CN103048458A - ELISA (enzyme-linked immunosorbent assay) method for quickly testing C-Myc by using monoclonal antibody - Google Patents

Abstract

The invention relates to an ELISA (enzyme-linked immunosorbent assay) method for quickly testing C-Myc by using a monoclonal antibody. According to the method, prokaryotic expression C-Myc protein is purified through protein purification technology; immune mouse spleen cells are performed cell fusion with SP2/0 myeloma cell, and a clone capable of stably secreting an antibody against C-myc IgG1 and a Cmyc IgM antibody clone are obtained based on ELISA screening and the subtype identification of subclone and monoclonal antibody Ig. A sandwich ELISA method and the two direct ELISA methods for quickly testing c-Myc are built by using an antibody against c-Myc IgG1 and an antibody against c-Myc Ig. With the method, a C-myc protein sample is accurately detected, the detected C-myc protein concentration range is 0.01-1000 pmol/L, and the detection limit is 0.01-0.05 pmol/L; the specificity and the sensitivity of C-Myc detection are greatly improved, the operation is simple, and the repeatability is high; and the method detects the expression level of C-Myc in tumor tissues, and provides experimental data for screening of malignant tumor.

Description

Utilize the ELISA method of monoclonal antibody fast detecting C-Myc

Technical field

The present invention relates to a kind of lesion detection kit developing field, more particularly, relate to a kind of ELISA method of utilizing monoclonal antibody fast detecting C-Myc.

Background technology

But proto-oncogene c-myc is a kind ofly to make the cell infinite multiplication, obtain the immortalization function, promote fissional regulatory gene, high expressed all in a lot of tumours ^[1]In cancerous tumor cell, the C-myc protein abnormal expression increases, and makes the adjusting restriction of cell detachment normal growth, transforms to the malignant phenotype, and canceration occurs.It is reported, excessively expressing with tumor development of C-myc gene is closely related, such as leukaemia, lung cancer, liver cancer, cancer of the stomach, breast cancer, colon cancer, cervical carcinoma, some neuroblast is sick, retinoblastoma, osteogenic sarcoma, chondrosarcoma, chordoma, embryonal-cell lipoma, rhabdomyosarcoma, Hodgkin's disease and head tumor etc. have amplification or the overexpression of C-myc gene ^[2]C-myc gene overexpression in all bone-marrow-derived lymphocyte leukaemia tissues particularly ^[3]Compare with normal cell, the expression of C-myc increases tens times in the tumour cell, is the important symbol that malignant tumour occurs.

At present, article and the patent of relevant C-Myc research are as follows:

（1）Dang?CV,Resar?LM,Emison?E,et?al.Function?of?the?C-myc?oncogenictranscription?factor[J].Exp?Cell?Res,1999,253(1):63-77.

（2）Riou?GF,Bourhis?J,Le?MG.The?C-myc?proto-oncogene?in?invasive?carcinomasof?the?uterine?cervix:clinical?relevance?of?over?expression?in?early?stages?of?thecancer[J].Anticancer?Res,1990,9-10(5A):1225-1231.

（3）May?PC,Foot?N,Dunn?R,et?al.Detection?of?cryptic?and?variant?IgH-MYCrearrangements?in?high-grade?non-Hodgkin's?lymphoma?by?fluorescence?in?situhybridization:implications?for?cytogenetic?testing[J].Cancer?Genet?Cytogenet,2010,198(1):71-75.

(4) for detection of the LSPR sensing chip (patent publication No.: CN102175649A) of oncogene C-myc recombinant protein

(5) a kind of fluorescence spectrometry method (patent publication No.: CN101893573A) of oncogene C-myc protein

Article 1-3 mainly study about the meaning of the high expressed of C-Myc in tumor tissues with and mechanism.

Patent 4 inventions provide a kind of LSPR sensing chip based on local surface plasma resonance (LSPR) spectral detection oncogene C-myc recombinant protein.Golden film surface-assembled last layer DTT unimolecular layer at the LSPR sensing chip, connect the golden nanometer particle layer, 3-mercaptopropionic acid unimolecular layer in the finishing of described golden nanometer particle layer, then activate by DMAP-EDC, 3-mercaptopropionic acid unimolecular layer is combined with the C-myc monoclonal antibody, be combined with the immune response of C-myc recombinant protein by the C-myc monoclonal antibody, cause the displacement that the golden nanometer particle surface produces the local surface plasma resonance absorption peak, detect the content of C-myc recombinant protein in the cancerous issue with this.

Patent 5 disclosure of the invention a kind of fluorescence spectrometry method of oncogene C-myc protein, it is measured according to being that C-myc albumen itself has fluorescence and is combined rear generation Fluorescence-quenching with gold nano colloidal sol, this fluorescent quenching intensity (Δ F) and C-myc protein concentration are linear corresponding relation between changing, and reach the purpose of measuring the C-myc protein content.

At present, utilize the ELISA of two kinds of monoclonal antibody fast detecting c-Myc to there is no report.

Summary of the invention

The present invention develops a kind of ELISA that utilizes monoclonal antibody fast detecting C-Myc, is intended to obtain stably excreting specificity and the very high anti-C-myc IgG of susceptibility ₁Monoclonal antibody and IgM monoclonal antibody, for kinds of tumors particularly the exploitation of bone-marrow-derived lymphocyte leukemia diagnosis kit solid data basis is provided.

In order to achieve the above object, a kind of ELISA method of utilizing monoclonal antibody fast detecting C-Myc of the present invention comprises the steps:

S1, preparation C-myc antigen, and purifying, the C-myc albumen of acquisition purifying;

S2, utilize the C-myc albumen of described purifying to carry out immune health Balb/c mouse;

S3, immune Balb/c mouse boosting cell and SP2/0 myeloma cell are carried out Fusion of Cells;

S4, screening positive hybridoma cell;

S5, described positive hybridoma cell is carried out subclone, obtaining 2 strains can the anti-C-myc IgG of stably excreting ₁The clone and the monoclonal antibody hybridoma cell strain of anti-C-myc IgM;

Two kinds of described monoclonal antibody hybridoma cell strains of injection prepare monoclonal antibody in a large number in S6, the Balb/c mouse peritoneal;

S7, select in following three kinds of methods any one, realize fast detecting C-Myc:

Method 1: utilize two kinds of anti-c-Myc monoclonal antibodies to detect the sandwich method ELISA method of tumour cell c-Myc expression.

Method 2: utilize anti-c-Myc IgM monoclonal antibody to detect the Direct ELISA method of tumour cell c-Myc expression.

Method 3: utilize anti-c-Myc IgG monoclonal antibody to detect the Direct ELISA method of tumour cell c-Myc expression.

