CN102128937B - Evaluating and monitoring method for effect of desensitization therapy, detection kit of allergen specificity IgG4 antibody and preparation method thereof - Google Patents
Evaluating and monitoring method for effect of desensitization therapy, detection kit of allergen specificity IgG4 antibody and preparation method thereof Download PDFInfo
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Abstract
The invention relates to an evaluating and monitoring method for the effect of desensitization therapy, and an Enzyme-Linked Immuno-Sorbent Assay (ELISA) detection kit of allergen specificity IgG4 antibody and a preparation method thereof. After the desensitization therapy is finished, the content of the allergen specificity IgG4 antibody in the blood serum is detected to judge the curing effect of the performed desensitization therapy; when the content of the allergen specificity IgG4 antibody in the blood serum is obviously increased, the curing effect of the performed desensitization therapy is considered to be obvious; when the content of the allergen specificity IgG4 antibody in the blood serum reaches the peak value, the performed desensitization therapy is considered to be successful. The evaluating and monitoring method for the effect of desensitization therapy, the detection kit of allergen specificity IgG4 antibody and the preparation method thereof have the following benefits: firstly, after the patient is cured by the performed desensitization therapy, a method for detecting specificity IgG4 antibody is provided for providing the evaluation index for the curing effect of the performed desensitization therapy; secondly, the method for evaluating and monitoring the effect of desensitization therapy, the detection kit of allergen specificity IgG4 antibody and the preparation method thereof have the advantages of being high in sensitivity, good in specificity, quick and convenient to use.
Description
Technical field
The present invention relates to Enzyme Linked Immunoadsorbent Assay (ELISA) detection kit of a kind of evaluation monitoring method for desensitization treatment effect and allergen specificity IgG4 and preparation method thereof.
background technology
Desensitization therapy (desensitization) is called again specific active immunotherapy (specific immunotherapy, SIT) or hyposensitization (hyposensitization).WHO bases in 1997 understanding to specific active immunotherapy mechanism and clinical efficacy for many years, having proposed new term is specificity allergic reaction bacterin treatment (specific allergy vaccination, SAV).SAV determines after anaphylactia patient's allergen clinically, this allergen is made to allergen extract and be mixed with the preparation of various variable concentrations, through repeatedly injecting or repeatedly contacting with patient by other method of administration, dosage is ascending, concentration from low to high, thereby improve patient to this kind of allergenic tolerance, in the time again contacting this kind of allergen, no longer produce allergic phenomena or allergic phenomena is alleviated.
But carry out after desensitization treatment, often desensitization treatment effect is not carried out the evaluation of science, cannot judge the effect of desensitization treatment.Successfully the remarkable rising of serological specificity IgG4 antibody is followed in immunization therapy conventionally.As far back as nineteen sixty-eight, just there is the correlation function of the IgG antibody that studies show that SIT induction generation.Rising, the especially rising of IgG4 hypotype antibody horizontal of anaphylactogen blocking-up property IgG antibody horizontal, in specific active immunotherapy (specific immunotherapy, SIT) process, brought into play important effect.The analysis of the IgG antibody subtype bringing out for immunization therapy shows that IgG1 and IgG4 have remarkable rising, especially IgG4.IgG4 antibody water product can increase 10 times～100 times.
IgG4 antibody is considered to catch anaphylactogen, and stop anaphylactogen to arrive the combination of effector cell and IgE, thereby stop the activation of mast cell and basophilic granulocyte, and active medium in release particles, cause I type hypersensitivity (Fc ε R I approach).And can, by the combination (Fc ε R II approach) of blocking-up anaphylactogen-IgE compound and low compatibility IgE acceptor, suppress activation and the aggravation of inflammation path in anaphylactia.These researchs have clearly illustrated that IgG4 antibody plays the effect of blocking antibody in immunization therapy.
The unique generally acknowledged index of evaluating now desensitization curative effect is the content detection of allergen specificity IgG4 antibody.Accepting after specific active immunotherapy (specific immunotherapy, SIT), allergen specificity IgG4 antibody content raises, and with the increase of desensitization, antibody content rises, become immunization therapy curative effect comment an index.By detecting allergen specificity IgG4 antibody horizontal, desensitization treatment effect is evaluated.
