CN102507943B - Double antibody sandwiched enzyme-linked immuno sorbent assay (ELISA) kit for detecting non small cell lung cancer (NSCLC), and preparation method for double antibody sandwiched ELISA kit - Google Patents
Double antibody sandwiched enzyme-linked immuno sorbent assay (ELISA) kit for detecting non small cell lung cancer (NSCLC), and preparation method for double antibody sandwiched ELISA kit Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57423—Specifically defined cancers of lung
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Abstract
The invention discloses a double antibody sandwiched enzyme-linked immuno sorbent assay (ELISA) kit for detecting non small cell lung cancer (NSCLC), and a preparation method for the double antibody sandwiched ELISA kit. The kit consists of anti human NSCLC monoclonal antibody NJ001-1 which is secreted by a hybridoma cell strain of which the collection number is CCTCC NO:C201172, a 96 ferment plate coated with anti SPC-A1 rabbit polyclonal antibody, goat anti mouse intravenous gamma globulin (IgG) labeled with horse radish peroxidase, antibody diluent, namely a 1 percent phosphate buffer solution (PBS), lotion, a stop solution 2M H2SO4, a tetramethylbenzidine (TMB) substrate color development solution, positive control, namely a SPC-A1 lysis solution, and negative control, namely human antibody (AB) serum or fetal calf serum and blank control, namely 1 percent PBS. Compared with the conventional detection method, a method for detecting the NSCLC by using the kit has the highest sensitivity (59.1 percent) and specificity (4.4 percent), and highest precision and stability.
Description
Technical field
The invention belongs to the biology sample detection field, relate to a kind of double-antibody sandwich elisa kit for detection of non-small cell lung cancer and preparation method thereof.
Background technology
Lung cancer is one of the highest malignant tumour of human M ﹠ M, and global annual lung cancer occurs to surpass 1,500,000 examples, and wherein 80% is non-small cell lung cancer (NSCLC).Non-small cell type lung cancer comprises squama cancer, gland cancer, large cell carcinoma, and compare its growth of cancer cells division with small cell carcinoma slower, and diffusion transfer is evening relatively.Although Diagnosis Technique makes rapid progress, 5 years survival rates of lung cancer are still less than 15%.Improve the survival rate of patients with lung cancer, early diagnosis is even more important.Monoclonal antibody has high degree of specificity, all becomes current study hotspot aspect tumor diagnosis and therapy.
Summary of the invention
The purpose of this invention is to provide a kind of double-antibody sandwich elisa kit for detection of non-small cell lung cancer.
Another object of the present invention provides the preparation method of this ELISA kit.
Purpose of the present invention can be achieved through the following technical solutions:
A kind of double-antibody sandwich elisa kit for detection of non-small cell lung cancer, comprise following composition: preserving number is the anti-human non-small cell lung carcinoma monoclonal antibody NJ001-1 of the hybridoma cell strain secretion of CCTCC NO:C201172, the 96 hole ELISA Plate that anti-SPC-A1 rabbit polyclonal antibody is coated, the horseradish peroxidase-labeled sheep anti-mouse igg, antibody diluent: 1%PBS, washing lotion, stop buffer 2M H
2SO
4, tmb substrate nitrite ion, positive control: SPC-A1 lysate, negative control: people AB serum or hyclone and blank: 1%PBS.
Described double-antibody sandwich elisa kit for detection of non-small cell lung cancer preferably comprises following composition:
Preserving number is the anti-human non-small cell lung carcinoma monoclonal antibody NJ001-1 5-10 μ L of the hybridoma cell strain secretion of CCTCC NO:C201172,1 of the ELISA Plate that anti-SPC-A1 rabbit polyclonal antibody is coated with, horseradish peroxidase-labeled sheep anti-mouse igg 5-10 μ L, antibody diluent is 1%PBS 40-50mL, washing lotion 40-50mL, stop buffer 2M H
2SO
48-10mL, tmb substrate nitrite ion A, each 8-10mL of B liquid, positive control: SPC-A1 lysate 400-600 μ L, negative control: people AB serum or hyclone 400-600 μ L and blank: 1%PBS 1-2mL.
Wherein, described preserving number is that the anti-human non-small cell lung carcinoma monoclonal antibody NJ001-1 concentration of the hybridoma cell strain secretion of CCTCC NO:C201172 is 200mg/L.
The by the following method preparation of described anti-SPC-A1 rabbit polyclonal antibody: first immunisation is with 1 * 10
8Individual SPC-A1 cell carries out the auricular vein injection to new zealand rabbit, and thereafter every all booster immunizations once, immunizing dose is 3 * 10
8Individual cell, all carry out ear edge vein exploitating blood before each immunity, indirect elisa method detects serum antibody titer, carries out the abdominal aorta bloodletting after antibody titer reaches more than or equal to 1: 300000,37 ℃ of rabbit blood are placed 1h, change again 4 ℃ over to and spend the night, fully shrink rear rapid separation of serum until blood, and in 4 ℃, the centrifugal 30min of 3000r/min, collect supernatant, utilize Protein A affinity chromatography to carry out purifying, tiring behind the purifying is 1: 150 000-1: 170 000.
The coated ELISA Plate of described anti-SPC-A1 rabbit polyclonal antibody prepares by the following method: with the carbonate of pH9.6 be coated with damping fluid with purifying after anti-SPC-A1 rabbit polyclonal antibody be diluted to purpose concentration 0.63 μ g/mL; Add in the micropore behind the antibody-solutions mixing that dilution is good, 100 μ L/ holes, 4 ℃ are spent the night; Wash plate 3 times; Add the 3%BSA confining liquid, 300 μ L/ holes, 4 ℃ are spent the night; Wash plate 3 times.
