CN101684145A - Antigen peptide identified by p53 antoantibody, kit and application thereof in preparing tumor detection kit - Google Patents
Antigen peptide identified by p53 antoantibody, kit and application thereof in preparing tumor detection kit Download PDFInfo
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- CN101684145A CN101684145A CN200810222871A CN200810222871A CN101684145A CN 101684145 A CN101684145 A CN 101684145A CN 200810222871 A CN200810222871 A CN 200810222871A CN 200810222871 A CN200810222871 A CN 200810222871A CN 101684145 A CN101684145 A CN 101684145A
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Abstract
The invention discloses an antigen peptide identified by p53 antoantibody, and an active fragment of the peptide is as follows: (1) a sequence shown in SEQ ID No.4 is F-S-D-L-W-K; (2) a sequence shownin SEQ ID No.5 is D-I-E-Q-W-F-T; (3) a sequence show in SEQ ID No.6 is Y-E-P-P-E. The invention finds out a linear antigenic determinant and a core sequence thereof identified by the p53 antoantibody. The kit of the invention synthesizes the antigen peptide identified by the p53 antoantibody into a 96 pore plate, can reduce synthesized cost of each sample; if the kit is produced in mass, the detection cost of each patient can be half reduced. Meanwhile, the peptide is shorter and can not be easily degraded, and the valid period of the kit can be prolonged to two years, thus having considerable value in use.
Description
Technical field
The present invention relates to the test kit of a peptide species and preparation thereof, the antigenic peptide test kit of p53 autoantibody identification specifically and the application in preparation detection tumor reagent box.
Background technology
Lung cancer is to cause main causes of death in the various malignant tumours.Most of patient has been in late period (III or IV phase) when finding, overall five year survival rate is less than 5%.Therefore, new quick, the easy method of early diagnosis of nonsmall-cell lung cancer of development has great importance.Tumour is owing to the cell that the cytodifferentiation proliferative abnormality causes is grown out of control, necessarily having new albumen in the malignant proliferation of tumor process occurs, simultaneously some normal protein matter also can overexpression, and then triggers the immune response reaction, causes the generation of serum autoantibody.Studies show that the autoantibody of tumour patient is expected to be used for clinical detection as new tumor marker.Wherein, the p53 gene is the important cancer suppressor gene of body, it plays a significant role at the aspects such as apoptosis, growth regulating, differentiation, aging and DNA reparation of cell, and the wild type p53 gene can stop normal cell to change tumour cell into, therefore is called as " gene bodyguard ".And in most of human tumors, all found the inactivation of p53 gene.Discover that the p53 autoantibody is present in various different types of tumors patients' the serum.In the nonsmall-cell lung cancer patient, the recall rate of serum p53 autoantibody is about 20%.Conventional detection mainly is the ELISA test kit, its principle be will reorganization wild type p53 albumen bag by to 96 hole flat boards, after hatching jointly with serum, p53 antibody in the serum will with the combination of p53 antigen protein, the anti-human IgG that adds horseradish peroxidase-labeled then, make the solution colour developing by developer, measure the OD value with microplate reader, and then measure the concentration of p53 autoantibody in the serum.This method needs to express the p53 pure protein earlier could prepare test kit, because expression, the purifying difficulty of pure protein are bigger, so preparation cost is higher.
Existing test kit is that the wild type p53 pure protein of will recombinate is coated in 96 orifice plates, because proteic expression of p53 and purge process more complicated, so the cost of this test kit is higher.Everyone part cost needs 160 yuan approximately.Owing to use whole protein to detect, degrade easily in addition, so its validity period is shorter, the validity period of two kinds of test kits that use was about about 1 year now.
Summary of the invention
The objective of the invention is to solve in the existing p53 test kit that the p53 pure protein is expressed, purifying difficulty height, the defective that the test kit cost is too high and propose the antigenic peptide of a kind of highly sensitive, the identification of specific p53 autoantibody.
Another object of the present invention is to provide the application of antigenic peptide in preparation detection tumor reagent box of above-mentioned p53 autoantibody identification.
Another object of the present invention is to provide the antigenic peptide of above-mentioned p53 autoantibody identification to detect tumour serum p53 antibody kit.
For achieving the above object, the inventor's invention thinking is: utilize the antigenic peptide of polypeptide array technique screening serum autoantibody identification, and carry out the autoantibody detection according to the core sequence of antigenic peptide.
