CN103323602A

CN103323602A - Double antibody sandwich ELISA detection method for TRPC6 protein and kit thereof

Info

Publication number: CN103323602A
Application number: CN201310215961XA
Authority: CN
Inventors: 张萃; 李红枝; 刘晓波; 刘若飞; 陈玉琼; 葛如意
Original assignee: Guangdong Pharmaceutical University
Current assignee: Guangdong Pharmaceutical University
Priority date: 2013-06-03
Filing date: 2013-06-03
Publication date: 2013-09-25

Abstract

The invention provides a double antibody sandwich ELISA detection method for TRPC6 protein and a kit thereof. The method comprises the following steps: coating a microwell plate with TRPC6 protein monoclonal antibody mAb-H8; sealing the microwell plate with a blocking reagent; respectively adding diluents of standard TRPC6 polypeptide and a sample to be tested; then adding TRPC6 protein monoclonal antibody pAb-T and an enzyme-labeled secondary antibody; adding a substrate for coloration after washing; measuring OD value at the wavelength of 490nm by the use of a microplate reader; drawing a standard curve of TRPC6 polypeptide; and calculating the concentration of TRPC6 protein in the sample to be tested by the use of the standard curve. The invention also provides an ELISA kit for detection of TRPC6 protein. The detection result of the method and the kit provided by the invention is accurate and stable and has high sensitivity. TRPC6 protein expression level of different tissue cells can be detected rapidly through an enzyme-labeled experiment. The superiority of the method is fully reflected by its advantages of convenience, simplicity and high sensitivity.

Description

A kind of double-antibody sandwich elisa detection method and kit of TRPC6 albumen

Technical field

The present invention relates to immunological technique and related application field thereof, be specifically related to a kind of double-antibody sandwich elisa detection method and kit of TRPC6 albumen.

Background technology

Transient receptor potential cationic channel 6(Transient Receptor Potential Channel, TRPC6) in the conduction of calcium signal path, bringing into play very important effect, the TRPC6 albumen of TRPC6 gene code can be by forming homotype or special-shaped condensate, form non-selective ion channel, calcium ion and sodion will enter in the cell through this passage.Calcium channel is the indispensable ion path of the every physiological activity of body, can keep the biopotential of cell membrane both sides, keep normal Nerve conduction, muscle stretches and diastolic function, nerve-muscle conduction function, also have the mechanism such as some hormonal actions all to show by the calcium channel molecule, TRPC6 is at heart, kidney, in the tissue corresponding expression is arranged all in the human body vitals such as muscle, and TRPC6 is as the important non-selective ion channel of calcium ion, its calcium signal path by regulating the intracellular free calcium level mediation is the various important physiological effect of balance the body again, research in recent years shows, the TRPC6 molecule can be used as the targeted molecular of some drugs effect, and the variation of its protein expression level is closely related with human various diseases.

For detection of the polyclonal antibody that mostly is of TRPC6 protein expression, the non-specific binding factor is many in application, is difficult to the evaluate and analyze result at present, and preparation TRPC6 monoclonal antibody has its advantage, and has overcome the limitation of polyclonal antibody.In addition, the research that relevant each histocyte TRPC6 expresses, be main mainly with detecting TRPC6 mRNA, have not yet to see analysis result on protein level, utilize the outer immunological technique method of TRPC6 monoclonal antibody construct, measure the expression of protein level with this, more be conducive to the auxiliary diagnosis of disease surveillance.

Enzyme Immunoassay is that it develops by early stage competition law, indirect method dual-antigen sandwich method, double antibody sandwich method and enzyme multiplied immunoassay method etc. till now with the high efficiency of the specificity of antigen-antibody reaction and substrate for enzymatic activity reaction and a kind of immunoassay technology that selectivity combines.Along with the enzyme labeling technical progress, the mark application of enzymes expands to alkaline phosphatase, beta galactosidase, urease etc. from peroxidase, but uses maximum horseradish peroxidases (HRP) that remain.This experiment is on the basis of the anti-TRPC6 monoclonal antibody hybridoma cell of the stably excreting of setting up, process Mitochondrion IgG subgroup identification is the IgG class, it is stronger with affinity on specificity that the antibody Biological characteristics filters out mAb-H8, recycle prepared anti-TRPC6 polyclonal antibody pAb-T, make up double antibody sandwich ELISA, finally under anti-IgG-HRP amplification, to be used for the mensuration of TRPC6 protein expression, the foundation of the method has actual application value for various histiocytic TRPC6 expression studies.

