CN1236312C - Human polycystic albumen -1 quantitative determination kit - Google Patents

Abstract

The present invention relates to a test kit used for quantitatively detecting the content of multisaccate protein-1 in human body fluid, which belongs to the technical field of medical immunology detection. The present invention has the technical scheme that an anti-human multisaccate protein-1 N-end monoclonal antibody and an anti-human multisaccate protein-1 N-end polyclonal antibody are used, a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) method used for quantitatively detecting the content of the multisaccate protein-1 in body fluid and a tissue lysis solution is established by an immunological technique; whether people are sick of autosomal dominant heritability multicystic kidney diseases is judged by contrasting the differences of the content of the multisaccate protein-1. The present invention has important reference value to the diseases for clinical diagnosis and provides an efficient way for preventing and curing the diseases early.

Description

Many capsules of people albumen-1 detection by quantitative kit

Technical field

The present invention relates to Medical Immunology detection technique field, is a kind of double-antibody sandwich elisa kit that is used for many capsules of detection by quantitative people body fluid albumen-1 content.

Background technology

Autosomal dominant polycystic kidney disease (autosomal dominant polycystic kidney disease, ADPKD) be one of human modal lethal single gene inheritance disease, involve all races, at the worldwide incidence of disease is 1/500 ~ 1/1000, has 1,300 ten thousand people to get involved approximately in the whole world.It is characterized in that the bilateral renal cyst is generation of carrying out property and increase, disease is made progress gradually, and the patient that is everlasting promptly develops into end stage renal failure in the later stage in middle age.The kidney failure that ADPKD causes accounts for more than 10% of renal replacement therapy patients sum.This cystic disease harm is very big, does not occur over just kidney, also often involves the outer organ of kidney, causes polycystic liver, ductus pancreaticus and cholangiectasis, colonic diverticula, intracranial aneurysm, heart valve disorders etc.

So far the clinical diagnosis index of ADPKD is not also sought unity of standard in the world, how according to family history, clinical manifestation, the result makes diagnosis in conjunction with imaging examination.The imaging examination of patient Chang Yin routine physical examination is found this disease, and the kidney tumour is often very obvious during discovery.The gene diagnosis result is definitely reliable, but technical conditions, auxiliary examination equipment requirements are higher, complex operation is gone back at present the body fluid diagnosis index of neither one ADPKD clinically, press for one simple, convenient, be easy to promote, the detection method of responsive and special clinical diagnosis ADPKD.

Summary of the invention

In view of ADPKD owing to No. 16 chromosomal PKD1 genes and (or) No. 4 chromosomal PKD2 gene mutations cause that its generation of 85 ~ 90% system is because coded product---the 26S Proteasome Structure and Function caused by abnormal of many capsules albumen-1 (PC-1) of PKD1.So, can be by the content of PC-1 in the detection by quantitative body fluid, tentative diagnosis ADPKD.PC-1 is the transmembrane glycoprotein of an about 460kD of molecular weight, is striden film district relative little endochylema tail with 1 (C end) and is formed by 1 huge extracellular region (N end), 11.The albumen motif of the numerous complicated that extracellular region had has determined the outer part of the born of the same parents of PC-1 that it is exercised the importance of normal physiological function.The present invention sets about PC-1 is detected from the N end of extracellular region.

The present invention utilizes anti-many capsules of people albumen-1N end monoclonal antibody and anti-many capsules of people albumen-1N to hold polyclonal antibody, adopt double-antibody sandwich enzyme-linked immunosorbent assay (ELlSA) method, prepared double-antibody sandwich PC-1-ELlSA kit, with the content of PC-1 in this kit detection by quantitative people body fluid.Judge whether to suffer from ADPKD by the difference that compares PC-1 content in the body fluid, provide an important reference frame for preventing and treating this disease early.

The main agents of kit of the present invention is anti-PC-1N end monoclonal antibody MA7B1 (being coated on the ELISA Plate) and anti-PC-1N end polyclonal antibody PAPC1, the antigen that is used to prepare two kinds of antibody is PC-1 extracellular region N end fusion PC-1-e2, and this fusion is corresponding to the 46th ~ 202 amino acids residue of PC-1.By the content of PC-1N end in the test sample, just can represent the level of PC-1 in being subjected to the sample product.