Concrete technical scheme of the present invention is:
A kind of antigenic peptide of p53 autoantibody identification, its polypeptide active fragment is:
(1) sequence: F-S-D-L-W-K shown in the SEQ ID No.4;
(2) sequence: D-I-E-Q-W-F-T shown in the SEQ ID No.5;
(3) sequence: Y-E-P-P-E shown in the SEQ ID No.6.
Above-mentioned SEQ ID No.4～6 are the core sequence of the antigenic peptide of p53 autoantibody identification.
The antigenic peptide of above-mentioned a kind of p53 autoantibody identification, it is the amino acid derived sequence that is formed through replacement, disappearance or the interpolation of amino-acid residue by aminoacid sequence shown in SEQ ID No.4, SEQ ID No.5 and the SEQ ID No.6.
The antigenic peptide of above-mentioned a kind of p53 autoantibody identification, the derived sequence of its polypeptide fragment (1) is SEQ ID No.1:Q-E-T-F-S-D-L-W-K-L-L-P.
The antigenic peptide of above-mentioned a kind of p53 autoantibody identification, the derived sequence of its polypeptide fragment (2) is SEQ ID No.2:S-P-D-D-I-E-Q-W-F-T-E-D.
The antigenic peptide of above-mentioned a kind of p53 autoantibody identification, the derived sequence of its polypeptide fragment (3) is SEQ ID No.3:V-V-P-Y-E-P-P-E-V.
The application of the antigenic peptide of p53 autoantibody identification in preparation detection tumour serum p53 antibody kit.
The application of the antigenic peptide of above-mentioned p53 autoantibody identification in preparation detection tumour serum p53 antibody kit, described tumour is a lung cancer.
A kind of detection tumour serum p53 antibody kit, it comprises that bag is by 96 orifice plates of specific polypeptide, reagent 1---dcq buffer liquid (PBS, 3M, PH 7.4), and reagent 2---diluted sample damping fluid (bovine serum albumin, 1mg/ml), reagent 3---tmb substrate solution, reagent 4---reaction terminating liquid (2M H
2SO
4), two of reagent 5---horseradish enzyme labelling resists reagent 6a---negative control sample (serum that p53 antibody is negative), reagent 6b～6e---standard model (the p53 antibody concentration is respectively 1-5-10-15U/ml); Wherein, it in this test kit enzyme plate hole antigenic peptide through the aforesaid p53 autoantibody identification of coating buffer and the processing of retardance liquid, 96 orifice plates comprise 8 row * 12 row, and as shown in Figure 7, each reacting hole endoperidium has the antigenic peptide chain of above-mentioned p53 autoantibody identification.The concentration of the antigenic peptide of p53 autoantibody identification is: each reacting hole 10 μ g/ml.
The core sequence that the contriver obtains is the minimum of blood-serum P 53 antibodies but most critical peptide sequence.Experimental study shows that this core sequence is that the identification of serum autoantibody is necessary, but is not sequence optimum in the application.A series of the deriving that obtains on the basis of core sequence of the present invention (SEQ ID No.4-6) all can be discerned (seeing Fig. 4-6 for details) by patients with lung cancer serum autoantibody, so derived sequence is not just given unnecessary details at this one by one.We know, exist in a large number at the antigenic antibody of difference in the serum, if use core sequence as detecting antigen, because core sequence is shorter, may cross reaction takes place and false positive occurs.As detecting antigen, can obtain quite good detecting sensitivity as long as discover 12 peptides that comprise core sequence; Simultaneously, use longer sequence can avoid the cross reaction of using short amino acid sequence to cause, improve specific degree.
The preservation of test kit:
1. test kit must keep in Dark Place at 2～8 ℃.
2. the reagent 1 after the dilution---dcq buffer liquid must be 2～8 ℃ of preservations, and use in one month.The testing sample precaution:
1. testing sample is a human serum, and sample must be fresh, or in the refrigerator below-40 ℃, preserve.
2. serum sample is forbidden multigelation.
The experiment precaution:
1. multiple hole preferably done by every routine test serum sample or average in 3 holes, so that reduce error.
2. should avoid in the experimentation interrupting, prevent the reacting hole drying.
3. abundant mixing before all reagent use.
The antigenic peptide of p53 autoantibody identification of the present invention can be used for preparation and detect tumour serum p53 antibody kit and used for preparing vaccine.