Under the optimal way, the concrete substep of step S1 is:

S11, the pET20b-C-myc that will contain prokaryotic expression carrier express bacterium E.coli BL21 and cultivate 170rpm 37 ℃ of concussions;

S12, after IPTG induces, collect thalline, nickel pillar chromatographic purifying is smashed, utilized to ultrasound wave.

Specifically, the concrete grammar of step S1 is: will contain prokaryotic expression carrier pET20b-C-myc and express the E.coli BL21 of bacterium at 37 ℃ of concussion cultivations, 170rpm; Bacterium liquid OD _600nmWhen value is 0.4-0.6, after 1-2mM IPTG induces 1-3 hour, the centrifugal 10min collecting cell of 6500rmp, suspend, broken Ultrasonic Cell Disruptor smudge cells 10min, 2-8M urea carries out repeatability behind the sex change 2h, with nickel ion affinity chromatograph post purifying protein, obtains at last the C-Myc albumen of the above purity of 90-95% through the AKATA instrument.

Under the optimal way, above-mentioned steps S2 concrete steps are: utilize the C-myc albumen of prokaryotic expression and purifying and isopyknic Freund's complete adjuvant fully emulsified, antigen is injected in the healthy Balb/c mouse peritoneal, 0.4ml/, the antigen amount is 50-100 μ g/; After the first immunisation 14 days, use incomplete Freunds adjuvant instead and carry out the 2nd immunity with the antigen amount of volume is fully emulsified, the antigen amount is constant; The 28th day, according to g/ antigen amount of 100-200 μ mouse is carried out for the third time immunity, the antibody titer of mouse has reached 1:64000-1: 1280000.

Under the optimal way, above-mentioned steps S3 concrete steps are: Balb/c mouse boosting cell and SP2/0 myeloma cell are mixed in the 50ml centrifuge tube with the ratio of 5:1-10:1,1200rpm, 5min are centrifugal, abandon supernatant, 50% the PEG1000 that splashes into rapidly 37 ℃ of preheatings in 25s-30s is 0.5ml and mixing gently approximately, 700rpm, 2min are centrifugal, the RPMI-1640 basal medium 15ml that slowly adds again 37 ℃ of preheatings along tube wall, test tube is done draw a circle sample approximately 2min that moves, centrifugal 1400rpm, 5min, abandon supernatant, clean again 1 time with method.The HT nutrient culture media 30ml that slowly adds 37 ℃ of preheatings along tube wall, gently mixing fused cell.

Under the optimal way, above-mentioned steps S5 concrete steps are: when hybridoma colony growth 40-60% is above, screen positive hole with the ELISA method; For positive hole, with HT nutrient culture media diluting cells, carry out subclone.Cultivated 8-9 days, and the supernatant in the monoclonal cell hole of mark was carried out ELISA detect screening positive clone.

Under the optimal way, above-mentioned steps S6 concrete steps are: the saxol in the Healthy female Balb/c mouse peritoneal behind the injection autoclaving, 0.4-0.6ml/ are only.After one week, to the hybridoma of its lumbar injection energy stably excreting C-myc antibody, 0.5ml/, cell number is about 2-5 * 10 again ⁶Individual; After 10-14 days, treat that mouse web portion obviously expands and when being slow in action, collect ascites, 3000rpm, 10min are centrifugal, discard fat and paraffin oil, collect middle level clarification ascites, and-20 ℃ frozen for subsequent use.2B2 and 2A9 clone's mouse ascites is tired and is respectively 1:64000,1:32000.

Under the optimal way, the concrete steps of method 1 are among the above-mentioned steps S7: be coated with the monoclonal antibody hybridoma cell strain of anti-C-myc IgM with the carbonate buffer solution dilution of pH 9.6, make concentration reach 5-20 μ g/ml; With PBS-T washing, 200 μ l/ holes, dry behind each 30s and wash plate liquid, triplicate; Add the tested tumour cell lysate that 1:10-1:50 doubly dilutes, 37 ℃ of reaction 1h; Wash with PBS-T; Add confining liquid 200 μ l/ holes, 37 ℃ of wet box temperature are bathed 1h; The monoclonal antibody hybridoma cell strain that adds the anti-C-myc IgM of 1:1000-1:2000 doubling dilution, 100 μ l/ holes, 37 ℃ of wet box temperature are bathed 1h; Wash with PBS-T; HRP enzyme mark goat anti-mouse igg is done the 1:5000 dilution, and 100 μ l/ holes add ELISA Plate, and 37 ℃ of wet box temperature are bathed 1h; Wash with PBS-T; Add o-phenylenediamine liquid 100 μ l/ holes, 37 ℃ of lucifuge reaction 15min; Add stop buffer, the 2mol/L sulfuric acid solution, 100 μ l/ holes, microplate reader is measured OD _492nmValue.

Under the optimal way, the concrete steps of method 2 are among the above-mentioned steps S7: the carbonate buffer solution dilution with pH 9.6 is coated with the tested tumour cell lysate that 1:10-1:50 doubly dilutes.With PBS-T washing, 200 μ l/ holes, dry behind each 30s and wash plate liquid, triplicate; Add confining liquid 200 μ l/ holes, 37 ℃ of temperature are bathed 1h; The anti-C-Myc IgM(2A9 that adds the 1:1000-1:2000 doubling dilution) 100 μ l/ holes, 37 ℃ of temperature are bathed 1h; Wash with PBS-T; HRP enzyme mark goat-anti Mouse IgM is done the 1:5000-1:10000 dilution, and 100 μ l/ holes add ELISA Plate, and 37 ℃ of temperature are bathed 1h; Wash with PBS-T; Add o-phenylenediamine (OPD) liquid 100 μ l/ holes, 37 ℃ of lucifuge reaction 15min; Add stop buffer (2mol/L sulfuric acid solution) 100 μ l/ holes, microplate reader is measured OD _492nmValue.

Under the optimal way, the concrete steps of method 3 are among the above-mentioned steps S7: the carbonate buffer solution dilution with pH 9.6 is coated with the tested tumour cell lysate that 1:10-1:50 doubly dilutes.37 ℃ of reaction 1h; Wash with PBS-T; Add confining liquid 200 μ l/ holes, 37 ℃ of temperature are bathed 1h; The anti-c-Myc IgG(2B2 that adds the 1:1000-1:2000 doubling dilution) 100 μ l/ holes, 37 ℃ of temperature are bathed 1h; Wash with PBS-T; HRP enzyme mark goat anti-mouse igg is done the 1:5000 dilution, and 100 μ l/ holes add ELISA Plate, and 37 ℃ of temperature are bathed 1h; Wash with PBS-T; Add o-phenylenediamine (OPD) liquid 100 μ l/ holes, 37 ℃ of lucifuge reaction 15min; Add stop buffer (2mol/L sulfuric acid solution) 100 μ l/ holes, microplate reader is measured OD _492nmValue.

