CN101158687B - Applications of polypeptides PSA2 for producing prostate gland carcinoma diagnose kit - Google Patents
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- CN101158687B CN101158687B CN200710055749A CN200710055749A CN101158687B CN 101158687 B CN101158687 B CN 101158687B CN 200710055749 A CN200710055749 A CN 200710055749A CN 200710055749 A CN200710055749 A CN 200710055749A CN 101158687 B CN101158687 B CN 101158687B
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Abstract
The invention relates to an application of human polypeptide PSA2 in a preparation of a prostate gland cancer diagnosis kit, which belongs to the biotechnology field, and the invention can be used for an early diagnosis of prostate gland cancer and an evaluation of treatment effect. The invention discloses a preparation of human recombinant polypeptide PSA2 diagnostic reagent kit, and the preparation comprises an antibody preparation, a labeled antibody and a preparation of peridium antibody polystyrene micro-plates. The invention has the advantages that the operation of the kit is simple, the test is sensitive and the reappearance is good. According to a plurality of experiments of the detecting of prostate gland cancer, and each milliliter human recombinant polypeptide PSA2 is counted as one micro-gramme, the detecting reference range of the prostate gland cancer is: when each milliliter human recombinant polypeptide PSA2 is lower than 5nanogram, the prostate gland cancer is negative; when each milliliter human recombinant polypeptide PSA2 is higher than 5nanogram, the prostate gland cancer is positive.
Description
Technical field
The invention belongs to biological technical field, this patent relates generally to the application of PSA2 in preparation albumen and gene diagnosis kit.Can be used for the early diagnosis of prostate cancer and the monitoring of result of treatment.
Background technology
Since, (prostate specific antigen PSA) has been used for since crowd's examination PSA, and epoch-making variation has taken place in the diagnosis of prostate cancer.The U.S. as far back as nineteen eighty-three at first Using P SA carry out the examination of crowd's prostate cancer, last 20 years and realized early stage diagnosis and treatment.Yet, only possess the specific PSA of prostata tissue and remain, 1. PSA in the group generaI investigation in following shortcoming>and have only the people of 25-30% to be diagnosed as prostate cancer among the crowd of 4.0ng/ml, invalid biopsy rate is up to 70%; 2. PSA is to the specific type cancer beyond the adenocarcinoma of prostate, and like no diagnostic values such as signet ring cell cancer squama like cell cancers, the case load of failing to pinpoint a disease in diagnosis accounts for 8% of carninomatosis example sum; 3. be difficult to differentiate that early prostate cancer and the caused PSA of benign prostatic hyperplasia slightly increase.Therefore, explore new prostate cancer marker protein and become the difficult problem that needs to be resolved hurrily.
PSA is that a kind of prostate epithelial cell produces the Tryase of justacrine in serum, has very high prostata tissue specificity.PSA exists with three kinds of forms in blood: promptly free molecule form (FPSA), with the composite form (PSA-ACT) of α 12 chymotrypsin inhibitors and with the composite form (PSA-α 2M) of α 22 macroglobulin enzymes, wherein have only few part to exist with the FPSA form.Under the normal circumstances, prostate epithelial cell excretory PSA gets in the prostate gland lumen of gland, gets rid of with prostatic fluid, and becomes one of important composition of seminal fluid.Because, there is basal cell layer to constitute barrier around the prostate epithelial cell, just often PSA only gets rid of through the tube chamber side, and blood-serum P SA content is lower than 4.0ng/ml.During prostate cancer, carcinous body of gland lacks basal cell layer, and excretory PSA directly gets between prostate gland to go into blood and blood-serum P SA content is increased through capillary vessel in the matter.Although PSA has above-mentioned shortcoming in prostatic cancer early diagnosis,, still there is not the marker protein that can replace it so far.
The discovery of PSA2
PSA2 uses the SELDI-TOF protein biochip technology, and a molecular weight of finding through serum differential protein spectroscopy study is 15868 prostate cancer high expression level albumen.