The application of the antigenic peptide of p53 autoantibody identification of the present invention in preparation detection tumour serum p53 antibody test paper.
Advantage of the present invention and beneficial effect:
Key point of the present invention is to find linear antigenic determinant and its core sequence of the identification of p53 autoantibody, is difficult to accurate location with existing additive method.Those skilled in the art think that all the time the antigenic peptide epi-position of p53 autoantibody identification is at random, and those skilled in the art do not recognize that always the antigenic determinant of tumour serum autoantibody identification is to be gathered in several hot zones; The present invention when the antigenic peptide of preparation screening p53 autoantibody identification as can be known the antigenic peptide epi-position be not at random, and be that fixed is discerned some specific zone.The present invention has overcome those skilled in the art's prejudice just and has further finished.
The polypeptide array is mainly used the linear polypeptide of determining monoclonal antibody identification technically, and the serum autoantibody is a kind of polyclonal antibody.Difficult technically, the present invention is to synthetic spot diameter, and the improvement of serum dilution ratio, dilution reagent and hybridization conditions makes this method can be applied to the polyclonal antibody screening, success screened the linear antigenic determinant of serum antibody.The present invention can determine above-mentioned specific epitope by polypeptide chip, and can further accurately determine its core sequence.It is not only similar to existing ELISA test kit with specificity to utilize the antigenic peptide core sequence of the p53 autoantibody identification that the present invention obtains to prepare the application-aware degree of detection of lung cancer serum p53 autoantibody test kit, and its serum Dilution ratio is higher 10 times than available reagent box, (test kit is 1: 100, the present invention is 1: 1000), can reduce the cost of test kit greatly.Polypeptide preparation method of the present invention is easy, has good application and market outlook.Kit designed of the present invention is that the antigenic peptide that the p53 autoantibody is discerned is synthesized in 96 orifice plates, can reduce the synthetic expense of each sample, if mass production, each patient's testing cost will reduce half.Greatly reduce use cost.Simultaneously, because polypeptide is shorter, be not easy degraded, the validity period of test kit can extend to 2 years.Has very considerable utility value.
The foregoing invention content is described in detail technical scheme of the present invention, and in order to make the clearer understanding the present invention of those skilled in the art, existing the present invention will be further described with embodiment in conjunction with the accompanying drawings.
Description of drawings
Fig. 1 is 32 * 4 synoptic diagram for the polypeptide array;
Fig. 2 is s2, s5, s7, s10, s15 serum specimen epitope figure;
Fig. 3 is s27, s80, s90, s95, s105 serum specimen epitope figure;
Fig. 4 is determine (the SEQ ID No.4) of SEQ ID No.1 core sequence;
Fig. 5 is determine (the SEQ ID No.5) of SEQ ID No.2 core sequence;
Fig. 6 is determine (the SEQ ID No6) of SEQ ID No.3 core sequence;
Fig. 7 is p53 test kit 96 orifice plate synoptic diagram of the present invention;
Fig. 8 is the interpretation of test kit detected result; The concentration that can read p53 antibody according to the clean absorption value of testing sample from the typical curve.
Embodiment
This embodiment agents useful for same and equipment source:
1, reagent source:
(1) 20 of the FMOC-radical protection kinds of natural amino acid dry powder: Germany, intavis company;
(2) 1-Methyl-2-Pyrrolidone: 1-Methyl-2-pyrrolidone, 〉=99.5%, NMP, Sigma company;
(3) dimethyl formamide: Dimethylformamide, 〉=99.8%, DMF, Sigma company; (4) piperidines: Piperidine, 〉=99.0%, traditional Chinese medicines group;
(5) ethanol: Ethanol, 〉=99.9%, Sigma company;
(6) diacetyl oxide: Acetic anhydride, 〉=98.5%, traditional Chinese medicines group;
(7) trifluoroacetic acid Trifluoroacetic acid, 〉=99.5%, TFA, Sigma company;
(8) methylene dichloride: Dichlormethane, 〉=99.9%, DCM, Sigma company;
(9) tri isopropyl silane: Triisopropylsilane, 〉=99.8%, Sigma company;
(10) I-hydroxybenzotriazole: Hydroxybenzotriazole, HOBt, Shanghai gill biochemistry;
(11) di-isopropyl carbodiimide: Diisopropyl carbodiimide, 〉=98.0%, DIC, Sigma company;
(12) tetrabromophenol sulfonphthalein: Bromphenol blue, BPB, Sigma company;
(13) p53 monoclonal antibody Sc-53394:Santa Cruz, the U.S.;
(14) p53 polyclonal antibody: doctor's moral company, Wuhan;
(15) ELISA test kit QIA53:Merck, Germany;
(17) horseradish peroxidase-labeled mountain sheep anti-mouse igg (H+L) ZB-2305: company of middle China fir Golden Bridge;
(18) horseradish peroxidase-labeled goat anti-rabbit igg (H+L) ZB-2301: company of middle China fir Golden Bridge;
(19) the anti-human IgG of horseradish peroxidase-labeled goat (H+L) ZB-2304: company of middle China fir Golden Bridge;
(20) ECL developer: Pierce company, the U.S.;
Serum: serum specimen 59 examples before the nonsmall-cell lung cancer patient art that BJ Chest Science Hospital's thoracic surgery is in hospital, in normal healthy controls person's serum specimen 30 examples of Physical Check-Ups.
