CN1641351A

CN1641351A - Human brain red protein detecting and diagnostic kit, and its preparing method and use

Info

Publication number: CN1641351A
Application number: CN 200310113502
Authority: CN
Inventors: 张成岗; 陈婷方; 王春丽; 高艳; 孙建伟; 韩洪彦; 徐文琳; 王航雁
Original assignee: Institute of Radiation Medicine of CAMMS
Current assignee: Institute of Radiation Medicine of CAMMS
Priority date: 2004-01-16
Filing date: 2004-01-16
Publication date: 2005-07-20
Anticipated expiration: 2024-01-16
Also published as: CN100489526C

Abstract

The invention provides a novel human NGB detecting and diagnosing reagent box, and its manufacturing method and use. This reagent box is composed of solid-phase carrier as well as monoclonal antibody, multiclonal antibody and standard NGB product, etc; its preparing method include the steps of preparing antigen and antibody, purifying antibody, packaging reagent box. It has higher sensitivity and can be applied to detecting the content of NGB in sample, and provide effective detecting means for early diagnosis and course of diseases such as epileptic dementia, cerebral infarction, traumatic brain damage, etc.

Description

A kind of Novel Human NGB detects and diagnostic kit, Preparation Method And The Use

Technical field the present invention relates to a kind of immune detection and diagnostic reagent, is specifically related to a kind of Novel Human NGB enzyme linked immunosorbent detection and diagnostic kit, also relates to Preparation Method And The Use.

The background technology NGB (Neuroglobin, NGB) be in the body the 3rd class except haemoglobin and myoglobins important take oxygen albumen, specifically expressing in nervous system is distributed widely in the brain tissue.The same with myoglobins, NGB can be reversibly in conjunction with oxygen, and with oxygen very high affinity is arranged.Myoglobins is atomic at the normal human serum intensive amount, and when cardiac muscle and Skeletal muscle injury, myoglobins can discharge from damaged cell, goes into blood fast, causes serum myoglobin to raise.Myoglobins has become responsive and special biochemical diagnosis index such as some important diseases such as miocardial infarction, renal failure clinically.NGB and myoglobins are in function and have strict similarity in nature: NGB has 151 amino acid, and molecular weight is 17 kilodaltons, and myoglobins then has 154 amino acid, and molecular weight also is 17 kilodaltons.In view of NGB and myoglobins have strict similarity on function, it is the oxygen supply that myoglobins is responsible for cardiac muscle and skeletal muscle, NGB then is responsible for the oxygen supply of brain, both all can be released into blood from damaging cells, therefore, NGB relates at senile dementia, cerebral infarction, traumatic brain injury etc. in the early diagnosis of diseases such as neure damage and sex change death has potential diagnostic value, the course of disease in the above-mentioned disease treatment process is followed the tracks of also to have important value simultaneously.

Summary of the invention the invention provides a kind of Novel Human NGB ELISA measuring reagent kit.This kit is by one of 96 hole ELISA Plate, 1 bottle of NGB standard items, 1 bottle of NGB monoclonal antibody, 1 bottle of stop buffer of concentrated cleaning solution (sulfuric acid solution (H of 2M that 1 bottle of NGB polyclonal antibody, horseradish peroxidase (HRP)-1 bottle of goat anti-rabbit immunoglobulin (IgG), o-phenylenediamine (OPD) are 2 bottles, 20 times ₂SO ₄)) 1 bottle of composition.Wherein the HRP-goat anti-rabbit igg is a middle mountain company product.The invention also discloses preparation method, using method of kit and uses thereof.

This kit of purposes has higher sensitivity, can be used for the content of NGB in the test sample, and can be senile dementia, cerebral infarction, traumatic brain injury etc. and relate to early diagnosis in the diseases such as neure damage and sex change death and the course of disease in the therapeutic process and follow the tracks of effective detection means is provided.

Below needing, the use of this kit of preparation method provides reagent for oneself:

1) wrap diluted liquid (the 0.05mol/L carbonate buffer solution, pH9.6): Na ₂CO ₃1.5g+NaHCO ₃2.9g be dissolved in distilled water, add distilled water and replenish volume to 1000ml.

2) phosphate buffer (PBS, 0.02M, pH7.4): NaCl 16.0g+NaH ₂PO ₄2H ₂O 0.593g+Na ₂HPO ₄12H ₂O 5.8g adds distilled water to 2000ml.

3) sample dilution: PBS 100ml+0.1g BSA+50 μ l Tween 20.

4) confining liquid (5% skimmed milk-PBS solution): 50g skimmed milk+PBS to 1000ml.