The carbonate of described pH 9.6 is coated with damping fluid: Na
2CO
31.59g, NaHCO
32.93g, add ddH
2O to 1L regulates pH to 9.6, mixing with 10M NaOH at last.
Described lotion prescription is: 2.0g NaCl; 0.2g KH
2PO
42.9g Na
2HPO
412H
2O; 0.2g KCl; 0.2g NaN
340mL ddH
2O; 0.5mL Tween-20 adds ddH before use
2O to 1L; SPC-A1 lysate preparation method is: SPC-A1 is laid in six orifice plates, and concentration is 1 * 10
6Individual/mL; After at the bottom of cell is paved with the hole, abandon nutrient solution, add 4 ℃ of precooling 1%PBS and wash twice, 2mL/ hole; Every hole adds lysate 150 μ L, places on the oscillator; Behind the 30min, with rifle head scraping attached cell, collect lysate in 1.5mL EP pipe, the centrifugal 10min of 13000g; Leave and take supernatant ,-20 ℃ of preservations after the packing.
The preparation method of described double-antibody sandwich elisa kit for detection of non-small cell lung cancer comprises following steps:
(1) preparation of monoclonal antibody NJ001-1: get female BALB/c mouse lumbar injection 0.5mL paraffin oil in age in 8-10 week, behind the 10d respectively the well-grown preserving number of lumbar injection be the hybridoma NM001-11 * 10 of CCTCC NO:C201172
6/ only, 1-2 aspirates ascites after week, behind 37 ℃ of 1h, 4 ℃ are spent the night, and next day is centrifugal with ascites, through Protein G affinity column purifying, obtain the monoclonal antibody NJ001-1 of purifying, antibody is that 1: 1 liquid dissolves through glycerine and water ratio, and final concentration is 200mg/L;
(2) preparation of anti-SPC-A1 rabbit polyclonal antibody: first immunisation is with 1 * 10
8Individual SPC-A1 cell carries out the auricular vein injection to new zealand rabbit, and thereafter every all booster immunizations once, immunizing dose is 3 * 10
8Individual cell all carries out ear edge vein exploitating blood before each immunity, and indirect elisa method detects serum antibody titer, after reaching more than or equal to 1: 300 000, antibody titer carries out the abdominal aorta bloodletting, 37 ℃ of rabbit blood are placed 1h, change again 4 ℃ over to and spend the night, fully shrink rear rapid separation of serum until blood, and in 4 ℃, the centrifugal 30min of 3 000r/min collects supernatant, utilizes Protein A affinity chromatography to carry out purifying, tiring behind the purifying is 1: 150 000-1: 170 000, and concentration is 14.77 μ g/mL;
(3) anti-SPC-A1 rabbit polyclonal antibody coated elisa plate: with the coated damping fluid of the carbonate of pH 9.6 with purifying after anti-SPC-A1 rabbit polyclonal antibody be diluted to purpose concentration 0.63 μ g/mL; Add in the micropore behind the abundant mixing of liquid that dilution is good, 100 μ L/ holes, 4 ℃ are spent the night; Wash plate 3 times, 200 μ L/ holes; Add the 3%BSA confining liquid, 300 μ L/ holes, 4 ℃ are spent the night; Wash plate 3 times, 200 μ L/ holes;-20 ℃ of preservations;
(4) preparation of washing lotion, SPC-A1 lysate, 1%PBS: washing lotion: 2.0g NaCl; 0.2g KH
2PO
42.9g Na
2HPO
412H
2O; 0.2g KCl; 0.2g NaN
340mL ddH
2O; 0.5mL Tween-20 adds ddH before use
2O to 1L; SPC-A1 lysate preparation method: SPC-A1 is laid in six orifice plates, and concentration is 1 * 10
6Individual/mL; After at the bottom of cell is paved with the hole, abandon nutrient solution, add 4 ℃ of precooling 1%PBS and wash twice, 2mL/ hole; Every hole adds lysate 150 μ L, places on the oscillator; Behind the 30min, with rifle head scraping attached cell, collect lysate in 1.5mL EP pipe, the centrifugal 10min of 13 000g; Leave and take supernatant ,-20 ℃ of preservations after the packing; 1%PBS:8g NaCl; 2.2g KCl; 1.44g Na
2HPO
40.24g KH
2PO
4Add ddH
2O to 800mL;
(5) kit assembling: the preserving number of above-mentioned preparation is the anti-human non-small cell lung carcinoma monoclonal antibody NJ001-1 5-10 μ L of the hybridoma cell strain secretion of CCTCC NO:C201172,1 of the ELISA Plate that anti-SPC-A1 rabbit polyclonal antibody is coated with, horseradish peroxidase-labeled sheep anti-mouse igg 5-10 μ L, antibody diluent is 1%PBS 40-50mL, washing lotion 40-50mL, stop buffer 2M H
2SO
48-10mL, tmb substrate nitrite ion A, each 8-10mL of B liquid, positive control: SPC-A1 lysate 400-600 μ L, negative control: people AB serum or hyclone 400-600 μ L and blank: 1%PBS 1-2mL is assembled into kit.
The carbonate of described pH 9.6 is coated with damping fluid: Na
2CO
31.59g, NaHCO
32.93g, add ddH
2O to 1L regulates pH to 9.6, mixing with 10M NaOH at last.