Peptide is synthetic: and the p53 antigenic peptide (Q-E-T-F-S-D-L-W-K-L-L-P, S-P-D-D-I-E-Q-W-F-T-E-D, V-V-P-Y-E-P-P-E-V)
2, major equipment:
ASP SL Peptide synthesizer Germany, intavis company
Nitrocellulose filter Germany, intavis company
TS-8 shaking table China, its woods Bel instrument manufacturing company
The screening of the linear antigen sequence of embodiment 1P53 autoantibody identification
1, the concrete implementation step of the screening process of core sequence:
According to the proteic aminoacid sequence of p53,3 amino acid in every interval adopt the some synthetic technology at the synthetic 12 galanin peptide chips in activated cellulose film surface.Because wild type p53 albumen contains 393 amino-acid residues,, promptly comprise 128 points on the polypeptide array so its total length comprises 12 galanin peptide chains of 128 overlapping 9 aminoacid sequences.Therefore, our the polypeptide array of design is 32 * 4, and promptly every row has 32 points, whenever shows 4 points, as shown in Figure 1.
According to the operation instructions of ASP SL Peptide synthesizer, the natural amino acid solution of 20 kinds of FMOC-radical protections is put into the position of correspondence on the machine.After preset program imported computer, Peptide synthesizer ASP SL (Germany, intavis company) can be under the control of MutilPep software program be added drop-wise to specific amino acid activatory nitrocellulose filter surface.Owing to will synthesize 12 galanin peptide chains; so carry out the reaction in 12 cycles, amino acid will pass through capping between each cycle, and a series of processes such as FMOC-group deprotection could combine with next amino acid; during last end cycle, amino acid whose side chain protected group be taken off.
The steps include: that Peptide synthesizer ASP SL is added drop-wise to certain location on the nitrocellulose filter with specified amino acid, 2% solution of acetic anhydride was washed film 5 minutes, and dimethyl formamide is washed film 5 times, each 2 minutes.20% piperidine solution is washed film and was sloughed the FMOC-blocking group in 10 minutes, and dimethyl formamide is washed film 5 times, each 2 minutes.Ethanol is washed film 2 times, each 2 minutes, dries.Repeat aforesaid operations, up to finishing 12 cycles, 20% piperidine solution is washed film 2 times during last end cycle, each 10 minutes.Dimethyl formamide is washed film 5 times, each 2 minutes.Ethanol is washed to dry behind the film 2 times and is spent the night.At last, the 10ml trifluoroacetic acid, 300 μ l methylene dichloride, 200 μ l water thorough mixing were washed film 1 hour with mixed solution, sloughed the side chain protected group, and methylene dichloride is washed film 5 times, each 2 minutes.Dimethyl formamide is washed film 5 times, each 2 minutes.Ethanol dries after washing film 2 times.The synthetic back of polypeptide chip is preserved standby-20 ℃ of sealings.
2, polypeptide chip and serum are hatched jointly:
(1) rehydration of film
1. film is immersed in 40ml 100% ethanol, shook on the shaking table 5 minutes.
2. film is immersed in 40ml 75% ethanol, shook on the shaking table 5 minutes.
3. film is immersed in 40ml 50% ethanol, shook on the shaking table 5 minutes.
4. add 150ml PBS and soak 30 minutes (fade be as the criterion fully with the white point on the film).
(2) sealing
5% skim-milk/PBS-T solution sealing, following 3 hours of room temperature.
(3) polypeptide array and sero-reaction.