Summary of the invention

The object of the invention is to provides a kind of double-antibody sandwich elisa detection method of TRPC6 albumen in order to overcome the deficiency of TRPC6 protein detection technology in the prior art.The method can be used for measuring various histocyte TRPC6 protein expression levels.

Another object of the present invention provides a kind of double-antibody sandwich elisa detection kit of TRPC6 albumen.

The present invention is achieved through the following technical solutions above-mentioned purpose:

A kind of double-antibody sandwich elisa detection method of TRPC6 albumen, comprise the steps: the coated microwell plate with TRPC6 protein monoclonal antibody mAb-H8, behind the confining liquid closed porosity plate, the dilution that adds respectively standard TRPC6 polypeptide and testing sample, add again TRPC6 protein polyclone antibody pAb-T, then add ELIAS secondary antibody, add the substrate colour developing after the washing, under the 490nm wavelength, measure the OD value with microplate reader, make the typical curve of standard TRPC6 polypeptide, calculate TRPC6 protein concentration in the testing sample by typical curve;

Described TRPC6 protein monoclonal antibody mAb-H8 is obtained by purifying after the hybridoma cell strain H8 secretion; Hybridoma cell strain H8 is prepared as antigen by the TRPC6 polypeptide, can secrete the monoclonal antibody of anti-TRPC6, be preserved in Chinese Typical Representative culture collection center, preservation address: China on March 7th, 2012. Wuhan. Wuhan University, deposit number is CCTCC-C201236.The amino acid sequence of TRPC6 polypeptide is: KDLTKVTLGDNVKYY(is shown in SEQ ID NO:1).The preparation method of hybridoma cell strain H8 is documented in application number: CN201210145560.7; Denomination of invention is: in the patent of the monoclonal antibody of TRPC6 antigen polypeptide and anti-TRPC6.

Preferably, the working concentration of described TRPC6 protein monoclonal antibody mAb-H8 when coated microwell plate is 5 ~ 20 μ g/mL; More preferably, the working concentration of described TRPC6 protein monoclonal antibody mAb-H8 when coated microwell plate is 10 μ g/mL.

Described TRPC6 protein polyclone antibody pAb-T by TRPC6 polypeptide-K LH conjugate immune rat after the separation of serum purifying obtain; The sequence of described TRPC6 polypeptide is: KDLTKVTLGDNVKYY, and shown in SEQ ID NO:1.The preparation method of described TRPC6 polypeptide-K LH conjugate can be with reference to this area conventional methods.The ELIAS secondary antibody of selecting is the conventional use in this area, and described marker enzyme is horseradish peroxidase.

Preferably, the working concentration of described TRPC6 protein polyclone antibody pAb-T is 2.5 ~ 10 μ g/mL; More preferably, the working concentration of described TRPC6 protein polyclone antibody pAb-T is 5 μ g/mL.

Preferably, described confining liquid is 37 ℃ of sealings of 5% skimmed milk power 1.5 ~ 2.0 hours; Perhaps, 37 ℃ of sealings of 5% bovine serum albumin(BSA) 1.0 ~ 1.5 hours, perhaps 37 ℃ of sealings of 5% hyclone are 1.5 ~ 2.0 hours.

More preferably, described confining liquid is 37 ℃ of sealings of 5% skimmed milk power 1.5 hours.

Preferably, described TRPC6 protein monoclonal antibody mAb-H8 and TRPC6 protein polyclone antibody pAb-T adopt the sad-ammonium sulfate salting-out process purifying of optimization to obtain, the optimal conditions of optimizing sad-ammonium sulfate salting-out process is that cells and supernatant and acetic acid-sodium acetate buffer volume ratio are 1:2 ~ 2:1, sad final concentration is 25 ~ 35 μ L/mL, the ammonium sulfate saturation degree is 35 ~ 45%, and the pH value is 7.0 ~ 7.4 during ammonium sulfate precipitation.More preferably, described optimization is sad-and the optimal conditions of ammonium sulfate salting-out process is that cells and supernatant and acetic acid-sodium acetate buffer volume ratio are 1:1, and sad final concentration is 30 μ L/mL, and the ammonium sulfate saturation degree is 40%, the pH value is 7.2 during ammonium sulfate precipitation.