Beneficial effect of the present invention:
The present invention has at first confirmed that by Western blot the expression of NJ001-1 associated target antigens in the patients with lung adenocarcinoma serum is apparently higher than physical examination of healthy population on the basis of the monoclonal antibody NJ001-1 that has successfully prepared energy specific recognition non-small cell lung cancer; Then immune new zealand rabbit preparation and purifying obtain the polyclonal antibody of the anti-SPC-A1 of high-titer, unite this resists with the NJ001-1 monoclonal antibody more and set up the double-antibody sandwich elisa kit, utilize at last this kit that a large amount of clinical samples is carried out determination and analysis.The result shows that the detection that this kit is used for non-small cell lung cancer has good specificity, precision and stability, is expected to play a significant role in clinical detection.The non-small cell lung cancer antigen ELISA detecting kit of ratifying without national FSDA on the market at present, adopt existing electrochemical luminescence method to detect non-small cell lung cancer, compare with this ELISA detection method, more responsive chemoluminescence method detects, and the result shows that susceptibility and specificity all were better than chemoluminescence method when kit of the present invention was used for the detection of non-small cell lung cancer.
Description of drawings
The indirect CELISA of Fig. 1 is analyzed the reactivity of monoclonal antibody NJ001-1 and kinds of tumor cells and non-tumor cell.
Protein immunoblot result and the gray analysis of NJ001-1 associated target antigens in Fig. 2 serum;
A:2,3 and 6 is the serum sample of three physical examination of healthy populations, and 4 and 5 is the serum sample of two patients with lung adenocarcinomas, and 1 is SPC-A1 cell pyrolysis liquid (positive control);
B:1 is patients with lung adenocarcinoma, and 2 is physical examination of healthy population, 3 positive contrasts.
The bioactivity of the anti-SPC-A1 polyclonal antibody of Fig. 3.
Fig. 4 chessboard method is determined the best effort concentration of coated antibody and NJ001-1.
The preservation information of biological material specimens
Hybridoma cell strain NM001-1 is preserved in Chinese Typical Representative culture collection center on August 31st, 2011, and the preservation address is Wuhan, China, Wuhan University, and preserving number is CCTCC NO:C201172.
Embodiment
1, related cell and main agents in following examples: human lung cancer cell line SPC-A1, A549, NCI-H460, NCI-H520, the human hepatoma cell line HepG2, MCF-7 ZR-75-30, CCL188 Colo205 and HELF are that WI-38 is available from Chinese Academy of Sciences's cell bank; The SP2/0 mouse myeloma cell line is preserved by this laboratory; Age in 6-8 week and 8-10 age in week female BALB/c mouse available from Shanghai Si Laike animal used as test company.RPMI 1640, DMEM, hyclone, 0.25% trypsase, polyglycol, hypoxanthine thymidine (HT), aminopterin-induced syndrome hypoxanthine thymidine (HAT), people AB serum and paraffin oil are available from U.S. Gibco Invitrogen company; The sheep anti-mouse igg of FITC mark and solvable type single component tmb substrate solution are available from sky, Beijing root company; Mouse Ig hypotype is measured kit available from U.S. Santa Cruz company; Western blot developer is available from U.S. Cell Signaling company; Inflammatory pseudotumor of lung tissue, cancerous lung tissue and breast cancer tissue are provided by Jiangsu Prov. People's Hospital pathology department; Model 550 type microplate reader are available from Bio-Rad company.2.5Kg male new zealand rabbit available from Shanghai Si Laike animal used as test company.BSA is available from Biosharp company; GAPDH antibody is available from green skies company; The horseradish peroxidase-labeled sheep anti-mouse igg is available from company of middle China fir Golden Bridge; Proteo Extract Albumin/IgG Removal Kit is available from Merck ﹠ Co., Inc..
2, related cell culture processes is in following examples: above-mentioned cell grows in respectively among the DMEM or RPMI1640 nutrient solution that contains 10% hyclone, penicillin and each 100U/mL of streptomysin, 37 ℃, 5%CO
2Cultivate in the constant incubator.Human peripheral blood single nucleus cell (PBMC) adopts conventional lymphocyte separation medium to separate available from the healthy blood donor.
3, specimen collection patients with lung cancer 303 examples that year June in October, 2007 to 2011, pathology department made a definite diagnosis, wherein 195 examples are adenocarcinoma of lung, as the adenocarcinoma of lung group among the embodiment 6-8,69 examples are lung squamous cancer, as the lung squamous cancer among the embodiment 6-8, adenocarcinoma of lung group and lung squamous cancer are combined as the non-small cell lung cancer group; 39 examples are small-cell carcinoma of the lung, as the small-cell carcinoma of the lung group among the embodiment 6-8; Collect simultaneously 50 routine lung benign disease patients, as the lung benign disease group among the embodiment 6-8 and 500 routine physical examination of healthy populations as the health examination group among the embodiment 6-8.Clinicopathologic features sees Table 1, follows up a case by regular visits to in October, 2011.
The clinicopathologic features of the different groups of table 1
4, sample disposal gathers venous blood 2mL with the serum separation gel vacuum test tube, and the centrifugal 10min of first room temperature 3000r/min shifts upper serum, again in 4 ℃, the centrifugal 10min of 16 000 * g, draws the every pipe 200 μ L of the acellular serum packing in upper strata, puts-70 ℃ of preservations.Aforesaid operations is finished in the 2h after collection of specimens.
5, statistical analysis adopts SPSS 16.0 statistics softwares that data are carried out statistical study.Relatively adopt the definite probabilistic method of Fisher ' s between the group of qualitative data, P＜0.05 o'clock has statistical significance.
The preparation of embodiment 1 monoclonal antibody
1.1 animal immune is got female BALB/c mouse of 6-8 week, every usefulness 2 * 10
6SPC-A1 cell/time lumbar injection immunity 3 times, every 2 weeks of minor tick.All carry out the blood sampling of mouse endocanthion before each immunity, CELISA detects the mice serum antibody titer indirectly, treats that the immune serum antibody titer reaches maximum and get mice spleen cell when no longer raising to merge, and the 3d booster immunization is 1 time before merging.