Enzyme Linked Immunoadsorbent Assay (ELISA) method, owing to only needing to use common microplate reader, has simple and safe, economy feature fast, is applicable to China's Multi-level hospital and carries out desensitization treatment effect assessment.
It is to utilize the fixing allergen protein of solid phase carrier in conjunction with the sIgG4 in serum sample that ELISA method detects allergen specificity IgG4 antibody (sIgG4) in serum sample, wash away the anti-human IgG4 antibody that adds enzyme labeling after unconjugated other albumen, be combined with allergen specificity IgG4 antibody, form anaphylactogen-sIgG4-hrp-antibody complex, by reacting with chromogenic enzyme substrate liquid, the enzyme amount that detects combination, detects sIgG4 kind and concentration in serum indirectly.
Summary of the invention
The object of the present invention is to provide a kind of evaluation monitoring method for desensitization treatment effect, this evaluation method is in desensitization treatment process, for doctor provides objective index, in order to adjust patient's desensitization treatment dosage, the desensitization treatment effect of before carrying out for it provides foundation; After desensitization treatment finishes the course for the treatment of, for desensitization treatment effect provides an objective data target, avoid the drawback that only relies on doctor's experience to be evaluated desensitization treatment effect.
Of the present invention also have object be for above-mentioned monitoring method propose a kind of highly sensitive, specificity good, use Enzyme Linked Immunoadsorbent Assay detection kit of allergen specificity IgG4 quickly and easily and preparation method thereof, judge desensitization treatment curative effect and patient's tolerance situation by detecting sIgG4 kind in serum and concentration, have highly sensitive, specificity good and use advantage quickly and easily.
The present invention for the solution that the problem of the above-mentioned proposition of solution adopts is:
For the evaluation monitoring method of desensitization treatment effect, it is characterized in that after desensitization treatment finishes, by detecting the content of allergen specificity IgG4 antibody in serum, judge the curative effect of carried out desensitization treatment, when allergen specificity IgG4 antibody in serum rises significantly, illustrate that desensitization treatment effect is remarkable, when allergen specificity IgG4 antibody in serum reaches after peak value, success of desensitization treatment is described.
In desensitization treatment process, allergen specificity IgG4 antibody has one significantly to increase, and successfully the remarkable rising of serological specificity IgG4 antibody is followed in immunization therapy conventionally.After allergen specificity IgG4 antibody in serum significantly raises, illustrate that desensitization treatment curative effect is obvious.
The Enzyme Linked Immunoadsorbent Assay detection kit of allergen specificity IgG4, is characterized in that including the ELISA Plate, enzyme labelled antibody, substrate nitrite ion A, substrate nitrite ion B, sample diluting liquid, concentrated cleaning solution and the reaction terminating liquid that are coated with allergic protein.
Press such scheme, the described ELISA Plate that is coated with allergic protein is the pre-coated plate that is coated with dermatophagoides pteronyssinus, be coated with the pre-coated plate of dog skin bits, be coated with cat fur bits pre-coated plate, be coated with short artemisiifolia pre-coated plate, be coated with argy wormwood pre-coated plate, be coated with the pre-coated plate of Egg-white or be coated with the pre-coated plate of milk.
Press such scheme, the anti-human IgG4 monoclonal antibody that described enzyme labelled antibody is horseradish peroxidase-labeled.
Press such scheme, described substrate nitrite ion A is TMB.
Press such scheme, described substrate nitrite ion B is urea peroxide.
Press such scheme, the solution of described sample diluting liquid for containing bovine serum albumin(BSA) (BSA), cell factor and TWEEN-20.
Press such scheme, described concentrated cleaning solution is the solution that contains phosphate-buffered salt and TWEEN-20.
Press such scheme, described reaction terminating liquid is sulfuric acid.
