CN202305538U

CN202305538U - Angiotensin II enzyme-linked immunosorbent assay (ELISA) reagent kit

Info

Publication number: CN202305538U
Application number: CN2011204270016U
Authority: CN
Inventors: 何峰容; 李华渊; 杨娟; 章秀波; 汪景
Original assignee: Uscn Life Science Inc Wuhan
Current assignee: Uscn Life Science Inc Wuhan
Priority date: 2011-10-18
Filing date: 2011-10-18
Publication date: 2012-07-04
Anticipated expiration: 2021-10-18

Abstract

The utility model provides an angiotensin II enzyme-linked immunosorbent assay (ELISA) reagent kit, which comprises a kit cover (4), an internal ELISA plate (5) and a kit body (6), wherein the ELISA plate (5) comprises a solid-phase carrier (1) and a covering layer (3), the solid-phase carrier (1) is a micro porous plate (2) provided with a plurality of micro pores, the covering layer (3) is attached onto the surfaces of the micro pores of the solid-phase carrier (1), the covering layer (3) is a composite layer formed by sequentially coating Ang II and bovine serum albumin onto the surfaces of the micro pores of the solid-phase carrier (1), and the solid-phase carrier (1) is a 96-pore polystyrene reagent plate. Antibody segments combined with the Ang II are obtained through sieving by a phage antibody display method, and in addition, crossed combined antibodies of Ang I and Ang III are removed through negative sieving, so the specificity of the antibodies is further improved.

Description

The Angiotensin II enzyme-linked immunosorbent assay kit

Technical field

The utility model relates to biological technical field, relates in particular to a kind of Angiotensin II (AngII) enzyme linked immunosorbent assay (ELISA) (ELISA) kit.

Background technology

RAS (RAS) plays a significant role in regulating physiological functions such as blood pressure, electrolyte and isohydria.Proangiotensin (AGT) is mainly synthetic and be released into blood by liver; Under the effect of feritin, generate 10 peptide compounds; Be angiotensin I (AngI), AngI generates 8 peptide AngII under Angiotensin-Converting (ACE) effect, and the latter produces the AngIII of 7 peptides again through the ACE effect.Wherein AngII is the main effects molecule of RAS; Through vasoactive anxiety plain II acceptor 1 (AT1) and AT2 acceptor, in regulating blood pressure, pipe ball feedback, the incidence and development of renal tubule, occupy critical role to heavily absorption, kidney growth and the angiocardiopathy of water sodium.Under the pathological state, AngII plays a significant role in processes such as hypertension, atherosclerotic and insecondary ventricular hypertrophy; Simultaneously, in the diabetic, AngII can suppress the insulin signaling approach directly or indirectly, increases the weight of insulin resistance.Simultaneously, this material also can be used as the hazards that cardiovascular event takes place the prediction diabetic.

Display technique of bacteriophage is to be carrier with the bacteriophage; With the directed bacteriophage coat protein gene regions of inserting of complete antibody variable region fragment; Make its product expression and be showed in phage surface, through the repetitive process of absorption-wash-out-amplification, screening obtains to express the bacteriophage of the scFv fragment that combines with the known antigens specificity; And making it become complete antibody through genetic modification, improved antibody promptly can be used as the raw material of preparation ELISA kit.Because present business-like phage antibody library storage capacity can reach 10 ¹⁰-10 ¹¹, make that being filtered into power increases greatly, can be applicable in the many antibody that are difficult to obtain of preparation through immune animal.

The direct method of estimating AngII at present has high performance liquid chromatography (HPLC), radioimmunology (RIA) and enzyme linked immunosorbent assay (ELISA).Wherein the high performance liquid chromatography sensing range is generally at 2.5-500pg/ml, and the sample recovery is more than 90%.But the defective that this method exists also is very tangible, exists detecting instrument expense height, complex operation, sample to need shortcomings such as pre-treatment.The radioimmunology detection sensitivity is higher, but owing to be applied to radiomaterial, higher for demand of laboratory, and the experiment waste liquid is hard to manage.By contrast, the ELISA method is easy and simple to handle, does not need large-scale instrument, simultaneously because the application of the system that Avidin-biotin amplifies has improved its sensitivity greatly.The important source material of preparation ELISA and RIA kit is exactly the specific antibody to AngII.Traditional preparation method for antibody is through immune animal; Obtain the bone-marrow-derived lymphocyte that can produce antibody; With the myeloma cell merge obtain hybridoma after, obtain producing the monoclonal B cell of high-affinity antibody through screening, immune animal generates the ascites that is rich in antibody again.This method and technology is ripe, but operation is more loaded down with trivial details, and immunogenicity of antigens is required high.Because AngII only contains 8 amino acid, its sequence is Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, and molecular weight is 1046.2, and less immunogenic is difficult for obtaining antibody through immune animal.