1.2 SPC-A1 cell 10 is inoculated in the cell ELISA test indirectly
5/ hole is on 96 orifice plates, merge to Growth of Cells and to reach 80%, 95% ethanol is fixed, PBS washes 3 times, the penetrating 20min of 0.2%Triton-X-100, with 37 ℃ of sealings of 50g/L BSA 2h, add successively different dilution immune serum 100 μ L, 37 ℃ of incubation 1h again, again after PBS washes 3 times, the sheep anti-mouse igg 100 μ L that add the HRP mark of dilution in 1: 1 000,37 ℃ of incubation 45min are after the PBS washing, add the TMB nitrite ion, cessation reaction behind 37 ℃ of incubation 10min, absorbance (OD) value when measuring 450nm with microplate reader, with immune serum (1: 1 000) not as negative control.
1.3 Fusion of Cells is got the grinding of immune mouse spleen and is made cell suspension, merge (Yao Xiaoling with the myeloma cell SP2/0 that is in exponential phase, Liu Xiaoyan, Wu Qiang, etc. the purifying [J] of the preparation of people's lung cancer related monoclonal antibody and antigen thereof. Chinese Journal of Immunology, 2006,22 (12): 1140-1145.), merge first 480 holes, cell clone occurs after merging for 1 week, growth hybridoma in 330 holes is arranged, and fusion rate is about 70%.Carry out indirect cell ELISA experiment sieving positive hybridoma cell (immune serum in the 1.2 indirect cell ELISA tests is replaced with the Hybridoma Cell Culture supernatant) according to the method in 1.2, carry out transferred species and 3 limiting dilution assay subclones, adopt and obtain the anti-SPC-A1 monoclonal antibody of stably excreting and positive the strongest hybridoma cell strain NM001-1.Hybridoma cell strain NM001-1 is preserved in Chinese Typical Representative culture collection center on August 31st, 2011, and the preservation address is Wuhan, Wuhan University, and preserving number is CCTCC NO:C201172.
1.4 the preparation of odd contradictive hydroperitoneum and purifying
Get 8-10 female BALB/c mouse lumbar injection 0.5mL paraffin oil in age in week, the difference well-grown hybridoma NM001-1 of lumbar injection (CCTCC NO:C201172) about 1 * 10 behind the 10d
6/ only, 1-2 aspirates ascites after week, and behind 37 ℃ of 1h, 4 ℃ are spent the night, and next day is centrifugal with ascites respectively, through Protein G affinity column purifying, obtains the monoclonal antibody NJ001-1 of purifying; Antibody is that 1: 1 liquid dissolves through glycerine and water ratio, and final concentration is 200mg/L.
1.5 the evaluation of monoclonal antibody
1.5.1 the evaluation of monoclonal antibody Ig subclass: purified monoclonal antibody is with PBS dilution in 1: 10 000, according to measuring the operation of kit instructions.The subclass of monoclonal antibody NJ001-1 is IgG, and light chain is the κ chain.
1.5.2 the mensuration that monoclonal antibody is tired: respectively with the monoclonal antibody NJ001-1 PBS doubling dilution of purifying, get respectively 100 μ L adding and be coated with in 96 orifice plates of SPCA1 cell, measure OD with indirect CELISA
450Immunoreactive monoclonal antibody greatest dilution can occur with coated cell and be it and tire in value.It is 4 * 10 that monoclonal antibody NJ001-1 tires
6, be used for the preparation of kit of the present invention.
1.5.3 the specific evaluation of monoclonal antibody: with monoclonal antibody NJ001-1 and above-mentioned 8 kinds of tumour cells (human lung cancer cell line: adenocarcinoma of lung SPC-A1 and A549 of purifying, maxicell lung cancer: NCI-H460, lung squamous cancer: NCI-H520, Bel7402: HepG2, MCF-7: ZR-75-30, CCL188: Colo205 and HELF system: WI-38) and Healthy People PBMC do respectively indirect cell ELISA analysis, observe and have or not positive reaction.The result shows, monoclonal antibody NJ001-1 only has more by force reaction to lung carcinoma cell (SPCA1, A549, NCI-H520, NCI-H460) antigen, to other tumour cells (HepG2, Colo 205, ZR-75-30) antigen, normal human embryonic lung cell's (WI-38) antigen and Healthy People PBMC all reactionless (seeing Fig. 1).
Select at random the serum of 3 routine patients with lung adenocarcinomas and 2 routine physical examination of healthy populations,, sample is concentrated through icy ketone method except albumin and IgG in the serum deprivation by Proteo Extract Albumin/IgG Removal kit.The SDS-PAGE glue of the sample application to 12% after concentrated is carried out Protein Separation, after with protein delivery to pvdf membrane, skim milk room temperature sealing 2h with 5%, again respectively with the NJ001-1 monoclonal antibody of dilution in 1: 1 000 and 4 ℃ of shaking table overnight incubation of GAPDH antibody of dilution in 1: 400, take out film with 1 * TBST rinsing 3 times, the sheep anti mouse two anti-room temperature shaking tables that add dilution in 1: 3 000 are hatched 2h, carry out the ECL colour developing after 1 * TBST rinsing 3 times.The expression of NJ001-1 associated target antigens is significantly higher than physical examination of healthy population in the patients with lung adenocarcinoma serum, sees Fig. 2 A, and gray analysis the results are shown in Figure 2B.