Preferably, described TRPC6 protein polyclone antibody pAb-T prepares by the following method: get 100 ~ 120 μ g TRPC6 polypeptide-K LH conjugates at every turn and adopt the subcutaneous multi-point injection method of mouse, 3 weeks of interval, mouse is carried out immune-treated three times, and for the third time immunity rear blood sampling of rear 1 week is surveyed it and is tired, and selection is tired greater than the booster immunization that carries out of 1:10000, booster immunization was got blood after 3 days, separation of serum is optimized sad-ammonium sulfate salting-out process antibody purification, saves backup; The sequence of described TRPC6 polypeptide is shown in SEQ ID NO:1.

Preferably, the double-antibody sandwich elisa detection method of described TRPC6 albumen comprises the steps:

S1. be the coated microwell plate of TRPC6 protein monoclonal antibody mAb-H8 of 10 μ g/ mL with working concentration, 100 ~ 120 μ L/ holes, 4 ℃ of coated spending the night; PBST washing 3 times, 350 μ L/ holes pat dry;

S2. add 5% skimmed milk power, 100 ~ 120 μ l/ holes, 37 ℃ are coated with 1.5 hours; PBST washing 3 times, 350 μ L/ holes pat dry;

S3. the dilution that adds respectively standard TRPC6 polypeptide and testing sample was hatched 1 hour for 37 ℃; PBST washing 3 times, 350 μ L/ holes pat dry;

S4. adding working concentration is the TRPC6 protein polyclone antibody pAb-T of 5 μ g/mL again, hatches 1 hour for 37 ℃; PBST washing 3 times, 350 μ L/ holes pat dry;

S4. add again sheep anti mouse HRP-IgG(1:5000 dilution) ELIAS secondary antibody, hatched 1 hour for 37 ℃ in 100 μ L/ holes; PBST washing 3 times, 350 μ L/ holes pat dry;

S5. add the OPD substrate solution, 100 μ L/ holes, 37 ℃ of lucifuge reaction 15min; Add 2M sulfuric acid cessation reaction, 100 μ L/ holes; Microplate reader 490nm place measures the OD value; The drawing standard curve calculates TRPC6 protein content in the sample to be checked.

A kind of ELISA kit that detects TRPC6 albumen includes ELISA Plate and sandwich antibody, and described ELISA Plate is coated with TRPC6 protein monoclonal antibody mAb-H8, and described sandwich antibody is TRPC6 protein polyclone antibody pAb-T; Described TRPC6 protein monoclonal antibody mAb-H8 is obtained by purifying after the hybridoma cell strain H8 secretion; Described enzyme mark TRPC6 protein polyclone antibody pAb-T is obtained carrying out enzyme labeling behind the TRPC6 protein polyclone antibody pAb-T by separation of serum purifying behind the TRPC6 polypeptide-K LH conjugate immune mouse again; The sequence of described TRPC6 polypeptide is shown in SEQ ID NO:1.

Compared with prior art, the present invention has following beneficial effect:

1. TRPC6 protein monoclonal antibody mAb-H8 of the present invention and TRPC6 protein polyclone antibody pAb-T adopt the sad-ammonium sulfate salting-out process purifying of optimization to obtain, the optimal conditions of optimizing sad-ammonium sulfate salting-out process is that supernatant-damping fluid volume is than being 1:2 ~ 2:1, sad final concentration is 25 ~ 35 μ L/mL, the ammonium sulfate saturation degree is 35 ~ 45%, and the pH value is 7.0 ~ 7.4 during ammonium sulfate precipitation.(supernatant-damping fluid volume is than being 1:1 under the condition of optimum, sad final concentration is 30 μ L/mL, the ammonium sulfate saturation degree is 40%, the pH value is 7.2 during ammonium sulfate precipitation), the concentration of the mAb-H8 that purifying obtains can reach 40 μ g/mL, the concentration of pAb-T can reach 25 μ g/mL, and monoclonal antibody and the polyclonal antibody that can obtain higher concentration are the prerequisites of setting up sandwich ELISA method.

