CN103175954A - Human IgE antibody detection kit, and making method and detection method thereof - Google Patents
Human IgE antibody detection kit, and making method and detection method thereof Download PDFInfo
- Publication number
- CN103175954A CN103175954A CN2013100738444A CN201310073844A CN103175954A CN 103175954 A CN103175954 A CN 103175954A CN 2013100738444 A CN2013100738444 A CN 2013100738444A CN 201310073844 A CN201310073844 A CN 201310073844A CN 103175954 A CN103175954 A CN 103175954A
- Authority
- CN
- China
- Prior art keywords
- dissolved
- preparation
- ige antibody
- antibody
- biotin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a human IgE antibody detection kit. The kit comprises a coated micro-porous plate or its making raw material, a sample diluent, a quality control serum, a concentrated cleaning solution, a substrate solution, a stopping solution, a biotin-labeled anti-IgE antibody binding solution, and a horseradish peroxidase-labeled avidin binding solution. The kit has a high sensitivity and a high specificity because of the adoption of a biotin-avidin amplification system. The invention also discloses a making method of the kit. The labeling efficiency can be improved through the SATA modification of the Fc end of the invariable region of an antibody and the connection of HPDP-LC-biotin and the modified antibody. The invention further discloses a detection method of the human IgE antibody. The human IgE antibody detection kit or the kit made through the making method has the advantages of low cost, simple operation, use convenience, and easy popularization application when the kit is used in detection.
Description
Technical field
The present invention relates to the enzyme linked immunosorbent detection field, particularly, relate to a kind of people IgE antibody assay kit, preparation method and detection method.
Background technology
Anaphylactia claims again allergic disease, after referring to that body is to some antigen primary response, and when again accepting same antigen and stimulating, generation a kind of take body physiological function disorder or tissue damage as main specific immune response.1966, at first Ishizaka etc. found and prove that IgE antibody is the main antibody of mediation type i allergic reaction.Clinical allergy mainly refers to type i allergic reaction, its main mechanism is can stimulate specific b cells to produce IgE antibody after anaphylactogen (allergen) enters body, the Fc section of specific IgE antibody can with mast cell and basophil after birth on Fc ε RI to combination, make body be in sensitization.When identical anaphylactogen enters the body of sensitization again, just can be combined and trigger cell activation with the mast cell of sensitization and the variable region of the specific IgE antibody on basophilic granulocyte, discharge the biologically active mediums such as histamine, leukotriene (LTs), platelet activating factor (PAF).These biologically active mediums can act on the effect organs such as skin, blood vessel, respiratory tract and alimentary canal, cause the allergic conditions such as smooth muscle spasm, telangiectasis, vasopermeability increase, glandular secretion increase, often show as clinically bronchial astehma, allergic rhinitis, allergic gastroenteritis and anaphylaxis dermatosis etc., can cause life-threatening anaphylactic shock when serious.
In recent years, along with socioeconomic development, the anaphylactia of IgE mediation increases sharply, and has affected a lot of people's quality of life, has brought heavy financial burden to society.According to bibliographical information, the type i allergic reaction crowd that gets involved of IgE mediation has reached 1/4th of world population sum.The World Health Organization (WHO) (WHO) classifies anaphylactia as 21 century and needs one of three large diseases of primary study and control.Anaphylactia has been regarded as unhealthful subject matter in developed country.And because the incidence of anaphylactia constantly rises, research and the concern of anaphylactia are pass by greatly to improve.Therefore, carry out the clinical needs with studying of the suitable current allergology of research of IgE antibody test new technology.
The IgE antibody detection method mainly contains radioimmunology, fluorescent immune method, band immunization, Western blot, euzymelinked immunosorbent assay (ELISA) (ELISA) etc.Radioimmunology has radioactive contamination, easily human body is damaged; Fluorescent immune method needs expensive instrument; Band immunization and Western blot complicated operation, accuracy is low; These methods all are not suitable for promoting on a large scale clinically.Comparatively speaking, euzymelinked immunosorbent assay (ELISA) is simple to operate, accuracy is high, need not expensive instrument, is the method for generally using clinically at present.
Because the content of IgE antibody in human body is very low, easily be subject to the interference of the antibody such as IgG, conventional ELISA detection method is due to insufficient sensitivity, and the accuracy of detection is relatively poor, and when especially detecting low value IgE antibody, result is more inaccurate.And present import reagent box occupies the status of home market front three, and the IgE antibody diagnosing reagent kit of import because of expensive, allows many medium and small hospitals hang back.Chinese market is sold and only is only limited to the use of part large hospital, can't significantly popularize use, especially domestic long-term dependence on import always, and external product is absolute monopolization and the driver's seat of market share at home.Therefore, domestic have independent intellectual property right in the urgent need to exploitation and gather sensitive high, high specificity, favorable reproducibility, production domesticization IgE antibody test reagent that cost is low, and this has been that the task of top priority needs problem demanding prompt solutions.
