CN104749371A - An enzyme-linked immunosorbent assay kit for human nephroblastoma-overexpressed gene encoded protein - Google Patents

Abstract

The invention discloses a double-antibody sandwich ELISA kit for human nephroblastoma-overexpressed gene encoded protein (NOV protein) and a preparing method thereof. The kit comprises an ELISA plate precoated with an NOV protein monoclonal antibody, an enzyme-labeled NOV protein rabbit polyclonal antibody, a washing liquid, a diluting liquid, a coloring liquid, a terminating liquid and a protein standard substance. The kit has characteristics of high sensitivity, high specificity, capability of being quantitative, accurate, simple and easy to use, good reproducibility, and the like, can rapidly detect a large number of samples simultaneously, can be used for qualitative and quantitative detection of the NOV protein in base and clinical research, and has a wide application prospect.

Description

People's nephroblastoma overepressed gene encoding proteins enzyme linked immunological kit

Technical field

The one that the present invention relates in technical field of immunoassay detects the double-antibody sandwich elisa kit of people's nephroblastoma overepressed gene encoding proteins (nov protein).

Background technology

People's nephroblastoma overepressed gene encoding proteins (Nephroblastoma-overexpressed geneprotein homolog, NOV), i.e. nov protein, also referred to as CCN3 albumen (CCN family member3) or insulin-like growth factor bindin 9 (Insulin-like growth factor-binding protein9, IGFBP9).Nov protein belongs to CCN family, and this family is made up of the secreting glycoprotein that one group of structure is similar, and family member known at present comprises CCN1 to CCN6 totally six kinds of albumen.CCN family protein cell propagation, break up and stick and vascularization, tumour occur and progression in all there is vital role.

NOV has multiple different physiological action, such as there is research display NOV to have to promote human nerve stem carefully to roar propagation and promote the effect of its Differentiating Into Neurons, delay the renal tubule transdifferentiation process caused by TGF-β 1 and play an important role in the generation of gestational diabetes mellitus.In addition, nov protein also can be used as the early diagnosis marker of pregnant pre-eclampsia.Meanwhile, existing many result of study display NOV are the marks of a novel tumour: NOV content is that positivity is relevant to melanomatous malignant degree; Overexpression nov protein can promote growth, the migration and invasion of esophageal cancer cell; The positive expression rate of NOV in cervical cancer tissues is apparently higher than the positive expression rate in normal cervical tissues, and the tumor size of the expression of NOV and cervical carcinoma and lymphatic metastasis etc. have significant correlation, illustrate that the expression of NOV is with the generation of cervical carcinoma with develop relevant, is considered to can be used as the diagnosis of cervical carcinoma and the molecular marker of clinical monitoring; NOV judges malignancy to have researcher to think, i.e. cell adhesion and the mark of invading profit.

NOV is as important biomarker, it detects research to tumour genesis mechanism, the tracking of development, Index for diagnosis and clinical treatment is quickly and accurately very crucial, thus need to set up a kind of easy, effective, can be used for all kinds of common sample as serum, blood plasma carry out the technical method that high flux detects fast.Compared with other technology, solid-phase enzyme-linked immune method because highly sensitive, specificity good, testing cost is low, simple to operate, be applicable to during analysis that the advantage such as pattern detection in enormous quantities is widely used in various important biomolecule mark detects.Current domestic conventional nov protein enzyme linked immunological kit mainly relies on import, expensive, the arrival cycle is long, and the domestic nov protein clinical diagnosis product still do not ratified through national Bureau of Drugs Supervision, therefore need exploitation associated antibodies and kit badly, support every fundamental research of domestic nov protein, break the corner on the market of imported product, and providing critical material for developing clinical in vitro diagnosis in vitro reagent further, expedite product is developed, and fills the domestic gaps.

Summary of the invention

The object of this invention is to provide that a kind of detection sensitivity is high, accuracy be strong, the double-antibody sandwich elisa kit of people's nephroblastoma overepressed gene encoding proteins (nov protein) of low cost.