The polyclonal antibody preparation of 2 anti-TRPC6 of the present invention, available TRPC6 polypeptide immune rat separates acquisition from serum, polyclonal antibody has its advantage, can identify a plurality of epi-positions of same antigen, in immune detection, have catch, enlarge-effect, the also fewer impact that is subject to the antigen conformation change.Therefore under the same conditions, utilize polyclonal antibody can improve the sensitivity of detection as detection antibody, especially some abundance albumen on the low side is more easily detected.

3. with the two sandwich ELISA methods of class methods its advantage is arranged, monoclonal antibody is as coated antibody, high specificity, and the sandwich antibody of the conduct of polyclonal antibody, and the capture antigen ability is strong, is rational matched combined using in detecting.Through the preliminary assay method of setting up relatively, how anti-monoclonal antibody-sandwich mode be higher than two monoclonal antibody sandwich mode mensuration susceptibilitys.

4. although sandwich ELISA method is a kind of very ripe detection technique, but, antibody for different antigen, even the dissimilar antibody of same antigen is when setting up sandwich ELISA detection method, the kind of the coated concentration of coated antibody, detection antibody working concentration and confining liquid can have a strong impact on the sensitivity of sandwich ELISA testing result, the present invention finds by a large amount of investigative tests, when the working concentration of envelope antigen mAb-H8 is 10 μ g/mL; When the working concentration of described TRPC6 protein polyclone antibody pAb-T was 5 μ g/mL, the valid analysing range of TRPC6 albumen was between 0.625 ~ 10 μ g/mL.The detection effect of different confining liquids is also different, and that the confining liquid effect is best is 37 ℃ of sealings of 5% skimmed milk power 1.5h, secondly is 5% bovine serum albumin(BSA), and 5% hyclone is a bit weaker.

5. ELISA method and the ELISA detection kit set up of the present invention can be used for the mensuration that various histocytes are expressed the TRPC6 albumen, on protein level so as to the monitoring histocyte sample relevant with the TRPC6 developed by molecule.

Description of drawings

SDS-PAGE behind Fig. 1 .TRPC6 protein monoclonal antibody mAb-H8 purifying identifies; 1,2,3 swimming lanes are three purifying samples of pAb-T, and 4 swimming lanes are serum before the purifying; 2,3,4 swimming lanes are three purifying samples of supernatant mAb-H8, and 1 swimming lane is supernatant before the purifying.

SDS-PAGE behind Fig. 2 .TRPC6 protein polyclone antibody pAb-T purifying identifies; 1,2,3 swimming lanes are three purifying samples of pAb-T, and 4 swimming lanes are serum before the purifying.

Fig. 3. mAb-H8-pAb-T sandwich mode.

Fig. 4. pAb-T-mAb-H8 sandwich mode.

Fig. 5. TRPC6 polypeptide protein concentration standard curve.

Embodiment

Further describe the present invention below in conjunction with the drawings and specific embodiments.Employed test method is conventional method if no special instructions among the following embodiment; Employed material, reagent etc. if no special instructions, are reagent and the material that can obtain from commercial channels.

Embodiment 1

The preparation of S1.TRPC6 protein monoclonal antibody (mAb-H8) and polyclonal antibody (pAb-T).

S11. TRPC6 protein monoclonal antibody mAb-H8 is obtained by purifying after the hybridoma cell strain H8 secretion; Hybridoma cell strain H8 is prepared as antigen by the TRPC6 polypeptide, can secrete the monoclonal antibody of anti-TRPC6, be preserved in Chinese Typical Representative culture collection center, preservation address: China on March 7th, 2012. Wuhan. Wuhan University, deposit number is CCTCC-C201236.The amino acid sequence of TRPC6 polypeptide is: KDLTKVTLGDNVKYY(is shown in SEQ ID NO:1).The preparation method of hybridoma cell strain H8 is documented in application number: CN201210145560.7; Denomination of invention is: in the patent of the monoclonal antibody of TRPC6 antigen polypeptide and anti-TRPC6.

S12. the preparation method of large mouse-anti TRPC6 polyclonal antibody (pAb-T) can reference: Zhu Liping, Chen Xueqing. immunology common experimental method [M], 1 edition, Beijing: People's Medical Officer Press, 2000:19-20. concrete steps are: three rats of primary immune response, get 100 μ g TRPC6 polypeptide-K LH conjugates at every turn and adopt the subcutaneous multi-point injection method of mouse, 3 weeks of interval, carry out three immunity, for the third time immunity rear blood sampling of rear 1 week is surveyed it and is tired, and selection high-titer person (more than the 1:10000) carries out booster immunization, and booster immunization was got blood after 3 days, separation of serum saves backup.The sequence of TRPC6 polypeptide is: KDLTKVTLGDNVKYY, and shown in SEQ ID NO:1.The preparation method of described TRPC6 polypeptide-K LH conjugate can be with reference to this area conventional methods.