Summary of the invention
The object of the present invention is to provide that a kind of sensitivity is high, high specificity, favorable reproducibility, people IgE antibody assay kit that cost is low.
Second purpose of the present invention is to provide the preparation method of the high people IgE antibody assay kit of a kind of labeling effciency.
The people IgE antibody detection method that provides a kind of cost low, simple to operate, easy to use is provided the 3rd purpose of the present invention.
For achieving the above object, the present invention adopts following technical scheme:
People IgE antibody assay kit, comprise: coated microwell plate or its raw materials and sample dilution, quality controlled serum, concentrated cleaning solutions, substrate solution, stop buffer, also comprise biotin labeled anti-IgE antibodies in conjunction with the Avidin of liquid, horseradish peroxidase-labeled in conjunction with liquid.
Further, described coated microwell plate is the microwell plate that is coated with anti human IgE antibody.
Further, described sample dilution is that carrier protein or animal blood serum are dissolved in phosphate buffer; The positive serum of described quality controlled serum is dissolved in phosphate buffer; Described concentrated cleaning solutions is that tween is dissolved in phosphate buffer; Described substrate solution is that tetramethyl benzidine and hydrogen peroxide are dissolved in citrate buffer solution; Described stop buffer is vitriolated solution; Also comprise standard serum, described standard serum is dissolved in phosphate buffer for high value people IgE antibody serum.
Further, described biotin labeled anti-IgE antibodies in conjunction with liquid is: biotin labeled anti-IgE antibodies and carrier protein or animal blood serum are dissolved in phosphate buffer; The Avidin of described horseradish peroxidase-labeled in conjunction with liquid is: the Streptavidin of horseradish peroxidase-labeled and carrier protein or animal blood serum are dissolved in phosphate buffer.
The preparation method of people IgE antibody assay kit comprises the preparation of following sample: coated microwell plate or its raw materials, sample dilution, quality controlled serum, concentrated cleaning solutions, substrate solution, stop buffer also comprise:
A, biotin labeled anti-IgE antibodies are in conjunction with the preparation of liquid:
(1) the constant region Fc end with anti-IgE antibodies carries out the SATA modification; (2) with the HPDP-LC-biotin with modify after anti-IgE antibodies be connected, form biotin labeled anti-IgE antibodies; (3) biotin labeled anti-IgE antibodies is dissolved in antibody diluent the biotin labeled anti-IgE antibodies of preparation in conjunction with liquid.
The Avidin of B, horseradish peroxidase-labeled is in conjunction with the preparation of liquid:
Horseradish peroxidase and Avidin are carried out mark, after the Avidin of horseradish peroxidase-labeled is dissolved in the Avidin of preparation horseradish peroxidase-labeled in the enzyme dilution in conjunction with liquid.
Further, described step (1) is: take in the phosphate buffer that anti human IgE antibody incorporates 0.1M, and PH7.0-7.6, the concentration that makes antibody is 1-5mg/ml; Take SATA and be dissolved in DMF or DMSO, making concentration is 5-10mg/ml; The SATA solution that adds 10-40 μ l in every milliliter of antibody-solutions, room temperature reaction 30-60min; Remove unreacted SATA solution with SephadexG25 post or dialysis; Described step (2) is: take the HPDP-LC-biotin and be dissolved in DMF or DMSO, making concentration is 1-5mg/ml; The HPDP-LC-biotin solution that adds 50-150 μ l in the antibody-solutions of every milliliter of SATA modification, room temperature reaction 60-120min; Remove unreacted HPDP-LC-biotin solution with SephadexG25 post or dialysis; Described step (3) is: take carrier protein or measure animal blood serum and be dissolved in phosphate buffer, and add protective agent preparation antibody diluent, biotin labeled anti human IgE antibody is dissolved in prepares biotin labeled anti-IgE antibodies in antibody diluent in conjunction with liquid; Described step B is: horseradish peroxidase and Streptavidin are carried out mark; take carrier protein or measure animal blood serum and be dissolved in phosphate buffer; and add protective agent preparation enzyme dilution, the Streptavidin of horseradish peroxidase-labeled is dissolved in the Avidin of preparation horseradish peroxidase-labeled in the enzyme dilution in conjunction with liquid.