Antibody is the key factor of decision ELISA kit quality, and its affinity, specificity are directly connected to sensitivity, accuracy and the accuracy of kit.Therefore, be necessary to select for use a kind of simple effective method to obtain high-quality antibody.The key issue that this technology will solve is that screening is directed against the specific scFv section of AngII from phage library.Because the catabolite of AGT has 3, is followed successively by AngI, AngII and AngIII have only 1-2 amino acid whose difference between every kind, therefore the antibody of screening to avoid with AngII outside the cross reaction of 2 kinds of fragments.For this reason; After bacteriophage being carried out conventional forward selection and enrichment; Carry out negative sense again and select to remove the antibody that can combine with AngI and AngIII, this measure can guarantee a little less than last gained antibody and this two analog no cross reactions or the reaction extremely, and is most important to guarantee reagent box detection accuracy.

Secondly, the scFv fragment that is merely antibody that phage library is expressed does not possess two valency integrated structures of traditional antibody, the antigen adhesion a little less than.For this reason; Through the gene fusion that AngII scFv and IgG3 upper reaches hinge area/p53 four are gathered functional domain; Utilize p53 four to gather the functional domain expression product AngII scFv antibody is assembled into the tetramer; Can combine four epitopes simultaneously, and can the phase mutual interference on the site, space, thus obtained antibody can be used for preparing the ELISA kit of anti-AngII.

The utility model content

The purpose of the utility model provides a kind of Angiotensin II enzyme-linked immunosorbent assay kit; The ELISA Plate and the box body that comprise lid, inside; Wherein ELISA Plate comprises solid phase carrier and encapsulates layer; Solid phase carrier is the microwell plate that is provided with a plurality of micropores, encapsulates the micropore surface of layer attached to said solid phase carrier, and encapsulating layer is to be coated on the composite bed that forms on the micropore surface of solid phase carrier successively by AngII and bovine serum albumin(BSA).

Preferably, the above-mentioned solid phase carrier is 96 hole polystyrene agent plate.

Preferably, the above-mentioned solid phase carrier is 48 hole polystyrene agent plate.

Preferably, the above-mentioned solid phase carrier is 60 hole polystyrene agent plate.

The utility model utilizes the phage antibody display technique to screen the AngII antibody that obtains high-affinity, is applied to then in the preparation and production of ELISA kit.This technical modelling the selection effect of humoral immunity of organism system; The antibody with different variable regions that is presented on phage surface screens through immobilization antigen, and then utilizes bacteriophage that specific antibody is obtained in colibacillary infection and protein expression fast.

Compare with classic method, this method can obtain specificity height, antibody that affinity is high in a short time, and the bacteriophage that obtains through screening can long preservation, in going down to posterity, also can keep the stability of gene structure.Antibody is the important source material of ELISA kit, and what high-quality antibody can the guarantee reagent box is high-quality, and especially aspects such as the specificity of kit, sensitivity have very big improvement.

Description of drawings

Fig. 1 is the utility model ELISA Plate structural representation;

Fig. 2 is the utility model ELISA Plate cut-open view;

Fig. 3 is the utility model kit structural representation;

Fig. 4 is the ELISA kit canonical plotting of the utility model AngII.

Among the figure: the 1-solid phase carrier; The 2-micropore; 3-encapsulates layer; The 4-lid; The 5-ELISA Plate; The 6-box body.

Embodiment

Below in conjunction with accompanying drawing the utility model is further described.

