of its content.It has easy, quick, sensitive, stable advantage, test kit of the present invention, coated antibody and the antibody of enzyme labelling and the prostate specific antigen in the sample form 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, when it gets back to stable ground state, can launch photon, utilize illumination instrument to measure the yield of photon, the yield of this photon and the helpless amount of the detection in the sample are directly proportional, thus Criterion curve and calculate the content of test substance in the sample.Kit test method has highly sensitive, and sensing range is wide, easy to operate, to the experimenter without injury, simultaneously production cost is low, has alleviated the burden of doctor and patient, therefore more is conducive to promoting the use of of prostate cancer clinical diagnosis.
[description of drawings]
What Fig. 1 showed is to survey serum titer (before the cytogamy) with indirect ELISA method;
What Fig. 2 showed is anti-t-PSA monoclonal antibody titre measuring result in the ELISA method that the hybridoma among the present invention produces;
What Fig. 3 showed is the anti-t-PSA monoclonal antibody titre measuring result of mark HRP;
What Fig. 4 showed is the detection sensitivity of sandwich method ELISA (S-ELISA) system;
Fig. 5 shows 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 only to be used for explanation the present invention and be not used in and limit the scope of the invention.In addition, should be understood that after having set forth content of the present invention that those skilled in the art can do various changes and modification to the present invention, these equivalent form of values are the scope defined in claims among the application equally.
Embodiment 1: animal immune
Select the male Balb/C healthy mice about 8 ages in week with used myeloma cell's homology, antigen is that protein content is that the prostate specific antigen of 100ug is mixed with Freund's complete adjuvant, PBS, and after the emulsification, every of 100ug is each fully, take the back multiple spot, reach oxter, inguinal region immunity.Immune programme for children: after 15 days, carry out same dose and the Freund's incomplete adjuvant mixed immunity second time; Carry out again for the third time same dose and Fu Shi Freund's complete adjuvant mixed immunity after 15 days; Afterbody is got blood and is surveyed serum titer (seeing accompanying drawing 1) with indirect ELISA method after 10 days, does not add the adjuvant booster immunization with the pure antigen of same dose; Extracting spleen cell merges after 3 days.
Table 1: use for the third time the antibody titers in the mice serum after the t-PSA immunity
Embodiment 2: the structure of hybridoma
1, the cultivation of myeloma cell strain and preparation
What (1) the present invention adopted is the SP2/0 myeloma cell strain, and this cell strain growth and fusion efficiencies are all good, and the doubling time is 10-12h.Select to be in logarithmic phase, cellular form and active good cell during fusion.The myeloma cell should do to adapt at substratum first before fusion and cultivate, and makes Growth of Cells to best state (being logarithmic phase);
(2) SP2/0 that cultivates is drawn in the pipe of 50mL, centrifugal, abandon supernatant, hang, add the substratum of 10mL, draw a small amount of 10 times of dilution countings.
2, the preparation of splenocyte
(1) mouse is placed in the sealing bag, fills CO2 and treat its death by suffocation;
(2) the mouse sterilization is fixed on the dissection plate, in Bechtop, gets spleen, be placed in the culture dish of 12mL substratum, peel adhesion organization off, grind spleen, until till the surplus white tissue, suction pipe all picks up, and slowly gets on the tube wall that makes the tissue block adhesion again, and is centrifugal, abandon supernatant, add the erythrocyte cracked liquid cracking 10min of 10mL, the substratum that adds again 20-25mL stops its reaction, after centrifugal, abandon supernatant, add the substratum of 10mL, draw a small amount of 10 times of dilution countings.
3, cytogamy
Doing cell behind the booster immunization in 3 days merges.