Further, in described step B, horseradish peroxidase and Streptavidin 3:1 carry out mark with the sodium periodate method.
Further, described coated microwell plate is the microwell plate that is coated with anti human IgE antibody.
Further, the microwell plate that preparation is coated: anti human IgE antibody is dissolved in carbonic acid or phosphoric acid, acetate buffer solution, mixing adds in microwell plate, hatches, after being dissolved in phosphate buffer and washing plate with tween, add again confining liquid in microwell plate, hatch, discard liquid in the hole, dry microwell plate is enclosed microwell plate in aluminium foil bag after drying; Preparation sample dilution: measure carrier protein or measure animal blood serum and be dissolved in phosphate buffer; Preparation quality controlled serum: measure positive serum and be dissolved in phosphate buffer, and add protective agent; Preparation concentrated cleaning solutions: measure tween and be dissolved in phosphate buffer; Preparation substrate solution: take tetramethyl benzidine and hydrogen peroxide and be dissolved in citrate buffer solution; Preparation stop buffer: measure sulfuric acid and be dissolved in pure water; Also comprise preparation standard serum: measure high value people IgE antibody serum and be dissolved in phosphate buffer, and add protective agent.
The detection method of people IgE antibody adopts above-mentioned people IgE antibody assay kit to detect, or adopts the kit of above-mentioned preparation method's preparation to detect.
Compared with prior art, the present invention has following beneficial effect:
1, adopt the biotin-avidin amplification system, because the affinity of biotin and Avidin is higher, and an Avidin molecule can be in conjunction with 4 biotin molecules, make detection sensitivity improve 4-5 doubly than conventional ELISA detection method, effectively solved the IgE antibody content low, conventional ELISA detection method is due to insufficient sensitivity, the inaccurate problem of testing result.
2, adopt double antibody to add heart method and detect people IgE antibody, can effectively eliminate the interference of the antibody such as IgG, IgM, IgA, further improved the accuracy that detects.
3, the molecular weight due to anti human IgE antibody is large, and the molecular weight of biotin is again little, and directly mark is easy to biotin is combined in the variable region Fab end of antibody, and antibody is lost activity, and reduces labeling effciency.The present invention first carries out the SATA modification with the constant region Fc end of antibody, be connected with antibody after modification with the HPDP-LC-biotin that adds arm again, biotin only is marked on the Fc end of antibody, and the variable region Fab of antibody end is unaffected, has kept the activity of antibody.The biotin molecule space of extension arm increases, and has improved the joint efficiency of biotin and Avidin, the sensitivity that has further improved product.
4, adopt kit of the present invention to carry out the IgE antibody test, cost is low, simple to operate, easy to use, need not expensive instrument and equipment, be easy to apply, and has extremely good social benefit and considerable economic benefit.
Embodiment
Below the preferred embodiments of the present invention are described, should be appreciated that preferred embodiment described herein only is used for description and interpretation the present invention, is not intended to limit the present invention.
Embodiment 1: the component of people IgE antibody assay kit
The component of people IgE antibody assay kit comprises: coated microwell plate or its raw materials and sample dilution, quality controlled serum, concentrated cleaning solutions, substrate solution, stop buffer, also comprise biotin labeled anti-IgE antibodies in conjunction with the Avidin of liquid, horseradish peroxidase-labeled in conjunction with liquid.
Wherein: coated microwell plate is the microwell plate that is coated with anti human IgE antibody.The sample dilution is that carrier protein or animal blood serum are dissolved in phosphate buffer; The positive serum of quality controlled serum is dissolved in phosphate buffer; Concentrated cleaning solutions is that tween is dissolved in phosphate buffer; Substrate solution is that tetramethyl benzidine and hydrogen peroxide are dissolved in citrate buffer solution; Stop buffer is vitriolated solution.
The component of mentioned reagent box also comprises standard serum, and standard serum is dissolved in phosphate buffer for high value people IgE antibody serum.
Above-mentioned biotin labeled anti-IgE antibodies in conjunction with liquid is: biotin labeled anti-IgE antibodies and carrier protein or animal blood serum are dissolved in phosphate buffer; The Avidin of above-mentioned horseradish peroxidase-labeled in conjunction with liquid is: the Streptavidin of horseradish peroxidase-labeled and carrier protein or animal blood serum are dissolved in phosphate buffer.
Embodiment 2: the preparation method of people IgE antibody assay kit
1, anti human IgE antibody is dissolved in carbonic acid (or phosphoric acid, the acetic acid) damping fluid of 50mM mixing by 1 μ g/ml.