Kit of the present invention is composed as follows:

1.96 1 of hole ELISA Plate, it has wrapped by anti-people PC-1N end monoclonal antibody MA7B1, and bag is by concentration 5 μ g/ml.

2. sample diluting liquid is 1 bottle, 5ml, and it is the PBS damping fluid that contains 0.05% Tween-20 and 1% bovine serum albumin(BSA).

3. detect 1 bottle of antibody working fluid, 10ml, it contains anti-people PC-1N end polyclonal antibody PAPC1 1 μ g/ml.

4. the enzyme labelled antibody working fluid is 1 bottle, 10ml, and it contains the goat anti-rabbit igg of horseradish peroxidase-labeled.

5. the substrate dilution is 1 bottle, 20ml, and it contains pH5.0 phosphate-citrate salts damping fluid and hydrogen peroxide (H ₂O ₂).

6. stop buffer is 1 bottle, 5ml, and it is 2M H ₂SO ₄

7. cleansing solution (20 *) is 1 bottle, 50ml, and it is the pH7.4 phosphate buffer that contains 1.0% Tween-20.

8. standard items 2 are managed, and every pipe contains PC-1-e2 freeze-dried powder 400ng.

9. o-phenylenediamine (OPD) sheet is 3, every 2mg.

10. coordinate paper is 1.

The preparation of kit ELISA Plate of the present invention and each reagent and see the embodiment part for details with the method for PC-1 content in this kit human body liquid sample.

Embodiment

The preparation and the source of each reagent in the people PC-1 detection by quantitative kit

1. standard items albumen is the preparation of fusion PC-1-e2

Standard items albumen behaviour PC-1N end fusion PC-1-e2 freeze-dried powder, this albumen is corresponding to the flank-LRR-flank district of people PC-1 extracellular region N end and the 46th ~ 202 amino acids residue in part WSC district, and purity is more than 97%.The preparation process of this standard items albumen sees the patent of invention " many capsules of people albumen-1N end fusion PC-1-e2 " of application on September 25th, 2003 for details, and number of patent application is 03151184.8.Now its preparation process is summarized as follows:

(1) the total RNA of preparation people's nephridial tissue

Get fresh health adult's nephridial tissue, with Shanghai Hua Shun company total RNA extraction agent box, guanidine isothiocyanate method routinely extracts total RNA.

(2) synthetic primer

Human PKD1 cDNA sequence (GenBank L33243) according to known designs and synthesizes primer.

Forward primer: 5 '-ATA TGCCGCGTCAAC-3 ', its 5 ' end is introduced BamH I restriction enzyme site;

Reverse primer: 5 '-TAT

CGTGGGCAGCTG-3 ', its 5 ' end is introduced the HindIII restriction enzyme site.

(3) RT-PCR amplification PKD1e2 gene

With the total RNA of people's nephridial tissue is template, with the PKD1 cDNA sequence PKD1e2 gene of above-mentioned primer by RT-PCR amplification coding PC-1N end flank-LRR-flank district and part WSC district, its total length 474bp.

(4) construction of expression vector pQE30-PKD1e2 and engineering bacteria M15/pQE30-PKD1e2

With fusion protein expression vector pQE30 (German Qiagen company product, its 5 ' end of reading frame contains the nucleotide sequence of 6 histidines of continuous programming code) and the PKD1e2 gene use BamH I and HindIII double digestion respectively, two kinds of enzymes are cut product glue reclaim back T4DNA ligase connection, transformed competence colibacillus Escherichia coli M15 then, its transformant recombinant plasmid is identified by BamHI/HindIII double digestion and dna sequence analysis, recombinant expression carrier is pQE30-PKD1e2, and engineering bacteria is M15/pQE30-PKD1e2.

(5) abduction delivering of fusion PC-1-e2

Engineering bacteria M15/pQE30-PKD1e2 is 37 ℃ of overnight incubation in the LB nutrient culture media, are seeded in 2 * YT nutrient culture media with 1: 20 volume ratio, continue to be cultured to OD ₆₀₀Value is 0.5 ~ 0.6, and adding isopropylthiogalactoside (IPTG) to final concentration is 1mM/L, 32 ℃ of abduction deliverings 4 ~ 5 hours.Behind broken bacterium and Ni-NTA Agarose purifying, row SDS-PAGE electrophoresis shows that the fusion protein molecule amount of expressing is 19.0kD, is fusion PC-1-e2.