Cytogamy is the key link of hybridoma technology, and basic step is that to get the Sp2/0 cell that is in logarithmic phase and splenocyte 1: 10 mixed, by polyoxyethylene glycol (PEG) method to obtain hybridoma, called after 3-20-1 and 3-26-1.The hybridoma that obtains is suspended in the HAT substratum that contains feeder cell, then joins in 96 orifice plates, and at 37 ℃, sealing was cultivated 12 days in the incubator of 5%CO2;
Embodiment 3: the preparation of monoclonal antibody and screening
1, the preparation of monoclonal antibody
The Kong Gezhong of the hybridoma that obtains from embodiment two reclaims the supernatant liquor of substratum, is chosen at the monoclonal antibody of reacting with antigenic peptide in the ELISA method.
2, the screening of monoclonal antibody
(1) with 100uL concentration be the t-PSA of 0.2ug/ml to each Kong Gezhong of 96 orifice plates, make it be fixed in solid phase in 4 ℃ after spending the night;
(2) be that 1% bovine serum albumin sealed 2 hours with 150uL concentration;
(3) medium supernatant with the 100uL hybridoma joins each Kong Gezhong, in 37 ℃ of reactions 2 hours, then adds the sheep anti-mouse antibody of dilution 10K horseradish peroxidase doubly in 37 ℃ of reactions 1 hour;
(4) use tetramethyl benzidine micropore peroxidase substrate (TMB) as the substrate 20min that develops the color;
(5) after interpolation 50uL concentration is the sulfuric acid termination reaction of 0.2N, measure the absorbancy of 450nm;
(6) select absorbancy and be approximately 3 3-20-1 and 3-26-1, and carry out subclone by limiting dilution assay.
3, a large amount of preparations of monoclonal antibody and and purifying
Cell behind the subclone is carried out enlarged culturing with cell-culturing rotating bottle, after about 20 days, collect supernatant, (Protein A) carries out affinitive layer purification with staphylococcal protein A,SPA.The monoclonal antibody that obtains is called after 3-20-1 and 3-26-1 respectively.
4, the mensuration of antibody titer
Tiring of 2 kinds of mAb that filter out measured by the ELISA method.Add respectively 3-20-1,3-26-1 (10ug/mL), after reaction, use horseradish peroxidase anti--mouse antibodies and TMB develop the color, result as shown in Figure 2 two kinds of mAb tires and reaches 10
-9More than.
Embodiment 4: the mark of monoclonal antibody and the mensuration of titre
To be purified into to such an extent that each antibody carries out the HRP mark according to a conventional method, the titre of the monoclonal antibody that mark is good is measured by following method, is that the t-PSA of 0.2ug/ml is fixed on (100uL/ hole) on the 96 hole microplates with concentration.The bovine serum albumin of use 1% sealed 2 hours, and tagged monoclonal antibody (100 times of the first hole dilutions) is done 4 times of dilutions since the second hole, and reaction is 2 hours under room temperature.After adding TMB, reaction was at room temperature carried out 20 minutes, with the sulfuric acid stopped reaction of 0.2N.Measurement obtains for the titre that is fixed on the antigen in the solid phase by the mode among the embodiment three in the absorbancy of 450nm.The result shows to have effective titre (result such as Fig. 3).
Embodiment 5: double-antibody sandwich elisa detects the foundation of t-PSA method
(1), coated with one of monoclonal antibody of described in twos pairing, the monoclonal antibody of 0.5ug/ml is added to microwell plate soil with the amount in 100uL/ hole, hatches in 4 ℃ and is fixed in solid phase in 24 hours;
(2), to use the pH value contain 0.1%Tween20 be that 7.4 20mM PBS (PBST) washes 2 times with the amount in 200uL/ hole to the hole lattice.Adding 1% bovine serum albumin with the amount in 150uL/ hole sealed 2 hours.
(3), Kong Geyong PBST washes 4 times with the amount in 200uL/ hole, adds continuous 4 times of dilution t-PSA by initial concentration 10ug/ml, in room temperature incubation 2 hours, then adds the strain antibody (1:4K of mark HRP; The 100uL/ hole) and in room temperature incubation 2 hours.