2, add in microwell plate by certain putting in order, every hole 100 μ l, 4 ℃ of overnight incubation after being dissolved in phosphate buffer and washing plate 3 times with tween, then add confining liquid (phosphate buffer that contains 2%BSA), every hole 200 μ l, 4 ℃ of overnight incubation in microwell plate.
3, discard liquid in the hole, the dry microwell plate of ambient temperature overnight is enclosed microwell plate in aluminium foil bag after drying.
4, take 10gBSA and be dissolved in preparation sample dilution in 1 liter of 50mM phosphate buffer, carry out packing by every bottle of 50ml.
5, the SATA of labelled antibody (N-Succinimidyl-S-acetylthioacetate) modifies.Take in the phosphate buffer that anti human IgE antibody incorporates 0.1M, PH7.2, the concentration that makes antibody is 2mg/ml.Take SATA and be dissolved in DMF, making concentration is 8mg/ml.The SATA solution that adds 20 μ l in every milliliter of antibody-solutions, room temperature reaction 40min.Remove unreacted SATA solution with the SephadexG25 post.
6, the mark of biotin and anti human IgE antibody.Take the HPDP-LC-biotin and be dissolved in DMF, making concentration is 2mg/ml.The HPDP-LC-biotin solution that adds 100 μ l in the antibody-solutions of every milliliter of SATA modification, room temperature reaction 90min.Remove unreacted HPDP-LC-biotin solution with the SephadexG25 post.
7, taking 10gBSA is dissolved in 1 liter of 50mM phosphate buffer; and add protective agent to prepare antibody diluent; biotin labeled anti human IgE antibody (1:2000) is dissolved in prepares biotin labeled anti human IgE antibody in antibody diluent in conjunction with liquid, carry out packing by every bottle of 20ml.
8, horseradish peroxidase and Streptavidin (3:1) are carried out mark with the sodium periodate method.
9, taking 10gBSA is dissolved in 1 liter of 50mM phosphate buffer; and add protective agent to prepare the enzyme dilution; the Avidin that the Avidin (1:5000) of horseradish peroxidase-labeled is dissolved in preparation horseradish peroxidase-labeled in the enzyme dilution carries out packing in conjunction with liquid by every bottle of 20ml.
10, take 0.2g tetramethyl benzidine (TMB) and 0.6ml hydrogen peroxide and be dissolved in 1 liter of citrate buffer solution and prepare substrate solution, carry out packing by every bottle of 15ml.
11, measure the 10ml tween and be dissolved in 1 liter of phosphate buffer and prepare concentrated cleaning solutions, carry out packing by every bottle of 50ml.
12, measure 50ml sulfuric acid and be dissolved in 1 liter of pure water and prepare stop buffer, carry out packing by every bottle of 15ml.
13, measure high value people IgE antibody serum and be dissolved in phosphate buffer, and add protective agent preparation standard serum, carry out packing by every bottle of 1ml.
14, measure positive serum and be dissolved in phosphate buffer, and add protective agent preparation quality controlled serum, carry out packing by every bottle of 1ml.
15, labelled according to the component of kit, coated microwell plate, sample dilution, biotin labeling antibody are put into the relevant position of kit in conjunction with the Avidin of liquid, horseradish peroxidase-labeled in conjunction with liquid, standard serum, quality controlled serum, concentrated washing lotion, substrate solution, stop buffer, instructions, be assembled into complete kit.
BSA in above-mentioned steps 4,7,9 is replaceable is animal blood serum, configures according to a conventional method corresponding dilution.
Embodiment 3: the preparation method of people IgE antibody assay kit
Method by embodiment 2 prepares people IgE antibody assay kit, and difference is:
In step 5, PH7.0, the concentration that makes antibody is 1mg/ml.Take SATA and be dissolved in DMSO, making concentration is 5mg/ml.The SATA solution that adds 10 μ l in every milliliter of antibody-solutions, room temperature reaction 30min.Remove unreacted SATA solution with dialysis.
In step 6, take the HPDP-LC-biotin and be dissolved in DMSO, making concentration is 1mg/ml.The HPDP-LC-biotin solution that adds 50 μ l in the antibody-solutions of every milliliter of SATA modification, room temperature reaction 60min.Remove unreacted HPDP-LC-biotin solution with dialysis.