(6) purifying of fusion Pc-1-e2 and SDS-PAGE analyze

Thalline behind 4 ℃ of centrifugal collection steps (5) abduction delivering, centrifugal after the abundant cracking of 8M/L urea lysate (pH8.0), supernatant and 50% Ni-NTA Agarose (German Qiagen company product) are mixed, with 8M urea pH gradient buffering liquid (pH6.3, pH5.9, pH4.5) washing and wash-out fusion PC-1-e2.12% SDS-PAGE analysis fusioning protein PC-1-e2 analyzes through the albumen thin layer scanning, and this fusion accounts for 47.11% of bacterial protein.

(7) the freeze-drying packing of fusion PC-1-e2

Fusion PC-1-e2 Lowry standard measure with purifying is sub-packed in several 1.5ml plastic centrifuge tubes, and making every pipe PC-1-e2 content is 400ng, then with the freeze dryer freeze-drying to becoming freeze-dried powder, put 4 ℃ of preservations.

2. the preparation of sample diluting liquid

Sample diluting liquid is that to contain its compound method of phosphate (PBS) damping fluid (pH7.4) of 0.05% Tween-20 and 1% bovine serum albumin(BSA) as follows:

(1) preparation PBS damping fluid (pH7.4):

NaCl 8.0g

KH ₂PO ₄ 0.2g

Na ₂HPO ₄□12H ₂O 2.9g

KCl 0.2g

Distilled water adds to 1000ml

Adjust pH to 7.4

(2) preparation contains the PBS damping fluid of 0.05% Tween-20 and 1% bovine serum albumin(BSA)

Tween-20 500 μ l

Bovine serum albumin(BSA) 10g

PBS adds to 1000ml

Be distributed into the 5ml/ bottle, put 4 ℃ of preservations.

3. the preparation of enzyme labelled antibody working fluid

The goat anti-rabbit igg of horseradish peroxidase-labeled is available from U.S. Sigma company.This antibody stoste is sub-packed in the bottle with after 10,000 times of the sample diluting liquid dilutions, and every bottle of 10ml puts 4 ℃ of preservations.

4. the preparation of substrate dilution

(1) preparation phosphate-citrate salts damping fluid (pH5.0):

0.1M citric acid (19.2g/1000ml) 24.3ml

0.2M Na ₂HPO ₄(28.4g/1000ml) 25.7ml

Distilled water 50ml

(2) preparation substrate dilution: in above-mentioned 100ml phosphate-citrate salts damping fluid, add 30%H ₂O ₂160 μ l, packing behind the mixing, every bottle of 20ml puts 4 ℃ of preservations.

5. the preparation of stop buffer

Stop buffer is 2M H ₂SO ₄Get H ₂SO ₄22.2ml, slowly add in the 177.8ml distilled water.Be distributed into every bottle of 5ml.

6. the preparation of cleansing solution (20 *)

Cleansing solution is to contain 1.0% to tell 20 * PBS damping fluid (pH7.4) of soil temperature-20.Its compound method is as follows:

NaCl 160.0g

KH ₂PO ₄ 4.0g

Na ₂HPO ₄□12H ₂O 58.0g

KCl 4.0g

Tween-20 10ml

Distilled water adds to 1000ml

Adjust pH to 7.4 is distributed into the 50ml/ bottle.When the preparation working fluid, only need concentrate is done 20 times of dilutions.

7. the bag quilt and the sealing of the preparation of monoclonal antibody MA7B1 and 96 hole ELISA Plate

Capture antibody is that (antibody subtype is IgG to mouse source anti-people PC-1N end monoclonal antibody MA7B1 ₁, purity is more than 97%) and its preparation employing hybridoma technology, method sees the patent of invention " anti-many capsules of people albumen-1N end monoclonal antibody MA7B1 " of application on September 25th, 2003 for details, and number of patent application is 03151183.X.Now its preparation process is summarized as follows:

(1) immune Balb/C mouse

With the subcutaneous routinely immune male Balb/C mouse of foregoing fusion PC-1-e2, repeatedly immunity reaches 1: 6,000 ~ 1 to tiring of mouse polyvalent antibody: promptly can be used for preparing monoclonal antibody at 10,000 o'clock.