(4), add TMB after, reaction was at room temperature carried out 20 minutes, the sulfuric acid that adds 0.2N comes stopped reaction and measures the absorbancy of 450nm.
(5) result shows that the sensitivity of detection is very high.(seeing shown in the accompanying drawing 4)
Embodiment 6: prepare prostate specific antigen quantitative determination reagent kit of the present invention (chemoluminescence method)
(1) sodium periodate oxidation mark horseradish peroxidase
(1) oxidation of enzyme (whole process lucifuge)
Taking by weighing 3-5mg HRP is dissolved among the 600 μ L ddH2O2;
B, adding freshly prepared 150uL 0.1M sodium periodate (NaIO4) (MW:213.89g/mol gets 0.22g and is dissolved in the 10mL 10mM pH7.0 sodium phosphate buffer);
C, mixing, room temperature, lucifuge is hatched 20min;
D, solution is dialysed with dialysis tubing, 3000rpm/min, 4 ℃, 20min, solution are 1.0mM pH4.0 sodium-acetate buffer, change 3-4 time;
E, dialyse to 800 μ L, to the EP pipe at last;
(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, adding 500 μ L 0.2M pH9.5 carbonate buffer solutions, mixing detects the pH value 9.0～9.5;
C, the good HRP solution of will dialysing mixes with monoclonal antibody solution immediately, and room temperature is rocked 2h, lucifuge;
D, the adding freshly prepared NaBH4 of 80 μ L (4.0mg/mL gets 40mg and is dissolved among the 10mL ddH2O2);
E, mixing (lucifuge) are hatched 1.5h for 4 ℃;
F, with PBS dialyse to volume be 2mL;
G, adding equal-volume glycerine, the 1mL/ pipe ,-20C preserves
(2) preparation of enzyme labelled antibody working fluid
(1) preparation of enzyme mark diluent
Accurately take by weighing Tutofusin tris 7.27g according to standard recipe, add technique water 800.0mL, after stirring makes abundant dissolving, it is an amount of to add concentrated hydrochloric acid, stir, the pH value of using the digital display acidometer to measure liquid is 7.1～7.3, add successively load weighted other recipe ingredients in the mentioned solution again, fully after the stirring and dissolving, use process water to be 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.
The enzyme conjugates damping fluid that is up to the standards adds anti--PSA-HRP according to the Dilution ratio of used antibody, stir clockwise fully mixed in 30 minutes after 2～8 ℃ save backup, during packing by 96 person-portions: 11.0mL/ bottle, validity period 18 months.
(2) preparation of enzyme labelled antibody working fluid
Through evidence, the best effort concentration of enzyme labelled antibody is 1: 2000, with (1) described diluent enzyme labelled antibody is diluted to needed working concentration.
(3) preparation of prostate specific antigen standard substance
Accurately take by weighing above-mentioned each component according to the standard recipe content, after adding the abundant dissolving of process water 800.0mL stirring, the pH value of using the digital display acidometer to measure liquid should be 7.1～7.3, be settled to 1000.0mL with process water, mix, with the prostate specific antigen sterling, add the standard substance diluent, prepare a high density standard substance dope, adopt the method by low dilution to be diluted to each concentration, be mixed with 0,2.5,5,15,45, the series standard product of 100ng/mL, 2～8 ℃ save backup.
During the PSA standard substance packing that is up to the standards, 0.5mL/ bottle, validity period 18 months.