S2. the purifying of TRPC6 protein monoclonal antibody (mAb-H8) and polyclonal antibody (pAb-T).

The present invention routine sad-ammonium sulfate salting-out process (conventional sad-ammonium sulfate salting-out process list of references: Zhu Liping, Chen Xueqing. immunology common experimental method [M], 1 edition, Beijing: People's Medical Officer Press, create on basis 2000:56-57) that a kind of suitable TRPC6 protein monoclonal antibody (mAb-H8) and polyclonal antibody (pAb-T) optimize sad-ammonium sulfate salting-out process, mAb-H8 in pAb-T in the serum and the cells and supernatant is carried out purifying, concrete optimal conditions is that cells and supernatant and acetic acid-sodium acetate buffer volume ratio are 1:1, sad final concentration is 30 μ L/mL, the ammonium sulfate saturation degree is 40%, and the pH value is 7.2 during ammonium sulfate precipitation.Concrete steps are: cells and supernatant is diluted (1:1) with 1 times of volumes of acetic acid-sodium acetate buffer 1.; 2. dropwise add sad (30 μ l/mL) down in stirring, add in 30 min, 4 ℃ leave standstill 2h, under 4 ℃ with 12000 rev/mins centrifugal 30 minutes, collect supernatant, abandon precipitation; 3. supernatant adds the 0.01mol/L PBS of 1/10 volume after 125 μ m nylon mesh are filtered, and transfers pH to 7.2 with 1 mol/L NaOH; 4. supernatant mixes with equal-volume saturated ammonium sulfate solution, and ammonium sulfate concentrations is 40%, be statically placed in 4 ℃ 2 hours, in 4 ℃ lower 12000 rev/mins centrifugal 30 minutes, collecting precipitation, and precipitation is dissolved among an amount of PBS; 5. repeat 40% saturation concentration ammonium sulfate precipitation step once, sediment is dissolved among the 1ml PBS the most at last, dialysed overnight in the bag filter of then packing into; 6. after dialysis finishes with dialysate in 4 ℃ lower 12000 rev/mins centrifugal 30 minutes, remove insoluble sediment, measure packing behind the protein content, frozen for subsequent use in-20 ℃.

Adopt above-mentioned optimal conditions, the content value behind the antibody purification is maximum, and mAb-H8 40 μ g/mL, pAb-T are 25 μ g/mL.The conventional SDS-PAGE electrophoresis of monoclonal antibody behind the purifying and how anti-employing identifies, qualification result is seen Fig. 1 and Fig. 2.

The mensuration of embodiment 2 mAb-H8-Ag saturation curves

Adopt indirect ELISA method to measure, with coating buffer Ag is diluted to respectively 1.25,2.5,5,20,40 μ g/mL, 4 ℃ of coated spending the night, seal 1.5h with 5% skimmed milk power after washing three times (each 5min), every hole adds 100 μ l dilution variable concentrations purifying mAb-H8 behind the repeated washing, hatch 1 h for 37 ℃, washing, add again the enzyme mark IgG antibody (1:5000) of determining optimum dilution degree and hatch 1 h, after the washing, behind OPD shading reaction 15min, microplate reader is measured the OD value under the 490nm, analyzes data and draws monoclonal antibody-antigen saturation concentration.

Interpretation of result shows: when antigen diluent during to 10 μ g/mL, response curve illustrates that near platform the combination of antigen and antibody reaches capacity, and drawing at last the saturation concentration that antigen is combined with mAb-H8 is 10 μ g/mL.

