CN102944682A - Crab type antibody repertoire detection kit - Google Patents
Crab type antibody repertoire detection kit Download PDFInfo
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- CN102944682A CN102944682A CN2012105056837A CN201210505683A CN102944682A CN 102944682 A CN102944682 A CN 102944682A CN 2012105056837 A CN2012105056837 A CN 2012105056837A CN 201210505683 A CN201210505683 A CN 201210505683A CN 102944682 A CN102944682 A CN 102944682A
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Abstract
The invention discloses a crab type antibody repertoire detection kit and belongs to an enzyme immunoblotting technique. The crab type antibody repertoire detection kit comprises a chromogenic substrate, a blot reaction slot and a mouse-anti-human IgE-AP labeled antibody, and the crab type antibody repertoire detection kit further comprises water concentrate, detection areas and quality control areas, wherein the water concentrate is used for sample dilution and enzyme labeled antibody dilution, the detection areas are respectively coated by crab proteins with different molecular weights, and the quality control areas are coated by rabbit-anti-mouse IgG antibodies and are labeled by C. According to the crab type antibody repertoire detection kit disclosed by the invention, crab protein coarse extract is transferred and coated on a PVDF (polyvinylidene fluoride) membrane after being directly subjected to SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), so that the crab proteins are separated and are reacted with blood serum to be detected based on naturally existing consistency; and therefore, the comprehensiveness of the specific IgE detection is ensured; above all, a detection result can respond specific antibodies of multiple allergen ingredients in the blood serum to be detected, the accuracy of clinical diagnosis can be improved, the crab type antibody repertoire detection kit is suitable for further confirmation of crab allergy, and a reasonable desensitization scheme is defined.
Description
Technical field
The present invention relates to the enzyme immunoblot assay, specifically a kind of detection crab autopath serological specificity IgE(sIgE) the antibody repertoire detection kit.
Background technology
The essence of food hypersenstivity is a kind of immune response of mistake, body is during to certain food hypersenstivity, can produce in vivo corresponding specific antibody (IgE and IgG), this antibody causes allergic reaction to self injury, clinically can show as skin, respiratory tract, symptom of digestive tract, even threat to life.For the detection of food hypersenstivity, mainly concentrate on medical history observation and in vivo studies (such as skin prick test, food provocative test) in early days; But these methods are not only expensive, consuming time, and exist the risk of bringing out severe allergic reaction, gradually are eliminated.At present, often adopt clinically detection specificity antibody to determine patient's food hypersenstivity situation, finally determine allergenic foods.
Adopt vitro detection crab class specific antibody to have not to be subjected to the advantages such as interfering effects of drug, safe devoid of risk, patient be acceptant.Specific IgE is measured based on the immunochemistry principle, namely measures unknown antibody (IgE) by known crab albumen (anaphylactogen).As detecting the antibody for crab albumen in the serum, can judge that namely detected object is irritated to crab.Serological specificity IgE need to adopt immuno-labelling technique, usually uses the things that serve as a mark such as enzyme, fluorescent material, i.e. enzyme linked immunosorbent assay, enzyme Western blot and luciferase immunization.No matter adopt which kind of labelling technique, all have the known antigens of high-quality to be the basis, the quality of envelope antigen directly affects testing result.
The existing diagnostic kit total protein compositions that extract from food that adopt as known antigens more, and the general tree total protein segments, and does not also consider the proportionate relationship between the each component.As envelope antigen, the food allergen diagnostic kit of detection specificity IgE all exists following defective or deficiency based on food total protein extract.Take crab as example: there is obvious individual difference in the food hypersenstivity reaction, and there is obvious individual difference in specific IgE in different autopath's serum; In other words, there are certain difference in kind and the concentration of specific IgE in the crab autopath serum, and this difference has great importance to clinical diagnosis and treatment.Existing food allergen kit can not be distinguished the heterogeneity of specific IgE owing to adopting total protein to be coated with.
Summary of the invention
Technical matters to be solved by this invention is, a kind of crab antibody-like spectrum diagnostic kit of multi-component specific IgE simultaneous determination is provided, solve present crab specific diagnosis kit and can only filter out the existence of crab specific IgE in the serum, and can not determine that the patient is to the difference of different allergen proteins in the crab.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is: a kind of crab antibody-like spectrum detection kit, comprise chromogenic substrate, marking reactive tank, mouse-anti people IgE-AP labelled antibody, also comprise the albumen that is coated with respectively each molecular weight of crab and the pvdf membrane detector bar of the anti-mouse IgE of rabbit antibody, be used for the concentrated washing lotion of sample dilution, enzyme labelled antibody dilution.