Proteic separation and purification of blood-serum P SA2 and evaluation
Separation and purification and mass spectrum are identified: get the proteic serum sample of high expression level PSA2; The IMAC-CU column purification obtains protein concentrated solution, and the SDS-PAGE electrophoresis cuts the protein band of corresponding molecular weight; MALDI-TOF identifies, similar albumen information (the big gene ltd of Beijing China) is provided;
15868 proteic N-end sequencings: the PROCISE491 sequenator of using American AB I company; Carried out this marker protein N-terminal order-checking (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences; Centralab accomplishes); Carry out data base querying according to sequencing result, prove that PSA2 is a fragment in the LZTR2 albumen.Because of 15868 fragments of people LZTR2 (Leucine Zipper Transcription Regulator2) have the complementary action with PSA in prostate cancer diagnosis, therefore, called after PSA2.LZTR2 (Leucine Zipper TranscriptionRegulator2) is called RGPR (Reducacion Gene Promotor Region Related Protein) again.
Summary of the invention
The present invention provides a kind of people's recombinant polypeptide PSA2 to detect the application in the prostate gland cancer reagent kit in preparation, with the lower problem of specificity that solves present PSA diagnostic kit.The present invention matches with blood-serum P SA value through detecting PSA2 content of peptides in the blood, improves the diagnosis efficiency of prostate cancer.
The concordance rate of PSA2 polypeptide and PSA diagnosis is 72.3%, and the complementary action of diagnosis is high.Only 5 examples are that < 4.0ng/ml, PSA2 then obviously increase the case that can diagnose out PSA to fail to pinpoint a disease in diagnosis exactly to PSA in 83 routine patients with prostate cancer; At all the other 78 routine PSA content>in the patients with prostate cancer of 4.Ong/ml, it is 17 examples that the PSA2 signal to noise ratio is lower than the value of defining 5, all accurately is diagnosed by PSA; All not increase the person be 61 examples for PSA and PSA2 in 83 routine patients with prostate cancer, and the diagnosis concordance rate is 72.3%, does not have 1 example by the two case of failing to pinpoint a disease in diagnosis.Because of it has in prostate cancer diagnosis and PSA height complementary action, called after PSA2.
Aminoacid sequence such as the SEQ ID NO:1 of people's recombinant polypeptide PSA2 of the present invention are said, YYGRKKATLEWAMKNHLWGHALFLSSKMDPQTYSWVMSGFTSTLALNDPLQTLFQL MSGRIPQAATCCGEKQWGDWRPHLAVILSNQAGDPELYQRAIVAIGDTLAGKGLVE AAHFCYLMAHVPFGHYTVKTDHLVLLG.
The application of short peptide sequence segment in the preparation prostatic cancer diagnostic reagent kit arbitrarily in the aminoacid sequence of human polypeptides PSA2.
The aminoacid sequence of three small peptide PSA2 is YYGRKKATLEWAMKNHLWGHC, MAHVPFGHYTVKTDHLVLLGC and MKNHLWGHALFLSSKMDPQTC.SEQ?IDNO：2、3、4。
Inventor's recombinant polypeptide PSA2 diagnostic kit is with people's recombinant polypeptide PSA2 antigen-immunized animal, obtains corresponding anti-people's recombinant polypeptide PSA2 antiserum(antisera), the diagnostic kit of taking immune labeled absorption technology antigen-antibody binding reaction principle to process.