1. film is put into a hybridization bag, by every cm
2Film adds 0.1ml serum, and the serum Dilution ratio is 1: 1000, removes all bubbles in the bag, seals up opening with film sealing machine, 4 ℃ of shaken overnight.
2. after reaction finishes, cut off hybridization bag, discard reaction solution.
(4) wash film
PBS-T 40ml washes film 6 times, each 10 minutes.
(5) second antibody reaction
1. film is put into hybridization bag, add the anti-human IgG solution of goat of dilution in 1: 10000, every cm
2Film adds 0.1ml.
2. seal hybridization bag, vibrated gently under the room temperature 2 hours.
(6) wash film
1. 40ml PBS-T washes film 3 times, each 10 minutes.
2. 40ml PBS washes film 3 times, each 10 minutes.
(7) development and image analysis.
Develop with ECL, exposure scans film, with Peptide AA V0.3.0 software system analysis image.
3, experimental result shows
Through the polypeptide array detection, in the 59 routine patients with lung cancer serum, 14 routine patients serum p53 antibody positives, all the other are negative, and positive rate is 23.73%; 30 routine normal healthy controls person's serum p53 antibody are all negative, and the result is identical with ELISA test kit detected result.Some common epitope of 14 routine patient p53 autoantibodies identification illustrates that the p53 autoantibody discerns some core sequence, rather than discerns the proteic any one section polypeptide of p53 at random.P53 autoantibody identification frequency antigens with higher epi-position among the 14 routine patients serums mainly contains three peptide sections, be respectively Q-E-T-F-S-D-L-W-K-L-L-P, S-P-D-D-I-E-Q-W-F-T-E-D, V-V-P-Y-E-P-P-E-V, sequence shown in SEQ No.1 promptly of the present invention, SEQ No.2 and the SEQ No.3.As Fig. 2, shown in Figure 3, s2, s5, s7, s10, s15, s27, s80, s90, s95, s105 represent different serum specimens respectively.P53 autoantibody identification frequency antigens with higher site is the 5th, 6,7 points as seen from the figure, the 15th, 16,17 points, the 72nd, 73 points, three polypeptide segments having represented this institute to propose respectively.More than some frequency that occurs the highest, these antigenic peptides of p53 autoantibody specific recognition are described.
Determining of embodiment 2p53 polypeptide core sequence
For determining the core sequence of SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 polypeptide, the contriver has used two kinds of methods: 1, based on full length sequence, reduce by an amino acid synthesizing series polypeptide successively from polypeptide N-terminal and C-terminal.Be used for determining the core sequence of sequence shown in SEQ ID No.1 and the SEQ ID No.3.2, at SEQ ID No.2 sequence, the contriver has adopted based on the 21 galanin peptide sequences that comprise core sequence, and 4-12 synthesis peptide library method is determined its core sequence.Synthetic series polypeptide is hatched, developed the color to determine the polypeptide core sequence according to above-mentioned experimental technique and positive serum.Two kinds of experimental techniques have successfully been determined the core sequence of three polypeptide.All experiments show simultaneously, add amino acids formed long peptide derived sequence at the core polypeptide two ends and also can be discerned by the P53 serum antibody, thereby can be used as the antigen prepd detection reagent.
See Fig. 4 for details, A1, B1 are SEQ ID NO.1 original series Q-E-T-F-S-D-L-W-K-L-L-P, and from amino acid of the terminal minimizing of the nitrogen of original series, B2-B10 reduces by an amino-acid residue from the one of carbon tip of original series to A2-A10 successively successively.Show with the positive serum results of hybridization: A1-4 is positive, and illustrates that the 4th amino acid of Q-E-T-F-S-D-L-W-K-L-L-P Polypetide Nitrogen end is that autoantibody identification is necessary; By that analogy, it is positive that B shows 4 points, illustrates that therefore the 4th amino-acid residue of polypeptide carbon teminal is that autoantibody identification is necessary, determine that the core sequence of this section polypeptide is: F-S-D-L-W-K.The result points out the 12 peptide A1-4 that comprise core sequence simultaneously, and B1-4 all can be by the clear antibody recognition of P53, thereby can be used as the antigen prepd detection reagent.
As shown in Figure 5, all amino acid that Fig. 5 comprises 15～18 correspondences of SEQ ID No.2 are 21 peptides, and its sequence is: L-M-L-S-P-D-D-I-E-Q-W-F-T-E-D-P-G-P-D-E-A (underscore partly is the No.2 sequence).