(2) preparation of monoclonal antibody MA7B1

Getting the successful Balb/c mouse spleen cell of above-mentioned immunity induces down and myeloma cell strain SP2/O carries out Fusion of Cells at Macrogol 4000 (PEG 4000), the HAT selectivity is cultivated and is filtered out fused cell, filter out the hybridoma of the anti-PC-1N end of secretion antibody with indirect enzyme-linked immunosorbent assay (indirect elisa method), carry out cloning with limiting dilution assay again and cultivate, obtain the hybridoma cell strain 7B1 of the anti-PC-1N end of stably excreting monoclonal antibody MA7B1.Routinely this strain 7B1 hybridoma is inoculated in the homology mouse peritoneal, induces generation ascites, extract ascites,, obtain the IgG of purifying by the affinity chromatography purifying ₁Type antibody MA7B1.After antibody done freeze-drying and handle the MA7B1 freeze-dried powder, put-80 ℃ and preserve 8. antibody sandwiches and sealing 96 hole ELISA Plate

96 hole ELISA Plate are available from Denmark NUNC company, and the bag quilt and the closed process of antibody are as follows:

(1) coated elisa plate

1. prepare the ELISA Plate bag and be cushioned liquid:

Na ₂CO ₃ 1.59g

NaHCO ₃ 2.93g

Distilled water adds to 1000ml

Adjust pH to 9.6.

2. be cushioned liquid with bag again monoclonal antibody MA7B1 freeze-dried powder is diluted to final concentration 5 μ g/ml (Lowry method protein quantification), it is added 100 μ l as capture antibody in the every hole of ELISA Plate wrap quilt, put 4 ℃ and spend the night.Can under 4 ℃ of conditions, preserve 6 months after the ELISA Plate sealing that bag is crossed.

(2) sealing

Use 1 * cleansing solution to wash routinely 3 times ELISA Plate, each 3 ~ 5 minutes, dry.Every again hole adds 200 μ l confining liquids (the PBS damping fluid that contains 1%BSA of pH7.4), and 37 ℃ of wet box sealings were spent the night in 2 hours or 4 ℃.

9. detect the preparation of antibody working fluid

Detecting antibody is rabbit source anti-people PC-1N end polyclonal antibody PAPC1 (purity is more than 95%).Be the sero-fast purifying thing that obtains behind the standard items albumen PC-1-e2 immunity new zealand white rabbit.Its preparation process sees the patent of invention " anti-many capsules of people albumen-1N end polyclonal antibody PAPC1 " of on September 25th, 2003 application for details, and number of patent application is 03151182.1 existing its preparation process to be summarized as follows:

With subcutaneous immune 3 the female new zealand white rabbits of foregoing fusion PC-1-e2 (code name is respectively A, B, C), repeatedly immunity to tiring of rabbit polyvalent antibody reaches 1.5 * 10 ^{5 ~}2.0 * 10 ⁵, get rabbit anteserum, again with the capable IgG purifying of affinity chromatography, obtain the polyclonal antibody PAPC1 of purifying.After this, with antibody Lowry standard measure, after the packing freeze-drying handle freeze-dried powder, put-80 ℃ of preservations.During preparation antibody working fluid, it is 1 μ g/ml that freeze-dried powder is diluted to concentration with sample diluting liquid, is distributed into every bottle of 10ml.

10. o-phenylenediamine (OPD) sheet

3, every 2mg is available from the magnificent biotech company in Shanghai.

The foundation of people PC-1 detection by quantitative kit typical curve

Set up the typical curve that detects people PC-1 content, this is one step of key of the double-antibody sandwich elisa kit of preparation, assembling detection by quantitative people PC-1 content.The concrete operations step is as follows:

(1) preparation of related reagent

1. cleansing solution: foregoing 20 * cleansing solution is done 20 times of dilutions with distilled water, and adjust pH to 7.4.

2. substrate solution: get foregoing substrate dilution 5ml, add 2mg OPD sheet, be 40% o-phenylenediamine (OPD)-H ₂O ₂Solution faces with preceding fresh preparation, uses immediately after joining.

(2) foundation of typical curve

1. the dilution of standard items PC-1-e2 and application of sample

To be washed 3 times with cleansing solution by good ELISA Plate with monoclonal antibody MA7B1 bag, each 3 ~ 5 minutes, dry.By certain gradient dilution PC-1-e2 freeze-dried powder, dilute 8 concentration: 1000.00ng/ml, 500.00ng/ml, 250.00ng/ml, 125.00ng/ml, 62.50ng/ml, 31.25ng/ml, 15.63ng/ml, 0.00ng/ml with antibody diluent altogether.The PC1-e2 of every kind of concentration draws 100 μ l and joins in the above-mentioned MA7B1 antibody sandwich hole (every kind of concentration is done 8 multiple holes), 37 ℃ of wet box incubations 2 hours.