Embodiment 4: the preparation method of people IgE antibody assay kit
Method by embodiment 2 prepares people IgE antibody assay kit, and difference is:
In step 5, PH7.6, the concentration that makes antibody is 5mg/ml.Take SATA and be dissolved in DMSO, making concentration is 10mg/ml.The SATA solution that adds 40 μ l in every milliliter of antibody-solutions, room temperature reaction 60min.Remove unreacted SATA solution with dialysis.
In step 6, take the HPDP-LC-biotin and be dissolved in DMSO, making concentration is 5mg/ml.The HPDP-LC-biotin solution that adds 150 μ l in the antibody-solutions of every milliliter of SATA modification, room temperature reaction 120min.Remove unreacted HPDP-LC-biotin solution with dialysis.
Embodiment 5: people IgE antibody detection method
Adopt the people IgE antibody assay kit of embodiment 1 to detect, or adopt the kit of embodiment 2,3,4 preparation method's preparations to detect.
Before using please with all the reagent balances in kit to room temperature.
1, concentrated cleaning solutions (1:25) 40ml is added in distilled water or deionized water, making final volume is 1000ml, and mixing is joined to get cleaning buffer solution.
2, prepare the drawing standard curve: get 100 μ l and join in the micropore that is coated with anti-IgE antibodies from every pipe standards serum, every kind of standard items repeat 2 holes.
3, add 90 μ l sample dilutions and 10 μ l quality controlled serum or clinical samples in the micropore of coated anti-IgE antibodies.
4, with sealed membrane or polybag closed porosity plate, put room temperature (22-25 ° of C) and hatched 1 hour.
5, hatch after, clean each reacting hole with 300 μ l cleaning buffer solutions, need to clean 3 times.If select automatic cleaning apparatus, please refer to producer's explanation and carry out wash cycles 3 times, and the cleaning capacity is made as 300 μ l.
6, add the 100 biotin labeled anti human IgE antibody of μ l in conjunction with liquid respectively in each micropore.
7, with sealed membrane or polybag closed porosity plate, put room temperature (22-25 ° of C) and hatched 1 hour.
8, repeating step 5 cleans microwell plate.
9, add the Streptavidin of 100 μ l horseradish peroxidase-labeled in conjunction with liquid respectively in each micropore.
10, putting room temperature (22-25 ° of C) hatched 30 minutes.
11, repeating step 5 cleans microwell plate.
12, add 100 μ l substrate solutions in all reacting holes.
13, closed porosity plate is put room temperature (22-25 ° of C) and was hatched 30 minutes.
14, add 100 μ l stop buffers (solution in reacting hole becomes yellow by blueness at this moment) in all reacting holes.
15, measure every hole at the absorbance at 450nm place with microplate reader.
After testing, with respect to common ELISA detection method, its sensitivity has improved 4-5 doubly.
It should be noted that at last: the above only is the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment, the present invention is had been described in detail, for a person skilled in the art, it still can be modified to the technical scheme that previous embodiment is put down in writing, and perhaps part technical characterictic wherein is equal to replacement.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. people IgE antibody assay kit, comprise: coated microwell plate or its raw materials and sample dilution, quality controlled serum, concentrated cleaning solutions, substrate solution, stop buffer is characterized in that: also comprise biotin labeled anti-IgE antibodies in conjunction with the Avidin of liquid, horseradish peroxidase-labeled in conjunction with liquid.
2. people IgE antibody assay kit according to claim 1, it is characterized in that: described coated microwell plate is the microwell plate that is coated with anti human IgE antibody.
3. people IgE antibody assay kit according to claim 2 is characterized in that:
Described sample dilution is that carrier protein or animal blood serum are dissolved in phosphate buffer;
The positive serum of described quality controlled serum is dissolved in phosphate buffer;
Described concentrated cleaning solutions is that tween is dissolved in phosphate buffer;
Described substrate solution is that tetramethyl benzidine and hydrogen peroxide are dissolved in citrate buffer solution;
Described stop buffer is vitriolated solution;
Also comprise standard serum, described standard serum is dissolved in phosphate buffer for high value people IgE antibody serum.
4. according to claim 1,2 or 3 described people IgE antibody assay kits, is characterized in that, described biotin labeled anti-IgE antibodies in conjunction with liquid is: biotin labeled anti-IgE antibodies and carrier protein or animal blood serum are dissolved in phosphate buffer; The Avidin of described horseradish peroxidase-labeled in conjunction with liquid is: the Streptavidin of horseradish peroxidase-labeled and carrier protein or animal blood serum are dissolved in phosphate buffer.