Each point that A is capable is the polypeptide segment of 12 peptides, and every adjacent two points move an amino-acid residue, up to comprising above-mentioned 21 amino-acid residues.Therefore, A is capable to have 10 points, and each some corresponding amino acid sequence is as follows.
A1 L-M-L-S-P-D-D-I-E-Q-W-F
A2 M-L-S-P-D-D-I-E-Q-W-F-T
A3 L-S-P-D-D-I-E-Q-W-F-T-E
A4 S-P-D-D-I-E-Q-W-F-T-E-D
A5 P-D-D-I-E-Q-W-F-T-E-D-P
A6 D-D-I-E-Q-W-F-T-E-D-P-G
A7 D-I-E-Q-W-F-T-E-D-P-G-P
A8 I-E-Q-W-F-T-E-D-P-G-P-D
A9 E-Q-W-F-T-E-D-P-G-P-D-E
A10 Q-W-F-T-E-D-P-G-P-D-E-A
In like manner, each point that B is capable is the polypeptide segment of 11 peptides, and every adjacent two points move-individual amino-acid residue, and up to comprising above-mentioned 21 amino-acid residues, therefore, B is capable to have 11 points.And the like, each point that C is capable is the polypeptide segment of 10 peptides, has 12 points; Each point that D is capable is the polypeptide segment of 9 peptides, has 13 points; Each point that G is capable is the polypeptide segment of 3 peptides, has 19 points.
Show that with the positive serum results of hybridization A is capable, and the 1st～8th point is positive, its consensus sequence is: I-E-Q-W-F.B is capable, and the 2nd～8th point is positive, C is capable, and the 3rd～8th point is positive, D is capable, and the 4th～8th point is positive, E is capable, and the 5th～8th point is positive, F is capable, and the 6th～8th point is positive, G is capable, and the 7th～8th point is positive, and its consensus sequence is: therefore I-E-Q-W-F, determine that the core sequence of polypeptide is: I-E-Q-W-F.
In addition, this experimental result be presented at A-G capable in, all comprise the polypeptide (6-12aa) of the different lengths of I-E-Q-W-F core sequence and can both be discerned by the serum autoantibody, the polypeptide of deriving that adds at the two ends of core sequence promptly that amino acid produces can be by the identification of blood-serum P 53 autoantibodies, thereby so these polypeptide can be as detecting antigen preparation detection reagent.
As shown in Figure 6, Fig. 6 A1, B1 are the 12 peptide sequence V-V-P-Y-E-P-P-E-V-G-S-D that comprise SEQ ID NO.3 original series V-V-P-Y-E-P-P-E-V, A2-A10 reduces by an amino acid from the one of carbon tip of original series successively, and B2-B10 is successively from amino-acid residue of the terminal minimizing of the nitrogen of original series.Show that with the positive serum results of hybridization A1-4 is positive, illustrate that the 4th amino acid of V-V-P-Y-E-P-P-E-V-G-S-D polypeptide carbon teminal is that autoantibody identification is necessary; By that analogy, it is positive that B shows 5 points, illustrates that the 5th amino-acid residue of Polypetide Nitrogen end is that autoantibody identification is necessary, therefore infers that the core sequence of this section polypeptide is: Y-E-P-P-E.Fig. 6 result also points out the 12 peptide A1-4 that comprise core sequence, and B1-5 all can be discerned by the P53 serum antibody, thereby can be used as the antigen prepd detection reagent.
The preparation and the using method of embodiment 3 p53 test kits of the present invention
Synthetic those skilled in the art of polypeptide of the present invention can entrust biotech firm to finish, and present embodiment is by Shanghai Huada Tianyuan Biotechnology Co.ltd's synthetic.
One, the preparation of test kit
1. according to above-mentioned core sequence synthetic antigen polypeptide.
2. use 0.05M, PH 9.0 carbonate bags are cushioned liquid antigenic peptide are diluted to 10 μ g/ml, add 0.1ml in the reacting hole of each enzyme plate, and 4 ℃ are spent the night.Discard solution in the hole next day, washes 3 times each 5 minutes with lavation buffer solution.
3. drying sealing preserves.
The preparation of reagent:
1. lavation buffer solution (PH7.4PBS 20 *): 3M
KH
2PO
40.2 gram
Na
2HPO
412H
2O 2.9 grams
NaCl 8.0 grams
KCl 0.2 gram
Tween-20?0.05％ 0.5ml
Adding distil water (should add the 950ml distilled water diluting during use to 50ml.)
