CN109709336A - A kind of detection kit and detection method of vascular endothelial growth factor - Google Patents

Abstract

The invention discloses a kind of vascular endothelial growth factor detection kit and detection method, kit of the invention includes: VEGF antibody, the VEGF standard items, chemiluminescence preexciting liquid, chemiluminescence exciting liquid, cleaning solution of the magnetic particle of Streptavidin coupling, the VEGF antibody of biotin labeling, chemiluminescent labels label.It is detected using the kit, high sensitivity, reaction time are short, easy to operate, have a good application prospect.

Description

A kind of detection kit and detection method of vascular endothelial growth factor

Technical field

The present invention relates to a kind of protein immunization detection methods, are used for blood vessel endothelium based on electrochemical luminescence principle about one kind The kit and method of growth factor (VEGF) detection.

Background technique

Growth, the transfer of tumour cell rely on the formation of new vessels, vascular endothelial growth factor (vascular Endothelial growth factor, VEGF) it is most effective angiogenic growth factor (Ferraraetal.Endocr.Rev.1997,18:4-25).Vascular endothelial growth factor is that recent researches are relatively broad A kind of completely new broad-spectrum tumor marker (Cancer biomarker), it is considered to be the label of most significant neoplastic hematologic disorder screening Object.There are four big functions according to the research achievement of our company and external document report vascular endothelial growth factor detection kit: VEGF peripheral blood cell counts can be used as auxiliary diagnosis (Kondo S, the Biochim Biophys of the screening of (1) tumour, (2) tumour Acta.1994Mar 31；1221 (2): 211-4), the assessment of (3) oncotherapy curative effect and monitoring, (4) tumor prognosis, Yi Jifu Hair monitoring (Marpg D Clin Cancer Res April2,2013；DOI:10.1158/1078-0432.CCR-12- 3409)。

VEGF relative molecular weight is 45kDa, is the glycoprotein dimer connected by disulfide bond, other areas by same N-terminal Domain is made of two polypeptide chains of certain differences.VEGF gene is located at chromosome 6p21.3, which cuts by transcriptional level It cuts, can produce 5 kinds of different transcriptons, be separately encoded 23,121,165,186 and 206 amino acid polypeptides, VEGF is a kind of allusion quotation The exocrine protein of type.The monomer that VEGF is decomposed is inactive, removes 2 glycosyl of N to biological effect without influence, but may be in cell It works in secretion.Vegf gene is made of 8 exons and 7 intrones, is positioned at chromosome 6p21.3, due to exon shearing difference and form different hypotypes, produce respectively VEGF121, VEGF145, At least five kinds of protein forms such as VEGF165, VEGF185, VEGF206, wherein VEGF121, VEGF145, VEGF165 are secreting types Soluble protein is expressed in the mankind, can be directly acted on vascular endothelial cell and be promoted vascular endothelial cell proliferation, increase blood vessel Permeability.

The research of labelling immunoassay technology and application development are rapid nearly ten years, are widely used to biomedical basis Theoretical research and each field of clinical disease diagnosis.Method for detecting serological index is mainly immune including radioactive isotope Analysis, enzyme linked immunosorbent assay and chemiluminescence immune assay.These methods both can be used as Primary Screening Test and can also be used as really Recognize test, wherein chemoluminescence method has many advantages, such as to detect that the range of linearity is wide, detecting instrument is simple and convenient to operate.

Chinese patent 201510115025.0 discloses a kind of vascular endothelial growth factor detection kit and its raw material system It is standby, it discloses and is coated with Streptavidin, biotinylated VEGF monoclonal antibody is added, it is affine with being formed after antigen-reactive in sample Element-Antibody-antigen complex adds the VEGF monoclonal antibody of another plant of HRP enzyme label, and it is anti-to ultimately form Avidin-Biotinization Body-antigen-HRP labelled antibody compound.The detection luminous intensity but realized using this method is not high enough, influences detection effect Fruit.

Summary of the invention

The purpose of the present invention is to provide a kind of vascular endothelial growth factor detection kit and detection method, luminous intensities It is high, easy to operate, it has a good application prospect.

A kind of detection kit of vascular endothelial growth factor, comprising: magnetic particle, the biotin of Streptavidin coupling VEGF antibody, the VEGF standard items, chemiluminescence preexciting liquid, chemistry that VEGF antibody, the chemiluminescent labels of label mark Shine exciting liquid, cleaning solution.