The major technology route of the Angiotensin II enzyme-linked immunosorbent assay kit preparation that the utility model provides is following:

1. the forward screening of anti-AngII specific antibody:

Buy the phage antibody library that commercialization builds, and carry out bacterium liquid and cultivate and carry out phage surface and appear.With purpose antigen, promptly screen in AngII antagonist storehouse, earlier antibody library and AngII is interacted; It is fully combined; Then non-specific bond bacteriophage is removed in washing, combines bacteriophage to elute amplification specificity at last, and then gets into the elutriation of next round.Generally need the process of repetition 2-3 wheel elutriation-wash-out-amplification, final elutriation goes out the phage antibody of the high specific bond AngII of affinity.

2. the negative sense screening of anti-AngII specific antibody:

Concrete operations are similar with the forward screening; Different is on solid phase carrier, to encapsulate AngI and AngIII respectively; Bacteriophage and the solid phase antigen that to go up the expression AngII antibody of step results are then hatched; Collect eluent behind the wash-out, the bacteriophage that does not promptly combine with AngI and AngII, the antibody of increase once more acquisition and AngI and III no cross reaction is showed bacteriophage.

3. the transformation of anti-AngII antibody scFv:

Because the antibody library selected for use is the scFv storehouse, screening gained antibody has only an antigen binding domain, the antigen adhesion a little less than, need further to transform.The gene fusion of gathering functional domain through AngII scFv and IgG3 upper reaches hinge area/p53 four can be assembled into the tetramer automatically owing to this gene expression product, becomes tetravalent antibody accordingly, can significantly improve the adhesion to antigen.To contain in plasmid transfection to Chinese hamster ovary (CHO) cell of fusion, adopt affinity chromatography purifying purpose antibody after the amplification cultivation, and its compatibility and specificity identified through methods such as Western-Blot and ELISA.

4.ELISA the development of kit:

This kit is used competition and is suppressed the level that enzyme-linked immunosorbent assay is measured AngII in serum or the plasma sample.The preparation process is following: use 96 hole polystyrene agent plate (PS) as solid phase carrier, be placed under the ultraviolet light irradiation earlier 2 hours, make the activation of PS plate.On the agent plate capillary strip, encapsulate certain density AngII in advance, 4 ℃ are spent the night, and process the ELISA Plate that encapsulates through washing after the sealing of plate and bovine serum albumin(BSA) is handled.During detection, add sample to be measured and biotinylated AngII antibody successively, form the AngII and the AngII in the sample that encapsulate on the ELISA Plate and compete the situation that combines anti-AngII antibody; Again through Avidin horseradish peroxidase (HRP) amplifying signal; With 3,3 ', 5; 5 '-tetramethyl benzidine (TMB) and sulfuric acid is respectively as substrate and stop buffer, accomplishes the preparation of Angiotensin II enzyme-linked immunosorbent assay kit.

The ELISA kit has plurality of advantages than the RIA kit; Through with Phoenix Pharmaceuticals; Inc. the RIA kit of producing compares, and visible its operation is simpler, need not special experiment condition and environment and operating personnel are influenced little, is applicable to the application of common lab.

ELISA kit and RIA kit comparative result are as shown in table 1.

Table 1

The specific detection of the Angiotensin II that the utility model provides (AngII) enzyme-linked immunosorbent assay kit.The cross reacting rate of other analogs is as shown in table 2:

Analog	Cross reacting rate
		AngI	＜0.5％
AngIII	＜1％
		Leu-Heptapeptide	＜0.3％
otensin?III	＜0.01％
		Neurotensin	＜0.01％
Substance?P	＜0.01％
		Vasopressin	＜0.01％
Endothel?in-1	＜0.01％
		BNP-32	＜0.01％
Alpha-ANP(1-28)	＜0.01％

Table 2

This shows that the kit specificity is higher, low with the analog cross reacting rate of AngII, strengthened the accuracy of testing result greatly.

The recovery of the Angiotensin II that the utility model provides (AngII) enzyme-linked immunosorbent assay kit detects.

Get the standard solution (AngII concentration is 50pg/ml, 200pg/ml, 800pg/ml) of variable concentrations, add respectively in the Quality Control negative serum, detect, and comparative measurements value and desired value obtain the recovery with this kit.Comparative result is as shown in table 3:

Table 3

Can find out that from result relatively the recovery all can reach more than 90%, and is less with the desired value deviation, receives the influence of sample content thing to be checked less, be fit to the detection of serum and plasma sample.