(4) the coated polystyrene board of prostate specific antigen monoclonal antibody
(1) with 0.05mol/L, the prostate specific antigen monoclonal antibody of the dipotassium hydrogen phosphate solution of pH9.6 and proper concn is mixed and made into coating buffer, and it is coated on the polystyrene board, places in the wet box, and it is coated to spend the night;
(2) Shen Di: wash plate hole 2 times with the PBS-Tween washing lotion;
(3) sealing:
The preparation of confining liquid:
Accurate weighing NaCl 8g, KCl 0.2g, Na
2HPO
412H
2O 2.9g, KH
2PO
40.2g, stirring, the pH value of using the digital display acidometer to measure liquid is 7.3～7.5, again load weighted other recipe ingredients are added in the mentioned solution successively, fully after the stirring and dissolving, use process water to be settled to 1000.0mL, 2～8 ℃ save backup, validity period 15 days.Every hole adds 150 μ L during use, and wet box is hatched 2h, gets rid of confining liquid, pats dry at clean thieving paper;
(4) drying: polystyrene board is placed on freeze-drying 2h on the 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 by weighing above-mentioned each component, add purified water 600.0mL, stir fully and be settled to 700.0mL after the 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 by weighing above-mentioned each component, add purified water 600.0mL, stir fully and be settled to 700.0mL after the 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 use in 1: 1 ratio.
(6) 10 * washingss
Accurately take by weighing above-mentioned each component, add process water and be settled to 800.0mL, stirring adds the technique water and is settled to 1000.0mL after fully dissolving, and 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 30.0mL/ bottle, validity period 18 months.
(7) composition of work in-process and finished product
The various annexes such as the various work in-process compositions of above-mentioned steps gained and product description form PSA quantitative determination reagent kits (chemoluminescence method).
Insolubilized antibody in the test kit is to be coated with in advance, do not need on-the-spot coated, during easy to use and joint; Calibration object is liquid; The enzyme labelling thing is the liquid that has been diluted to working concentration, also can directly use.
Embodiment 7: prostate specific antigen quantitative determination reagent kit of the present invention (chemoluminescence method) working method is as follows:
(1) prepares
Will serum sample to be checked and detection kit return to room temperature (18-25 ℃) (approximately 15min), opaque polystyrene board in the test kit of the present invention is fixed on t-PSA antibody in the plate hole, can directly use, need not be current coated, very easy to use.
(2) working method:
1, the coated lath with requirement is placed on the support;
2, the standard substance and the blood sample that in coated hole, add respectively 10 μ L
3, every hole adds the enzyme labelling thing of 100 μ L, and vibration mixes it a little; Put in the wet box and hatch 1h;
4, discard liquid in the hole, with the washings after the dilution, automatic washer or manual wash plate 4 times buckle dried at last at clean thieving paper;
5, luminous substrate A, B equal proportion are mixed to this used volume, every hole adds mixed luminous substrate 100 μ L, and vibration mixes it a little, lucifuge room temperature reaction 2min,
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 (referring to accompanying drawing 5) is found the concentration value of t-PSA in the serum with the value of test serum RLU at typical curve, calculates detected result.
8, statistical study detected result.
Measure used time weak point, only needed more than one hour just can all finish fast and easy according to above-mentioned steps with mentioned reagent box of the present invention.
Methodology index during embodiment eight kit measurement of the present invention is as follows:
1, sensing range: 0-100ng/mL:
2. normal reference value:＜4.0ng/mL;
3, sensitivity: minimum detection limit is 0.04ng/mL;
4, precision: precision (detects basic, normal, high three groups of samples (n=10) all less than 5% in analyzing, precision between analysis (detects basic, normal, high three groups of samples (n=10) all less than 15%, be higher than national standard, illustrate that test kit of the present invention has good repeatability in detecting test;
5, linearity: r 〉=0.99;
6, specificity: and the kinds of tumor mark no cross reactions such as CEA, AFP, PAP.
The used serum amount of test kit of the present invention is few, only needs 10 μ L, vitro detection to the patient without any side effect; What simultaneously the present invention used is chemiluminescence immune analysis method, indices also is better than ELISA adsorption analysis method (ELISA), therefore the present invention more can satisfy demand clinically for clinical detection t-PSA provides a kind of easier, method fast and accurately.