The preparation method of the pvdf membrane detector bar of the described albumen that is coated with respectively each molecular weight of crab and the anti-mouse IgE of rabbit antibody may further comprise the steps:
(1) being prepared as follows of protein crude extract:
Get fresh and alive sea crab, shell, the pure musculature of picking, grind to form homogenate, the ratio of 1:10～1:20 adds-20 ℃ cold acetone by volume, puts 4 ℃ of refrigerator degreasings, magnetic agitation was 30 minutes in per 2 hours, elimination acetone after 24 hours, aeration-drying, the dried crab albumen of weighing, add 10～20 milliliters of PH7.4,0.01M PBS by every gram sample, put 4 ℃ of refrigerators, stirred 1-2 hour, after 24 hours, 12000 rev/mins centrifugal 20 minutes, collect supernatant, packing-20 ℃ preservation is to be ready for use on the albumen of electrophoretic separation different molecular weight;
(2) with the preparation crab albumen carry out the SDS-PAGE electrophoresis after, the different bands of the technology of utilizing transferring film after with electrophoresis are transferred on the polystyrene pvdf membrane, become the detection zone of the albumen of each molecular weight, simultaneously, the anti-mouse IgE of rabbit antibody on another pvdf membrane is coated, as the Quality Control district of test, these two pvdf membranes are fixed on the same end liner.
The preparation method of described step (2) is:
1. detection zone: protein crude extract is behind the SDS-PAGE electrophoresis, pvdf membrane soaks through methyl alcohol, distilled water, transferring film damping fluid successively, afterwards, driedly turned under 15 volts of voltages of instrument transferring film 30 minutes, more successively with methyl alcohol, distilled water, 0.01MTBST flushing, the skimmed milk sealing of mass percent 5% is spent the night, discard confining liquid, 0.01M TBST rinsing 3 times, each 10 minutes, air-dry, for subsequent use;
2. control zone: it is the 1.6-2.4 mg/ml that the anti-mouse IgE of rabbit antibody is regulated concentration, point sample is on pvdf membrane, room temperature incubation 2 hours, with after methyl alcohol, distilled water, the 0.01M TBST flushing, 5% skimmed milk sealing is spent the night, and discards confining liquid successively, 0.01M TBST rinsing 3 times, each 10 minutes, air-dry, for subsequent use;
3. will be 1. and the detection zone that 2. prepares and control zone be fixed on the end liner, for subsequent use.
Described concentrated lotion prescription for sample dilution, enzyme labelled antibody dilution is:
Tris 1.21g
Sodium chloride 5.84g
Distilled water 80ml
Transfer PH to 8.0, add 1ml mass percent concentration 30%TWEEN-20, after fully stirring, 4 ℃ are settled to 100mL.
The invention has the beneficial effects as follows: after the present invention has taked the SDS-PAGE electrophoresis, transferring film is coated in protein ingredient on the pvdf membrane, different protein ingredients in the crab class are separated, like this, just can clearly find out which exists plant the specific antibody (IgE) of irritated component in the serum to be checked in the testing result, can improve the accuracy of clinical diagnosis, be suitable for the further affirmation of crab class allergy, and can formulate the desensitization treatment scheme that is fit to according to the specific IgE kind that different patients are contained.
Description of drawings
Fig. 1 uses the patients serum of crab antibody-like spectrum detection kit of the present invention and crab zone of protein marking result (to annotate: M: molecular weight standard Por: protein electrophoresis 1～9: single routine patients serum 10: the negative serum contrast).
Fig. 2 is crab antibody-like spectrum detection kit experimentation schematic diagram of the present invention.
Embodiment
Below in conjunction with the drawings and specific embodiments the present invention is described in further detail:
As shown in Figure 2, crab antibody-like spectrum diagnostic kit of the present invention, comprise chromogenic substrate, marking reactive tank, mouse-anti people IgE-AP labelled antibody, also comprise the albumen that is coated with respectively each molecular weight of crab and pvdf membrane detector bar, sample diluent, enzyme labelled antibody dilution and the concentrated washing lotion of the anti-mouse IgE of rabbit antibody.