The Angiotensin II enzyme-linked immunosorbent assay kit that uses the utility model to produce detects normal human serum and plasma sample.

1. concrete operations step is following:

1.1 reagent is prepared: standard items are diluted to 1000pg/ml, 333.33pg/ml, 111.11pg/ml, 37.04pg/ml, 12.35pg/ml successively; Prepare each 10 example of serum and plasma sample, all gather from the normal person.The preparation of detection liquid A need be used corresponding dilution will detect liquid A stock solution in 1: 100 ratio and be diluted to working concentration;

1.2 application of sample: establish gauge orifice, testing sample hole, blank well respectively.If gauge orifice 5 holes add the standard items of 50 μ L variable concentrations successively, blank well adds 50 μ L standard items dilutions; Surplus hole adds testing sample 50 μ L, and every immediately then hole adds detects solution A working fluid 50 μ L, the vibration mixing; ELISA Plate adds overlay film, 37oC incubation 1 hour;

1.3 wash plate: discard liquid in the hole, soaked 1-2 minute with the cleansing solution washing of 350 μ L in every hole, gets rid of liquid in the plate, repeats to wash plate 3 times.After the last washing, dry the cleansing solution in the hole fully;

Detect liquid B 1.4 add: detect liquid B stock solution and can use after the dilution proportion by 1: 100.Every hole adds detects solution B working fluid 100 μ L, covers overlay film, 37oC incubation 30 minutes;

1.5 wash plate: discard liquid in the hole, dry, wash plate 5 times, method is with step 2;

1.6 colour developing: every hole adds substrate solution 90 μ L, and ELISA Plate adds overlay film, the colour developing of 37oC lucifuge;

1.7 cessation reaction and reading: every hole adds stop bath 50 μ L when treating that the typical curve gradient is obvious, cessation reaction, and use the optical density (O.D. value) of ELIASA immediately in each hole of 450nm wavelength measurement.

2. the gained experimental result is following:

2.1 Angiotensin II (AngII) enzyme-linked immunosorbent assay kit typical curve is as shown in Figure 4; Each item index of Angiotensin II (AngII) enzyme-linked immunosorbent assay kit is as shown in table 4.

Table 4

2.2 the testing result of AngII content is as shown in table 5 in the sample:

Table 5

The method screening that the utility model is showed through phage antibody obtains the antibody fragment that combines with AngII, and screens the antibody of removal and AngI and AngIII cross coupled through negative sense, has further improved the specificity of antibody thus; Utilize the characteristic of p53 that the gained antibody fragment is assembled into the tetramer simultaneously, its affinity is increased.The acquisition of this antibody is simple to operation, and the cycle is shorter, and satisfies the requirement of preparation ELISA kit fully.ELISA kit specificity, accuracy and degree of accuracy through experiment showed, this Antibody Preparation of use are all higher, can be applied to this index and detect.

The embodiment of the above is the preferred embodiments of the utility model; It is not the practical implementation scope that limits the utility model with this; The scope of the utility model comprises and is not limited to this embodiment that all shapes according to the utility model, the equivalence that structure is done change all in the protection domain of the utility model.

Claims

1. Angiotensin II enzyme-linked immunosorbent assay kit; The ELISA Plate (5) and the box body (6) that comprise lid (4), inside; Wherein ELISA Plate (5) comprises solid phase carrier (1) and encapsulates layer (3); It is characterized in that: said solid phase carrier (1) is for being provided with the microwell plate (2) of a plurality of micropores; The said layer (3) that encapsulates is attached to the micropore surface of said solid phase carrier (1), and said to encapsulate layer (3) be to be coated on the composite bed that forms on the micropore surface of solid phase carrier (1) successively by AngII and bovine serum albumin(BSA).

2. Angiotensin II enzyme-linked immunosorbent assay kit according to claim 1 is characterized in that, said solid phase carrier (1) is 96 hole polystyrene agent plate.

3. Angiotensin II enzyme-linked immunosorbent assay kit according to claim 1 is characterized in that, said solid phase carrier (1) is 48 hole polystyrene agent plate.

4. Angiotensin II enzyme-linked immunosorbent assay kit according to claim 1 is characterized in that, said solid phase carrier (1) is 60 hole polystyrene agent plate.