The aminoacid sequence of described human polypeptides PSA2 is included in the LZTR2 aminoacid sequence, its aminoacid sequence SEQ ID NO:5:
1melwapqrlp?qtrgkataps?kdpdrgfrrd?ghhrpvphsw?hngerfhqwq?dnrgspqpqq
61epradhqqqp?hyasrpgdwh?qpvsgvdyye?ggymqlysr?pgyensyqsy?qsptmreeya
121ygsyyyhghp?qwlqeervpr?qrspyiwhed?yreqkyldeh?hyenqhspfg?tnsethfqsn
181srnpckdspa?snsgqewpge?lfpgsllaea?qknkpslase?snllqqresg?lssssyelsq
241yirdaperdd?ppasaawspv?qadvssagpk?apmkfyiphv?pvsfgpggql?vhvgpssptd
301gqaalvelhs?mevilndsee?qeemrsfsgp?liredvhkvd?imtfcqqkaa?qscksetlgs
361rdsallwqll?vllcrqngsm?vgsdiaellm?qdckklekyk?rqppvanlin?Itdedwpvls
421sgtpnlltge?ippsvetpaq?ivekftrlly?ygrkkealew?amknhlwgha?Iflsskmdpq
481tyswvmsgft?stlalndplq?tlfqlmsgri?pqaatccgek?qwgdwrphla?vilsnqagdp
541elyqraivai?gdtlagkglv?eaahfcylma?hvpfghytvk?tdhlvllgss?hsqeflkfat
601teaiqrteif?eycqmlgrpk?sfipsfqvyk?llyasrlady?glvsqalhyc?eaigaavlsq
661gesshpvllv?eliklaeklk?lsdplvlerr?sgdrdlepdw?laqlrrqleq?kvagdigdph
721ptrsdisgag?gtttentfyq?dfsgcqgyse?apgyrsalwl?tpeqtcllqp?spqqpfplqp
781gsypagggag?qtgtprpfys?vpethlpgtg?ssvavteatg?gtvweemlqt?hlgpgentvs
841qetsqppdgq?eviskpqtpl?aarprsises?sassakedek?essdeadkns?prntaqrgkl
901gdgkehtkss?gfgwfswfrs?kptknaspag?dedssdspds?eetprassph?qaglglsltp
961spespplpdv?safsrgrggg?egrgsassgg?aaagagvggl?sgpesvsfel?csnpgvllpp
1021palkgavply?npsqvpqlpt?atslnrpnrl?aqrryptqpc
People's recombinant polypeptide PSA2 antigen of the present invention is meant people's recombinant polypeptide PSA2 pure protein antigen; Described immune animal comprises various immune animals such as horse, sheep, rabbit, cavy etc.
The people's recombinant polypeptide PSA2 antibody that adopts in inventor's recombinant polypeptide PSA2 diagnostic kit is people's recombinant polypeptide PSA2 protein antibodies.This antibody comprises monoclonal antibody and polyclonal antibody.
Polypeptide PSA2 protein antibodies, the application of humanized antibody in preparation prostate cancer therapy medicine that this antibody derives for the basis.
The detection kit of a kind of prostatic cancer specific albumen PSA2; Comprise the enzyme plate, ELIAS secondary antibody, common reagent Tween-20 and the chromogenic substrate TMB TMB that are coated with antibody; It is characterized in that being the PSA2 fusion rotein antibody of handling through coating buffer and retardance liquid is handled in the enzyme plate hole of this test kit, other is equipped with the anti-PSA2 polypeptide antibody of the rabbit of horseradish peroxidase HRP mark.
The cDNA of polypeptide PSA2 coding and place albumen full-length cDNA are to it being the application of basic design synthetic primer in the preparation prostatic cancer diagnostic reagent kit.
To the cDNA and the place albumen full-length cDNA basic design synthetic antisense nucleic acid of PSA2 coding, siRNA is to the application in the prostate cancer diagnosis treatment.