Preferably, the chemiluminescent labels are acridinium ester or acridine ester derivant, and the acridine ester derivant includes Acridinium ester acid-NHS ester, acridinic acid propane sulfonic acid sodium salt or 9- azetidinecarboxylic acid.

Preferably, the chemiluminescence preexciting liquid is the ethyl alcohol containing mass percent 0.1-1% and quality percentage The mass fraction of Triton X-100 and/or Tween-20 than 0.1-1% are the H of 0.1-1%₂O₂Solution, the chemiluminescence Exciting liquid be the ethyl alcohol containing mass percent 0.1-1% and mass percent 0.1-1% Triton X-100 and/or The molar concentration of Tween-20 is the NaOH solution of 0.05-1mol/L.

Preferably, the magnetic particle of Streptavidin coupling by pH be 6-8, molar concentration 0.01-0.2mol/L Phosphate buffer or Tris buffer form, partial size be 0.05-2 μm.

It is highly preferred that the magnetic particle kernel is ferroso-ferric oxide or di-iron trioxide, the surface modification of magnetic particle Group is carboxyl.

Preferably, the VEGF standard items are that it is (excellent that VEGF antigen sterling is added using bovine serum albumin(BSA) buffer as solvent It selects purity to be not less than 90%) to be formulated.

It is highly preferred that the VEGF standard items are the VEGF standard items with multiple concentration gradients being made into.

Preferably, the cleaning solution includes the Triton X-100 and/or Tween 20, pH of mass percent 0.5-10% The phosphate buffer or Tris buffer for being 0.1-0.5mol/L for 6-8, molar concentration, mass percent 0.5-5%'s Proclin 300。

The present invention also provides the detection methods using mentioned reagent box, comprising:

(1) the VEGF antibody for taking VEGF antibody, sample to be tested, the chemiluminescent labels of biotin labeling to mark is in anti- It answers in container, mixes well, and in 37 DEG C of 10~30min of incubation, while preparing standard items；

(2) into reaction system be added Streptavidin coupling magnetic particle, mix well, and in 37 DEG C be incubated for 3~ 10min, rear cleaning separation magnetic particle；The mixed of chemiluminescence preexciting liquid and chemiluminescence exciting liquid is added in magnetropism particle Solution is closed, reacts and measures luminous intensity；

(3) standard curve is drawn according to standard concentration and luminous intensity, sample luminous intensity is corresponding on standard curve Concentration value is the measurement concentration of sample to be tested.

Beneficial effects of the present invention are as follows:

(1) present invention selects magnetic particle to provide not only as reaction carriers using the detection architecture of electrochemical luminescence It is a kind of close to homogeneous reaction system, and overcome the technological deficiency of existing enzyme-linked immunization.

(2) present invention selects acridinium ester or derivatives thereof as marker, and reaction does not need catalyst, as long as alkaline environment Can carry out, be swift in response, can the photon that generates of catching reaction completely, background luminescence is low, and signal-to-noise ratio is high, and disturbing factor is few, Reagent stability is good, and system is simple, and exciting liquid is at low cost.

(3) present invention adds ethyl alcohol as stabilizer in chemiluminescence preexciting liquid and chemiluminescence exciting liquid, adds Add TritonX-100 or Tween-20 as reinforcing agent, can effectively enhance the luminous intensity of acridinium ester or derivatives thereof, together When also can be reduced luminescent system and the chance of bubble occur；The present invention also increases 300 preservative of Proclin in cleaning solution, can Conducive to further maintenance electrode.

(4) present invention is realized by full-automatic electrochemical luminescence immune detection system, and the addition of reagent and sample is equal It is completed by instrument, the operation is more convenient, reduces artificial error.

Specific embodiment

Below by specific embodiment, the present invention is described in detail, but the purposes of these exemplary embodiments and Purpose is only used to enumerate the present invention, not constitutes any type of any restriction to real protection scope of the invention, more non-to incite somebody to action Protection scope of the present invention is confined to this.

Reagent used in the following embodiment, such as no special explanation, can be from buying on the market.

Embodiment

The invention mainly includes steps:

(1) preparation of monoclonal antibody

1, the expression and purification of VEGF165 fusion protein

1) it is complete that people VEGF 165 building of VEGF gene cloning and prokaryotic expression plasmid: is chosen from human placenta cDNA library Long sequence pGEX-4T-1 is as carrier, which contains glutathione S-transferase label, and the expression of albumen is by tac promoter control System.Tac promoter is induced by IPTG.Cloning site chooses EcoRI and NotI.