5) substrate buffer solution: A liquid (0.1mol/L citric acid solution): citric acid 19.2g adds distilled water to 1000ml; B liquid (0.2mol/L Na ₂HPO ₄): Na ₂HPO ₄12H ₂O 71.7g adds distilled water to 1000ml; Face with before getting A liquid 24.3ml, B liquid 25.7ml and mix.

6)30％H ₂O ₂?500ml。

The preparation method that this kit is concrete:

1) preparation of antigen: the prokaryotic expression carrier pGEX-4T-2 transformed into escherichia coli BL21 that will be built with the NGB gene coding region, by isopropyl-(isopropyl-1-thio-D-galactopyranoside, IPTG) abduction delivering, obtain the solubility expression product, with behind glutathione S-transferase (GST) the affinity column purifying as antigen (Fig. 1, Fig. 2).

2) the antigen immune Balb/c mouse of preparation prepares anti-NGB monoclonal antibody by traditional cell fusion method the preparation of antibody: with 1), simultaneously immunizing rabbit prepare anti-NGB polyclonal antibody (Fig. 3, Fig. 4).

3) purifying antibody: with 2) antibody of gained through centrifugal, precipitation, desalination, slightly carry antibody, through Protein G post affinity purification, promptly get antibody purification then.

4) preparation of kit:, be divided in bottle or the conical centrifuge tube by the kit requirement with 8 kinds of compositions of kit.

5) specificity of identification kit, sensitivity, precision, qualified stability (Fig. 7).

The using method of this kit of using method is as follows:

1) the bag quilt of antibody: the monoclonal antibody (10 μ g/ml) of the anti-NGB of coating buffer dilution is added in the 96 hole ELISA Plate, every hole 100 μ l, 4 ℃ are spent the night, cleansing solution washing 1 time, each 3 minutes.Add confining liquid 200 μ l/ holes, 40 ℃ of sealings 50～60 minutes (or 4 ℃ spend the night).Sealing finishes the back and washes plate 3 times with cleansing solution, each 3 minutes.

2) add sample: will contain the testing sample and the standard NGB albumen (5.76ng/ml, 28.8ng/ml, 144ng/ml, 720ng/ml, 3.6 μ g/ml, 18 μ g/ml) that suitably dilute by 0.1% BSA and 0.05% tween (Tween), 20 sample diluting liquids and add enzyme reaction plate (100 μ l/ hole) respectively, place 40 ℃ of incubations 1 hour.Discard liquid in the hole, cleansing solution is washed plate 3 times, each 3 minutes.

3) add the anti-NGB polyclonal antibody of rabbit: get and contain the anti-NGB polyclonal antibody of rabbit (1: 500) of suitable dilution of 0.1% BSA and 0.05% polysorbas20 sample diluting liquid, add in the ELISA Plate (100 μ l/ hole), placed 40 ℃ of incubations 30 minutes.Discard liquid in the hole, cleansing solution is washed plate 3 times, each 3 minutes.

4) add the HRP-goat anti-rabbit igg: get the suitableeest dilution HRP-goat anti-rabbit igg (1: 5000), add 100 μ l/ holes in the ELISA Plate, placed 40 ℃ of incubations 40 minutes.Discard liquid in the hole, cleansing solution is washed plate 3 times, each 3 minutes.

5) colour developing: get substrate solution A liquid 24.3ml and B liquid 25.7ml and mix, add o-phenylenediamine, add 30%H at last to concentration 4～10mg/ml ₂O ₂Solution 25～50 μ l.Get freshly prepared substrate buffer solution 100 μ l/ holes and add reaction plate.Room temperature lucifuge reaction 5～10 minutes.

6) cessation reaction: when positive control obvious change color or negative control occurred and slightly develops the color, every hole added stop buffer 50 μ l cessation reactions.

7) result calculates: measure absorbance value (A) at the 492nm place.With standard protein concentration as horizontal ordinate, the standard protein hole A that records _492nmValue is ordinate, drawing standard curve.Check in the NGB content of testing sample by typical curve, multiply by extension rate again, promptly get the actual content in the testing sample.

Description of drawings

Fig. 1: the expression of results of fusion GST-NGB.Swimming lane 1: the total protein of expressing among the Escherichia coli E.coli (DE3) behind the transfection empty carrier pGEX-4T-2; Swimming lane 2:, obtain the GST protein sample with expressed proteins among the Escherichia coli E.coli (DE3) behind the GST affinity column purifying transfection empty carrier pGEX-4T-2.Swimming lane 3: the total protein of expressing among the Escherichia coli E.coli (DE3) behind the transfection expression carrier pGEX-4T-2/NGB; Swimming lane 4: with the GST-NGB fusion of GST affinity column purifying acquisition; Swimming lane 5: molecular weight marker in the protein.