The preparation method of people's recombinant polypeptide PSA2 disclosed by the invention is following:
Adopt histidine-tagged expressing fusion protein human polypeptides PSA2:
Adopt polymerase chain reaction (PCR) method human cloning recombinant polypeptide PSA2 gene protein encoding sequence; Make up this gene Fusion expression plasmid, import in the intestinal bacteria of BL21, I PTG induces its expression in intestinal bacteria; SDS-PAGE electrophoresis; Western blot identifies, with histidine-tagged fusion rotein purification kit purified fusion protein, with a large amount of isolation and purification protein of HPLC; Resist and the mouse monoclonal antibody with the preparation rabbit, preparation is used for people's recombinant polypeptide PSA2 gene diagnosis kit of prostatic cancer early diagnosis more.Specific as follows:
According to the histidine-tagged reading frame design primer of prokaryotic expression carrier, make the coding region reading frame of people's recombinant polypeptide PSA2 gene consistent with histidine-tagged reading frame.With the plasmid that contains the PSA2 gene is template (0.1ug), adds each 10pmol of above-mentioned primer and increases, and reclaims PCR product band.Cut PCR with Sma 1 I enzyme and reclaim product and expression vector, produce the sticking end of coupling.Little glue reclaims the carrier that reagent reclaims linear 4.9kb, PSA2 coding region fragment.Expression vector is connected with people's recombinant polypeptide PSA2 gene, is built into in-frame histidine-tagged PSA2 fusion gene.With containing the recombinant plasmid transformed intestinal bacteria BL2 1 of fusion gene, prepare DNA in a small amount, Sal I and Not I enzyme are cut evaluation and whether are contained the insertion fragment, check order with automatic dna sequencer after containing the segmental positive colony DNA purifying of insertion.Design synthesizing recombined human polypeptide PSA2 primer sequence:
1TACTACGGTCGCAAAAAAGCG 21
2TTTCATCGCCCATTCCAGAGTCGCTTTTTTGCGACCGTAG 40
3CTCTGGAATGGGCGATGAAAAATCACCTGTGGGGTCACGC 40
4TCCATCTTAGAGGAGAGGAACAGCGCGTGACCCCACAGGT 40
5TGTTCCTCTCCTCTAAGATGGACCCGCAGACCTACTCCTG 40
6AGAGGTGAAGCCAGACATAACCCAGGAGTAGGTCTGCGGG 40
7GTTATGTCTGGCTTCACCTCTACGCTGGCTCTCAATGACC 40
8AGCTGGAACAGGGTCTGCAGCGGGTCATTGAGAGCCAGCG 40
9GCAGACCCTGTTCCAGCTGATGTCCGGTCGTATTCCACAG 40
10TTCACCGCAGCAAGTCGCCGCCTGTGGAATACGACCGGAC 40
11CGACTTGCTGCGGTGAAAAACAGTGGGGTGACTGGCGTCC 40
12TTGCTCAGGATAACAGCCAGGTGCGGACGCCAGTCACCCC 40
13CTGGCTGTTATCCTGAGCAACCAGGCTGGTGACCCGGAGC 40
14TAGCAACGATGGCACGCTGGTAGAGCTCCGGGTCACCAGC 40
15GCGTGCCATCGTTGCTATCGGTGATACCCTCGCGGGTAAA 40
16GTGCGCCGCTTCAACCAGACCTTTACCCGCGAGGGTATCA 40
17GGTTGAAGCGGCGCACTTCTGCTACCTGATGGCTCATGTT 40
18ACCGTGTAGTGGCCGAACGGAACATGAGCCATCAGGTAGC 40
19TTCGGCCACTACACGGTCAAAACCGACCATCTGGTTCTGC 40
20ACCCAGCAGAACCAGATGGTCGG 23
Single positive colony inoculation goes into to contain in the 2ml LB nutrient solution of acillin (100ug/ml), and 37.C, incubated overnight.The 400ul overnight culture is inoculated into 40ml respectively contains in the LB training base of acillin (100ug/ml), in 37 ℃ of cultivation 7h, adding IPTG is 1mmol/L to final concentration, and 37 ℃, cultivation 2.5h is transferred in the 50ml centrifuge tube.The centrifugal 10min of room temperature 12000 * g removes most supernatant, places on ice, is resuspended among the PBS of 600ul of precooling.Adding final concentration is N,O-Diacetylmuramidase, the 10ug/ml DNa seI of 100ug/ml, and with the multigelation method rupture of membranes of liquid nitrogen and warm water mediation, the centrifugal 10min of 11000rp collects supernatant.Resin in the resuspended histidine-tagged Mi croSpin purification column is opened lid and bottom, places the centrifuge tube of a clean 1.5ml, and the centrifugal 1min of 735 * g abandons liquid, covers bottom, adds the bacterial lysate of 600ul in the post.Cover tight loam cake, mixing is gently opened lid and bottom after placing room temperature 5-10min, places the centrifuge tube of a clean 1.5ml, and the centrifugal 1min of 735 * g washs 2 times with identical method with 400ul PBS again.Add the Triptide elutriant of 200ul in the post, cover tight loam cake, mixing places room temperature 5～10min gently.The centrifugal 1min of 735 * g collects effluent, obtains human polypeptides PSA2 (See Figure).