The preparation of embodiment 3 anti-SPC-A1 polyclonal antibodies
First immunisation is with 1 * 10
8Individual SPC-A1 cell carries out the auricular vein injection to new zealand rabbit, and thereafter every all booster immunizations once, immunizing dose is 3 * 10
8Individual cell.All carry out ear edge vein exploitating blood before each immunity, (with SPC-A1 cell bed board, concentration is 1 * 10 to indirect elisa method
5Individual/hole, since the serum of preimmune serum and this time collection is carried out respectively application of sample behind the doubling dilution in 1: 5 000, anti-as two with the horseradish peroxidase-labeled goat anti-rabbit igg, with OD
450Greater than highly diluted multiple the tiring as serum of the serum of 2.1 times of negative control holes) detect serum antibody titer.After reaching more than or equal to 1: 300 000, antibody titer carries out the abdominal aorta bloodletting, 37 ℃ of rabbit blood are placed 1h, change again 4 ℃ over to and spend the night, fully shrink rear rapid separation of serum until blood, and in 4 ℃, the centrifugal 30min of 3 000r/min, collect supernatant, utilize Protein A affinity chromatography to carry out purifying, tiring behind the purifying is 1: 160 000, see Fig. 3, concentration is 14.77 μ g/mL.
Determining of embodiment 4 coated antibodies and primary antibodie best effort concentration
Determine the working concentration of coated antibody and primary antibodie with the chessboard titration experiments.With coated damping fluid (pH 9.6) with coated antibody (anti-SPC-A1 rabbit is how anti-, 14.77mg/mL) is diluted to 2.50,1.25,0.63 and 0.31 μ g/mL coated elisa plate, the every hole of 100 μ L, 4 ℃ are spent the night; Wash plate 3 times with washing lotion; The every hole 300 μ L of 3%BSA, 4 ℃ of sealings are spent the night, and obtain the coated ELISA Plate of anti-SPC-A1 polyclonal antibody; It is the same to wash plate with washing lotion, adds serum to be checked, and 2h is hatched for 37 ℃ in the every hole of 50 μ L, washes plate 5 times; Use respectively 1%PBS (antibody diluent) to carry out the dilution of 1: 3 000,1: 4 000,1: 5 000 and 1: 6 000 primary antibodie (monoclonal antibody NJ001-1,200mg/L), every hole adds 100 μ L, hatches 1h for 37 ℃; Wash plate 5 times; The horseradish peroxidase-labeled sheep anti-mouse igg is diluted to 1: 3 000 with 1%PBS (antibody diluent), and every hole adds 100 μ L, hatches 30min for 37 ℃, washes plate 5 times with washing lotion; Every hole adds freshly prepared tmb substrate nitrite ion 100 μ L (except the blank well), and room temperature lucifuge colour developing 10min is with 2M H
2SO
4The stop buffer cessation reaction.Read the OD value, reading wavelength 450nm with Model550 type enzyme connection instrument (Bio-Rad company).The positive, feminine gender and blank are respectively the SPC-A1 lysate, people AB serum or hyclone and 1%PBS.Select positive hole OD
450Value/negative hole OD
450The concentration of the value corresponding coated antibody of (P/N value) largest hole and primary antibodie is best effort concentration.The result shows: when the concentration of coated antibody is 0.63 μ g/mL, the primary antibodie dilutability is 1: 4 000 o'clock, can guarantee that as much as possible in conjunction with target antigen, P/N value maximum (9.8) is seen Fig. 4 simultaneously.
The compound method of each reagent that relates in the said method:
Stop buffer, tmb substrate nitrite ion: available from Shanghai Xiamen Kehua
Washing lotion: 2.0g NaCl; 0.2g KH
2PO
42.9g Na
2HPO
412H
2O; 0.2g KCl; 0.2g NaN
340mL ddH
2O; 0.5mL Tween-20 adds ddH before use
2O to 1L.
SPC-A1 lysate preparation method: SPC-A1 is laid on (concentration is 1 * 10 in six orifice plates
6Individual/mL); After at the bottom of cell is paved with the hole, abandon nutrient solution, add 4 ℃ of precooling 1%PBS and wash twice, 2mL/ hole; Every hole adds lysate 150 μ L, places on the oscillator; Behind the 30min, with rifle head scraping attached cell, collect lysate in 1.5mL EP pipe, the centrifugal 10min of 13000g; Leave and take supernatant ,-20 ℃ of preservations after the packing.
Antibody diluent 1%PBS:8g NaCl; 2.2g KCl; 1.44g Na
2HPO
40.24g KH
2PO
4Add ddH
2O to 800mL.The carbonate of pH9.6 is coated with damping fluid: Na
2CO
31.59g NaHCO3 2.93g, adding distil water regulate pH to 9.6 with 10M NaOH at last to 1L, fully mixing.
Double-antibody sandwich elisa kit for detection of non-small cell lung cancer, comprise following component: the preserving number of above-mentioned preparation is the anti-human non-small cell lung carcinoma monoclonal antibody NJ001-1 5 μ L of the hybridoma cell strain secretion of CCTCC NO:C201172, the 96 hole ELISA Plate that anti-SPC-A1 rabbit polyclonal antibody is coated, horseradish peroxidase-labeled sheep anti-mouse igg 5 μ L, washing lotion 40mL, stop buffer 2M H
2SO
48mL, tmb substrate nitrite ion A, each 8mL of B liquid, positive control: SPC-A1 lysate 500 μ L, negative control: people AB serum or hyclone 500 μ L and blanks: 1%PBS 1mL, antibody diluent are 1%PBS 50mL.
The monoclonal antibody that uses in the kit, how preparation method anti-and each reagent sees embodiment 1,3,4.Stop buffer, tmb substrate nitrite ion: available from Shanghai Kehua Bio-engineering Co., Ltd.