The present invention utilizes the sandwich method ELISA of monoclonal antibody fast detecting C-Myc, at first by the protein purification technology prokaryotic expression C-Myc albumen is purified.The mouse boosting cell and the SP2/0 myeloma cell that utilize purifying C-myc to carry out immunity carry out Fusion of Cells, through the screening of ELISA method, and subclone and the evaluation of monoclonal antibody Ig hypotype, obtaining 2 strains can the anti-C-myc IgG of stably excreting ₁Clone (2B2) and anti-C-myc IgM(2A9) clone.Utilize anti-C-myc IgG ₁Antibody and anti-C-myc IgM antibody are set up the sandwich method ELISA of fast detecting c-Myc.Use the present invention the C-myc protein sample is carried out Accurate Determining, detectable C-myc protein concentration scope is 0.01～1000pmol/L, detects down and is limited to 0.01～0.05pmol/L.The present invention has improved specificity and the susceptibility that detects C-Myc greatly, and is simple to operate, repeatability is high, and the present invention detects the expression of the C-Myc in the tumor tissues, for the screening of malignant tumour provides experimental data.The present invention can be developed to the sandwich method ELISA that utilizes monoclonal antibody fast detecting C-Myc in the novel detection tumor reagent box with independent intellectual property right and push industrialization to, benefits the whole mankind, has great social effect and market significance widely.

Description of drawings

Fig. 1 is C-myc protein purification figure; Wherein, swimming lane 1 is protein marker; Swimming lane 2 is cellular lysate liquid supernatant; Swimming lane 3 is cellular lysate liquid inclusion body; The C-myc albumen of purifying;

Fig. 2 is the antibody titer in the serum after 3 immunity of mouse;

Fig. 3 is the hybridoma cell strain (200 times) that merged the 9th day;

Fig. 4 is that the hypotype of monoclonal antibody is identified;

Fig. 5 is the comparison of measuring c-Myc content in health tissues and the tumor tissues.

Embodiment

Technical scheme of the present invention is: the present invention is prokaryotic expression c-Myc albumen at first, passes through the protein purification technology with c-Myc protein extraction, purifying again.The mouse boosting cell and the SP2/0 myeloma cell that utilize purifying C-myc to carry out immunity carry out Fusion of Cells, through the screening of ELISA method, and subclone and the evaluation of monoclonal antibody Ig hypotype, obtaining 2 strains can the anti-C-myc IgG of stably excreting ₁Clone (2B2) and anti-C-myc IgM(2A9) clone.Utilize anti-C-myc IgG ₁Monoclonal antibody and anti-C-myc IgM monoclonal antibody are set up the sandwich method ELISA of fast detecting c-Myc.

Embodiment 1:

The concrete grammar step is as follows:

1.C-myc the preparation and purification of antigen

Bacterium liquid OD _600nmValue is 0.4 o'clock, after IPTG induces 1 hour, the centrifugal 10min collecting cell of 6500rmp, suspend, Ultrasonic Cell Disruptor smudge cells (20% power, broken 10min), 2-8M urea carry out repeatability behind the sex change 2h, through AKATA instrument nickel ion affinity chromatograph post purifying protein, obtain at last the c-Myc albumen (Fig. 1) of 95% above purity.

2. the preparation of monoclonal antibody

2.1 animal immune

Utilize C-myc albumen and isopyknic Freund's complete adjuvant of prokaryotic expression and purifying fully emulsified, antigen is injected in the healthy Balb/c mouse peritoneal, 0.4ml/, the antigen amount is 50 μ g/; After the first immunisation 14 days, use incomplete Freunds adjuvant instead and carry out the 2nd immunity with the antigen amount of volume is fully emulsified, the antigen amount is constant; The 28th day, according to 100 a μ g/ antigen amount mouse is carried out booster immunization.After the immunity, the antibody titer of mouse has reached 1:640000, sees Fig. 2 for the third time, and the immune programme for children that presentation of results is set up is rationally effective.

2.2 Fusion of Cells

Be mixed in the 50ml centrifuge tube with the ratio of 10:1 with SP2/0 myeloma cell after collecting immune Balb/c mouse boosting cell, 1200rpm, 5min are centrifugal, abandon supernatant, 50% the PEG1000 that splashes into rapidly 37 ℃ of preheatings when 25s is 0.5ml and mixing gently approximately, and 700rpm, 2min are centrifugal, slowly add the RPMI-1640 basal medium 15ml of 37 ℃ of preheatings along tube wall again, test tube is done draw a circle sample approximately 2min that moves, 1400rpm, 5min are centrifugal, abandon supernatant, clean 1 time with method again.Slowly add the HT nutrient culture media 30ml of 37 ℃ of preheatings along tube wall, the mixing fused cell is added drop-wise in 96 orifice plates that are covered with in advance the trophocyte gently, and 100 μ l/ holes place 37 ℃, 5%CO ₂Incubator is cultivated.Add HAT nutrient culture media [complete medium that contains hypoxanthine (H), aminopterin (A), thymidine (T) and glycocoll] 50 μ l/ holes next day; The 4th day, every hole was inhaled and is abandoned 200 μ l, adds HAT nutrient culture media 100 μ l/ holes again; The 7th day, 100 μ l HAT nutrient culture media were added in every hole again.The last merging the 9th day, Jing Xiahui observed and does not merge or splenocyte and SP2/0 myeloma cell that self merges can be all dead, and hybridoma survival (Fig. 3).

2.3 the screening of positive hybridoma cell and subclone

When hybridoma colony growth 50% is above, screen positive hole with the ELISA method.For positive hole, with HT nutrient culture media [nutrient culture media that contains hypoxanthine (H), thymidine (T)] diluting cells, be added drop-wise in another 96 orifice plates that have been covered with trophocyte, carry out subclone.Subclone the 5th day, the hole and half amount that are marked with monoclonal cell are changed the HT nutrient culture media.The 9th day, the supernatant in the monoclonal cell hole of mark is carried out ELISA detect, obtain the monoclonal antibody hybridoma cell strain that 2 strains can the anti-C-myc of stably excreting, 2B2 and 2A9 clone.

Detect through the Mouse Monoclonal Antibody Isotyping Reagents with Sigma, the Antibody types that demonstrates 2B2 and 2A9 clone is respectively IgG ₁Subclass and IgM(Fig. 4).

2.4 a large amount of preparations of monoclonal antibody

Saxol in Healthy female Balb/c mouse peritoneal behind the injection autoclaving, 0.4ml/ are only.After one week, to the hybridoma of its lumbar injection energy stably excreting C-myc antibody, only (cell number is about 2 * 10 to 0.5ml/ again ⁶Individual).After 10-14 days, treat that mouse web portion obviously expands and when being slow in action, collect ascites, 3000rpm, 10min are centrifugal, discard fat and paraffin oil, collect middle level clarification ascites, and-20 ℃ frozen for subsequent use.2B2 and 2A9 clone's mouse ascites is tired and is respectively 1:64000,1:32000, shows that the monoclonal antibody of acquisition has higher susceptibility.