The preparation of human polypeptides PSA2 diagnostic kit disclosed by the invention comprises the following steps:
1, Antibody Preparation
Get healthy immune animal, carry out immunity with human polypeptides PSA2 antigen, the first time adds BCG-CWS with antigen and freund adjuvant grinds well; Injection animal lymphonodi cervicales or subcutaneous muscle tissue, per two weeks once are total to immune 4-5 time; Antigen amount antigen amount increases and decreases according to the weight of animals, the last two weeks back blood sampling of immunity, the animal AHS with the saturated ammonium sulphate two-stage precipitation after last albumen A/G affinity chromatography; Obtain animal and resist everybody recombinant polypeptide PSA2IgG antibody, mensuration is tired, and is freezing subsequent use;
The antigenic synthesis of polypeptide antibody of the preparation anti-human polypeptides PSA2 of rabbit:
1., the aminoacid sequence of basis, design and 21 amino acid whose PSA2 polypeptide of chemosynthesis, the sequence of this polypeptide is: YYGRKKATLEWAMKNHLWGH
C, with peptide C end and keyhole limpet hemocyanin (keyhole limpet hemocyanin, KLH) crosslinked to increase immunogenicity.
2., polypeptide is dissolved in phosphoric acid salt (PBS) damping fluid, be injected to new zealand rabbit, initial dose is 300ug/kg, and the dosage of booster immunization is about about 1/4 of initial dose.Per 2～3 all booster immunizations once are total to immunity 4 times, and the antiserum(antisera) of acquisition is purified into the IgG of anti-PSA2 with the affinitive layer purification test kit (the concrete steps strictness is undertaken by the specification sheets of Pierce company) of Pierce company;
2, antibody labeling
Get people's recombinant polypeptide PSA2IgG antibody and adopt conventional sodium periodate method mark horseradish peroxidase;
3, coated antibody polystyrene micropore plate preparation
Get people's recombinant polypeptide PSA2IgG antibody of mark, working concentration adds polystyrene micropore in 50mM under the pH9.5 carbonate condition, every hole 100 microlitres, and placed about 40 hours in 2-8 ℃ of condition; Taking-up is with saline water washing 5 times, and oven dry packs with aluminium foil bag.
4, other uses reagent:
(1) coating buffer: 0.05molpH9.5 carbonate solution;
(2) washings: 0.8% sodium-chlor--0.05%Tween20;
(3) diluent: 0.15molpH7.2 phosphoric acid salt monochlor(in)ate sodium solution;
(4) colour developing damping fluid: 0.05molpH5.0 phosphoric acid salt one citric acid solution;
(5) developer; Adjacent benzene two ammoniums;
(6) stop buffer: 2M sulphuric acid soln.
The concrete preparation method of people's recombinant polypeptide PSA2 diagnostic kit:
Every coated antibody polystyrene micropore plate of each experiment is established people's recombinant polypeptide PSA2 reference standard article (0,5,10,20,50 nanograms/milliliter) 5 holes and blank well 1 hole, and all the other each holes add human serum sample to be checked, every hole application of sample 100 microlitres.Put 37 ℃, kept 1 hour, take out, clap and do with washings washing 5 times.Except that blank well, all the other each holes add enzymic-labelled antibody 100 microlitres.Put 37 ℃, kept 1 hour, take out with washings washing 5 times, bat.Each hole adds substrate colour developing liquid 100 microlitres.Put 37 ℃, kept 15 minutes, each hole adds stop buffer 50 microlitres.
The result judges
(1) adopt enzyme mark detector, predominant wavelength 492nm, commplementary wave length 630nm, each hole absorbance is measured in standard substance 0 nanograms/milliliter school zero.With people's recombinant polypeptide PSA2 reference;
(2) be X-coordinate with people's recombinant polypeptide PSA2 reference standard position marks indicating value, absorbance is an ordinate zou, the drawing standard curve;
(3) look into typical curve with the absorbance in each hole to be checked and draw the relevant detection result.