After Fig. 2: GST-NGB cuts through the fibrin ferment enzyme, the NGB albumen that obtains.Swimming lane 1: molecular weight marker in the protein; After swimming lane 2:GST-NGB cuts through the fibrin ferment enzyme, the NGB sample that obtains.

Fig. 3: the Quality Identification of mouse anti NGB monoclonal antibody.Using Western blotting technology detects the monoclonal antibody that obtains, the proof monoclonal antibody has very high specificity, each strain antibody all can combine with NGB and GST-NGB, and reactionless with GST and mycoprotein, and detects the eukaryotic expression product (GFP-NGB) of NGB specifically.

Fig. 4: the hypotype of prepared mouse anti NGB monoclonal antibody is identified in this kit.Through identifying, confirm the anti-NGB monoclonal antibody of four strains 3G8,3H2,2E7, the heavy chain of 1E12 is IgG1, and light chain is the κ chain.

Fig. 5: the Quality Identification of the anti-NGB polyclonal antibody of rabbit: the anti-NGB polyclonal antibody of rabbit can detect the NGB albumen of expressing in the eukaryotic.Method: after using expression vector pcDNA3.1/V5/6 * His/NGB, pcDNA3.1/V5/6 * His/LacZ and empty carrier transfection COS-7 cell respectively, the total protein of cell behind the collection transfection 48h, Western blotting detects.Swimming lane 1: the anti-NGB polyclonal antibody of rabbit can detect the band of about 20kDa; Swimming lane 2: negative control, the empty carrier rotaring redyeing COS 7 cell fails to detect any band with the anti-NGB polyclonal antibody of rabbit; Swimming lane 3: can detect NGB-6 * His amalgamation and expression albumen with anti-6 * His monoclonal antibody; Swimming lane 4: positive control: can detect LacZ-6 * His amalgamation and expression albumen with anti-6 * His monoclonal antibody.

Fig. 6: the Quality Identification of the anti-NGB polyclonal antibody of rabbit.Detect the Tissue distribution of NGB in Adult Rat Brain with the anti-NGB polyclonal antibody of rabbit prepared in this kit, visible NGB immune response positive cell is distributed widely in each district (CA of hippocampus ₁, CA ₂, CA ₃, CA ₄) in pyramidal cell layer and cerebellum purkinje cell and the cell process thereof.

Fig. 7: the typical curve of this kit.The concentration range that can detect NGB concentration is at 5.76ng/ml～18 μ g/ml.

Claims

1. a Novel Human NGB detects and diagnostic kit, it is characterized in that this kit is mainly by 1 of 96 hole ELISA Plate, 1 bottle of NGB standard items, 1 bottle of NGB monoclonal antibody, 1 bottle of NGB polyclonal antibody, 1 bottle of negative control, the goat anti-rabbit immunoglobulin G1 bottle of horseradish peroxidase-labeled, 1 bottle of 2 bottles, 20 times concentrated cleaning solution of o-phenylenediamine and stop buffer (2M sulfuric acid solution (H ₂SO ₄)) 1 bottle of composition.

2. the preparation method of the described kit of claim 1 is characterized in that comprising the steps:

1) preparation of antigen: will be built with the prokaryotic expression carrier transformed into escherichia coli of NGB gene coding region, obtain the solubility expression product, behind the affinity column purifying as antigen.

2) the antigen immune Balb/c mouse of preparation prepares anti-NGB monoclonal antibody by traditional cell fusion method the preparation of antibody: with 1), and immunizing rabbit prepares anti-NGB polyclonal antibody simultaneously.

3) purifying antibody: with 2) monoclonal antibody of gained through centrifugal, precipitation, desalination, slightly carry antibody, then through affinity purification, promptly get antibody purification.

4) assembling of kit: will form 8 kinds of compositions of kit, and be divided in bottle or the conical centrifuge tube by the kit requirement.

5) specificity of identification kit, sensitivity, precision, stability.

3. the described a kind of Novel Human NGB of claim 1 detects and diagnostic kit, it is characterized in that the concentration range of the NGB that can detect be: 5.76ng/ml～18 μ g/ml.

4. the described a kind of Novel Human NGB of claim 1 detects and diagnostic kit, it is characterized in that wherein said negative controls is a normal human serum, and dense cleansing solution is phosphoric acid-Tween-20.

5. the described a kind of Novel Human NGB of claim 1 detects and diagnostic kit the purposes in detecting NGB.