2) VEGF expressing fusion protein: pGEX-4T-1/VEGF165 is converted to BL21 (DE3) pLysS competent cell (Escherichia coli), are stablized and efficient expression strain.LB culture medium culture, 37 DEG C of expression GST-VEGF165 fusion protein packets Contain body.

3) VEGF165 fusion protein purification: recombinant is induced in LB liquid medium through IPTG, is expressed 2 hours Afterwards, thallus is collected, after ultrasonication, Sarkosyl is added, mixes.Fusion protein supernatant will be contained, Glpgtathione is added After rinsing, reduced glutathione elution is added in Sepharose 4B gel.Collect albumen.50% glycerin storage in- 20℃。

2, mouse immune and monoclonal antibody preparation

1) it mouse immunization protocol: selects 6-8 week old female BALA/c mouse to be immunized in three times, antigen: using large intestine Bacillus source VEGF；Immunologic adjuvant uses Freund's adjuvant；Immunization ways: subcutaneous multi-point injection and intraperitoneal injection, primary immunization； Subcutaneous multi-point injection after 20 μ g of VEGF antigen and adjuvant are fully emulsified；Secondary immunity: with behind primary immunization interval 3 weeks, VEGF is anti- Subcutaneous multi-point injection (avoiding primary immunization injection point), is immunized: three times between secondary immunity after 20 μ g of original and adjuvant are fully emulsified Every after two weeks, 20 μ g of VEGF antigen and adjuvant it is fully emulsified after intraperitoneal injection.

2) bioactivity: 10-14 days venous blood samplings after being immunized three times detect antibody titer, reach 10⁶It is above to carry out cell Fusion.

3) cell fusion

3-1) prepared by myeloma cell: 30 days or so the recovery myeloma cell strains before doing cell fusion use RPMI-1640 Culture medium culture to state it is preferable after, then in the miscellaneous azaguanine of 8- (8-AG) culture medium culture culture 1 week, after use instead again Solid tumor is beaten to mouse after RPMI-1640 culture medium culture 3-4 days.Neck is drawn to put to death taking-up mouse within 12-18 days after beating oncocyte Oncocyte, oncocyte are ground after taking out, and are being isolated and purified with lymphocyte separation medium and to cell count；

3-2) the preparation of splenocyte

Immunized mice plucks eyeball execution, takes spleen, grinds, and counts to splenocyte.

3-3) oncocyte is merged with splenocyte

Splenocyte and oncocyte are merged in the ratio of cell count 5: 1, are induced using PEG1200, are added to and complete after fusion 96 orifice plate culture 5-7 days of feeder layer, after measured with HAT culture medium half and change liquid, CLIA method screens positive cell strain.

3-4) cloning and cell determine pearl

Cloning method: limiting dilution assay, the positive cell strain selected carry out limiting dilution, repeatedly 2-3 limiting dilution, The cell of 95% or more positive rate of selection determines pearl, and cultivates amplification freeze-stored cell strain

3-5) monoclonal antibody is cultivated

Select 6-8 week old female BALA/c mouse peritoneal injection 10⁷A cell for having determined pearl, takes mouse ascites in 5-10 days, Supernatant is taken after ascites centrifugation,

It is 3-6) monoclonal antibody-purified

Select GE company proteinG purification column；Purification column is balanced with 20mM PB buffer, ascites loading is added, is used PH2.70.1mol glycine HCI buffer is eluted, and is collected with the EP pipe containing PH8.71mol tris buffer, 0.05mM PB dialysis, concentration freeze.

3-7) monoclonal antibody Property Identification: Property Identification is detected including the use of Sigma CLIA antibody typing reagent.It is single Anti-cell strain supernatant detects antibody specificity using immunoblotting analysis test and competitive assay.Utilize competition CLIA detection antibody parent And power.

(2) preparation of the magnetic particle of Streptavidin coupling

1, by 10mL iron oxide (Fe₃O₄) nanoparticle (partial size is about 1.5 μm) is dissolved in isometric pH 6.0, In 0.05mol/L borate buffer solution, 1- ethyl-(the 3- diformazan for being 150: 1 with the molar ratio of ferric oxide nano particles is added Base aminopropyl) phosphinylidyne diimine and N- hydroxy thiosuccinimide, 37 DEG C are reacted 2.5 hours, by iron oxide nano-granule Sublist face converting carboxylate groups are the active ester of stable under acidic conditions.

2, the solution of streptavidin for being 10: 1 with ferric oxide nano particles molar ratio is added, is adjusted with 1M NaOH solution PH value of solution reacts at room temperature 3 hours to 8.5, Streptavidin is coupled to ferric oxide nano particles surface.