The coated ELISA Plate of described anti-SPC-A1 rabbit polyclonal antibody prepares by the following method: with the carbonate of pH9.6 be coated with damping fluid with purifying after anti-SPC-A1 rabbit polyclonal antibody be diluted to purpose concentration 0.63 μ g/mL; Add in the micropore behind the antibody-solutions mixing that dilution is good, 100 μ L/ holes, 4 ℃ are spent the night; Wash plate 3 times, 200 μ L/ holes; Add the 3%BSA confining liquid, 300 μ L/ holes, 4 ℃ are spent the night; Wash plate 3 times, 200 μ L/ holes;-20 ℃ of preservations.
Take out the kit of embodiment 5 preparations of-20 ℃ of preservations from refrigerator, test serum, positive control (SPC-A1 lysate) and negative control (people AB serum or hyclone) place on ice and melt; Take out coated elisa plate and related reagent and return to room temperature; After serum melts, put upside down mixing, centrifugal; Get test serum supernatant 50 μ L and add in the micropore, add simultaneously positive control, negative control, each 50 μ L/ hole of blank; Hatch 2h for 37 ℃, take out elisa plate and wash plate 5 times with washing lotion, 200 μ L/ holes, each 2min; By 1: 4 000 dilution primary antibodie NJ001-1,100 μ L/ holes added micropore with antibody diluent 1%PBS; Hatch 1h for 37 ℃, it is the same to wash plate; According to 1: 3 000 dilution two anti-horseradish peroxidase-labeled sheep anti-mouse iggs, 100 μ L/ holes added micropore with antibody diluent 1%PBS; Hatch 30min for 37 ℃, it is the same to wash plate; Tmb substrate nitrite ion A, B were mixed by 1: 1, and 100 μ L/ holes add in the micropore (the blank hole does not add substrate), and room temperature lucifuge 10min adds stop buffer, 50 μ L/ holes; Model 550 type enzymes connection instrument detects OD
450Value.
The double antibody sandwich ELISA testing result shows that the Positive rate of (table 2) adenocarcinoma of lung, lung squamous cancer, small-cell carcinoma of the lung and lung benign disease group is respectively 63.1% (123/195), 47.8% (33/69), 20.5% (8/39) and 12.0% (6/50) all is significantly higher than health examination group (4.4%, P＜0.05).The positive rate of non-small cell lung cancer group (adenocarcinoma of lung and lung squamous cancer) is significantly higher than small-cell carcinoma of the lung group (59.1%vs 20.5%, P＜0.05) and lung benign disease group (59.1%vs 12.0%, P＜0.05).In addition, the positive rate of adenocarcinoma of lung group is significantly higher than lung squamous cancer group (63.1%vs 47.8%, P＜0.05) in the non-small cell lung cancer.Clear and definite pathological staging is 124 examples in the 195 routine patients with lung adenocarcinomas, wherein the positive rate of group (I/II) and late period group (III/IV) reaches respectively 63.3% (19/30) and 79.8% (75/94) in early days, the result shows, this method namely has higher recall rate in the adenocarcinoma of lung case in early days, helps the early diagnosis of adenocarcinoma of lung.
Table 2 double antibody sandwich ELISA detects the positive rate of each group
Annotate:
*Compare P＜0.05 with adenocarcinoma of lung group positive rate; △ and lung squamous cancer group positive rate compare, P＜0.05; Zero with small-cell carcinoma of the lung group positive rate relatively, P＜0.05; and lung benign disease group positive rate compare, P＜0.05
Embodiment 7
By the chemoluminescence method than ELISA method sensitivity sample is carried out the synchronous detection of change of serum C EA, NSE and CYFRA21-1, compare with double-antibodies sandwich ELISA based on double-antibody sandwich elisa kit of the present invention.
1. susceptibility: be 59.1% (156/264) based on the double antibody sandwich ELISA of double-antibody sandwich elisa kit of the present invention to the Positive rate of Patients with Non-small-cell Lung in the table 3, be significantly higher than the positive rate (41.3%, 17.4% and 37.1%) of CEA, NSE and CYFRA21-1, have higher susceptibility so kit of the present invention detects respective target antigen to the detection of NJ001-1 associated target antigens than CEA, NSE and CYFRA21-1;
2. specificity: based on the double antibody sandwich ELISA of double-antibody sandwich elisa kit of the present invention to Patients With Small Cell Carcinoma of The Lung, the Positive rate of lung benign disease patient and physical examination of healthy population is respectively 20.5%, 12.0% and 4.4%, all be starkly lower than CEA, (wherein the CEA positive rate is 25.6% to the positive rate of NSE and CYFRA21-1,14.0% and 5.8%, the positive rate of NSE is 56.4%, 18.0% and 9.8%, the positive rate of CYFRA21-1 is 23.1%, 16.0% and 6.4%), thus this kit to the detection of NJ001-1 associated target antigens than CEA, NSE and CYFRA21-1 detect respective target antigen and have better specificity.
The ELISA method detects the positive rate of NJ001-1 associated target antigens and three kinds of tumor markerses of chemoluminescence method detection in each group of table 3
Annotate:
*Compare P＜0.05 with the NJ001-1 associated target antigens; Zero with the NJ001-1 associated target antigens relatively, P＜0.05; △ and NJ001-1 associated target antigens compare, P＜0.05;
3. for adenocarcinoma of lung: the positive rate that detects NJ001-1 associated target antigens in the 195 routine patients with lung adenocarcinoma serum based on the double antibody sandwich ELISA of double-antibody sandwich elisa kit of the present invention in the table 4 is 63.1%, (CEA 45.1% to be significantly higher than electrochemical luminescence method, NSE 15.9%, with CYFRA21-1 33.8%), P＜0.05.