3 utilize two kinds of anti-c-Myc monoclonal antibodies to detect the sandwich method ELISA method of tumour cell c-Myc expression

3.1 coated

Coated anti-c-Myc IgM(2A9), make concentration reach 5 μ g/ml, every hole 50 μ l coated elisa plates, 37 ℃ of reaction 1-2h.

3.2 wash plate

Dry coating buffer, with PBST washing, 200 μ l/ holes, dry behind each 30s and wash plate liquid, triplicate.

3.3 sealing

Add confining liquid 200 μ l/ holes, 37 ℃ of wet box temperature are bathed 30min.

3.4 wash plate

With PBST washing, 200 μ l/ holes, dry behind each 30s and wash plate liquid, triplicate.

3.5 add tested tumour cell Raji cell lysate

Add the tested tumour cell lysate that 1:10 doubly dilutes.37 ℃ of reaction 1h.Negative control is the normal cell lysate; Positive control is the Raji cell pyrolysis liquid.

3.6 wash plate

With PBST washing, 200 μ l/ holes, dry behind each 30s and wash plate liquid, triplicate.

3.7 primary antibodie reaction

The anti-C-myc IgM(2B2 that adds the 1:1000 doubling dilution) 100 μ l/ holes, 37 ℃ of wet box temperature are bathed 1h.

3.8 wash plate

Dry primary antibodie, with PBST washing, 200 μ l/ holes, dry behind each 30s and wash plate liquid, triplicate.

3.9 two anti-reactions

HRP enzyme mark goat anti-mouse igg is done the 1:5000 dilution, and 100 μ l/ holes add ELISA Plate, and 37 ℃ of wet box temperature are bathed 1h.

3.10 wash plate

Dry two and resist, with PBST washing, 200 μ l/ holes, dry behind each 30s and wash plate liquid, triplicate.

3.11 substrate

Add o-phenylenediamine (OPD) liquid 100 μ l/ holes, 37 ℃ of lucifuge reaction 15min.

3.12 stop

Add stop buffer (2mol/L sulfuric acid solution) 100 μ l/ holes, microplate reader is measured OD _492nmValue.

This experiment preparation bone-marrow-derived lymphocyte leukaemia lysate carries out the sandwich method ELISA experiment, measures c-Myc content in health tissues and the tumor tissues.Experimental result is with the poor expression of mean ± standard, and * * represents and P＜0.01 relatively of immune group not, the results are shown in Figure 5.As seen from Figure 5, c-Myc content is apparently higher than health tissues in the tumor tissues, and difference has statistical significance (P＜0.01).

Anti-C-Myc IgM(2A9 is used in this experiment) and anti-c-Myc IgG1(2B2) c-Myc antigen in the monoclonal antibody fast detecting tumour cell, having high degree of specificity and susceptibility, required time is 4.5-6h, the positive detection rate reaches 100%.Detectable c-Myc protein concentration scope is 0.01～1000pmo l/L, detects down and is limited to 0.01～0.05pmo l/L.

Embodiment 2:

The concrete grammar step is as follows:

1.C-myc the preparation and purification of antigen

Bacterium liquid OD _600nmValue is 0.6 o'clock, after IPTG induces 1 hour, the centrifugal 10min collecting cell of 6500rmp, suspend, Ultrasonic Cell Disruptor smudge cells (20% power, broken 10min), 2-8M urea carry out repeatability behind the sex change 2h, through AKATA instrument nickel ion affinity chromatograph post purifying protein, obtain at last the c-Myc albumen (Fig. 1) of 95% above purity.

2. the preparation of monoclonal antibody

2.1 animal immune

Utilize C-myc albumen and isopyknic Freund's complete adjuvant of prokaryotic expression and purifying fully emulsified, antigen is injected in the healthy Balb/c mouse peritoneal, 0.6ml/, the antigen amount is 100 μ g/; After the first immunisation 14 days, use incomplete Freunds adjuvant instead and carry out the 2nd immunity with the antigen amount of volume is fully emulsified, the antigen amount is constant; The 28th day, according to 200 a μ g/ antigen amount mouse is carried out booster immunization.After the immunity, the antibody titer of mouse has reached 1:1280000, sees Fig. 2 for the third time, and the immune programme for children that presentation of results is set up is rationally effective.

2.2 Fusion of Cells

Be mixed in the 50ml centrifuge tube with the ratio of 5:1 with SP2/0 myeloma cell after collecting immune Balb/c mouse boosting cell, 1200rpm, 5min are centrifugal, abandon supernatant, 50% the PEG1000 that splashes into rapidly 37 ℃ of preheatings when 30s is 0.5ml and mixing gently approximately, and 700rpm, 2min are centrifugal, slowly add the RPMI-1640 basal medium 15ml of 37 ℃ of preheatings along tube wall again, test tube is done draw a circle sample approximately 2min that moves, 1400rpm, 5min are centrifugal, abandon supernatant, clean 1 time with method again.Slowly add the HT nutrient culture media 30ml of 37 ℃ of preheatings along tube wall, the mixing fused cell is added drop-wise in 96 orifice plates that are covered with in advance the trophocyte gently, and 100 μ l/ holes place 37 ℃, 5%CO ₂Incubator is cultivated.Add HAT nutrient culture media 50 μ l/ holes next day; The 4th day, every hole was inhaled and is abandoned 200 μ l, adds HAT nutrient culture media 100 μ l/ holes again; The 7th day, 100 μ l HAT nutrient culture media were added in every hole again.The last merging the 9th day, Jing Xiahui observed and does not merge or splenocyte and SP2/0 myeloma cell that self merges can be all dead, and hybridoma survival (Fig. 3).

2.3 the screening of positive hybridoma cell and subclone

When hybridoma colony growth 50% is above, screen positive hole with the ELISA method.For positive hole, with HT nutrient culture media diluting cells, be added drop-wise in another 96 orifice plates that have been covered with trophocyte, carry out subclone.Subclone the 5th day, the hole and half amount that are marked with monoclonal cell are changed the HT nutrient culture media.The 9th day, the supernatant in the monoclonal cell hole of mark is carried out ELISA detect, obtain the monoclonal antibody hybridoma cell strain that 2 strains can the anti-C-myc of stably excreting, 2B2 and 2A9 clone.

Detect through the Mouse Monoclonal Antibody Isotyping Reagents with Sigma, the Antibody types that demonstrates 2B2 and 2A9 clone is respectively IgG ₁Subclass and IgM(Fig. 4).