Described crab antibody-like spectrum diagnostic reagent box preparation method (following percent concentration is mass percent concentration): 1. the preparation method of protein crude extract is as follows:
Get fresh and alive swimming crab (sea crab), shell, the pure musculature of picking is ground, grind to form homogenate as far as possible, the ratio of 1:10～1:20 adds cold acetone (20 ℃ of preservations) by volume, puts 4 ℃ of refrigerator degreasings, during about 2 hours of magnetic agitation, can stir 30 minutes at every turn, elimination acetone after 24 hours, aeration-drying, the dried crab albumen of weighing, add 0.01M PBS(PH7.4 by every gram sample) 10～20 milliliters, put 4 ℃ of refrigerators, during stirred about 2 hours, after 24 hours, 12000 rev/mins centrifugal 20 minutes, collect supernatant, packing-20 ℃ preservation is to be ready for use on the albumen of electrophoretic separation different molecular weight.
2. after the crab albumen for preparing being carried out the SDS-PAGE electrophoresis, the different bands of the technology of utilizing transferring film after with electrophoresis are transferred to (pvdf membrane on the polystyrene film, hereinafter to be referred as pvdf membrane), become the detection zone (T district) of the albumen of each molecular weight, simultaneously, the anti-mouse IgE of rabbit antibody on another pvdf membrane is coated, as the Quality Control district (C district) of test, these two pvdf membranes are fixed on the same end liner.The detailed process of preparation is: 1. detection zone: protein crude extract is behind the SDS-PAGE electrophoresis, pvdf membrane soaks through methyl alcohol, distilled water, transferring film damping fluid successively, afterwards, driedly turned under 15 volts of voltages of instrument transferring film 30 minutes, more successively with methyl alcohol, distilled water, 0.01M TBST flushing, 5% skimmed milk sealing is spent the night, discard confining liquid, 0.01M TBST rinsing 3 times, each 10 minutes, air-dry, for subsequent use.2. control zone: it is the 1.6-2.4 mg/ml that the anti-mouse IgE of rabbit antibody is regulated concentration, point sample is on pvdf membrane, room temperature incubation 2 hours, with after methyl alcohol, distilled water, the 0.01M TBST flushing, 5% skimmed milk sealing is spent the night, and discards confining liquid successively, 0.01M TBST rinsing 3 times, each 10 minutes, air-dry, for subsequent use.3. will be 1. and the detection zone that 2. prepares and control zone be fixed on the end liner, for subsequent use.
1. solution preparation
⑴
Magnetic agitation fully after the dissolving, is transferred pH to 7.4, adds ddH
2O is settled to 4L.
⑵ transferring film damping fluid
Magnetic agitation fully is settled to 2L after the dissolving, and 4 ℃ save backup.
⑶ TBST(0.1M pH8.0) (the concentrated washing lotion that is used for sample dilution, enzyme labelled antibody dilution)
Tris 1.21g
Sodium chloride (NaCl) 5.84g
Distilled water (ddH
2O) 80ml
Transfer PH to 8.0, add 1ml mass percent concentration 30%TWEEN-20, be settled to 100mL after fully stirring, 4 ℃ for subsequent use.
⑷ 0.01M TBST(is used for the washing lotion of sample dilution, enzyme labelled antibody dilution)
⑶ the TBST of 0.1M in is diluted 10 times
⑸ chromogenic substrate
Adopt nitroblue tetrazolium (NBT) (NBT) and 5-bromo-4-chloro-3-indyl-phosphoric acid (BCIP) mixed liquor, be commercialization reagent, U.S. Sigma company is on sale.
2. kit preparation (following concentration is mass percent concentration)
⑴ film is processed: get a pvdf membrane, be soaked in successively methyl alcohol, distilled water, the dried damping fluid that turns, soaked approximately 2-3 minute at every turn; Polyvinylidene fluoride (PVDF) film (U.S. Millipore company).
⑵ sample preparation: protein crude extract is according to the SDS-PAGE electrophoretic techniques of maturation, concentrated glue is 5%, separation gel is 10%, and sample concentration is 3.5 mg/ml, and sample-loading buffer mixes (volume ratio) with 3:2, boiled 5 minutes, every hole loading 15 microlitres, 65 volts of voltages were piled up 30 minutes, and 135 volts of voltages separate, treat that the bromophenol blue indication arrives the bottom, electrophoresis is complete.
⑶ transferring film: the gel behind the electrophoresis cuts concentrated glue, balance in the transferring film damping fluid; With the pvdf membrane of having processed, form the pattern of 4 metafiltration paper-pvdf membrane-gel-4 metafiltration paper, driedly turned under 15 volts of voltages of instrument transfer printing 30 minutes.