The invention has the advantages that: obtain corresponding prostatic cancer diagnostic reagent kit with human polypeptides PSA2 antigen.This test kit is easy and simple to handle, and test is sensitive, favorable reproducibility.Detecting the mensuration terms of reference that obtain prostate cancers through a large amount of tests is (in every milliliter of people's recombinant polypeptide PSA2 nanogram): negative below the 5ng/ml, more than positive.
Embodiment:
Embodiment 1, goat-anti people recombinant polypeptide PSA2IgG Antibody Preparation
Get the heavily adult sheep of 50kg of healthy male, personnel selection recombinant polypeptide PSA2 purifying protein antigen/synthetic polypeptide carries out immunity.The first time adds BCG-CWS with antigen and freund adjuvant grinds well, injection sheep lymphonodi cervicales and subcutaneous muscle tissue, and per two weeks once are total to antigen amount 5mg/ time immune 4-5 time.The last two weeks back blood sampling of immunity, goat-anti human serum with the saturated ammonium sulphate two-stage precipitation after last ion exchange column, obtain goat-anti people recombinant polypeptide PSA2IgG antibody, mensuration is tired, and is freezing subsequent use.
Embodiment 2, rabbit resist everybody recombinant polypeptide PSA2IgG Antibody Preparation
Get the adult rabbit (5kg) of healthy male, personnel selection recombinant polypeptide PSA2 purifying protein antigen/synthetic polypeptide carries out immunity.The first time adds BCG-CWS with antigen and freund adjuvant grinds well, muscle tissue under the injection rabbit skin, and per two weeks, once immunity was 4 times altogether, antigen amount 0.5mg/ time.The two weeks back blood sampling that immunity is last.The goat-anti human serum is gone up ion exchange column after with the saturated ammonium sulphate two-stage precipitation, obtains anti-everybody the recombinant polypeptide PSA2IgG antibody of rabbit, and mensuration is tired, and is freezing subsequent use.
Embodiment 3, mouse-anti people recombinant polypeptide PSA2IgG Antibody Preparation
Get the adult cavy of healthy male, personnel selection recombinant polypeptide PSA2 purifying protein antigen/synthetic polypeptide carries out immunity.The first time adds BCG-CWS with antigen and freund adjuvant grinds well, the subcutaneous muscle tissue of injection cavy, and per two weeks, once immunity was 4 times altogether, antigen amount 0.1mg/ time.The two weeks back blood sampling that immunity is last.The mouse-anti human serum is gone up ion exchange column after with the saturated ammonium sulphate two-stage precipitation, obtains mouse-anti people recombinant polypeptide PSA2IgG antibody, and mensuration is tired, and is freezing subsequent use.
Embodiment 4, antibody labeling
Get people's recombinant polypeptide PSA2IgG protein antibodies that arbitrary method obtains among the embodiment 1-3 and adopt conventional sodium periodate method mark horseradish peroxidase,
(1) people's recombinant polypeptide PSA2 antibody (2mg/ml) 0.9ml adds 0.1mol pH9.5NaAc;
(2) horseradish peroxidase 10mg is dissolved in 1.0mil.0mmolpH4.0NaAc.
(3) add 0.1mol sodium periodate 0.1ml, 1.6mmol terepthaloyl moietie 0.1ml is in above-mentioned horseradish peroxidase solution.
(4) above-mentioned horseradish peroxidase solution is mixed stirring 1-2 hour with antibody.
(5) add 0.1mol sodium tetrahydroborate 0.1ml static 30 minutes in 4～C.
(6) with 50% saturated ammonium sulphate separation marking thing.
(7) affinity tag is used 50% USP Kosher------PBS dissolving, and in keeping in cold storage.
Embodiment 5, the preparation of coated antibody polystyrene micropore plate
Get people's recombinant polypeptide PSA2IgG antibody of the mark that embodiment 4 obtains, working concentration adds polystyrene micropore in 50mM under the pH9.5 carbonate condition, every hole 100 microlitres, and placed about 40 hours in 2-8 ℃ of condition.Taking-up is with saline water washing 5 times, and oven dry packs with aluminium foil bag.