5. the preparation method of people IgE antibody assay kit comprises the preparation of following sample: coated microwell plate or its raw materials, sample dilution, quality controlled serum, concentrated cleaning solutions, substrate solution, stop buffer, it is characterized in that, and also comprise:
A, biotin labeled anti-IgE antibodies are in conjunction with the preparation of liquid:
(1) the constant region Fc end with anti-IgE antibodies carries out the SATA modification;
(2) with the HPDP-LC-biotin with modify after anti-IgE antibodies be connected, form biotin labeled anti-IgE antibodies;
(3) biotin labeled anti-IgE antibodies is dissolved in antibody diluent the biotin labeled anti-IgE antibodies of preparation in conjunction with liquid;
The Avidin of B, horseradish peroxidase-labeled is in conjunction with the preparation of liquid:
Horseradish peroxidase and Avidin are carried out mark, after the Avidin of horseradish peroxidase-labeled is dissolved in the Avidin of preparation horseradish peroxidase-labeled in the enzyme dilution in conjunction with liquid.
6. the preparation method of people IgE antibody assay kit according to claim 5 is characterized in that:
Described step (1) is: takes in the phosphate buffer that anti human IgE antibody incorporates 0.1M, and PH7.0-7.6, the concentration that makes antibody is 1-5mg/ml; Take SATA and be dissolved in DMF or DMSO, making concentration is 5-10mg/ml; The SATA solution that adds 10-40 μ l in every milliliter of antibody-solutions, room temperature reaction 30-60min; Remove unreacted SATA solution with SephadexG25 post or dialysis;
Described step (2) is: take the HPDP-LC-biotin and be dissolved in DMF or DMSO, making concentration is 1-5mg/ml; The HPDP-LC-biotin solution that adds 50-150 μ l in the antibody-solutions of every milliliter of SATA modification, room temperature reaction 60-120min; Remove unreacted HPDP-LC-biotin solution with SephadexG25 post or dialysis;
Described step (3) is: take carrier protein or measure animal blood serum and be dissolved in phosphate buffer, and add protective agent preparation antibody diluent, biotin labeled anti human IgE antibody is dissolved in prepares biotin labeled anti-IgE antibodies in antibody diluent in conjunction with liquid;
Described step B is: horseradish peroxidase and Streptavidin are carried out mark; take carrier protein or measure animal blood serum and be dissolved in phosphate buffer; and add protective agent preparation enzyme dilution, the Streptavidin of horseradish peroxidase-labeled is dissolved in the Avidin of preparation horseradish peroxidase-labeled in the enzyme dilution in conjunction with liquid.
7. the preparation method of people IgE antibody assay kit according to claim 6, it is characterized in that: in described step B, horseradish peroxidase and Streptavidin 3:1 carry out mark with the sodium periodate method.
8. the preparation method of according to claim 5,6 or 7 described people IgE antibody assay kits, it is characterized in that: described coated microwell plate is the microwell plate that is coated with anti human IgE antibody.
9. the preparation method of people IgE antibody assay kit according to claim 8 is characterized in that:
The microwell plate that preparation is coated: anti human IgE antibody is dissolved in carbonic acid or phosphoric acid, acetate buffer solution, mixing, add in microwell plate, hatch, after being dissolved in phosphate buffer and washing plate with tween, then add confining liquid in microwell plate, hatch, discard liquid in the hole, dry microwell plate is enclosed microwell plate in aluminium foil bag after drying;
Preparation sample dilution: measure carrier protein or measure animal blood serum and be dissolved in phosphate buffer;
Preparation quality controlled serum: measure positive serum and be dissolved in phosphate buffer, and add protective agent;
Preparation concentrated cleaning solutions: measure tween and be dissolved in phosphate buffer;
Preparation substrate solution: take tetramethyl benzidine and hydrogen peroxide and be dissolved in citrate buffer solution;
Preparation stop buffer: measure sulfuric acid and be dissolved in pure water;
Also comprise preparation standard serum: measure high value people IgE antibody serum and be dissolved in phosphate buffer, and add protective agent.