⑷ sealing: take out the pvdf membrane that takes a turn for the better, soak through methyl alcohol, distilled water, transferring film damping fluid successively, afterwards 5% skimmed milk sealing is spent the night.
⑸ rinsing: discard confining liquid, 0.01M TBST rinsing three times, each 10 minutes.
⑹ air-dry: pvdf membrane places on the dry filter paper, automatic drying, and labelling paper, for subsequent use.
⑺ the preparation of control zone (C district): it is the 1.6-2.4 mg/ml that the anti-mouse IgE of rabbit antibody is regulated concentration, point sample is on pvdf membrane, room temperature incubation 2 hours, with after methyl alcohol, distilled water, the 0.01M TBST flushing, 5% skimmed milk sealing is spent the night, and discards confining liquid successively, 0.01M TBST rinsing 3 times, each 10 minutes, air-dry, for subsequent use.
⑻ fix: the detection zone and the control zone detector bar that prepare are fixed on the end liner.
4. enzyme labeling mouse anti human IgE antibody
Mouse-anti people IgE-Ap(alkaline phosphatase) antibody is commercialization reagent, available from U.S. R﹠amp; D company.Product description provides best dilute concentration 1:1000, scope 1:800-1:12000.
5. using method of the present invention (determination step)
⑴ put into marking reactive tank with the pvdf membrane detector bar, adds the 1ml washing lotion; Be placed on and shake 10 minutes on the shaking table, make detector bar fully wetting.
⑵ blot the washing lotion in the groove, adds the test serum (serum specimen dilutes 10 times with sample diluent) after 0.5ml dilutes, put on the shaking table, and room temperature reaction 3.5 hours, this moment, Serum Antibody was combined with corresponding anaphylactogen.
⑶ blot the serum in the groove, adds the 1ml washing lotion, puts in the shaking table to shake 10 minutes; Repeated washing 3 times.
⑷ blot the washing lotion in the groove, add 0.5ml mouse-anti people's IgE-AP labelled antibody (mouse-anti people IgE-AP being done 250 times dilution with the enzyme labelled antibody dilution), put on the shaking table, room temperature reaction 0.5 hour, the anti-mouse IgE of C district rabbit antibody is combined with enzymic-labelled antibody and is formed compound, and the T district then forms anaphylactogen-specific IgE antibody-enzymic-labelled antibody compound.
⑸ blot enzyme labelled antibody, adds the 1ml washing lotion, puts in the shaking table to shake 10 minutes; Repeated washing 3 times.
⑹ add the 0.5ml chromogenic substrate, and lucifuge was reacted about 15-20 minute.
⑺ outwell substrate, adds the 1ml washing lotion, puts in the shaking table to shake 3-5 minute; Repeated washing 3 times.
⑻ dry detector bar is judged testing result according to detection zone colour developing situation, confirms whether the patients serum exists the kind of specific IgE and IgE.
⑼ as a result interpretation: with crab protein band be reference, if the specific antibody of crab class is arranged in the serum then corresponding band occurs, can report out irritated albumen; If without corresponding antibodies, then this detection zone is without colour developing in the serum; No matter the Quality Control district has or not specific antibody in the serum, and enzymic-labelled antibody all can be combined with the anti-mouse IgE of the rabbit on pvdf membrane surface antibody, the colour developing band occurs.As the Quality Control district do not develop the color the explanation this detection go wrong, can not report the result.Validity with this design test.
Clinical application effect
Utilize this kit to detect clinical definite to crab class autopath 30 people, select simultaneously Normal group 10 examples of crab class without allergy, the result shows that 10 routine Normal groups do not produce the band of colour developing, and 30 routine irritated patient's groups have all produced the colour developing band, just the position is different with the painted depth, the antibody that has all detected crab albumen allergy is described, just type is different with intensity, the antibody repertoire (as shown in Figure 1) that has shown crab allergy, analyze 30 routine autopath's seroreaction results, several different patterns appear: 1. take a kind of monospecific antibody wherein as main, be main for a certain albumen only namely, mainly respectively with 94KD, 48KD, 38KD and 36KD are main, such as 1～5 among Fig. 1; 2. several antibody exist simultaneously, namely for several albumen, such as the routine serum in 6 among Fig. 1～8 3; 3. without the reaction type of comparatively outstanding protein band, there are several lower antibody of tiring, such as No. 9 serum among Fig. 1.Accordingly, clinically can make targetedly therapeutic scheme.