2.4 a large amount of preparations of monoclonal antibody

Saxol in Healthy female Balb/c mouse peritoneal behind the injection autoclaving, 0.6ml/ are only.After one week, to the hybridoma of its lumbar injection energy stably excreting C-myc antibody, only (cell number is about 5 * 10 to 0.5ml/ again ⁶Individual).After 10-14 days, treat that mouse web portion obviously expands and when being slow in action, collect ascites, 3000rpm, 10min are centrifugal, discard fat and paraffin oil, collect middle level clarification ascites, and-20 ℃ frozen for subsequent use.2B2 and 2A9 clone's mouse ascites is tired and is respectively 1:64000,1:32000, shows that the monoclonal antibody of acquisition has higher susceptibility

3 utilize anti-c-Myc IgM monoclonal antibody to detect the Direct ELISA method of tumour cell c-Myc expression

3.1 coated

The tested tumour cell lysate of coated 0.01～1000pmo l/L.37 ℃ of reaction 1h.Negative control is the normal cell lysate; Positive control is the Raji cell pyrolysis liquid.

3.2 wash plate

Dry coating buffer, with PBST washing, 200 μ l/ holes, dry behind each 30s and wash plate liquid, triplicate.

3.3 sealing

Add confining liquid 200 μ l/ holes, 37 ℃ of wet box temperature are bathed 30min.

3.4 wash plate

With PBST washing, 200 μ l/ holes, dry behind each 30s and wash plate liquid, triplicate.

3.5 primary antibodie reaction

Add the anti-C-Myc IgM(2A9 that 1:2000 doubly dilutes) 100 μ l/ holes, 37 ℃ of temperature are bathed 1h.

3.6 wash plate

Dry primary antibodie, with PBST washing, 200 μ l/ holes, dry behind each 30s and wash plate liquid, triplicate.

3.7 two anti-reactions

HRP enzyme mark sheep anti mouse IgM is done the 1:5000 dilution, and 100 μ l/ holes add ELISA Plate, and 37 ℃ of wet box temperature are bathed 1h.

3.8 wash plate

Dry two and resist, with PBST washing, 200 μ l/ holes, dry behind each 30s and wash plate liquid, triplicate.

3.9 substrate

Add substrate solution 100 μ l/ holes, 37 ℃ of lucifuge reaction 15min.

3.10 stop

Add stop buffer 100 μ l/ holes, microplate reader is measured OD _492nmValue.

The anti-C-Myc IgM(2A9 of this experiment use) the c-Myc antigen in the monoclonal antibody fast detecting tumour cell has high degree of specificity and susceptibility, and required time is 2.5-3h, and the positive detection rate reaches 100%.Detectable c-Myc protein concentration scope is 0.01～1000pmo l/L, detects down and is limited to 0.01～0.05pmol/L.

Embodiment 3:

The concrete grammar step is as follows:

1.C-myc the preparation and purification of antigen

Bacterium liquid OD _600nmValue is 0.6 o'clock, after IPTG induces 1 hour, the centrifugal 10min collecting cell of 6500rmp, suspend, Ultrasonic Cell Disruptor smudge cells (20% power, broken 10min), 2-8M urea carry out repeatability behind the sex change 2h, through AKATA instrument nickel ion affinity chromatograph post purifying protein, obtain at last the c-Myc albumen (Fig. 1) of 95% above purity.

2. the preparation of monoclonal antibody

2.1 animal immune

Utilize C-myc albumen and isopyknic Freund's complete adjuvant of prokaryotic expression and purifying fully emulsified, antigen is injected in the healthy Balb/c mouse peritoneal, 0.6ml/, the antigen amount is 100 μ g/; After the first immunisation 14 days, use incomplete Freunds adjuvant instead and carry out the 2nd immunity with the antigen amount of volume is fully emulsified, the antigen amount is constant; The 28th day, according to 200 a μ g/ antigen amount mouse is carried out booster immunization.After the immunity, the antibody titer of mouse has reached 1:1280000, sees Fig. 2 for the third time, and the immune programme for children that presentation of results is set up is rationally effective.

2.2 Fusion of Cells

Be mixed in the 50ml centrifuge tube with the ratio of 5:1 with SP2/0 myeloma cell after collecting immune Balb/c mouse boosting cell, 1200rpm, 5min are centrifugal, abandon supernatant, 50% the PEG1000 that splashes into rapidly 37 ℃ of preheatings when 30s is 0.5ml and mixing gently approximately, and 700rpm, 2min are centrifugal, slowly add the RPMI-1640 basal medium 15ml of 37 ℃ of preheatings along tube wall again, test tube is done draw a circle sample approximately 2min that moves, 1400rpm, 5min are centrifugal, abandon supernatant, clean 1 time with method again.Slowly add the HT nutrient culture media 30ml of 37 ℃ of preheatings along tube wall, the mixing fused cell is added drop-wise in 96 orifice plates that are covered with in advance the trophocyte gently, and 100 μ l/ holes place 37 ℃, 5%CO ₂Incubator is cultivated.Add HAT nutrient culture media 50 μ l/ holes next day; The 4th day, every hole was inhaled and is abandoned 200 μ l, adds HAT nutrient culture media 100 μ l/ holes again; The 7th day, 100 μ l HAT nutrient culture media were added in every hole again.The last merging the 9th day, Jing Xiahui observed and does not merge or splenocyte and SP2/0 myeloma cell that self merges can be all dead, and hybridoma survival (Fig. 3).

2.3 the screening of positive hybridoma cell and subclone

When hybridoma colony growth 50% is above, screen positive hole with the ELISA method.For positive hole, with HT nutrient culture media diluting cells, be added drop-wise in another 96 orifice plates that have been covered with trophocyte, carry out subclone.Subclone the 5th day, the hole and half amount that are marked with monoclonal cell are changed the HT nutrient culture media.The 9th day, the supernatant in the monoclonal cell hole of mark is carried out ELISA detect, obtain the monoclonal antibody hybridoma cell strain that 2 strains can the anti-C-myc of stably excreting, 2B2 and 2A9 clone.

Detect through the Mouse Monoclonal Antibody Isotyping Reagents with Sigma, the Antibody types that demonstrates 2B2 and 2A9 clone is respectively IgG ₁Subclass and IgM(Fig. 4).

2.4 a large amount of preparations of monoclonal antibody

Saxol in Healthy female Balb/c mouse peritoneal behind the injection autoclaving, 0.6ml/ are only.After one week, to the hybridoma of its lumbar injection energy stably excreting C-myc antibody, only (cell number is about 5 * 10 to 0.5ml/ again ⁶Individual).After 10-14 days, treat that mouse web portion obviously expands and when being slow in action, collect ascites, 3000rpm, 10min are centrifugal, discard fat and paraffin oil, collect middle level clarification ascites, and-20 ℃ frozen for subsequent use.2B2 and 2A9 clone's mouse ascites is tired and is respectively 1:64000,1:32000, shows that the monoclonal antibody of acquisition has higher susceptibility

3 utilize anti-c-Myc IgG monoclonal antibody to detect the Direct ELISA method of tumour cell c-Myc expression

3.1 coated

The tested tumour cell lysate of coated 0.01～1000pmo l/L.37 ℃ of reaction 1h.Negative control is the normal cell lysate; Positive control is Raj i cell pyrolysis liquid.