The preparation method of the Enzyme Linked Immunoadsorbent Assay detection kit of allergen specificity IgG4 of the present invention, is characterized in that mainly including following steps:
1) prepare substrate nitrite ion A, substrate nitrite ion B, sample diluting liquid, concentrated cleaning solution and reaction terminating liquid;
2) be coated with the preparation of the ELISA Plate of allergic protein: a) preparation of allergic protein: by allergen protein powder with 1:15-25(w/v) amount add sodium bicarbonate-sodium chloride damping fluid (pH8.0-8.4) of 0.03-3mol/l, wherein 250 revs/min of the rotating speeds of magnetic stirring apparatus, at 4 ℃, spend the night, get at supernatant-20 ℃ and preserve; B) coated elisa plate: 1. with coating buffer, all kinds of allergic proteins are diluted to the coated concentration of 0.05ug/ml-10ug/ml, mix the anaphylactogen solution after being diluted, the every hole in ELISA Plate is added to the anaphylactogen solution after 100ul dilution; 2. will after ELISA Plate sealer, at 4 ℃, react and spend the night; 3. the anaphylactogen solution after the dilution in ELISA Plate is poured out, washed plate, every hole of ELISA Plate adds 200ul confining liquid to seal, and at 4 ℃, reacts and spends the night; 4. the confining liquid in ELISA Plate is poured out, patted dry, after vacuum drying, be encapsulated in the aluminium foil bag that drying agent is housed, at 4 ℃, store;
3) preparation of enzyme labelled antibody: the anti-human IgG4 monoclonal antibody that described enzyme labelled antibody is horseradish peroxidase-labeled, its preparation method is:
A) monoclonal cell strain is expelled in mouse body, produces ascites;
B) collect ascites, extract also purifying and obtain monoclonal antibody solution;
C) monoclonal antibody solution is mixed into row labels with activation horseradish peroxidase;
D) dialysed overnight, obtains the anti-human IgG4 monoclonal antibody of horseradish peroxidase-labeled.
Press such scheme, the sodium bicarbonate buffer solution that described coating buffer is pH9.0-9.8, the Tris-Hcl buffer solution that described confining liquid is the BSA that contains 1-10% (w/v).
Using method of the present invention, includes following steps:
(1) equilibrium temperature: before using, all reagent and ELISA Plate all should return to room temperature, and reagent must fully shake up before use;
(2) the preparation of cleansing solution: concentrated cleaning solution is diluted according to extension rate;
(3) hatch for the first time: will test required ELISA Plate from packing interior taking-up, numbering or mark patient's name, except blank every hole adds sample diluting liquid 2 (100ul) and serum 1ul, notice that often adding a duplicate samples just changes an ozzle, hatches 45min in 37 ℃ after mixing;
(4) wash plate: use up the liquid in ELISA Plate, every hole adds 200ul～300ul cleansing solution, leaves standstill 1min, use up liquid, so repeatedly wash 5 times, rock gently and can strengthen cleaning performance;
(5) secondary is hatched: in every hole, add 2 (100ul) enzyme labelled antibodies, after mixing, hatch 45min in 37 ℃;
(6) wash plate: with step (4);
(7) colour developing: every hole adds a substrate nitrite ion A, and a substrate nitrite ion B, hatches 15min in 37 ℃ after mixing;
(8) stop: every hole adds a reaction terminating liquid, measures every hole OD value (absorbance).
Beneficial effect of the present invention is:
1,, after patient passes through desensitization treatment, provide a kind of method of detection specificity IgG4 antibody, for desensitization treatment curative effect provides evaluation index; In desensitization treatment, allergen specificity IgG4 antibody has one significantly to increase, and successfully the remarkable rising of serological specificity IgG4 antibody is followed in immunization therapy conventionally.When allergen specificity IgG4 antibody in serum reaches after peak value (plateau), desensitization treatment success is described.By the content of allergen specificity IgG4 antibody in human body, judge the curative effect of carried out desensitization treatment;
2, have highly sensitive, specificity good, use advantage quickly and easily:
Sensitivity: detection sensitivity is 1ng/ml
Specificity: negative match-rate is 95.1%
Positive coincidence rate is 98.3%
Total coincidence rate is 96.7%
Simple operating steps, can adapt to common laboratory and detect less than 2 hours total detection time.
Embodiment
Below in conjunction with embodiment, the present invention is described further, but this explanation can not be construed as limiting the invention.
Embodiment 1
1) prepare substrate nitrite ion A, substrate nitrite ion B, sample diluting liquid, concentrated cleaning solution and reaction terminating liquid;
Described substrate nitrite ion A is TMB.
Described substrate nitrite ion B is urea peroxide.