2. drip and detect antibody PAPC1

ELISA Plate is washed 3 times with cleansing solution, each 3 ~ 5 minutes, dry.Every hole adds and detects antibody PAPCl 100 μ l in containing the ELISA Plate hole of standard items PC-1-e2,37 ℃ of wet box incubations 60 minutes.

5. dropping enzyme labelled antibody

ELISA Plate is washed 3 times with cleansing solution, each 3 ~ 5 minutes, dry.The every hole of ELISA Plate adds enzyme labelled antibody working fluid (goat anti-rabbit igg of horseradish peroxidase-labeled) 100 μ l, 37 ℃ of wet box incubations 60 minutes.

6. the detection of chromogenic reaction and optical density value

ELISA Plate is washed 3 times with cleansing solution, each 3 ~ 5 minutes, dry.The every hole of ELISA Plate adds 40% OPD substrate solution, 100 μ l, and low frequency oscillation is 15 seconds on the earthquake device, and the reaction of room temperature lucifuge is after 15 minutes, and Dropwise 50 μ l stop buffer in every hole was measured OD with enzyme connection detector in 5 minutes ₄₉₂Value.

7. set up typical curve

With standard items (PC-1-e2) concentration 1000.00,500.00,250.00,125.00,62.50,31.25,15.63,0.00ng/ml is horizontal ordinate, with the OD of respective concentration ₄₉₂Be ordinate after deducting blank value, on coordinate paper, map, the typical curve that draws, this curve linear scope is more satisfactory.This kit can detect 88 parts of humoral specimens altogether except that there being 8 holes to be used to set up the typical curve.

Detect the method for PC-1 content in ADPKD patient and the normal control group body fluid with kit of the present invention

One, the preparation of humoral specimen

Whole blood sample is put 4 ℃ of refrigerator overnight, and centrifugal 10 minutes of refrigerated centrifuge (4 ℃) 3000g collects serum ,-80 ℃ of preservations after the packing.For capsule liquid sample in urine and liver, the renal cyst, all at 4 ℃, centrifugal 10 minutes of 3000g collects supernatant, after the packing in-80 ℃ of preservations.

Two, the detection by quantitative of PC-1 in ADPKD patient and the normal control group body fluid

1. preparation reagent

(1) cleansing solution: 20 * cleansing solution is done dilution in 1: 20 with distilled water.

(2) standard items liquid preparation: add the dissolving of 200 μ l distilled waters before using in the standard QC, the PC-1-e2 solution concentration is 2ng/ μ l.

(3) preparation of substrate working fluid: before using the OPD sheet is put into the substrate dilution and dissolve, every adds solution 5ml.

2. the application of sample of standard items

Establish gauge orifice 8 holes on the ELISA Plate, every hole adds sample dilution 100 μ l earlier, adds standard items PC-1-e2 solution 100 μ l again in the 1st hole, moves to the 2nd hole with sample injector sucking-off 100 μ l behind the mixing.Oppose successively and doubly be diluted to the 7th hole, last, sucking-off 100 μ l discard from the 7th hole, and every hole is 100 μ l.The 8th hole is a blank.37 ℃ of wet box incubations 2 hours.

3. the application of sample of humoral specimen

It is some to establish the testing sample hole as required, and humoral specimen is added the testing sample hole respectively, and applied sample amount is 100 μ l/ holes, and negative control hole adds the equal-volume antibody diluent, the rearmounted 37 ℃ of wet box incubations of application of sample 2 hours.

4. drip and detect antibody

Above-mentioned ELISA Plate is washed 3 times with cleansing solution, each 3 ~ 5 minutes, dry.Add 100 μ l in the every hole of ELISA Plate and detect the antibody working fluid, 37 ℃ of wet box incubations 60 minutes.

5. dropping enzyme labelled antibody

Above-mentioned ELISA Plate is washed 3 times with cleansing solution, each 3 ~ 5 minutes, dry.Add enzyme labelled antibody working fluid 100 μ l/ holes again, 37 ℃ of wet box incubations 60 minutes.