Detection kit provided by the present invention comprises specific nov protein coated antibody, and the nov protein of enzyme labeling detects the standard items of antibody and nov protein.

Wherein said nov protein coated antibody is mouse monoclonal antibody, and its light chain and heavy chain amino acid sequence are respectively SEQ ID NO:1 and SEQ ID NO:2, and it is rabbit polyclonal antibody that described enzyme mark detects antibody, and described nov protein standard items are restructuring nov protein.

Described monoclonal antibody adopts restructuring nov protein immune animal, utilizes hybridoma technology to prepare.Described polyclonal antibody adopts restructuring nov protein immune animal, is prepared by Protein A purification and antigen affinity purification technology.Enzyme labelled antibody conventionally utilizes horseradish peroxidase (Horseradishperoxidase, HRP) labelled antibody to obtain.

Technical scheme of the present invention is preferred high sensitivity, the monoclonal antibody of high specific and polyclonal antibody carry out combinations of pairs, monoclonal antibody is adsorbed on solid phase carrier as coated antibody, coated antibody specificly can catch nov protein in sample and nov protein standard items, after adding enzyme mark detection antibody, form coated antibody, antigen, detection antibody complex, stop after corresponding substrate colour developing, read sample light absorption value, compare the content that can draw nov protein with typical curve.

The present invention adopts the nov protein level in the legal or quantitative detection sample of double antibodies sandwich, the method of inspection is convenient and easy, detection sensitivity and accuracy is high, high specificity, simultaneously can detect sample in enormous quantities fast, bag quilt and the detection antibody of kit chief component are independent research, therefore cost is low, reliability strong, plays a significant role in the relevant rudimentary and clinical research of nov protein.

Accompanying drawing explanation

The examination criteria curve map of Fig. 1 .NOV enzyme linked immunological kit

Embodiment

Below in conjunction with specific embodiment, the present invention will be further described and explanation.

The component preparation of embodiment 1 enzyme-linked immunologic detecting kit

1, the preparation of restructuring nov protein standard items

Standard items in described kit are restructuring nov protein, from Sino Biological Inc. (article No. is: 10264-H08B), this albumen utilizes insect cell to express in vitro, then the high purity N OV albumen of purifying acquisition, have biologically active, concrete activity and purity data can see the explanation of Yi Qiao Divine Land company's site for this product.

2, the preparation of nov protein mouse monoclonal antibody

1) animal immune

Select Balb/c mouse as immune animal, with above-mentioned restructuring nov protein for immunogene carries out immunity, immunizing dose is the nov protein of every mouse immune 50 μ g at every turn.During first immunisation, the complete Freund's adjuvant of immunogene and equivalent is made emulsifying agent, subcutaneous abdomen multi-point injection, interval gets same dose immunogene after 2 ~ 3 weeks and equivalent incomplete Freund's adjuvant makes emulsifying agent, booster immunization twice, indirect elisa method is used to measure serum titer after three immunity, serum titer reaches after 1: 16000, and once, after 4 days, extracting spleen cell carries out Fusion of Cells to the mouse peritoneal booster immunization that selection is tired the highest.

2) Fusion of Cells and cloning

Get mouse spleen, the rear single splenocyte suspension of centrifugal acquisition is filtered in grinding, after cell count, ratio in 5: 1 or 10: 1 mixes with the SP2/0 murine myeloma cell being in exponential phase, adopts polyglycol (PEG) method to carry out Fusion of Cells, bed board, by the effect of Selective agar medium, myeloma cell and splenocyte etc. do not merge or the cell of fusion not yet in effect cannot grow, and the hybridoma of effective integration will grow, breed in culture hole, and secretory antibody.After changing liquid three times, within 9-12 days, cells and supernatant is got after merging, using nov protein of recombinating as envelope antigen, indirect elisa method is utilized to measure supernatant, screen positive hole, and by limiting dilution assay, colonized culture is carried out to positive cell, until obtain the monoclonal hybridoma strain of the anti-nov protein specific antibody of stably excreting.Above Fusion of Cells and cloning method are the conventional classical way in immunology monoclonal antibody technique.