The solution of described sample diluting liquid for containing bovine serum albumin(BSA) (BSA), cell factor and TWEEN-20.
Described concentrated cleaning solution is the solution that contains phosphate-buffered salt and TWEEN-20.
Described reaction terminating liquid is sulfuric acid.
2) be coated with the preparation of the ELISA Plate of allergic protein: a) preparation of allergic protein: by dirt mite protein powder with 1:15(w/v) amount add sodium bicarbonate-sodium chloride damping fluid (pH8.0-8.4) of 0.03mol/l, wherein 250 revs/min of the rotating speeds of magnetic stirring apparatus, at 4 ℃, spend the night, get at supernatant-20 ℃ and preserve; B) coated elisa plate: 1. with coating buffer, all kinds of allergic proteins are diluted to the coated concentration of 0.05ug/ml, mix the anaphylactogen solution after being diluted, the every hole in ELISA Plate is added to the anaphylactogen solution after 100ul dilution; 2. will after ELISA Plate sealer, at 4 ℃, react and spend the night; 3. the anaphylactogen solution after the dilution in ELISA Plate is poured out, washed plate, every hole of ELISA Plate adds 200ul confining liquid to seal, and at 4 ℃, reacts and spends the night; 4. the confining liquid in ELISA Plate is poured out, patted dry, after vacuum drying, be encapsulated in the aluminium foil bag that drying agent is housed, at 4 ℃, store; Described coating buffer is the sodium bicarbonate buffer solution of pH9.0-9.8, the Tris-Hcl buffer solution that described confining liquid is the BSA that contains 1-10% (w/v);
3) preparation of enzyme labelled antibody: the anti-human IgG4 monoclonal antibody that described enzyme labelled antibody is horseradish peroxidase-labeled, its preparation method is:
A) monoclonal cell strain is expelled in mouse body, produces ascites;
B) collect ascites, extract also purifying and obtain monoclonal antibody solution;
C) monoclonal antibody solution is mixed into row labels with activation horseradish peroxidase;
D) dialysed overnight, obtains the anti-human IgG4 monoclonal antibody of horseradish peroxidase-labeled.
Embodiment 2
1) prepare substrate nitrite ion A, substrate nitrite ion B, sample diluting liquid, concentrated cleaning solution and reaction terminating liquid;
Described substrate nitrite ion A is TMB.
Described substrate nitrite ion B is urea peroxide.
The solution of described sample diluting liquid for containing bovine serum albumin(BSA) (BSA), cell factor and TWEEN-20.
Described concentrated cleaning solution is the solution that contains phosphate-buffered salt and TWEEN-20.
Described reaction terminating liquid is sulfuric acid.
2) be coated with the preparation of the ELISA Plate of allergic protein: a) preparation of allergic protein: consider dog skin to be worth doing protein powder with 1:20(w/v) amount add sodium bicarbonate-sodium chloride damping fluid (pH8.0-8.4) of 1.5mol/l, wherein 250 revs/min of the rotating speeds of magnetic stirring apparatus, at 4 ℃, spend the night, get at supernatant-20 ℃ and preserve; B) coated elisa plate: 1. with coating buffer, all kinds of allergic proteins are diluted to the coated concentration of 5ug/ml, mix the anaphylactogen solution after being diluted, the every hole in ELISA Plate is added to the anaphylactogen solution after 100ul dilution; 2. will after ELISA Plate sealer, at 4 ℃, react and spend the night; 3. the anaphylactogen solution after the dilution in ELISA Plate is poured out, washed plate, every hole of ELISA Plate adds 200ul confining liquid to seal, and at 4 ℃, reacts and spends the night; 4. the confining liquid in ELISA Plate is poured out, patted dry, after vacuum drying, be encapsulated in the aluminium foil bag that drying agent is housed, at 4 ℃, store; Described coating buffer is the sodium bicarbonate buffer solution of pH9.0-9.8, the Tris-Hcl buffer solution that described confining liquid is the BSA that contains 1-10% (w/v);
3) preparation of enzyme labelled antibody: the anti-human IgG4 monoclonal antibody that described enzyme labelled antibody is horseradish peroxidase-labeled, its preparation method is:
A) monoclonal cell strain is expelled in mouse body, produces ascites;
B) collect ascites, extract also purifying and obtain monoclonal antibody solution;
C) monoclonal antibody solution is mixed into row labels with activation horseradish peroxidase;
D) dialysed overnight, obtains the anti-human IgG4 monoclonal antibody of horseradish peroxidase-labeled.