2. diluent:
Bovine serum albumin (BSA) 0.1 gram
Add lavation buffer solution to 100ml
(3.TMB tetramethyl benzidine) solution:
TMB (10mg/5ml dehydrated alcohol) 1ml
Substrate buffer solution (PH5.5) 20ml
0.75％H
2O
2 64μl
4. stop buffer (2M H
2SO
4):
Distilled water 22.3ml dropwise adds the vitriol oil (98%) 2.7ml
5. the anti-human IgG of horseradish enzyme labelling.
6.p53 the human serum of negative antibody.
7. standard serum sample (the p53 antibody concentration is respectively 1-5-10-15U/ml).
Two, test kit using method:
Reagent is prepared before the experiment:
1.50ml reagent 1 adding distil water to 1000ml, dilutes at 1: 20.
2. 5 μ l test serum samples, reagent 6a～6e are added respectively in the 500 μ l reagent 2, fully mixing.
3. 24 μ l reagent 5 are added in the 12ml reagent 2, fully mixing.
The basic operational steps of this test kit is:
1. the test serum sample after will diluting, negative control sample, standard model add respectively in each reacting hole of enzyme plate, every hole 100 μ l.Add 100 μ l dilution buffer liquid in the blank hole, under 20-25 ℃ of condition, hatched 60 minutes.
2. each reacting hole of usefulness dcq buffer liquid cleaning of enzyme target is 4 times, each every hole 350 μ l.
3. the reagent 5 after will diluting adds each hole of enzyme plate, every hole 100 μ l.Under 20-25 ℃ of condition, hatched 60 minutes.
4. repeating step 2.
5. TMB solution is added in each hole of enzyme plate every hole 100 μ l.Under 20-25 ℃ of condition, lucifuge reaction 30 minutes.
6. each hole that reaction terminating liquid is added enzyme plate, every hole 100 μ l.Vibrate 30 seconds, fully mixing.
7. read the absorption value of each hole with microplate reader at 450nm.
Interpretation as a result:
1. read blank well respectively, negative control sample, standard model, the absorption value of test serum sample.
2. calculate the clean absorption value in each hole:
The absorption value of the absorption value-blank well of the clean absorption value=sample of sample.
3. the clean absorption value with standard model 6b～6e is an ordinate, is X-coordinate drawing standard curve with the concentration (1-5-10-15U/ml) of its p53 antibody.Typical curve is a linear regression curve, as shown in Figure 8.The concentration that can read p53 antibody according to the clean absorption value of testing sample from the typical curve.
Attention: the concentration that records p53 antibody for assurance is true, reliable, must satisfy several conditions:
1. the absorption value of blank well≤0.1.
2. absorption value≤2.5 of standard model 6e (15U/ml).
3. the p53 antibody concentration≤0.4U/ml of negative control sample.
4. if the p53 antibody concentration＞15U/ml of test serum sample should double to redeterminate after the dilution.
5. negative control sample 6a, standard model 6b～6e are only applicable to the test kit of same packing, and the test kit of Different Package can not be used with.
Use this test kit and detect the 59 routine nonsmall-cell lung cancer patients serum p53 autoantibodies for the treatment of in BJ Chest Science Hospital's thoracic surgery, 14 routine patient p53 antibody positives are arranged, positive rate is 23.73%.
Sequence table
＜110〉Yue Wen of Beijing Tuberculosis and Thoracic Tumor Research Institute great waves
＜120〉antigenic peptide of p53 autoantibody identification, test kit and the application in preparation detection tumor reagent box
<213>
＜400〉artificial sequence
QETFSDLWKL?LP 12
<212>PRT
<211>12
1
＜400〉artificial sequence
SPDDIEQWFT?ED 12
<212>PRT
<211>12
2
＜400〉artificial sequence
<212>PRT
<211>9
3
＜400〉artificial sequence
<212>PRT
<211>6
4
＜400〉artificial sequence
<212>PRT
<211>7
5
＜400〉artificial sequence
<212>PRT
<211>5
6
Claims (10)
1, a kind of antigenic peptide of p53 autoantibody identification is characterized in that its polypeptide active fragment is:
(1) sequence: F-S-D-L-W-K shown in the SEQ ID No.4;
(2) sequence: D-I-E-Q-W-F-T shown in the SEQ ID No.5;
(3) sequence: Y-E-P-P-E shown in the SEQ ID No.6.