4. for lung squamous cancer: similar to the above results, the positive rate that detects NJ001-1 associated target antigens in the 69 routine patients with lung adenocarcinoma serum based on the double antibody sandwich ELISA of double-antibody sandwich elisa kit of the present invention in the table 4 is 47.8%, apparently higher than electrochemical luminescence method (CEA 30.4%, NSE 21.7% and CYFRA21-1 46.4%).
5. for three joint-detection: adopt electrochemical luminescence method associating CEA, NSE, and the positive rate that three indexs of CYFRA21-1 detect 264 routine Patients with Non-small-cell Lung serum is 59.5% (157/264); And only adopt the double antibody sandwich ELISA detection NJ001-1 associated target antigens individual event detection based on double-antibody sandwich elisa kit of the present invention can reach intimate identical positive rate 59.1% (156/264).
6. for four joint-detection: add the present invention on three joint-detection bases and detect, namely the positive rate of four associatings (NJ001-1 associated target antigens, CEA, NSE and CYFRA21-1) in adenocarcinoma of lung and From Lung Squamous Carcinoma Patients serum is respectively 85.1%, 75.4%, be significantly higher than the positive rate 60.5% of three associatings (CEA, NSE and CYFRA21-1), 56.6%, improve significantly the positive rate of (improving about 20% approximately) non-small cell lung cancer, see Table 4.
The positive rate of individual event and joint-detection NJ001-1 associated target antigens and three kinds of tumor markerses in table 4 adenocarcinoma of lung and the lung squamous cancer group
Annotate:
*With four unite group relatively, P＜0.01; Zero expression is compared P＜0.05 with the NJ001-1 associated target antigens; △ represents to unite group relatively, P＜0.05 with four; represents to compare P＜0.05. with the NJ001-1 associated target antigens
Precision: the serum that mixes 10 parts of Patients with Non-small-cell Lungs, the double-antibody sandwich elisa kit that utilizes embodiment 5 preparation is to its continuous detecting 20 times, detect every day in addition 1 time, continuous detecting 20 days, result show, in batch and batch between CV% be respectively 2.24% and 2.52%, all in the permissible error scope (batch in CV%＜2.5%, CV%＜3.3% between batch), so this method precision is high, repeatability better.
Stability: place-20 ℃ to preserve respectively 30d, 90d and 270d the double-antibody sandwich elisa kit of embodiment 5 preparations, respectively high-level sample (pooled serums of 10 parts of Patients with Non-small-cell Lungs) and low-level sample (pooled serums of 10 parts of physical examination of healthy populations) are detected again, the CV% of high level sample is 1.83%, the CV% of low-level sample is 2.11%, all less than 3.3%, illustrate that this kit has good stability.
Claims (3)
1. double-antibody sandwich elisa kit for detection of non-small cell lung cancer, it is characterized in that comprising following composition: preserving number is the anti-human non-small cell lung carcinoma monoclonal antibody NJ001-1 5-10 μ L of the hybridoma cell strain secretion of CCTCC NO:C201172,1 of the 96 hole ELISA Plate that anti-SPC-A1 rabbit polyclonal antibody is coated with, horseradish peroxidase-labeled sheep anti-mouse igg 5-10 μ L, antibody diluent 1% PBS 40-50 mL, washing lotion 40-50 mL, stop buffer 2 M H
2SO
48-10 mL, tmb substrate nitrite ion A, each 8-10 mL of B liquid, positive control: SPC-A1 lysate 400-600 μ L, negative control: people AB serum or hyclone 400-600 μ L and blank: 1% PBS 1-2 mL; Wherein, described preserving number is that the anti-human non-small cell lung carcinoma monoclonal antibody NJ001-1 concentration of the hybridoma cell strain secretion of CCTCC NO:C201172 is 200 mg/L, and dilutability is 1:4 000 when using;
The by the following method preparation of described anti-SPC-A1 rabbit polyclonal antibody: first immunisation is with 1 * 10
8Individual SPC-A1 cell carries out the auricular vein injection to new zealand rabbit, and thereafter every all booster immunizations once, immunizing dose is 3 * 10
8Individual cell, all carry out ear edge vein exploitating blood before each immunity, indirect elisa method detects serum antibody titer, carries out the abdominal aorta bloodletting after antibody titer reaches more than or equal to 1:300 000,37 ℃ of rabbit blood are placed 1 h, change again 4 ℃ over to and spend the night, fully shrink rear rapid separation of serum until blood, and in 4 ℃, centrifugal 30 min of 3000 r/min, collect supernatant, utilize Protein A affinity chromatography to carry out purifying, tiring behind the purifying is 1:150 000-170 000;
The coated ELISA Plate of described anti-SPC-A1 rabbit polyclonal antibody prepares by the following method: with the carbonate of pH 9.6 be coated with damping fluid with purifying after anti-SPC-A1 rabbit polyclonal antibody be diluted to purpose concentration 0.63 μ g/mL; Add in the micropore behind the antibody-solutions mixing that dilution is good, 100 μ L/ holes, 4 ℃ are spent the night; Wash plate 3 times; Add 3% BSA confining liquid, 300 μ L/ holes, 4 ℃ are spent the night; Wash plate 3 times; The carbonate of described pH9.6 is coated with damping fluid: Na
2CO
31.59 g, NaHCO
32.93 g adds ddH
2O to 1 L regulates pH to 9.6, mixing with 10 M NaOH at last; Described lotion prescription is: 2.0 g NaCl; 0.2 g KH
2PO
42.9 g Na
2HPO
412H
2O; 0.2 g KCl; 0.2 g NaN
340 mL ddH
2O; 0.5 the mL Tween-20 adds ddH before use
2O to 1 L; SPC-A1 lysate preparation method is: SPC-A1 is laid in six orifice plates, and concentration is 1 * 10
6Individual/mL; After at the bottom of cell is paved with the hole, abandon nutrient solution, add 4 ℃ of precooling 1% PBS and wash twice, 2 mL/ hole; Every hole adds lysate 150 μ L, places on the oscillator; Behind 30 min, with rifle head scraping attached cell, collect lysate in 1.5 mL EP pipes, centrifugal 10 min of 13 000 g; Leave and take supernatant ,-20 ℃ of preservations after the packing.