The clinical blood sample measured value of embodiment nine test kits of the present invention
Take hospital clinical and collect 66 parts of patients serum's 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 | 0 | 91299 |
| S1 | 2.46 | 2.5 | 779419 |
| S2 | 5.07 | 5 | 1485374 |
| S3 | 15.01 | 15 | 3899760 |
| S4 | 44.87 | 45 | 9925272 |
| S5 | 99.99 | 100 | 16907330 |
At first to the standard substance concentration in the upper table and relative light unit (RLU) value of taking the logarithm, the Criterion curve, typical curve coefficient R value is 0.99, and in typical curve, find the concentration value of t-PSA in the serum with the value of test serum RLU, the statistical study detected result, compare with clinical case, coincidence rate reaches 99%.
In sum, test kit of the present invention simple to operate, low price, to the patient have no side effect, fast, high accuracy for examination, be more suitable for promoting the use of.
Claims (8)
1. the hybridoma cell strain of anti-prostate specific antigen, its preserving number is respectively CCTCC C201202 and CCTCCC201208.
2. the monoclonal antibody of anti-prostate specific antigen is that the hybridoma cell line of CCTCC C201202 and CCTCC C201208 is secreted by preserving number respectively, described monoclonal antibody called after 3-20-1 and 3-26-1.
3. the preparation method of hybridoma cell strain as claimed in claim 1, it is characterized in that: behind Balb/c mouse immune 3 times, 3 heaven-made last booster immunizations before cytogamy, adopt the ELISA method to divide 2 stepping row filter fused cells: the first step adopts indirect elisa method to filter out the positive hole of anti-prostate specific antigen PSA; Second step adopts the indirect competitive ELISA method that the positive hole nutrient solution that the first step filters out is detected, select absorbancy and the higher positive hole of sensitivity, adopt limiting dilution assay to clone, clone and adopted same two step screening method to detect in rear about 10 days, after so repeating 3 times, filter out at last 2 strain of hybridoma system.
4. the application of anti-prostate specific antigen monoclonal antibody as claimed in claim 2, namely utilize the DAS-ELISA of described monoclonal antibody to detect t-PSA, the antibody sources that sandwich assay matches in twos is in cell strain 3-26-1 and 3-20-1, and its step comprises:
(1) coated with one of described monoclonal antibody of matching in twos;
(2) adding testing sample hatches;
(3) anti-as two with another strain monoclonal antibody of the described in twos HRP mark of pairing, add reaction system;
(4) add enzyme reaction substrate after the washing, read the OD value with 450nm;
(5) result shows that the sensitivity of detection is very high.
5. a test kit that detects t-PSA content in the serum comprises: the opaque polystyrene board that is coated with anti-prostate specific antigen antibody; Prostate specific antigen series standard product; Another strain monoclonal antibody of the prostate specific antigen of enzyme labelling; The chemical luminous substrate A liquid of above-mentioned enzyme effect and B liquid and washings.
6. test kit as claimed in claim 5 is characterized in that: opaque polystyrene board adopts direct physical absorption method Sheet clonal antibody, and coating buffer is in the phosphate buffered saline buffer; Another strain prostate specific antigen monoclonal antibody and horseradish peroxidase coupling form enzymic-labelled antibody, employing be that the sodium periodate method of improvement is carried out mark; Prostate specific antigen series standard product add the configuration of prostate specific antigen sterling and form take calf serum as matrix; Luminous substrate A, B are the HRP-Luminol luminescence system.
7. test kit as claimed in claim 5, it is characterized in that: the used marker enzyme of traget antibody is horseradish peroxidase, usefulness be sodium periodate oxidation, the working concentration of gained enzymic-labelled antibody the best is 1: 2000; What be coated with that the opaque polystyrene board of anti-prostate antigen antibody adopts is that the 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 the technique water and is settled to 700mL, keeps in Dark Place; Luminous substrate B liquid is comprised of following ingredients: borax 7.99g, boric acid 3.46g, urea peroxide 0.07g add the technique water and are settled to 700mL.