3.2 wash plate

With PBST washing, 200 μ l/ holes, dry behind each 30s and wash plate liquid, triplicate.

3.3 sealing

Add confining liquid 200 μ l/ holes, 37 ℃ of wet box temperature are bathed 30min.

3.4 wash plate

With PBST washing, 200 μ l/ holes, dry behind each 30s and wash plate liquid, triplicate.

3.5 primary antibodie reaction

Add the anti-c-Myc IgG(2B2 that 1:2000 doubly dilutes) 100 μ l/ holes, 37 ℃ of wet box temperature are bathed 1h.

3.6 wash plate

Dry primary antibodie, with PBST washing, 200 μ l/ holes, dry behind each 30s and wash plate liquid, triplicate.

3.7 two anti-reactions

HRP enzyme mark sheep anti-mouse igg is done the 1:5000 dilution, and 100 μ l/ holes add ELISA Plate, and 37 ℃ of wet box temperature are bathed 1h.

3.8 wash plate

Dry two and resist, with PBST washing, 200 μ l/ holes, dry behind each 30s and wash plate liquid, triplicate.

3.9 substrate

Add substrate solution 100 μ l/ holes, 37 ℃ of lucifuge reaction 15min.

3.10 stop

Add stop buffer 100 μ l/ holes, microplate reader is measured 0D _492nmValue.

The anti-c-Myc IgG(2B2 of this experiment use) the c-Myc antigen in the monoclonal antibody fast detecting tumour cell has high degree of specificity and susceptibility, and required time is 2.5-3h, and the positive detection rate reaches 100%.Detectable c-Myc protein concentration scope is 0.01～1000pmol/L, detects down and is limited to 0.01～0.05pmol/L.

According to above-mentioned three embodiment, the present invention utilizes the ELISA method of monoclonal antibody fast detecting C-Myc, can be summarized as following steps:

The preparation and purification of S1, C-myc antigen, concrete substep is as follows:

S11, the expression bacterium E.coli BL21 that will contain prokaryotic expression carrier (pET20b-C-myc expresses bacterium) cultivate (170rpm) 37 ℃ of concussions;

S12, after IPTG induces, collect thalline, nickel pillar chromatographic purifying is smashed, utilized to ultrasound wave;

S2, the anti-C-myc monoclonal antibody of preparation, concrete substep is as follows:

S21, will utilize the C-myc albumen of purifying to carry out immune health Balb/c mouse;

S22, immune Balb/c mouse boosting cell and SP2/0 myeloma cell are carried out Fusion of Cells;

S23,3-4 days, utilize the HAT nutrient culture media to screen.

S24, positive hybridoma cell is carried out subclone, obtain the monoclonal antibody hybridoma cell strain that 2 strains can the anti-C-myc of stably excreting, 2B2 and 2A9 clone.

S25,2B2 and 2A9 clone's antibody subtype is respectively IgG ₁Subclass and IgM.

Injection 2B2 and 2A9 clonal cell line prepare monoclonal antibody in a large number in S26, the Balb/c mouse peritoneal.

S27, select in following three kinds of methods any one, realize fast detecting C-Myc:

Method 1: utilize two kinds of anti-c-Myc monoclonal antibodies to detect the sandwich method ELISA method of tumour cell c-Myc expression.

Method 2: utilize anti-c-Myc IgM monoclonal antibody to detect the Direct ELISA method of tumour cell c-Myc expression.

Method 3: utilize anti-c-Myc IgG monoclonal antibody to detect the Direct ELISA method of tumour cell c-Myc expression.

Under the optimal way, the concrete steps of step S1 are:

The E.coli BL21 that will contain prokaryotic expression carrier (pET20b-C-myc expresses bacterium) cultivates (170rpm), bacterium liquid OD 37 ℃ of concussions _600nmWhen value is 0.4-0.6, after 1-2mM IPTG induces 1-3 hour, the centrifugal 10min collecting cell of 6500rmp, suspend, Ultrasonic Cell Disruptor smudge cells (20% power, broken 10min), 2-8M urea carry out repeatability behind the sex change 2h, through AKATA instrument nickel ion affinity chromatograph post purifying protein, obtain at last the C-Myc albumen of the above purity of 90-95%.

Under the optimal way, step S21 concrete steps are: utilize the C-myc albumen of prokaryotic expression and purifying and isopyknic Freund's complete adjuvant fully emulsified, antigen is injected in the healthy Balb/c mouse peritoneal, 0.4ml/, the antigen amount is 50-100 μ g/; After the first immunisation 14 days, use incomplete Freunds adjuvant instead and carry out the 2nd immunity with the antigen amount of volume is fully emulsified, the antigen amount is constant; The 28th day, according to g/ antigen amount of 100-200 μ mouse is carried out for the third time immunity, the antibody titer of mouse has reached 1:64000-1:1280000.

Under the optimal way, in step S22 concrete steps be: Balb/c mouse boosting cell and SP2/0 myeloma cell are mixed in the 50ml centrifuge tube with the ratio of 5:1-10:1,1200rpm, 5min are centrifugal, abandon supernatant, 50% the PEG1000 that splashes into rapidly 37 ℃ of preheatings in 25s-30s is 0.5ml and mixing gently approximately, 700rpm, 2min are centrifugal, the RPMI-1640 basal medium 15ml that slowly adds again 37 ℃ of preheatings along tube wall, test tube is done draw a circle sample approximately 2min that moves, centrifugal 1400rpm, 5min, abandon supernatant, clean again 1 time with method.The HT nutrient culture media 30ml that slowly adds 37 ℃ of preheatings along tube wall, gently mixing fused cell.

Under the optimal way, in step S24 concrete steps be: when hybridoma colony growth 40-60% is above, screen positive hole with the ELISA method.For positive hole, with HT nutrient culture media diluting cells, carry out subclone.Cultivated 8-9 days, and the supernatant in the monoclonal cell hole of mark was carried out ELISA detect screening positive clone.

Under the optimal way, in step S26 concrete steps be: the saxol in the Healthy female Balb/c mouse peritoneal behind the injection autoclaving, 0.4-0.6ml/ are only.After one week, to the hybridoma of its lumbar injection energy stably excreting C-myc antibody, only (cell number is about 2-5 * 10 to 0.5ml/ again ⁶Individual).After 10-14 days, treat that mouse web portion obviously expands and when being slow in action, collect ascites, 3000rpm, 10min are centrifugal, discard fat and paraffin oil, collect middle level clarification ascites, and-20 ℃ frozen for subsequent use.2B2 and 2A9 clone's mouse ascites is tired and is respectively 1:64000,1:32000.