The pairing of embodiment 3. mAb-H8 and pAb-T is selected

To select screening, purpose be to determine best coated antibody and sandwich antibody to mAb-H8 through matching with pAb-T behind the Purification.Adopt the sandwich ELISA method, bioassay standard Ag, concentration is 0.1,0.5,1,5,10,20 μ g/mL; MAb-H8 is 10 μ g/mL, and pAb-T is 5 μ g/mL.Set 2 kinds of patterns: the A pattern is coated with mAb-H8, and pAb-T is sandwich antibody, i.e. mAb-H8-pAb-T sandwich mode; The B pattern is coated with how anti-pAb-T, and mAb-H8 is sandwich antibody, i.e. pAb-T-mAb-H8 sandwich mode.Experimental result shows highly sensitive than pAb-T-mAb-H8 sandwich mode (B) of mAb-H8-pAb-T sandwich mode (A), illustrate and set mAb-H8 to be that coated antibody has specificity high, and the pattern take how anti-pAb-T as sandwich antibody has amplification, so sandwich ELISA method of the present invention is selected the A pattern, the specificity of two kinds of patterns the results are shown in Figure 3 and Fig. 4.

The selection of embodiment 4 coated antibodies and antiserum best effort concentration

The concentration of coated antibody mAb-H8 is chosen 40,20,10,5,2.5 μ g/mL, many anti-pAb-T concentration choose 40,20,10,5,2.5, learn by the square formation titration, the working concentration of coated antibody mAb-H8 is 10 μ g/mL, when many anti-working concentrations were 5 μ g/mL, the valid analysing range of TRPC6 albumen was between 0.625 ~ 10 μ g/mL.

Determining of the selection of embodiment 5 sealers and off-period

With the suitableeest monoclonal antibody concentration (coated elisa plate of 10 μ g/mL), seal with 5% skimmed milk power, 3 kinds of different confining liquids of 5% hyclone and 5% bovine serum albumin(BSA) respectively after the washing, selected sealer is all used 0.05% polysorbas20-PBS (pH 7.4) preparation.The sealing mode is selected 37 ℃ of sealings 1.0h, 1.5 h and 2.0h, and sealing finishes with adding standard TRPC6 protein 10 μ g/mL after the PBST washing, and 37 ℃ of 1.0h washings add 37 ℃ of fixed positive monoclonal antibodies, negative serum again and hatch 1 h; Add enzymic-labelled antibody IgG after the washing, 37 ℃ of 1.0h washings, OPD colour developing are carried out microplate reader and are detected more different sealing effects.The result show three kinds of confining liquid effects best be 37 ℃ of sealings of 5% skimmed milk power 1.5h, secondly be 5% bovine serum albumin(BSA), 5% hyclone is a bit weaker, the results are shown in Table 1.

Selection and the time of the best confining liquid of table 1 double antibodies sandwich method are determined (OD _490nm)

Figure 201310215961X100002DEST_PATH_IMAGE002

Sensitivity and the specific assay of embodiment 6 monoclonal antibodies-how anti-sandwich method

For sensitivity and the specificity of further verification method, carry out determining between detection zone.By selected best of breed and working concentration, examination criteria TRPC6 polypeptide antigen is done typical curve with TRPC6 polypeptide antigen and the corresponding OD value of variable concentrations, and mensuration TRPC6 polypeptide protein concentration is between 1.0 ~ 20 μ g/mL the time, and curve is irregular S type, R ²=0.9697.TRPC6 polypeptide protein concentration is between 0.625 ~ 10 μ g/mL the time, and curve is line style, R ²=0.992, Fig. 5.According to the typical curve of measuring, determine that the valid analysing range of sample is between 0.625 ~ 10 μ g/mL.

The reaction of embodiment 7 structure sandwich ELISA methods mensuration systems is as follows:

S1. be coated with: join the flat miniature ELISA Plate in 96 holes after being diluted to optium concentration (10 μ g/mL) with monoclonal antibody mAb-H8 with coating buffer, every hole 100 μ L, 4 ℃ are spent the night; With the every hole 350 μ L washing of PBST 3 times;

S2. sealing: every hole adds 100 μ L and prepares 5% skimmed milk power confining liquid sealase target with PBST, washs 3 times with PBST behind 37 ℃ of 1.5h;

S3. the determined antigen with 0.01 mol/L PBS dilution adds ELISA Plate, and every hole 100 μ L are hatched 1 h for 37 ℃, identical washing lotion washing 3 times; Establish simultaneously standard positive, negative control, wash 3 times;

S4. the how anti-pAb-T(5 μ g/mL that dilutes with 0.01 mol/L PBS) add ELISA Plate, every hole 100 μ L are hatched 1 h for 37 ℃, identical washing lotion washing 3 times;

S5. with sheep anti mouse HRP-IgG(1:5000 dilution) add ELISA Plate, every hole 100 μ L, 37 ℃ of effect 1 h wash 3 times;

S6. add OPD substrate nitrite ion, every hole 100 μ L, lucifuge 15 min that develop the color; Add stop buffer: the H of 2 mol/L ₂SO ₄Cessation reaction, every hole 100 μ L; Microplate reader preheating 30 min, reading OD value under 490 nm.