10. the detection method of people IgE antibody, is characterized in that: adopt the described people IgE of claim 1-4 any one antibody assay kit to detect, or adopt the kit of the described preparation method's preparation of claim 5-9 any one to detect.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013100738444A CN103175954A (en) | 2013-03-08 | 2013-03-08 | Human IgE antibody detection kit, and making method and detection method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013100738444A CN103175954A (en) | 2013-03-08 | 2013-03-08 | Human IgE antibody detection kit, and making method and detection method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103175954A true CN103175954A (en) | 2013-06-26 |
Family
ID=48635953
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013100738444A Pending CN103175954A (en) | 2013-03-08 | 2013-03-08 | Human IgE antibody detection kit, and making method and detection method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103175954A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107383200A (en) * | 2016-05-17 | 2017-11-24 | 深圳先进技术研究院 | The preparation method and applications of mouse source anti human IgE monoclonal antibody |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002042771A2 (en) * | 2000-11-24 | 2002-05-30 | University Of North Carolina At Chapel Hill | Detection of binding reactions using catalytic electrochemical labels |
| CN1869699A (en) * | 2006-06-09 | 2006-11-29 | 深圳市博卡生物技术有限公司 | Method of anaphylactogen screening |
| CN101178406A (en) * | 2007-12-05 | 2008-05-14 | 杭州浙大生物基因工程有限公司 | Allergen specificity antibody IgE ELISA detection reagent box and method of producing the same |
| CN101201354A (en) * | 2006-12-14 | 2008-06-18 | 北京科美东雅生物技术有限公司 | Thyroxine chemiluminescence immune analysis quantitative measuring reagent kit and method for preparing the same |
| WO2009026074A1 (en) * | 2007-08-20 | 2009-02-26 | Bio-Rad Laboratories, Inc. | Simultaneous assay for determining drugs |
| CN101398429A (en) * | 2007-09-26 | 2009-04-01 | 北京科美东雅生物技术有限公司 | Tumor-associated antigen 242 magnetic microparticle chemiluminescence immune assay determination kit and method for making same |
| CN101522714A (en) * | 2004-05-20 | 2009-09-02 | 圣玛莉医院国民健康保险基金 | Ige-retargeting, function-altering molecules (ERFAM) for treatment of allergic diseases |
| CN102083460A (en) * | 2008-01-18 | 2011-06-01 | 米迪缪尼有限公司 | Cysteine engineered antibodies for site-specific conjugation |
| CN102167745A (en) * | 2010-02-25 | 2011-08-31 | 百迈博药业有限公司 | Human monoclonal antibody against IgE, preparation method and purpose thereof |
-
2013
- 2013-03-08 CN CN2013100738444A patent/CN103175954A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002042771A2 (en) * | 2000-11-24 | 2002-05-30 | University Of North Carolina At Chapel Hill | Detection of binding reactions using catalytic electrochemical labels |
| CN101522714A (en) * | 2004-05-20 | 2009-09-02 | 圣玛莉医院国民健康保险基金 | Ige-retargeting, function-altering molecules (ERFAM) for treatment of allergic diseases |
| CN1869699A (en) * | 2006-06-09 | 2006-11-29 | 深圳市博卡生物技术有限公司 | Method of anaphylactogen screening |
| CN101201354A (en) * | 2006-12-14 | 2008-06-18 | 北京科美东雅生物技术有限公司 | Thyroxine chemiluminescence immune analysis quantitative measuring reagent kit and method for preparing the same |
| WO2009026074A1 (en) * | 2007-08-20 | 2009-02-26 | Bio-Rad Laboratories, Inc. | Simultaneous assay for determining drugs |
| CN101398429A (en) * | 2007-09-26 | 2009-04-01 | 北京科美东雅生物技术有限公司 | Tumor-associated antigen 242 magnetic microparticle chemiluminescence immune assay determination kit and method for making same |
| CN101178406A (en) * | 2007-12-05 | 2008-05-14 | 杭州浙大生物基因工程有限公司 | Allergen specificity antibody IgE ELISA detection reagent box and method of producing the same |
| CN102083460A (en) * | 2008-01-18 | 2011-06-01 | 米迪缪尼有限公司 | Cysteine engineered antibodies for site-specific conjugation |
| CN102167745A (en) * | 2010-02-25 | 2011-08-31 | 百迈博药业有限公司 | Human monoclonal antibody against IgE, preparation method and purpose thereof |
Non-Patent Citations (7)
| Title |
|---|
| R.J. SHAW ET AL.: "Production and characterisation of monoclonal antibodies recognising ovine IgE", 《VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY》, vol. 51, 30 June 1996 (1996-06-30), pages 235 - 251, XP 002308849, DOI: doi:10.1016/0165-2427(95)05530-4 * |