3, final concentration of 3% single-ended amino polyethylene glycol (HO-PEG-NH is added into solution₃), by solution ph It adjusts to 5.0,18000rpm (28,000g-30,000g), 4 DEG C of centrifugation 30min, abandons supernatant, precipitating pH is 6.8, concentration For the Tris buffer of 0.1mol/L, to be resuspended to concentration be 0.5mg/mL, and the buffer contains the bovine serum albumin of 0.5wt% White, 0.05wt% TritonX-100,0.05%proclin300 and 0.05% Sodium azide.

(3) the VEGF Antibody preparation of biotin labeling

400 μ L5mg/mL VEGF monoclonal antibodies are taken, are stirred at 2-8 DEG C with 0.1mol/L, pH9.3 carbonate buffer solution Dialysis 8 hours is mixed, a not good liquor is changed in centre；Antibody of the ratio for being 1: 25 according to antibody molecule and biotin molecule ratio after dialysis Biotin solution is added in solution, and DMSO to final concentration of 10% is added, mixes；Slow oscillation, 37 DEG C are protected from light 2h；Add Enter the 3mol/L ethanolamine solutions of 80 μ L, room temperature is protected from light 30min；Stir dialysis at 2-8 DEG C with 0.01M PBS solution, 6 Hour changes a not good liquor, changes altogether liquid 3 times；Isometric glycerol is added to the antibody-solutions after dialysis, mixes, packing, concentration is 0.5mg/mL, -20 DEG C of preservations.

(4) preparation of the VEGF antibody of acridinium ester label

50 μ L5mg/mL VEGF monoclonal antibodies are taken, the carbonate buffer solution of 150 μ L0.05M pH 9.3 is added, are mixed, Then the acridinium ester that 5 μ L2mg/mL are added mixes, and is protected from light, takes out at room temperature after 2h, is handled with the super filter tube of 50Kd, ultrafiltration It is handled respectively with pure water and PBS buffer solution first in the process, the VEGF antibody for the acridinium ester label being eventually adding Solution, collects liquid in centrifuge tube to saving pipe, packing be stored in 4 DEG C it is spare.

(5) preparation of VEGF standard items and calibration

In the present embodiment, international agreement reference material concentration is compareed by standard items raw material VEGF, goes out series with PBS doubling dilution 5 concentration points of standard items, and prepare S0 point.It is subject to working standard, this series of products standard items is surveyed with chemoluminescence method Fixed each concentration point actual concentrations value, and each concentration point is finely tuned to target concentration value (0,50,100,200,400,800pg/mL), Within calculating linearly dependent coefficient r=0.9992, accuracy 90%-110%.Standard is dispensed with the pipe-produced glass bottle of 2ml specification Product solution, dispensed loading amount are 0.5ml/ bottles.It is placed in 2-8 DEG C of preservation.

(6) preparation of chemiluminescence preexciting liquid, chemiluminescence exciting liquid

(1) chemiluminescence preexciting liquid by mass fraction be 1% H₂O₂In solution be added 0.5wt% ethyl alcohol and The Triton X-100 of 0.25wt% is made, 2-8 DEG C be kept in dark place it is spare.

(2) chemiluminescence exciting liquid by molar concentration be 1M NaOH solution in be added 0.5wt% ethyl alcohol and The Tween-20 of 0.5wt% is made, 2-8 DEG C be kept in dark place it is spare.

(7) preparation of cleaning solution

Prepare pH 7.0, the phosphate buffer of 0.5mol/L, the buffer contain 10wt% Tween 20, The Proclin 300 of 5wt%, 2-8 DEG C be kept in dark place it is spare.

(8) it detects

With electrochemical luminescence automatic lmunoassays analyzer (601 type of Roche Cobas e) for detection instrument, 50 μ are sequentially added The VEGF antibody of the biotin labeling of L, the sample to be tested of 50 μ L, 50 μ L acridinium ester label VEGF antibody, make biotin mark The final concentration of 0.1-2 μ g/ml of the VEGF antibody of note, the final concentration of 0.1-5 μ g/ml of the VEGF antibody of acridinium ester label, reaction After 15min, the 20 μ L of Streptavidin magnetic particle that partial size is 1.5 μm is added, after reacting 5min, carries out Magneto separate, instrument will Reaction mixture be sent into darkroom, sequentially add 100 μ L chemiluminescence preexciting liquid, 100 μ L chemiluminescence exciting liquids carry out it is luminous Reaction, finally records luminous intensity, the content of sample is calculated from standard curve.