Under the optimal way, the concrete steps of method 1 are in step S27: with the coated anti-C-myc IgM(2A9 of carbonate buffer solution dilution of pH 9.6), make concentration reach 5-20 μ g/ml; With PBS-T washing, 200 μ l/ holes, dry behind each 30s and wash plate liquid, triplicate; Add the tested tumour cell lysate that 1:10-1:50 doubly dilutes.37 ℃ of reaction 1h; Wash with PBS-T; Add confining liquid 200 μ l/ holes, 37 ℃ of wet box temperature are bathed 1h; The anti-C-myc IgM(2B2 that adds the 1:1000-1:2000 doubling dilution) 100 μ l/ holes, 37 ℃ of wet box temperature are bathed 1h; Wash with PBS-T; HRP enzyme mark goat anti-mouse igg is done the 1:5000 dilution, and 100 μ l/ holes add ELISA Plate, and 37 ℃ of wet box temperature are bathed 1h; Wash with PBS-T; Add o-phenylenediamine (OPD) liquid 100 μ l/ holes, 37 ℃ of lucifuge reaction 15min; Add stop buffer (2mol/L sulfuric acid solution) 100 μ l/ holes, microplate reader is measured OD _492nmValue.

Under the optimal way, the concrete steps of method 2 are in step S27:

Carbonate buffer solution dilution with pH 9.6 is coated with the tested tumour cell lysate that 1:10-1:50 doubly dilutes.With PBS-T washing, 200 μ l/ holes, dry behind each 30s and wash plate liquid, triplicate; Add confining liquid 200 μ l/ holes, 37 ℃ of temperature are bathed 1h; The anti-C-Myc IgM(2A9 that adds the 1:1000-1:2000 doubling dilution) 100 μ l/ holes, 37 ℃ of temperature are bathed 1h; Wash with PBS-T; HRP enzyme mark goat-anti Mouse IgM is done the 1:5000-1:10000 dilution, and 100 μ l/ holes add ELISA Plate, and 37 ℃ of temperature are bathed 1h; Wash with PBS-T; Add o-phenylenediamine (OPD) liquid 100 μ l/ holes, 37 ℃ of lucifuge reaction 15min; Add stop buffer (2mol/L sulfuric acid solution) 100 μ l/ holes, microplate reader is measured OD _492nmValue.

Under the optimal way, the concrete steps of method 3 are in step S27:

Carbonate buffer solution dilution with pH 9.6 is coated with the tested tumour cell lysate that 1:10-1:50 doubly dilutes.37 ℃ of reaction 1h; Wash with PBS-T; Add confining liquid 200 μ l/ holes, 37 ℃ of temperature are bathed 1h; The anti-c-Myc IgG(2B2 that adds the 1:1000-1:2000 doubling dilution) 100 μ l/ holes, 37 ℃ of temperature are bathed 1h; Wash with PBS-T; HRP enzyme mark goat anti-mouse igg is done the 1:5000 dilution, and 100 μ l/ holes add ELISA Plate, and 37 ℃ of temperature are bathed 1h; Wash with PBS-T; Add o-phenylenediamine (OPD) liquid 100 μ l/ holes, 37 ℃ of lucifuge reaction 15min; Add stop buffer (2mol/L sulfuric acid solution) 100 μ l/ holes, microplate reader is measured OD _492nmValue.

The present invention also provides a kind of ELISA solution preparation method of utilizing monoclonal antibody to detect tumour cell c-Myc expression, comprising:

Cleansing solution, PBS-T damping fluid (the 20mM phosphate buffer adds 0.1%Tween20);

Antibody diluent, 2% skimmed milk power PBS solution;

Coating buffer, pH 9.6 carbonate buffer solutions;

Confining liquid, 5% skimmed milk power PBS solution;

Substrate, o-phenylenediamine (OPD) liquid;

Stop buffer, 2mo l/L sulfuric acid solution.

That three kinds of ELISA assay methods of the present invention have is easy and simple to handle, highly sensitive, detection limit is low, stability and high repeatability and other advantages, and the screening of malignant tumour is significant.

The above; only be the better embodiment of the present invention; but protection scope of the present invention is not limited to this; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses; be equal to replacement or change according to technical scheme of the present invention and inventive concept thereof, all should be encompassed within protection scope of the present invention.

Claims (10)

1. an ELISA method of utilizing monoclonal antibody fast detecting C-Myc is characterized in that, comprises the steps:

S1, preparation C-myc antigen, and purifying, the C-myc albumen of acquisition purifying;

S2, utilize the C-myc albumen of described purifying to carry out immune health Balb/c mouse;

S3, immune Balb/c mouse boosting cell and SP2/0 myeloma cell are carried out Fusion of Cells;

S4, screening positive hybridoma cell;

S5, described positive hybridoma cell is carried out subclone, obtaining 2 strains can the anti-C-myc IgG of stably excreting ₁The clone and the monoclonal antibody hybridoma cell strain of anti-C-myc IgM;

Two kinds of described monoclonal antibody hybridoma cell strains of injection prepare monoclonal antibody in a large number in S6, the Balb/c mouse peritoneal;

S7, select in following three kinds of methods any one, realize fast detecting C-Myc:

Method 1: utilize two kinds of anti-c-Myc monoclonal antibodies to detect the sandwich method ELISA method of tumour cell c-Myc expression.

Method 2: utilize anti-c-Myc IgM monoclonal antibody to detect the Direct ELISA method of tumour cell c-Myc expression.

Method 3: utilize anti-c-Myc IgG monoclonal antibody to detect the Direct ELISA method of tumour cell c-Myc expression.

2. the described ELISA method of utilizing monoclonal antibody fast detecting C-Myc according to claim 1 is characterized in that, the concrete grammar of step S1 is:

S11, the pET20b-C-myc that will contain prokaryotic expression carrier express bacterium E.coli BL21 and cultivate 170rpm 37 ℃ of concussions;

S12, after IPTG induces, collect thalline, nickel pillar chromatographic purifying is smashed, utilized to ultrasound wave.

3. the described ELISA method of utilizing monoclonal antibody fast detecting C-Myc according to claim 2 is characterized in that, the concrete grammar of S1 is: will contain the E.coliBL21 that prokaryotic expression carrier pET20b-C-myc expresses bacterium and cultivate 170rpm 37 ℃ of concussions; Bacterium liquid OD _600nmWhen value is 0.4-0.6, after 1-2mM IPTG induces 1-3 hour, the centrifugal 10min collecting cell of 6500rmp, suspend, broken Ultrasonic Cell Disruptor smudge cells 10min, 2-8M urea carries out repeatability behind the sex change 2h, with nickel ion affinity chromatograph post purifying protein, obtains at last the C-Myc albumen of the above purity of 90-95% through the AKATA instrument.