Embodiment 8 TRPC6 double antibodies sandwich ELISA detection methods of the present invention are in the application that detects rat, mouse brain albumen TRPC6 content

Above method can be used for the mensuration that TRPC6 crosses expression tissue.Because normal rat brain albumen is the highest tissue of TRPC6 expression thought of research institute not long ago, at first uses the method for setting up to measure, and measures in the lump the TRPC6 protein expression level of mouse brain albumen tissue simultaneously.Extract the brain tissue total protein, rat quantitatively is 13.21 μ g/mL, mouse quantitatively is 11.02 μ g/mL, then measures, with the negative contrast of normal mouse serum, the positive contrast of standard TRPC6, measure simultaneously the TRPC6 typical curve, measure sample, the positive and negative control and the results are shown in Table 2, sample TRPC6 is to obtain at typical curve quantitatively, statistical significance is arranged, * p＜0.05.

Table 2 is measured large mouse brain albumen TRPC6 content

(annotate: the brain protein concentration refers to the brain tissue total protein concentration that extracts, and ultraviolet spectrophotometer is measured concentration; The TRPC6 polypeptide is 10 μ g/mL, negative control normal mouse serum dilution 1:100, * p＜0.05.）

Two anti-(monoclonal antibody-how anti-) sandwich ELISA detection methods of embodiment 9 TRPC6 of the present invention and two sandwich ELISA methods detect the comparison of TRPC6 content in rat, the mouse brain albumen

Adopt this chamber to set up two monoclonal antibodies (monoclonal antibody of hybridoma cell strains H8 secretion) sandwich ELISA method and measure comparison, mouse is 0.82 ± 0.02; Rat is 1.22 ± 0.05; Negative control is 0.26 ± 0.004, the result shows that how anti-monoclonal antibody-sandwich ELISA method be relatively higher than the TRPC6 content value of simple two sandwich ELISA methods mensuration, many anti-high-affinity advantage places when antigen is combined, so with monoclonal antibody as capture antibody, with how anti-as detecting antibody, the enlarge-effect that so more is conducive to signal reduces the undetected of sample in actual applications.

SEQUENCE LISTING

＜110〉Guangdong Pharmaceutical University

＜120〉a kind of double-antibody sandwich elisa detection method and kit of TRPC6 albumen

<130>

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 15

<212> PRT

＜213〉TRPC6 polypeptide

<400> 1

Lys Asp Leu Thr Lys Val Thr Leu Gly Asp Asn Val Lys Tyr Tyr

1 5 10 15

Claims

1. the double-antibody sandwich elisa detection method of a TRPC6 albumen, it is characterized in that, comprise the steps: the coated microwell plate with TRPC6 protein monoclonal antibody mAb-H8, behind the confining liquid closed porosity plate, the dilution that adds respectively standard TRPC6 polypeptide and testing sample adds TRPC6 protein polyclone antibody pAb-T and ELIAS secondary antibody again, adds the substrate colour developing after the washing, measure the OD value, calculate TRPC6 protein concentration in the testing sample;

Described TRPC6 protein monoclonal antibody mAb-H8 is obtained by purifying after the hybridoma cell strain H8 secretion; The working concentration of mAb-H8 is 5 ~ 20 μ g/mL;

Described TRPC6 protein polyclone antibody pAb-T by TRPC6 polypeptide-K LH conjugate immune mouse after the separation of serum purifying obtain; The sequence of TRPC6 polypeptide is shown in SEQ ID NO:1; The working concentration of pAb-T is 2.5 ~ 10 μ g/mL;

Described confining liquid is 37 ℃ of sealings of 5% skimmed milk power 1.5 ~ 2.0 hours; Perhaps, 37 ℃ of sealings of 5% bovine serum albumin(BSA) 1.0 ~ 1.5 hours, perhaps 37 ℃ of sealings of 5% hyclone are 1.5 ~ 2.0 hours.