| R.J. SHAW ET AL.: "Quantification of total sheep IgE concentration using anti-ovine IgE monoclonal antibodies in an enzyme immunoassay", 《VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY》, vol. 57, 31 July 1997 (1997-07-31), pages 253 - 265 * |
| 党华 等: "变应原特异性IgE抗体检测试剂盒的研制", 《临床检验杂志》, vol. 28, no. 4, 31 August 2010 (2010-08-31), pages 269 - 270 * |
| 宋婷 等: "抗体介导的免疫脂质体的研究进展", 《药学与临床研究》, vol. 20, no. 2, 30 April 2012 (2012-04-30), pages 120 - 124 * |
| 毛露甜 等: "ABC－ELISA法检测牛乳过敏小鼠模型中的特异性IgE", 《生物技术通讯》, vol. 17, no. 3, 30 June 2006 (2006-06-30), pages 389 - 391 * |
| 王运刚 等: "Derf1血清特异性IgE ELISA检测方法的建立", 《中国病原生物学杂志》, vol. 7, no. 6, 30 June 2012 (2012-06-30), pages 436 - 439 * |
| 甘晓玲，郑风英，魏仲香主编: "《免疫学检验技术 》", 31 August 2012, article "免疫学检验技术", pages: 152-153 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107383200A (en) * | 2016-05-17 | 2017-11-24 | 深圳先进技术研究院 | The preparation method and applications of mouse source anti human IgE monoclonal antibody |
| CN107383200B (en) * | 2016-05-17 | 2021-04-09 | 深圳先进技术研究院 | Preparation method and application of mouse-derived anti-human IgE monoclonal antibody |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104142401A (en) | Bladder tumor-associated antigen detection kit | |
| CN106771233A (en) | ZnT8A autoantibody detection kits | |
| CN101571546A (en) | Human adiponectin enzyme-linked immunosorbent detection kit, preparation method and application thereof | |
| WO2021169664A1 (en) | Antigen for 2019 novel coronavirus and detection use thereof | |
| CN103439502B (en) | A kind of rapid specific antibody IgE detection kit and preparation method thereof | |
| CN101825636B (en) | Reagent strip for joint detection of syphilis specific IgM antibody and specific total antibody and preparation method thereof | |
| CN101825635B (en) | Reagent strip for joint detection of syphilis specific IgG antibody and specific total antibody and preparation method thereof | |
| CN102367270A (en) | Preparation method of cyclosporin A haptin and enzymelinked immunosorbent quantitative detection kit of cyclosporin A | |
| CN103389374B (en) | Micro whole blood application of sample method detects mycoplasma pneumoniae colloidal gold kit | |
| CN102128937B (en) | Evaluating and monitoring method for effect of desensitization therapy, detection kit of allergen specificity IgG4 antibody and preparation method thereof | |
| CN103175954A (en) | Human IgE antibody detection kit, and making method and detection method thereof | |
| CN1580772A (en) | SARS virus antibody double antigen sandwich ELISA detecting method | |
| BR112014024296B1 (en) | METHOD TO DETECT IN A SAMPLE THE PROBABILITY OF A PATIENT DEVELOPING TO A LIFE-THREATENING SEVERE DENGUE DISEASE, PROGNOSIS KIT, USE OF THE SAME REAGENTS AND IN VITRO METHOD OF DATA ACQUISITION ON A DENGUE VIRUS INFECTION | |
| CN114994306B (en) | Application of protein PKNOX1 in preparation of reagent for diagnosing alcoholic cardiomyopathy and diagnostic kit | |
| CN103033616A (en) | Detection method of hemorrhagic fever with renal syndrome IgM antibodies and reagent kit | |
| CN102253220B (en) | Kit used for diagnosing acute exacerbation period of chronic obstructive pulmonary disease | |
| CN204359795U (en) | Mite specific IgG4 subclass antibodies detection kit | |
| JP7081861B1 (en) | A kit for testing urothelial cancer that identifies Neu5Gc in urine modified with UMOD based on LIP, and a method for producing the same. | |
| CN103926406A (en) | Marker for indicating hepatitis infection and application of marker | |
| CN100476432C (en) | Detection reagent for sperm antibody and preparation method thereof | |
| CN102401831A (en) | Competitive enzyme-linked immunosorbent assay kit for liver cirrhosis and assay method for competitive enzyme-linked immunosorbent assay kit | |
| CN201697920U (en) | Syphilis specificity IgM antibody and specificity total antibody combined testing reagent strip | |
| CN201852840U (en) | Test paper card for quickly diagnosing human echinococcosis adopting colloid gold immunochromatography to test urine | |
| CN201955343U (en) | Syphilis cardiolipin and specific antibody IgM western-blot kit | |
| CN201926662U (en) | Syphilis specificity total antibody immunoblotting kit box |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| AD01 | Patent right deemed abandoned |
Effective date of abandoning: 20160914 |
|
| C20 | Patent right or utility model deemed to be abandoned or is abandoned |