(9) kit performance detection

It is measured according to performance of the conventional kit performance detection mode to the kit, as a result as follows:

1, linearity test

Kit Plays product are taken, measure 3 times, is averaged, and as a result makees regression straight line with expected concentration, calculates at every To regression coefficient r > 0.99, the range of linearity: 0-800pg/mL.

2, detection range

The VEGF standard items of the present embodiment have luminescence phenomenon, show this hair in the range of concentration is 0.1-10pg/ml Bright kit detection range is 0.1-10pg/ml.

3, precision

In the kit batch and difference between batch is respectively less than 10%.

4, sensitivity

This kit is 3.5pg/mL to VEGF sensitivity for analysis.

5, stability: kit is stored in 2-8 DEG C of light protected environment, and validity period is 12 months；It uncaps and is stored in 2-8 DEG C and keeps away In luminous environment, validity period is 30 days.

6, the rate of recovery: 90%-110%.

Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although Present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: it still may be used To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features； And these are modified or replaceed, technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution spirit and Range.

Claims (9)

1. a kind of detection kit of vascular endothelial growth factor, comprising: magnetic particle, the biotin mark of Streptavidin coupling The VEGF antibody of note, the VEGF antibody of chemiluminescent labels label, VEGF standard items, chemiluminescence preexciting liquid, chemistry hair Light exciting liquid, cleaning solution.

2. a kind of detection kit of vascular endothelial growth factor according to claim 1, which is characterized in that the chemistry Luminous marker is acridinium ester or acridine ester derivant, and the acridine ester derivant includes acridinium ester acid-NHS ester, acridinic acid third Sulfonate sodium or 9- azetidinecarboxylic acid.

3. a kind of detection kit of vascular endothelial growth factor according to claim 1, which is characterized in that the chemistry Shine the Triton X- that preexciting liquid is the ethyl alcohol and mass percent 0.1-1% that are 0.1-1% containing mass percent The mass fraction of 100 and/or Tween-20 is the H of 0.1-1%₂O₂Solution, the chemiluminescence exciting liquid are to contain quality percentage The molar concentration of the Triton X-100 and/or Tween-20 of ethyl alcohol and mass percent 0.1-1% than 0.1-1% is The NaOH solution of 0.05-lmol/L.

4. a kind of detection kit of vascular endothelial growth factor according to claim 1, which is characterized in that the strepto- The magnetic particle of Avidin coupling by pH is 6-8, the phosphate buffer that molar concentration is 0.01-0.2mol/L or Tris buffering Liquid is formulated, and partial size is 0.05-2 μm.

5. a kind of detection kit of vascular endothelial growth factor according to claim 1, which is characterized in that the magnetism Particle core is ferroso-ferric oxide or di-iron trioxide, and the surface modification group of magnetic particle is carboxyl.

6. a kind of detection kit of vascular endothelial growth factor according to claim 1, which is characterized in that the VEGF Standard items are that VEGF antigen sterling is added and is formulated using bovine serum albumin(BSA) buffer as solvent.

7. a kind of detection kit of vascular endothelial growth factor according to claim 6, which is characterized in that described VEGF standard items are the VEGF standard items with multiple concentration gradients being made into.

8. a kind of detection kit of vascular endothelial growth factor according to claim 1, which is characterized in that the cleaning Liquid includes the Triton X-100 and/or Tween 20 of mass percent 0.5-10%, pH 6-8, molar concentration 0.1- The phosphate buffer or Tris buffer of 0.5mol/L and the Proclin 300 of mass percent 0.5-5%.

9. a kind of described in any item detection methods of the detection kit of vascular endothelial growth factor of claim 1-8 are used, Include:

(1) the VEGF antibody for taking the VEGF antibody, sample to be tested, chemiluminescent labels of biotin labeling to mark is in reaction vessel In, it mixes well, and in 37 DEG C of 10~30min of incubation, while preparing VEGF standard items；

(2) into reaction system be added Streptavidin coupling magnetic particle, mix well, and in 37 DEG C be incubated for 3~ 10min, rear cleaning separation magnetic particle；The mixed of chemiluminescence preexciting liquid and chemiluminescence exciting liquid is added in magnetropism particle Solution is closed, reacts and measures luminous intensity；

(3) standard curve is drawn according to VEGF standard concentration and luminous intensity, sample luminous intensity is corresponding on standard curve Concentration value is the measurement concentration of sample to be tested.