Embodiment 6, people's recombinant polypeptide PSA2 test kit
Reagent
Coated antibody polystyrene micropore plate: the anti-people's recombinant polypeptide of solid phase PSA2 antibody.
Enzymic-labelled antibody: the anti-people's recombinant polypeptide of enzyme labelling PSA2 antibody, 0.15molpH7.2 phosphoric acid salt monochlor(in)ate sodium solution.
Washings (10X): 8% sodium-chlor---0.5%Tween20.
Colour developing damping fluid: 0.05molpH5.0 phosphoric acid salt one citric acid solution.
Developer: adjacent benzene two ammoniums.
Stop buffer: 2mol sulphuric acid soln.
Instrument
Enzyme mark detector (absorbance measurement precision 0.0～2.0A ± 0.001A).
Analytical procedure: double antibody sandwich method principle quantitatively determined
Sample requirement: human serum.
Technical requirements: (ELISA method) adopts enzyme mark detector, predominant wavelength 492nm commplementary wave length 630nm.
Method of calculation: with human polypeptides PSA2 reference standard position marks indicating value is X-coordinate, and absorbance is the ordinate zou directrix curve.The drawing standard curve.Look into typical curve with the absorbance in each hole to be checked and draw the relevant detection result.
Performance requriements
Physical behavior: outward appearance: each liquid components should be Clear & Transparent, the packing ne-leakage, and sealing bag does not have gas leakage.
The reagent blank absorbancy: at predominant wavelength 492nm commplementary wave length 630nm place, room temperature, absorbancy should < 0.15A.
Sensitivity for analysis: 15 minutes, reagent blank; Get every milliliter 0.16 microgram of human polypeptides PSA2 standard and detect detected result ± 20%.
Accuracy: get every milliliter 0.33 microgram of people's recombinant polypeptide PSA2 standard, detected result ± 15%.
Precision: 1.0 microgram CV～15%.
Measurement range: be linear in the 2.0 microgram scopes, linearity error answers≤15%, measures the correlation coefficient r of absorbance and sign value>0.98.
Experimental technique
Physical behavior: visual observation agent box each component outward appearance.
The reagent blank absorbancy: to contain 10% calf serum saline water is that blank sample detects, by predetermined operation, and with the blank school zero of chromogenic substrate, the record absorbancy, triplicate is averaged.
Sensitivity for analysis: detect every milliliter 5 nanogram of people's recombinant polypeptide PSA2 standard, repeat 3 times and average.
Accuracy: the examination criteria article, every milliliter 10 nanogram of people's recombinant polypeptide PSA2 standard,, repeat 3 times, average.
Precision (repeatability): get human polypeptides PSA2 content approximately the serum of every milliliter 20 nanogram detect and to carry out 20 times and measure, (N=20), obtain 20 absorbancy change mean X and standard deviation S, be calculated as follows the variation coefficient (CV), CV%=S/X
Measurement range: get every milliliter 5 nanogram of people's recombinant polypeptide PSA2 standard, 10 nanograms, 20 nanograms, 50 nanograms, each concentration duplicate detection 3 times, average.
Test case
Basic parameter: adopt enzyme mark detector, predominant wavelength 492nm commplementary wave length 630nm with the brilliant 0 mcg/ml school zero of people's recombinant polypeptide PSA2 reference standard, measures each hole absorbance.
Method of use:
(1) test every coated antibody polystyrene micropore plate at every turn establish brilliant (0,5 nanograms, 10 nanograms, 20 nanograms, 50 nanograms/milliliter) 5 holes of people's recombinant polypeptide PSA2 reference standard and blank well 1 hole, all the other each holes add serum sample to be checked, every hole application of sample 100 microlitres.Put 37 ℃ 1 hour, take out with washings washing 5 times, clap and do.
(2) except that blank well, all the other each holes add enzymic-labelled antibody 100 microlitres.Put 37 ℃, kept 1 hour, take out, clap and do with washings washing 5 times.