In sum, content of the present invention is not limited in the above-described embodiment, and the knowledgeable people in the same area can propose other embodiment easily within technological guidance's thought of the present invention, but this embodiment comprises within the scope of the present invention.
Claims (4)
1. a crab antibody-like is composed detection kit, comprise chromogenic substrate, marking reactive tank, mouse-anti people IgE-AP labelled antibody, it is characterized in that, also comprise the albumen that is coated with respectively each molecular weight of crab and the pvdf membrane detector bar of the anti-mouse IgE of rabbit antibody, be used for the concentrated washing lotion of sample dilution, enzyme labelled antibody dilution.
2. crab antibody-like according to claim 1 spectrum detection kit is characterized in that, the preparation method of the pvdf membrane detector bar of the described albumen that is coated with respectively each molecular weight of crab and the anti-mouse IgE of rabbit antibody may further comprise the steps:
(1) being prepared as follows of protein crude extract:
Get fresh and alive sea crab, shell, the pure musculature of picking, grind to form homogenate, the ratio of 1:10～1:20 adds-20 ℃ cold acetone by volume, puts 4 ℃ of refrigerator degreasings, magnetic agitation was 30 minutes in per 2 hours, elimination acetone after 24 hours, aeration-drying, the dried crab albumen of weighing, add 10～20 milliliters of PH7.4,0.01M PBS by every gram sample, put 4 ℃ of refrigerators, stirred 1-2 hour, after 24 hours, 12000 rev/mins centrifugal 20 minutes, collect supernatant, packing-20 ℃ preservation is to be ready for use on the albumen of electrophoretic separation different molecular weight;
(2) with the preparation crab albumen carry out the SDS-PAGE electrophoresis after, the different bands of the technology of utilizing transferring film after with electrophoresis are transferred on the polystyrene pvdf membrane, become the detection zone of the albumen of each molecular weight, simultaneously, the anti-mouse IgE of rabbit antibody on another pvdf membrane is coated, as the Quality Control district of test, these two pvdf membranes are fixed on the same end liner.
3. crab antibody-like spectrum detection kit according to claim 2 is characterized in that, the preparation method of described step (2) is:
1. detection zone: protein crude extract is behind the SDS-PAGE electrophoresis, pvdf membrane soaks through methyl alcohol, distilled water, transferring film damping fluid successively, afterwards, driedly turned under 15 volts of voltages of instrument transferring film 30 minutes, more successively with methyl alcohol, distilled water, 0.01MTBST flushing, the skimmed milk sealing of mass percent 5% is spent the night, discard confining liquid, 0.01M TBST rinsing 3 times, each 10 minutes, air-dry, for subsequent use;
2. control zone: it is the 1.6-2.4 mg/ml that the anti-mouse IgE of rabbit antibody is regulated concentration, point sample is on pvdf membrane, room temperature incubation 2 hours, with after methyl alcohol, distilled water, the 0.01M TBST flushing, 5% skimmed milk sealing is spent the night, and discards confining liquid successively, 0.01M TBST rinsing 3 times, each 10 minutes, air-dry, for subsequent use;
3. will be 1. and the detection zone that 2. prepares and control zone be fixed on the end liner, for subsequent use.
4. crab antibody-like spectrum detection kit according to claim 1 is characterized in that, described concentrated lotion prescription for sample dilution, enzyme labelled antibody dilution is:
Tris 1.21g
Sodium chloride 5.84g
Distilled water 80ml
Transfer PH to 8.0, add 1ml mass percent concentration 30%TWEEN-20, after fully stirring, 4 ℃ are settled to 100mL.
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| CN103995110A (en) * | 2014-05-28 | 2014-08-20 | 中生北控生物科技股份有限公司 | Test strip for screening specific aquatic products allergens IgE (Immunoglobulin E) and preparation method thereof |
| CN108061795A (en) * | 2017-11-24 | 2018-05-22 | 大连医科大学 | Improve the protein immunoblotting method of detection sensitivity |
| CN111596070A (en) * | 2020-06-10 | 2020-08-28 | 宁波大学 | Application of portunus trituberculatus tropomyosin allergy detection reagent |
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| CN111596070A (en) * | 2020-06-10 | 2020-08-28 | 宁波大学 | Application of portunus trituberculatus tropomyosin allergy detection reagent |
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Application publication date: 20130227 |