Embodiment 3
1) prepare substrate nitrite ion A, substrate nitrite ion B, sample diluting liquid, concentrated cleaning solution and reaction terminating liquid;
Described substrate nitrite ion A is TMB.
Described substrate nitrite ion B is urea peroxide.
The solution of described sample diluting liquid for containing bovine serum albumin(BSA) (BSA), cell factor and TWEEN-20.
Described concentrated cleaning solution is the solution that contains phosphate-buffered salt and TWEEN-20.
Described reaction terminating liquid is sulfuric acid.
2) be coated with the preparation of the ELISA Plate of allergic protein: a) preparation of allergic protein: by argy wormwood protein powder with 1:25(w/v) amount add sodium bicarbonate-sodium chloride damping fluid (pH8.0-8.4) of 3mol/l, wherein 250 revs/min of the rotating speeds of magnetic stirring apparatus, at 4 ℃, spend the night, get at supernatant-20 ℃ and preserve; B) coated elisa plate: 1. with coating buffer, all kinds of allergic proteins are diluted to the coated concentration of 10ug/ml, mix the anaphylactogen solution after being diluted, the every hole in ELISA Plate is added to the anaphylactogen solution after 100ul dilution; 2. will after ELISA Plate sealer, at 4 ℃, react and spend the night; 3. the anaphylactogen solution after the dilution in ELISA Plate is poured out, washed plate, every hole of ELISA Plate adds 200ul confining liquid to seal, and at 4 ℃, reacts and spends the night; 4. the confining liquid in ELISA Plate is poured out, patted dry, after vacuum drying, be encapsulated in the aluminium foil bag that drying agent is housed, at 4 ℃, store; Described coating buffer is the sodium bicarbonate buffer solution of pH9.0-9.8, the Tris-Hcl buffer solution that described confining liquid is the BSA that contains 1-10% (w/v);
3) preparation of enzyme labelled antibody: the anti-human IgG4 monoclonal antibody that described enzyme labelled antibody is horseradish peroxidase-labeled, its preparation method is:
A) monoclonal cell strain is expelled in mouse body, produces ascites;
B) collect ascites, extract also purifying and obtain monoclonal antibody solution;
C) monoclonal antibody solution is mixed into row labels with activation horseradish peroxidase;
D) dialysed overnight, obtains the anti-human IgG4 monoclonal antibody of horseradish peroxidase-labeled.
The using method of embodiment 1 allergen specificity IgG4 antibody ELISA detection kit:
(1) equilibrium temperature: before using, all reagent and ELISA Plate all should return to room temperature, and reagent must fully mix before use;
(2) the preparation of cleansing solution: cleansing solution is diluted according to extension rate;
(3) hatch for the first time: will test required ELISA Plate from packing interior taking-up, numbering or mark patient's name, except blank every hole adds sample diluting liquid 2 (100ul) and serum 1ul, notice that often adding a duplicate samples just changes an ozzle, hatches 45 minutes in 37 ℃ after mixing;
(4) wash plate: use up the liquid in ELISA Plate, every hole adds 200ul～300ul cleansing solution, leaves standstill 1min, use up liquid, so repeatedly wash 5 times, rock gently and can strengthen cleaning performance;
(5) secondary is hatched: in every hole, add the anti-human IgG4 antibody of 1 (50ul) HRP mark, after mixing, hatch 45 minutes in 37 ℃;
(6) wash plate: with step (4);
(7) colour developing: every hole adds a substrate nitrite ion A, and a substrate nitrite ion B, hatches 15min in 37 ℃ after mixing;
(8) stop: every hole adds a stop buffer, measures every hole OD value (absorbance).