(3) each hole adds substrate colour developing liquid 100 microlitres.Put the room temperature lucifuge, kept 15 minutes, add stop buffer 50 microlitres, absorbancy (OD) the 0.80 sample people recombinant polypeptide PSA2 content 5.2 nanograms/ml that tables look-up.
The result judges:
(1) adopt enzyme mark detector, predominant wavelength 492nm commplementary wave length 630nm with the brilliant 0 nanograms/milliliter school zero of people's recombinant polypeptide PSA2 reference standard, measures each hole absorbance.
(2) be X-coordinate with the brilliant sign value of people's recombinant polypeptide PSA2 reference standard, absorbance is an ordinate zou, the drawing standard curve.
(3) sample OD (absorbancy) 0.80 looks into canonical plotting, sample people recombinant polypeptide PSA2 value: 5.2 nanograms/ml, and people's recombinant polypeptide PSA2 content 5.2 nanograms/ml among this patients serum, prostate cancer is positive.
(4) the 47 routine normal serums and the 83 routine prostate cancer serum such as the following tables of process measuring
| Numbering | Type | PSA2 |
| 1 | Normally | 0.1 |
| 2 | Normally | 3.2 |
| 3 | Normally | 0.4 |
| 4 | Normally | 2.1 |
| 5 | Normally | 4.2 |
| 6 | Prostate cancer | 12.8 |
| 7 | Prostate cancer | 25.6 |
| 8 | Prostate cancer | 19.8 |
| 9 | Prostate cancer | 58.4 |
| 10 | Prostate cancer | 20.6 |
| 11 | Prostate cancer | 33 |
| 12 | Prostate cancer | 28.8 |
| 13 | Prostate cancer | 11.0 |
| 14 | Prostate cancer | 31.5 |
| 15 | Prostate cancer | 7.59 |
Sequence table
< 110>Jilin University
< 120>application of polypeptide PSA2 in the preparation prostatic cancer diagnostic reagent kit
<130>pyz2007
<160>5
<170>Patentln?version3.2
<210>1
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<212>PRT
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Claims (1)
1. the application of polypeptide PSA2 in the preparation prostatic cancer diagnostic reagent kit, the aminoacid sequence of described polypeptide PSA2 such as SEQ ID NO:1 are said.
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| CN105646665A (en) * | 2014-03-24 | 2016-06-08 | 朱育盼 | Short-chain polypeptides PCP14 capable of being specifically bound to prostate cancer cells and application of short-chain polypeptides PCP14 |
| CN105541972A (en) * | 2014-03-24 | 2016-05-04 | 朱育盼 | Short peptides PCP7 specifically binding to prostatic cancer cells and application of short peptides PCP7 |
| CN113527434A (en) * | 2021-07-14 | 2021-10-22 | 呈诺再生医学科技(珠海横琴新区)有限公司 | WTN polypeptide and application thereof in detection of prostate cancer |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1387539A (en) * | 1999-07-29 | 2002-12-25 | 米德列斯公司 | Human monoclonal antibodies to prostate specific membrane antigen |
| WO2005085292A2 (en) * | 2004-03-03 | 2005-09-15 | Biomerieux | Method for detecting the activatable free form of psa and the use thereof for diagnosing benign pathologies of the prostate and adenocarcinoma of the prostate |
| CN1715921A (en) * | 2005-06-17 | 2006-01-04 | 吉林大学 | Use of human RGPR protein in preparing prostatic cancer diagnostic reagent kit |
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2007
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1387539A (en) * | 1999-07-29 | 2002-12-25 | 米德列斯公司 | Human monoclonal antibodies to prostate specific membrane antigen |
| WO2005085292A2 (en) * | 2004-03-03 | 2005-09-15 | Biomerieux | Method for detecting the activatable free form of psa and the use thereof for diagnosing benign pathologies of the prostate and adenocarcinoma of the prostate |
| CN1715921A (en) * | 2005-06-17 | 2006-01-04 | 吉林大学 | Use of human RGPR protein in preparing prostatic cancer diagnostic reagent kit |
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