2. the double-antibody sandwich elisa detection method of described TRPC6 albumen according to claim 1 is characterized in that, the working concentration of described mAb-H8 is 10 μ g/mL; The working concentration of described pAb-T is 5 μ g/mL.

3. the double-antibody sandwich elisa detection method of described TRPC6 albumen according to claim 1 is characterized in that, described confining liquid is 37 ℃ of sealings of 5% skimmed milk power 1.5 hours.

4. the double-antibody sandwich elisa detection method of described TRPC6 albumen according to claim 1, it is characterized in that, described mAb-H8 and pAb-T adopt the sad-ammonium sulfate salting-out process purifying of optimization to obtain, the optimal conditions of optimizing sad-ammonium sulfate salting-out process is that cells and supernatant and acetic acid-sodium acetate buffer volume ratio are 1:2 ~ 2:1, sad final concentration is 25 ~ 35 μ L/mL, the ammonium sulfate saturation degree is 35 ~ 45%, and the pH value is 7.0 ~ 7.4 during ammonium sulfate precipitation.

5. the double-antibody sandwich elisa detection method of described TRPC6 albumen according to claim 4, it is characterized in that, described optimization is sad-and the optimal conditions of ammonium sulfate salting-out process is that cells and supernatant and acetic acid-sodium acetate buffer volume ratio are 1:1, sad final concentration is 30 μ L/mL, the ammonium sulfate saturation degree is 40%, and the pH value is 7.2 during ammonium sulfate precipitation.

6. the double-antibody sandwich elisa detection method of described TRPC6 albumen according to claim 1, it is characterized in that, described pAb-T prepares by the following method: get 100 ~ 120 μ g TRPC6 polypeptide-K LH conjugates at every turn and adopt the subcutaneous multi-point injection method of mouse, 3 weeks of interval, mouse is carried out immune-treated three times, for the third time immunity rear blood sampling of rear 1 week is surveyed it and is tired, selection is tired greater than the booster immunization that carries out of 1:10000, booster immunization was got blood after 3 days, separation of serum, optimize sad-ammonium sulfate salting-out process antibody purification, save backup; The sequence of described TRPC6 polypeptide is shown in SEQ ID NO:1.

7. the double-antibody sandwich elisa detection method of described TRPC6 albumen according to claim 1 is characterized in that, comprises the steps:

S1. be the coated microwell plate of TRPC6 protein monoclonal antibody mAb-H8 of 10 μ g/mL with working concentration, 100 ~ 120 μ L/ holes, 4 ℃ of coated spending the night; PBST washing 3 times, 350 μ L/ holes pat dry;

S3. the dilution that adds respectively standard TRPC6 polypeptide and testing sample, hatched 1 hour for 37 ℃ in 100 μ L/ holes; PBST washing 3 times, 350, pat dry;

S4. adding working concentration is the TRPC6 protein polyclone antibody pAb-T of 5 μ g/mL again, hatches 1 hour for 37 ℃; PBST washing 3 times, 350, pat dry;

S5. add again the sheep anti mouse HRP-IgG of 1:5000 dilution, hatched 1 hour for 37 ℃; PBST washing 3 times, 350, pat dry;

S6. add the OPD substrate solution, 100 μ L/ holes, 37 ℃ of lucifuge reaction 15min; Add 2M sulfuric acid cessation reaction, 100 μ L/ holes; Microplate reader 490nm place measures the OD value; The drawing standard curve calculates TRPC6 protein content in the sample to be checked.

8. an ELISA kit that detects TRPC6 albumen is characterized in that, comprises ELISA Plate and sandwich antibody, and described ELISA Plate is coated with TRPC6 protein monoclonal antibody mAb-H8, and described sandwich antibody is TRPC6 protein polyclone antibody pAb-T;

Described TRPC6 protein monoclonal antibody mAb-H8 is obtained by purifying after the hybridoma cell strain H8 secretion; Described enzyme mark TRPC6 protein polyclone antibody pAb-T is obtained carrying out enzyme labeling behind the TRPC6 protein polyclone antibody pAb-T by separation of serum purifying behind the TRPC6 polypeptide-K LH conjugate immune mouse again; The sequence of described TRPC6 polypeptide is shown in SEQ ID NO:1.

9. described ELISA kit according to claim 8 is characterized in that, the marker enzyme of described enzyme labelled antibody is horseradish peroxidase.