2, the antigenic peptide of a kind of p53 autoantibody identification according to claim 1, it is characterized in that it is through replacement, disappearance or the interpolation of amino-acid residue and the amino acid derived sequence that forms by aminoacid sequence shown in SEQ ID No.4, SEQ ID No.5 and the SEQ ID No.6.
3, the antigenic peptide of a kind of p53 autoantibody identification according to claim 1 and 2 is characterized in that the derived sequence of polypeptide fragment (1) is SEQ ID No.1:Q-E-T-F-S-D-L-W-K-L-L-P.
4, the antigenic peptide of a kind of p53 autoantibody identification according to claim 1 is characterized in that the derived sequence of polypeptide fragment (2) is SEQ ID No.2:S-P-D-D-I-E-Q-W-F-T-E-D.
5, the antigenic peptide of a kind of p53 autoantibody identification according to claim 1 is characterized in that the derived sequence of polypeptide fragment (3) is SEQ ID No.3:V-V-P-Y-E-P-P-E-V.
6, the application of the antigenic peptide of any described p53 autoantibody identification in preparation detection tumour serum p53 antibody kit in the claim 1 to 5.
7, the application of the antigenic peptide of p53 autoantibody identification according to claim 6 in preparation detection tumour serum p53 antibody kit is characterized in that described tumour is a lung cancer.
8, a kind of detection tumour serum p53 antibody kit, it comprises bag by 96 orifice plates of specific polypeptide, dcq buffer liquid, the diluted sample damping fluid, tmb substrate solution, reaction terminating liquid, two of horseradish enzyme labelling resists negative control sample, standard model; It is characterized in that described negative control sample is the negative serum of p53 antibody; Standard model is respectively the serum of 1-5-10-15U/ml for the p53 antibody concentration; It in this test kit enzyme plate hole antigenic peptide as any described p53 autoantibody identification in the claim 1 to 5 through coating buffer and the processing of retardance liquid.
9, a kind of detection tumour serum p53 antibody kit according to claim 8 is characterized in that, the concentration of the antigenic peptide of described p53 autoantibody identification is: each reacting hole 10 μ g/ml.
10, according to Claim 8 or 9 described a kind of detection tumour serum p53 antibody kits, it is characterized in that described tumour is a lung cancer.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111175505A (en) * | 2020-01-08 | 2020-05-19 | 浙江省肿瘤医院 | P53 autoantibody detection kit and application thereof |
| CN112748242A (en) * | 2020-12-21 | 2021-05-04 | 珠海碳云智能科技有限公司 | Secondary antibody buffer confining liquid for polypeptide chip technology platform detection, kit comprising same and application thereof |
| CN113403286A (en) * | 2021-06-24 | 2021-09-17 | 新乡学院 | Targeted three-display phage and preparation method and application thereof |
| CN114019165A (en) * | 2022-01-05 | 2022-02-08 | 首都医科大学附属北京妇产医院 | Polypeptide chip or kit and application thereof in diagnosing non-small cell lung cancer |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100491399C (en) * | 2005-12-27 | 2009-05-27 | 东北师范大学 | Hybrid protein of p53 protein epitope and filobactivirus gene 8 protein and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111175505A (en) * | 2020-01-08 | 2020-05-19 | 浙江省肿瘤医院 | P53 autoantibody detection kit and application thereof |
| CN112748242A (en) * | 2020-12-21 | 2021-05-04 | 珠海碳云智能科技有限公司 | Secondary antibody buffer confining liquid for polypeptide chip technology platform detection, kit comprising same and application thereof |
| CN113403286A (en) * | 2021-06-24 | 2021-09-17 | 新乡学院 | Targeted three-display phage and preparation method and application thereof |
| CN113403286B (en) * | 2021-06-24 | 2024-01-16 | 新乡学院 | Targeting three-display phage and preparation method and application thereof |
| CN114019165A (en) * | 2022-01-05 | 2022-02-08 | 首都医科大学附属北京妇产医院 | Polypeptide chip or kit and application thereof in diagnosing non-small cell lung cancer |
| CN114019165B (en) * | 2022-01-05 | 2022-04-01 | 首都医科大学附属北京妇产医院 | Polypeptide chip or kit and application thereof in diagnosing non-small cell lung cancer |
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