4. the described ELISA method of utilizing monoclonal antibody fast detecting C-Myc according to claim 3, it is characterized in that, the S2 concrete steps are: utilize the C-myc albumen of prokaryotic expression and purifying and isopyknic Freund's complete adjuvant fully emulsified, antigen is injected in the healthy Balb/c mouse peritoneal, 0.4ml/ only, the antigen amount is 50-100 μ g/; After the first immunisation 14 days, use incomplete Freunds adjuvant instead and carry out the 2nd immunity with the antigen amount of volume is fully emulsified, the antigen amount is constant; The 28th day, according to g/ antigen amount of 100-200 μ mouse is carried out for the third time immunity, the antibody titer of mouse has reached 1:64000-1: 1280000.

5. the described ELISA method of utilizing monoclonal antibody fast detecting C-Myc according to claim 4, it is characterized in that, the S3 concrete steps are: Balb/c mouse boosting cell and SP2/0 myeloma cell are mixed in the 50ml centrifuge tube with the ratio of 5:1-10:1,1200rpm, 5min is centrifugal, abandon supernatant, 50% the PEG1000 that splashes into rapidly 37 ℃ of preheatings in 25s-30s is 0.5ml and mixing gently approximately, 700rpm, 2min is centrifugal, the RPMI-1640 basal medium 15ml that slowly adds again 37 ℃ of preheatings along tube wall, test tube is done draw a circle sample approximately 2min that moves, centrifugal 1400rpm, 5min abandons supernatant, cleans 1 time with method again.The HT nutrient culture media 30ml that slowly adds 37 ℃ of preheatings along tube wall, gently mixing fused cell.

6. the described ELISA method of utilizing monoclonal antibody fast detecting C-Myc according to claim 5 is characterized in that, the S5 concrete steps are: when hybridoma colony growth 40-60% is above, screen positive hole with the ELISA method;

For positive hole, with HT nutrient culture media diluting cells, carry out subclone.Cultivated 8-9 days, and the supernatant in the monoclonal cell hole of mark was carried out ELISA detect screening positive clone.

7. the described ELISA method of utilizing monoclonal antibody fast detecting C-Myc according to claim 6 is characterized in that, the S6 concrete steps are: the saxol in the Healthy female Balb/c mouse peritoneal behind the injection autoclaving, 0.4-0.6ml/ only.After one week, to the hybridoma of its lumbar injection energy stably excreting C-myc antibody, 0.5ml/, cell number is about 2-5 * 10 again ⁶Individual;

After 10-14 days, treat that mouse web portion obviously expands and when being slow in action, collect ascites, 3000rpm, 10min are centrifugal, discard fat and paraffin oil, collect middle level clarification ascites, and-20 ℃ frozen for subsequent use.2B2 and 2A9 clone's mouse ascites is tired and is respectively 1:64000,1:32000.

8. the described ELISA method of utilizing monoclonal antibody fast detecting C-Myc according to claim 7 is characterized in that, the concrete steps of method 1 are among the S7:

Be coated with the monoclonal antibody hybridoma cell strain of anti-C-myc IgM with the carbonate buffer solution dilution of pH 9.6, make concentration reach 5-20 μ g/ml;

With PBS-T washing, 200 μ l/ holes, dry behind each 30s and wash plate liquid, triplicate;

Add the tested tumour cell lysate that 1:10-1:50 doubly dilutes, 37 ℃ of reaction 1h;

Wash with PBS-T; Add confining liquid 200 μ l/ holes, 37 ℃ of wet box temperature are bathed 1h;

The monoclonal antibody hybridoma cell strain that adds the anti-C-myc IgM of 1:1000-1:2000 doubling dilution, 100 μ l/ holes, 37 ℃ of wet box temperature are bathed 1h;

Wash with PBS-T; HRP enzyme mark goat anti-mouse igg is done the 1:5000 dilution, and 100 μ l/ holes add ELISA Plate, and 37 ℃ of wet box temperature are bathed 1h;

Wash with PBS-T; Add o-phenylenediamine liquid 100 μ l/ holes, 37 ℃ of lucifuge reaction 15min;

Add stop buffer, the 2mol/L sulfuric acid solution, 100 μ l/ holes, microplate reader is measured OD _492nmValue.

9. the described ELISA method of utilizing monoclonal antibody fast detecting C-Myc according to claim 7 is characterized in that, the concrete steps of method 2 are among the S7:

Carbonate buffer solution dilution with pH 9.6 is coated with the tested tumour cell lysate that 1:10-1:50 doubly dilutes.With PBS-T washing, 200 μ l/ holes, dry behind each 30s and wash plate liquid, triplicate; Add confining liquid 200 μ l/ holes, 37 ℃ of temperature are bathed 1h; The anti-C-Myc IgM(2A9 that adds the 1:1000-1:2000 doubling dilution) 100 μ l/ holes, 37 ℃ of temperature are bathed 1h; Wash with PBS-T; HRP enzyme mark goat-anti Mouse IgM is done the 1:5000-1:10000 dilution, and 100 μ l/ holes add ELISA Plate, and 37 ℃ of temperature are bathed 1h; Wash with PBS-T; Add o-phenylenediamine (OPD) liquid 100 μ l/ holes, 37 ℃ of lucifuge reaction 15min; Add stop buffer (2mo l/L sulfuric acid solution) 100 μ l/ holes, microplate reader is measured OD _492nmValue.

10. the described ELISA method of utilizing monoclonal antibody fast detecting C-Myc according to claim 7 is characterized in that, the concrete steps of method 3 are in step S7:

Carbonate buffer solution dilution with pH 9.6 is coated with the tested tumour cell lysate that 1:10-1:50 doubly dilutes.37 ℃ of reaction 1h; Wash with PBS-T; Add confining liquid 200 μ l/ holes, 37 ℃ of temperature are bathed 1h; The anti-c-Myc IgG(2B2 that adds the 1:1000-1:2000 doubling dilution) 100 μ l/ holes, 37 ℃ of temperature are bathed 1h; Wash with PBS-T; HRP enzyme mark goat anti-mouse igg is done the 1:5000 dilution, and 100 μ l/ holes add ELISA Plate, and 37 ℃ of temperature are bathed 1h; Wash with PBS-T; Add o-phenylenediamine (OPD) liquid 100 μ l/ holes, 37 ℃ of lucifuge reaction 15min; Add stop buffer (2mol/L sulfuric acid solution) 100 μ l/ holes, microplate reader is measured OD _492nmValue.

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