3) production of monoclonal antibody and purifying

Choose hybridoma cell strain, culture flask or bio-reactor is utilized to carry out cell chulture, collecting cell culture supernatant, routine protein A affinity column is utilized to carry out purifying, the antibody obtained is by packing after SDS-PAGE electrophoresis and indirect elisa method qualification antibody purity and specificity, for subsequent use in-20 DEG C of Cord blood.

4) sequencing of monoclonal antibody

Hybridoma is preserved for a long time and positive colony may be caused to lose due to unstable after repeatedly going down to posterity and pollution problem, for solving the problem, in process of the present invention, Protocols in Molecular Biology is utilized to be extracted light chain of antibody and the heavy chain gene of positive colony respectively, carry out sequencing, the plasmid including antibody gene can steady in a long-termly under-20 DEG C of conditions be preserved, simultaneously, according to antibody gene sequences, the technician in this professional domain conveniently can obtain identical monoclonal antibody by molecular biology method clonal expression.

It is as follows that antibody gene extracts amplification concrete grammar: collect the hybridoma that growth conditions is good, hybridoma total serum IgE is extracted by the operation scheme of the classical total RNA isolation kit instructions of BBI company, by electrophoresis detection quality, UV measures concentration.Be cDNA by the operation scheme of the MMLV first strand cDNAsynthesis kit instructions of BBI company by mRNA reverse transcription ,-20 DEG C frozen for subsequent use.Reverse transcription reaction system is: 11 μ l RNA (2.7 μ g), 5 × reaction buffer4 μ l, RNase inhibitor (20U/ μ l) 1 μ l, dNTP mix (10mmol/L) 2 μ l, M-MuLV reverse transcriptase1 μ l, total reaction volume is 20 μ l.According to list of references design primer, take cDNA as light chain and the heavy chain fragment that template difference PCR obtains antibody.PCR reaction system is: 2.0 μ l10 × Pyrobest buffer, 1.6 μ l2.5mM dNTP, 1.4 μ l10 μM primer pairs, 0.4 μ l hybridoma cDNA, and 0.2 μ l5U/ μ l Pyrobest DNAPolymerase, reaction system is 20 μ l.Amplification condition: sex change 94 DEG C of 4min; Sex change 94 DEG C of 1min, anneal 58 DEG C of 1min, extends 72 DEG C of 1min, 30 circulations; Extend 72 DEG C of 5min.Light chain and heavy chain fragment are inserted on pcDNA3T carrier, build pcDNA3-anti-NOV-L and pcDNA3-anti-NOV-H carrier.By vector Escherichia coli, picking positive colony, carries out order-checking qualification, analyzes sequencing result, obtain correct light chain and heavy chain amino acid sequence.The light-chain amino acid sequence that measurement result and sequential analysis show this antibody is SEQ ID NO:1; Heavy chain amino acid sequence is SEQ ID NO:2, refers to sequence table.Specific experiment operation is with reference to [Sa curtain Brooker J etc., " molecular cloning texts guide ", Beijing Science Press] well known to those skilled in the art.

3, the preparation of nov protein rabbit polyclonal antibody

Choose new zealand rabbit as immune animal, carry out immunity with nov protein of recombinating for immunogene, immunizing dose is the nov protein of every rabbit immune 500 μ g at every turn.The complete Freund's adjuvant of immunogene and equivalent is made emulsifying agent by first immunisation, neck dorsal sc multi-point injection, interval gets same dose immunogene in 2 ~ 3 weeks and equivalent incomplete Freund's adjuvant makes emulsifying agent, booster immunization, immune 4-5 time altogether, indirect elisa method measures serum titer and reaches after 1: 25000, heart extracting blood, by obtaining the polyclonal antibody of purifying after routine protein A affinity column and nov protein antigen affinity column two-step purifying, packing, is used for the preparation of enzyme labelled antibody in-20 DEG C of Cord blood.Wherein nov protein antigen affinity purification concrete steps are as follows:

A) Ago-Gel (GE company) of 0.7g cyanogen bromide-activated is claimed, swelling with 1mM HCl, then wash three times with 1mM HCl, for subsequent use;

B) nov protein getting 2mg utilizes hyperfiltration process albumen buffer exchange to be become 0.1M NaHCO3, pH8.3, and to control volume be 1 ~ 2ml;

C) joined by protein solution in the activated sepharose that step a) washed, room temperature shakes 4h;

D) with the Tris buffer blind unreacted group of pH8.0,0.1M;

E) fill gravity post, bed volume is about 2ml, rinses, and balance with PBS;

F) get Protein A purification how anti-cross post, rinse non-binding antibody with PBS, with the antibody of pH3.0 citrate buffer solution wash-out specific bond, with in Tris damping fluid and eluent to pH7.0-7.5.

4, the preparation of enzyme labelled antibody

A) HRP taking 5mg is dissolved in 0.5mL distilled water;

B) NaIO of the 0.1M that 0.5mL newly joins is added ₄solution, under room temperature, lucifuge stirs 20 minutes;

C) loaded in bag filter by above-mentioned solution, the sodium-acetate buffer (pH4.4) of 1mM is dialysed 4 DEG C and is spent the night;

D) get the polyclonal antibody of 5mg affinity purification, join in 1mL0.01M carbonate buffer solution, for subsequent use;

E) in c) liquid, add 0.2M carbonate buffer solution (pH9.5), make pH be elevated to 9.0 ~ 9.5, then add immediately d) in liquid, room temperature lucifuge stirs 2 hours gently;

F) 4mg/mLNaBH that 0.2mL now joins is added ₄liquid, mixing, places 2 hours in 4 DEG C;

G) loaded in bag filter by above-mentioned liquid, dialyse in the PBS of pH7.4,0.15M, 4 DEG C are spent the night.

Embodiment 2 detects the establishment of the enzyme linked immunological kit of nov protein

Set up the enzyme linked immunological kit detecting nov protein, comprise following reagent:

A) anti-nov protein mouse monoclonal antibody;

B) the anti-nov protein rabbit polyclonal antibody of HRP mark;

C) restructuring nov protein standard items;

D) bag is buffered liquid: the carbonate buffer solution of pH9.6,0.05mol/L;

E) confining liquid: the Tris damping fluid containing 2% bovine serum albumin(BSA);

F) sample diluting liquid: the phosphate buffer containing 0.1% bovine serum albumin(BSA);

G) cleansing solution: the phosphate buffer containing 0.1% tween;

H) substrate nitrite ion: be made up of nitrite ion A and nitrite ion B, nitrite ion A is hydrogen peroxide or urea peroxide, and nitrite ion B is tetramethyl biphenyl;

The sulfuric acid of i) stop buffer: 2mol/L.

The preparation of embodiment 3NOV enzyme linked immunological kit

1. utilize orthogonal test to grope optimum antibody combination and the working concentration of enzyme linked immunological kit

Ultraviolet spectrophotometer method is used to measure the concentration of antibody and antigen.Orthogonal test method is adopted to grope optimum antibody combination and optimum antibody working concentration, being diluted by anti-for difference nov protein mouse monoclonal antibody is 4 μ g/mL, 2 μ g/mL, 1 μ g/mL and 0.5 μ g/mL, the anti-nov protein rabbit polyclonal antibody dilution of HRP mark is 2 μ g/mL, 1 μ g/mL, 0.5 μ g/mL and 0.25 μ g/mL, and standard concentration is 1000pg/mL, 100pg/mL and 0pg/mL.Consider the absorbance value in blank well background and positive test hole, optimize a strain mouse monoclonal antibody as coated antibody, and confirm that its best effort concentration is that to mark the best effort concentration of polyclonal antibody be 0.5 μ g/mL for 2 μ g/mL, HRP.