8. such as the preparation method of test kit as described in the claim 5-7, may further comprise the steps:
(1) correction of the preparation of standard substance and concentration
With prostate specific antigen t-PSA sterling, add the standard substance diluent, prepare a high density standard substance dope, adopt the method by low dilution to be diluted to each concentration, be mixed with 0,2.5,5,15,45, the series standard product of 100ng/mL, standard substance are proofreaied and correct with national standard.
(2) coated
Adopt 0.5mol/L, pH value be the prostate specific antigen monoclonal antibody of 9.5 carbonate buffer solution and proper concn to be mixed into antibody concentration be 1 μ g/mL coating buffer, be coated on the opaque polystyrene board 4 ℃ of overnight incubation with 100 μ L/ holes;
(3) wash plate
Wash plate hole 2 times with the PBS-T washing lotion;
(4) sealing
Confining liquid comprises NaCl 8g, KCl 0.2g, and Na2HPO4.12H2O 2.9g, KH2PO4 0.2g, sucrose 20.00g, proclin300 1.00ml, BSA 20.00g, the pH value of confining liquid are 7.3-7.5, the sealing of 150 μ L/ holes, wet box is hatched 2h;
(5) drying
Remove confining liquid, dried overnight.
(6) be assembled into finished product
The polystyrene plank is put into aluminium foil bag and put into siccative, and sealing is preserved.
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Cited By (5)
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| CN103589689A (en) * | 2013-11-12 | 2014-02-19 | 北京利德曼生化股份有限公司 | PSA (prostate specific antigen) monoclonal antibodies and hybridoma cell strains with function of secreting antibodies |
| CN103773738A (en) * | 2014-01-15 | 2014-05-07 | 东北林业大学 | Hybridoma for generating anti-free-prostate-specific-antigen (anti-f-PSA) monoclonal antibody and preparation of chemiluminescence immunoassay kit |
| CN103789271A (en) * | 2014-01-26 | 2014-05-14 | 东北林业大学 | Hybridoma for resisting generation of CA242 monoclonal antibody and preparation of chemiluminescence immune assay kit |
| CN113533744A (en) * | 2021-07-20 | 2021-10-22 | 郑州大学 | ELISA kit for detecting human sorted tubulin 17 and preparation method thereof |
| CN115856304A (en) * | 2022-11-11 | 2023-03-28 | 厦门英博迈生物科技有限公司 | Method for screening high-sensitivity monoclonal antibody pairing |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103589689A (en) * | 2013-11-12 | 2014-02-19 | 北京利德曼生化股份有限公司 | PSA (prostate specific antigen) monoclonal antibodies and hybridoma cell strains with function of secreting antibodies |
| CN103589689B (en) * | 2013-11-12 | 2015-10-14 | 北京利德曼生化股份有限公司 | PSA monoclonal antibody and secrete the hybridoma cell strain of this antibody |
| CN103773738A (en) * | 2014-01-15 | 2014-05-07 | 东北林业大学 | Hybridoma for generating anti-free-prostate-specific-antigen (anti-f-PSA) monoclonal antibody and preparation of chemiluminescence immunoassay kit |
| CN103789271A (en) * | 2014-01-26 | 2014-05-14 | 东北林业大学 | Hybridoma for resisting generation of CA242 monoclonal antibody and preparation of chemiluminescence immune assay kit |
| CN113533744A (en) * | 2021-07-20 | 2021-10-22 | 郑州大学 | ELISA kit for detecting human sorted tubulin 17 and preparation method thereof |
| CN115856304A (en) * | 2022-11-11 | 2023-03-28 | 厦门英博迈生物科技有限公司 | Method for screening high-sensitivity monoclonal antibody pairing |
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