Mensuration obtains different OD values:
| Serum sample this shop | 1 | 2 | 3 | 4 | 5 | 6 |
| Desensitization past mite specific IgG4 detects OD value | 0.220 | 0.147 | 0.245 | 0.146 | 0.154 | 0.039 |
| Desensitization past mite specific IgG 4 antibody concentration (mg/L) | 0.293 | 0.196 | 0.326 | 0.194 | 0.205 | 0.052 |
| Desensitization dust kicked up by somebody walking in front mite specific IgG4 detects OD value | 3.161 | 3.157 | 3.009 | 2.112 | 2.013 | 1.163 |
| Desensitization dust kicked up by somebody walking in front mite specific IgG 4 antibody concentration (mg/L) | 4.204 | 4.199 | 4.002 | 2.809 | 2.677 | 1.547 |
| Serum sample this shop | 7 | 8 | 9 | 10 | 11 | 12 |
| Desensitization past mite specific IgG4 detects OD value | 0.275 | 0.332 | 0.190 | 0.120 | 0.074 | 0.295 |
| Desensitization past mite specific IgG 4 antibody concentration (mg/L) | 0.366 | 0.442 | 0.253 | 0.200 | 0.098 | 0.392 |
| Desensitization dust kicked up by somebody walking in front mite specific IgG4 detects OD value | 3.388 | 2.824 | 2.761 | 2.639 | 1.961 | 3.391 |
| Desensitization dust kicked up by somebody walking in front mite specific IgG 4 antibody concentration (m/L) | 4.506 | 3.756 | 3.672 | 3.510 | 2.608 | 4.510 |
Adopt the kit of embodiment 1 to carry out the detection of IgG4 antibody, draw table data.Upper table data declaration:
Choose 12 parts of desensitization treatment clinical effectiveness remarkable, the serum of desensitization treatment successful patient, the detection of dirt mite specific IgG 4 antibody before and after desensitizing, for 12 parts of dust mite allergy patients serums, after accepting desensitization treatment, in serum, dirt mite specific IgG 4 antibody concentration significantly increases, and illustrates and uses kit of the present invention can detect easy, quickly and accurately the dirt mite specific IgG 4 antibody content in sample.And dirt mite specific IgG 4 antibody content can be used as the index of curative effect in immunization therapy process.
Claims (1)
1. the Enzyme Linked Immunoadsorbent Assay of allergen specificity IgG4 detects the application of reagent for the preparation of the kit of the evaluation monitoring method of desensitization treatment effect, by detecting the content of allergen specificity IgG4 antibody in serum, judge the curative effect of carried out desensitization treatment, when allergen specificity IgG4 antibody in serum rises significantly, illustrate that desensitization treatment effect is remarkable, when allergen specificity IgG4 antibody in serum reaches after peak value, success of desensitization treatment is described, the Enzyme Linked Immunoadsorbent Assay detection kit of described allergen specificity IgG4, include the ELISA Plate that is coated with allergic protein, enzyme labelled antibody, substrate nitrite ion A, substrate nitrite ion B, sample diluting liquid, concentrated cleaning solution and reaction terminating liquid,
The described ELISA Plate that is coated with allergic protein is the pre-coated plate that is coated with dermatophagoides pteronyssinus, be coated with the pre-coated plate of dog skin bits, be coated with cat fur bits pre-coated plate, be coated with short artemisiifolia pre-coated plate, be coated with argy wormwood pre-coated plate, be coated with the pre-coated plate of Egg-white or be coated with the pre-coated plate of milk; Described enzyme labelled antibody is the anti-human IgG4 monoclonal antibody of horseradish peroxidase-labeled; Described substrate nitrite ion A is TMB; Described substrate nitrite ion B is urea peroxide; Described sample diluting liquid is the solution that contains bovine serum albumin(BSA), cell factor and TWEEN-20; Described concentrated cleaning solution is the solution that contains phosphate-buffered salt and TWEEN-20;
describedthe preparation method of the Enzyme Linked Immunoadsorbent Assay detection kit of allergen specificity IgG4, includes following steps:
1) prepare substrate nitrite ion A, substrate nitrite ion B, sample diluting liquid, concentrated cleaning solution and reaction terminating liquid;