2. the batch preparation of kit

1) a large amount of preparations of ELISA Plate:

Anti-nov protein antibody dilution to concentration is 2 μ g/mL by coated elisa plate: be buffered liquid with carbonate bag, and 100 μ L/ holes, wrap by 96 hole ELISA Plate, 4 DEG C of overnight incubation; Plate is washed 1 time, 200 μ L/ holes with cleansing solution; Close: every hole adds 300 μ L confining liquids and closes nonspecific binding site, incubated at room 1 hour; Then plate is washed 2 times with cleansing solution, 200 μ L/ holes; Pat dry rear vacuum packing machine packaging, 4 DEG C save backup.

2) a large amount of preparations of protein standard substance: dilute nov protein with sample diluting liquid, freeze-drying after packing, 4 DEG C save backup.

3) a large amount of preparations of enzyme labelled antibody: by how anti-for the anti-nov protein after affinity purification with HRP mark, add 50% glycerine, after packing ,-20 DEG C save backup.

The mensuration of embodiment 4NOV enzyme linked immunological kit major parameter

The major parameter of kit is measured, comprises the specificity of kit, accuracy and precision, concrete grammar and result as follows:

1. the specific detection of kit

Recombinant human protein EGF, TNFa, IL33, AFP, TIMP1, BAFF, HGF, TGF-β 1, γ-IFN and IGF1 are diluted to 100ng/mL, detect with nov protein enzyme-linked immunologic detecting kit, experiment results proved, people EGF, TNFa, IL33, AFP, TIMP1, BAFF, HGF, TGF-β 1 of this kit and 100ng/mL, the equal no cross reaction of γ-IFN and IGF1, show this kit specificity better.

2, the accuracy determination of kit

Carry out the accuracy of detection kit by adding recovery experiment, in human serum sample, add restructuring nov protein, the nov protein concentration of adding is 1.6ng/mL, 0.4ng/mL, 0.05ng/mL and 0ng/mL.Then with prepared kit, sample after interpolation is detected.Calculate the nov protein content in each sample according to typical curve, namely the nov protein content deducted in blank serum obtain measured nov protein content, and the protein content added divided by theory is TIANZHU XINGNAO Capsul.The results are shown in Table 1, result display TIANZHU XINGNAO Capsul is 95%-104%, and the prepared good accuracy of kit is described, serotonin is verified and detected without obviously interference.

Table 1NOV protein reagent box accuracy determination

The restructuring nov protein concentration (ng/mL) of adding	Measured value	The recovery
			1.6	1.664	104％
0.4	0.415	104％
			0.05	0.048	95％
0	0.108	-

3, the precision of kit measures

1) replica test

Select 5 parts of blood serum samples containing different N OV protein concentration, parallel 3 pieces of ELISA Plate detections, 5, each sample repetition on every block plate, each plate arranges standard control curve respectively, calculate the coefficient of variation (C.V) of each testing result respectively, in table 2, average coefficient of variation is 6.6%, illustrates that prepared kit has good repeatability.

Batch interior replica test of table 2NOV protein reagent box

2) differences between batches detect

Select 5 parts of blood serum samples containing different N OV protein concentration, duplicate detection 7 times in batches, 3 repetitions established by each each sample, arrange standard control curve in each plate.Calculate the coefficient of variation (C.V) between same increment product 7 testing results, in table 3, the coefficient of variation average out to 13.0% of 5 increment product, 7 testing results, meets in field and generally acknowledges differences between batches standard.

Table 3NOV protein reagent box differences between batches detect

The detecting step of embodiment 5NOV enzyme linked immunological kit

1. the collection of sample, process and preservation

1) cell culture supernatant: 1000 × g removes particle and polymkeric substance in centrifugal 10 minutes, and collect supernatant ,-20 DEG C save backup.

2) serum: venous blood collection, use after not collecting blood containing thermal source and endotoxic test tube, at 4 DEG C, centrifugal 15 minutes of 1000 × g, avoids haemolysis, and collect serum ,-20 DEG C save backup.