2. the preparation method of the double-antibody sandwich elisa kit for detection of non-small cell lung cancer claimed in claim 1 is characterized in that comprising following steps:
(1) preparation of monoclonal antibody NJ001-1: get female BALB/c mouse lumbar injection 0.5 mL paraffin oil in age in 8-10 week, behind 10 d respectively the well-grown preserving number of lumbar injection be the hybridoma NM001-1 1 * 10 of CCTCC NO:C201172
6/ only, 1-2 aspirates ascites after week, behind 37 ℃ of 1 h, 4 ℃ are spent the night, and next day is centrifugal with ascites, through Protein G affinity column purifying, obtain the monoclonal antibody NJ001-1 of purifying, antibody is that the liquid of 1:1 dissolves through glycerine and water ratio, and final concentration is 200 mg/L;
(2) preparation of anti-SPC-A1 rabbit polyclonal antibody: first immunisation is with 1 * 10
8Individual SPC-A1 cell carries out the auricular vein injection to new zealand rabbit, and thereafter every all booster immunizations once, immunizing dose is 3 * 10
8Individual cell, all carry out ear edge vein exploitating blood before each immunity, indirect elisa method detects serum antibody titer, after reaching more than or equal to 1:300 000, antibody titer carries out the abdominal aorta bloodletting, 37 ℃ of rabbit blood are placed 1 h, changing 4 ℃ over to spends the night again, fully shrink rear rapid separation of serum until blood, and in 4 ℃, centrifugal 30 min of 3 000 r/min collect supernatant, utilize Protein A affinity chromatography to carry out purifying, tire behind the purifying and be that 1:150 000-1:170 000, concentration are 14.77 μ g/mL;
(3) anti-SPC-A1 rabbit polyclonal antibody coated elisa plate: with the coated damping fluid of the carbonate of pH9.6 with purifying after anti-SPC-A1 rabbit polyclonal antibody be diluted to purpose concentration 0.63 μ g/mL; Add in the micropore behind the abundant mixing of liquid that dilution is good, 100 μ L/ holes, 4 ℃ are spent the night; Wash plate 3 times, 200 μ L/ holes; Add 3% BSA confining liquid, 300 μ L/ holes, 4 ℃ are spent the night; Wash plate 3 times, 200 μ L/ holes;-20 ℃ of preservations;
(4) preparation of washing lotion, SPC-A1 lysate, 1%PBS:
Washing lotion: 2.0 g NaCl; 0.2 g KH
2PO
42.9 g Na
2HPO
412H
2O; 0.2 g KCl; 0.2 g NaN
340 mL ddH
2O; 0.5 the mL Tween-20 adds ddH before use
2O to 1 L;
SPC-A1 lysate preparation method: SPC-A1 is laid in six orifice plates, and concentration is 1 * 10
6Individual/mL; After at the bottom of cell is paved with the hole, abandon nutrient solution, add 4 ℃ of precooling 1% PBS and wash twice, 2 mL/ hole; Every hole adds lysate 150 μ L, places on the oscillator; Behind 30 min, with rifle head scraping attached cell, collect lysate in 1.5 mL EP pipes, centrifugal 10 min of 13 000 g; Leave and take supernatant ,-20 ℃ of preservations after the packing;
1% PBS:8 g NaCl; 2.2 g KCl; 1.44 g Na
2HPO
40.24 g KH
2PO
4Add ddH
2O to 800 mL;
(5) kit assembling: the preserving number of above-mentioned preparation is the anti-human non-small cell lung carcinoma monoclonal antibody NJ001-1 5-10 μ L of the hybridoma cell strain secretion of CCTCC NO:C201172,1 of the ELISA Plate that anti-SPC-A1 rabbit polyclonal antibody is coated with, horseradish peroxidase-labeled sheep anti-mouse igg 5-10 μ L, antibody diluent 1% PBS 50 mL, washing lotion 40-50 mL, stop buffer 2 M H
2SO
48-10 mL, tmb substrate nitrite ion A, each 8-10 mL of B liquid, positive control: SPC-A1 lysate 400-600 μ L, negative control: people AB serum or hyclone 400-600 μ L and blank: 1%PBS 1-2 mL is assembled into kit.
3. the preparation method of the double-antibody sandwich elisa kit for detection of non-small cell lung cancer according to claim 2 is characterized in that coated damping fluid: the Na of carbonate of described pH 9.6
2CO
31.59 g, NaHCO
32.93 g adds ddH
2O to 1 L regulates pH to 9.6, mixing with 10 M NaOH at last.
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| PCT/CN2012/070303 WO2013063876A1 (en) | 2011-11-03 | 2012-01-13 | Double antibody sandwiched elisa kit for detecting non-small cell lung cancer and preparation method thereof |
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| CN105510585A (en) * | 2015-12-01 | 2016-04-20 | 苏州市博力生物科技有限公司 | Double-antibody sandwich ELISA kit for human beta3 acetylglucosaminyltransferase 8 (beta3GnT8) |
| CN111273029B (en) * | 2020-02-25 | 2023-03-31 | 芜湖天明生物技术有限公司 | rhTSG-6 double-antibody sandwich ELISA quantitative detection kit and use method and application thereof |
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