2) be coated with the preparation of the ELISA Plate of allergic protein: a) preparation of allergic protein: by allergen protein powder with 1:15-25(w/v) amount add sodium bicarbonate-sodium chloride damping fluid of 0.03-3mol/l, wherein 250 revs/min of the rotating speeds of magnetic stirring apparatus, at 4 ℃, spend the night, get at supernatant-20 ℃ and preserve; B) coated elisa plate: 1. with coating buffer, all kinds of allergic proteins are diluted to the coated concentration of 0.05ug/ml-10ug/ml, mix the anaphylactogen solution after being diluted, the every hole in ELISA Plate is added to the anaphylactogen solution after 100ul dilution; 2. will after ELISA Plate sealer, at 4 ℃, react and spend the night; 3. the anaphylactogen solution after the dilution in ELISA Plate is poured out, washed plate, every hole of ELISA Plate adds 200ul confining liquid to seal, and at 4 ℃, reacts and spends the night; 4. the confining liquid in ELISA Plate is poured out, patted dry, after vacuum drying, be encapsulated in the aluminium foil bag that drying agent is housed, at 4 ℃, store;
3) preparation of enzyme labelled antibody: the anti-human IgG4 monoclonal antibody that described enzyme labelled antibody is horseradish peroxidase-labeled, its preparation method is:
A) monoclonal cell strain is expelled in mouse body, produces ascites;
B) collect ascites, extract also purifying and obtain monoclonal antibody solution;
C) monoclonal antibody solution is mixed into row labels with activation horseradish peroxidase;
D) dialysed overnight, obtains the anti-human IgG4 monoclonal antibody of horseradish peroxidase-labeled; Described coating buffer is the sodium bicarbonate buffer solution of pH9.0-9.8, the Tris-Hcl buffer solution that described confining liquid is the BSA that contains 1-10% (w/v).
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| CN103837686A (en) * | 2014-03-06 | 2014-06-04 | 上海北加生化试剂有限公司 | Kit for detecting human immunoglobulin G4 as well as preparation method of kit |
| CN104535773B (en) * | 2015-01-07 | 2016-11-23 | 武汉中敏泰克生物科技有限公司 | A kind of dust mite specific IgG4 subclass antibodies calibration object and preparation method thereof |
| CN108646023A (en) * | 2018-05-03 | 2018-10-12 | 辽宁汇普源生物医学科技开发有限责任公司 | The ELISA kit of anaphylactogen is passed based on IgG4 antibody test gas |
| CN109030836A (en) * | 2018-10-25 | 2018-12-18 | 苏州大学附属儿童医院 | Food proteins Specific IgA antibody detects ELISA kit and its application |
| CN113125753B (en) * | 2021-04-16 | 2022-07-26 | 杭州浙大迪迅生物基因工程有限公司 | Kit for detecting specific antibody of dust mite component |
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|---|---|---|---|---|
| CN1343221A (en) * | 1999-01-15 | 2002-04-03 | 杰南技术公司 | Polypeptide variants with altered effector function |
| WO2007045477A2 (en) * | 2005-10-21 | 2007-04-26 | Novartis Ag | Human antibodies against il-13 and therapeutic uses |
| CN101109750A (en) * | 2007-07-27 | 2008-01-23 | 杭州浙大生科生物技术有限公司 | Reagent kit for detecting food allergen specificity IgG ELISA and preparing method thereof |
| CN101256186A (en) * | 2008-03-28 | 2008-09-03 | 杭州浙大生科生物技术有限公司 | Food anaphylactogen specificity IgG4 antibody ELISA detection kit and preparation method thereof |
| WO2010065929A2 (en) * | 2008-12-04 | 2010-06-10 | Massachusetts Institute Of Technology | Method for diagnosing allergic reactions |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1343221A (en) * | 1999-01-15 | 2002-04-03 | 杰南技术公司 | Polypeptide variants with altered effector function |
| WO2007045477A2 (en) * | 2005-10-21 | 2007-04-26 | Novartis Ag | Human antibodies against il-13 and therapeutic uses |
| CN101109750A (en) * | 2007-07-27 | 2008-01-23 | 杭州浙大生科生物技术有限公司 | Reagent kit for detecting food allergen specificity IgG ELISA and preparing method thereof |
| CN101256186A (en) * | 2008-03-28 | 2008-09-03 | 杭州浙大生科生物技术有限公司 | Food anaphylactogen specificity IgG4 antibody ELISA detection kit and preparation method thereof |
| WO2010065929A2 (en) * | 2008-12-04 | 2010-06-10 | Massachusetts Institute Of Technology | Method for diagnosing allergic reactions |
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