3) blood plasma: venous blood collection, use EDTA test tube to collect blood, centrifugal 15 minutes of 1000 × g at 4 DEG C, remove cell and particle ,-20 DEG C save backup.

2. application of sample

1) standard items wrapped by good ELISA Plate and freeze-drying are taken out, add 1mL sample diluting liquid standard items are dissolved, ambient temperatare puts 20 minutes, by standard items from 2000pg/mL, do the doubling dilution of 2 times, dilute 7 points, dilution is respectively got 100 μ L and add in 96 hole ELISA Plate according to the position of following table;

2) get test serum or plasma sample, add in reacting hole, 100 μ L/ holes, incubated at room temperature 2 hours;

3) cleansing solution washes plate 3 times, and 200 μ L/ holes, pat dry ELISA Plate.

3. add detection antibody

1) HRP labelled antibody sample diluting liquid is diluted to 0.5 μ g/mL, adds in reacting hole, 100 μ L/ holes, incubated at room temperature 1 hour;

2) cleansing solution washes plate 3 times, and 200 μ L/ holes, pat dry ELISA Plate.

4. develop the color

1) add the freshly prepared substrate nitrite ion of 200 μ L, room temperature reaction 20 minutes, then add 50 μ L stop buffer cessation reactions;

2) light absorption value is read under microplate reader 450nm wavelength.

5. the foundation of typical curve

With the concentration of nov protein standard items for horizontal ordinate, light absorption value is ordinate Criterion curve (Fig. 1), can calculate the nov protein content obtained in sample according to the sample light absorption value recorded.

Embodiment 6 kit detects the application of nov protein content in human serum

Nov protein enzyme-linked immunologic detecting kit prepared by utilization measures human serum nov protein content, detects the blood serum sample of 13 Healthy Peoples altogether, the results are shown in Table 4, and in the healthy population detected, the nov protein content range of serum is 5.5 ~ 42.4ng/mL.Above result shows, utilize kit of the present invention quantitatively can detect the content of nov protein in Healthy Human Serum sample, according to existing bibliographical information, the nov protein level of tumor patient is higher than normal population, therefore, the ELISA kit sensitivity that the present invention relates to can meet research needs completely, carries out accurate quantitative analysis mensuration to the nov protein content of normal population and tumor patient.

Table 4 Healthy Human Serum nov protein assay

Healthy Human Serum	Nov protein content (ng/mL)
		Serum 1	17.7
Serum 2	11.9
		Serum 3	11.1
Serum 4	13.2
		Serum 5	5.9
Serum 6	42.4
		Serum 7	14.7
Serum 8	14
		Serum 9	9.4
Serum 10	10.6
		Serum 11	11.4
Serum 12	5.5
		Serum 13	10.5

Claims (3)

1. people's nephroblastoma overepressed gene encoding proteins (nov protein) double-antibody sandwich elisa kit, it comprises:

(1) bag is by the ELISA Plate of nov protein monoclonal antibody;

(2) the nov protein polyclonal antibody of enzyme labeling;

Wherein, the light chain of the described nov protein monoclonal antibody for coated elisa plate and heavy chain amino acid sequence are respectively SEQ ID NO:1 and SEQ ID NO:2; The NOV polyclonal antibody of described enzyme labeling is the rabbit polyclonal antibody of horseradish peroxidase-labeled.

2. nov protein double-antibody sandwich elisa kit as claimed in claim 1, is characterized in that, also comprise following reagent:

(1) nov protein standard items: recombinant expressed nov protein;

(2) cleansing solution: the phosphate buffer containing 0.1% tween;

(3) sample diluting liquid: the phosphate buffer containing 0.1% bovine serum albumin(BSA);

(4) substrate nitrite ion: be made up of A liquid and B liquid, wherein substrate nitrite ion A is hydrogen peroxide or urea peroxide; Substrate nitrite ion B is tetramethyl benzidine;

(5) sulfuric acid of stop buffer: 2mol/L.

3. the double-antibody sandwich elisa kit of nov protein as claimed in claim 1 detects the application in the reagent of people's nov protein content in preparation.