6. the detection of chromogenic reaction and optical density value

Above-mentioned ELISA Plate is washed 3 times with cleansing solution, each 3 ~ 5 minutes, dry.The every hole of ELISA Plate adds substrate working fluid 100 μ l, and low frequency oscillation is 15 seconds on the earthquake device, room temperature lucifuge reaction 15 minutes, and Dropwise 50 μ l stop buffer in every hole joined detector bioassay standard product and testing sample OD with enzyme in 5 minutes ₄₉₂Value.

7. the result calculates and judges

(1) set up typical curve: with the concentration of standard items PC-1-e2 (0.00,15.63,31.25,62.50,125.00,250.00,500.00,1000.00ng/ml) is horizontal ordinate, the OD that microplate reader records ₄₉₂Value is ordinate after deducting blank value, sets up typical curve.On typical curve, along with the rising of standard items concentration, the OD that records ₄₉₂Value also increases.

(2) calculating of humoral specimen sample detection value: according to test sample OD ₄₉₂On above-mentioned typical curve, find corresponding PC-1 content behind the value subduction blank value.As blank well OD ₄₉₂Mean value is 0.201, test sample OD ₄₉₂Value is 0.54, and deducting behind the blank value is 0.339, and this value corresponding PC-1 content on typical curve is 82.68ng/ml.

The detection of kit quality inspection index of correlation of the present invention

1. the range of linearity: the range of linearity that this kit detects PC-1 content is 370pg/ml ~ 1000ng/ml.

2. the recovery: the PC-1-e2 of 60ng/ml is added in the test sample, and the recovery is 98.94% (n=8).

3. difference in criticizing: average out to 12.02%.

4. differences between batches: average out to 13.15%.

5. susceptibility: detecting ADPKD urine specimen susceptibility is 91.25%, and detecting ADPKD serum specimen susceptibility is 91.67%.

6. specificity: detecting normal group urine specificity is 90.10%; Detecting the normal group serological specificity is 88.7%.

Through clinic trial, it is as shown in table 1 that this kit detects the result of 110 routine normal persons and 101 routine ADPKD patients serum PC-1 and 114 routine normal persons and 120 routine ADPKD patient's urine PC-1.

The testing result of PC-1 in table 1 serum and the urine (x ± s, ng/ml)

	The normal person	ADPKD patient	P
				Serum (ng/ml)	98.76±37.36(n＝111)	53.19±16.90(n＝101)	＜0.05
Urine (ng/ml)	47.31±35.71(n＝114)	222.10±146.22(n＝120)	＜0.05

N: routine number

Table 1 result shows: in ADPKD patient's urine PC-1 content apparently higher than the normal person, significant difference (p＜0.05=; PC-1 content is starkly lower than the normal person among the ADPKD patients serum, significant difference (p＜0.05=.Above result shows, utilize kit energy detection by quantitative of the present invention to go out the content of PC-1 in the humoral specimen, clinical detection ADPKD is had higher susceptibility and specificity, be worth, provide an effective way for preventing and treating this disease early to diagnosing this disease to have important references.

Claims (1)

1. many capsules of people albumen-1 detection by quantitative kit, it is composed as follows:

1 of (1) 96 hole ELISA Plate has been wrapped by anti-many capsules of people albumen-1N end monoclonal antibody MA781;

(2) sample diluting liquid is 1 bottle, 5ml, and it is the PBS damping fluid that contains 0.05% Tween-20 and 1% bovine serum albumin(BSA);

(3) detect 1 bottle of antibody working fluid, 10ml, it contains anti-many capsules of people albumen-1N end polyclonal antibody PAPC1 1 μ g/ml;

(4) the enzyme labelled antibody working fluid is 1 bottle, 10ml, and it contains the goat anti-rabbit igg of horseradish peroxidase-labeled;

(5) the substrate dilution is 1 bottle, 20ml, and it contains pH5.0 phosphate-citrate salts damping fluid and hydrogen peroxide;

(6) stop buffer is 1 bottle, and 5ml is 2M H ₂SO ₄

1 bottle of (7) 20 * cleansing solution, 50ml, it is the pH7.4 phosphate buffer that contains 1.0% Tween-20;

(8) standard items 2 pipes, every pipe contain many capsules of people albumen-1 extracellular region N end fusion PC-1-e2 freeze-dried powder 400ng;

(9) the o-phenylenediamine sheet is 3, every 2mg;

(10) coordinate paper is 1.