CN102775486B - The novel agent of monitoring injury of the kidney and test kit - Google Patents
The novel agent of monitoring injury of the kidney and test kit Download PDFInfo
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Abstract
The present invention relates to novel agent and the test kit of monitoring injury of the kidney.The present inventor identifies crucial antigenic determinant from the NGAL of total length, using the short-peptide mixture containing these critical antigen determinants as immunogen, obtains the polyclonal antibody of the anti-NGAL of specificity.Antigen fragment provided by the invention and polyclonal antibody thereof, preparation method is simple, height of tiring, and high specificity is highly sensitive.
Description
Technical field
The invention belongs to biotechnology and immunity field; More specifically, the present invention relates to novel agent and the test kit of monitoring injury of the kidney.
Background technology
Neutrophilic granulocyte gelatinase-associated fat fortune albumen (NGAL) is in the past known as a kind of neutrophil activation mark, and when invading micro-organism causes neutrophil leucocyte retting conditions and granule protein exocytosis, NGAL is released in blood.Afterwards, NGAL be can be used as urine biomarker by report, was used for detecting the early onset thereof of renal tubular cell injury.U.S. Patent application 2005/0272101 to disclose in serum NGAL for same object.PCT application WO2006/066587 discloses and how to utilize the NGAL in urine or blood plasma or serum to detect.Therefore, NGAL as a kind of marker of seizure of disease by people's investigation and application.Human neutrophil genatinase associated lipocalin comprises 198 amino acid altogether.
Although NGAL as the mark of medical diagnosis on disease by people are understood, but due to its immunogenicity of full-length proteins own unsatisfactory, immunity obtains in the process of antibody also exists antibody titer difference, unstable defect.
Therefore, this area there is a need to optimize further for the above antibody detecting NGAL, to improving the accuracy rate of injury of the kidney diagnosis.
Summary of the invention
The object of the present invention is to provide a kind of novel agent and test kit of monitoring injury of the kidney.
In a first aspect of the present invention, provide a kind of isolated polypeptide, it is the polypeptide of aminoacid sequence as shown in SEQIDNO:1 or SEQIDNO:2, and described polypeptide derives from gelatinase-associated fat fortune albumen (NGAL) of neutrophilic granulocyte.
In another aspect of this invention, provide a kind of polynucleotide of separation, it coding described in polypeptide.
In another aspect of this invention, provide a kind of polypeptide mixture, it is mixed by the polypeptide of aminoacid sequence shown in SEQIDNO:1 and SEQIDNO:2.
In a preference, the ratio of the polypeptide mixing of aminoacid sequence shown in SEQIDNO:1 and SEQIDNO:2 is 1: 5 ~ 5: 1 according to weight ratio.
Preferably, the ratio of the polypeptide mixing of aminoacid sequence shown in SEQIDNO:1 and SEQIDNO:2 is 1: 3 ~ 3: 1 according to weight ratio.
In another aspect of this invention, provide the purposes of described polypeptide mixture, for the preparation of the antibody of the anti-neutrophilic granulocyte of specificity gelatinase-associated fat fortune albumen.
In another aspect of this invention, provide a kind of antibody for detecting injury of the kidney, it is obtained by described polypeptide mixture immune animal.
In a preference, described antibody is polyclonal antibody.
In another aspect of this invention, provide a kind of method of Dispersal risk, described method comprises: with described polypeptide mixture immune animal, isolates the antibody of the anti-neutrophilic granulocyte of specificity gelatinase-associated fat fortune albumen in the animal body after immunity.
In another aspect of this invention, a kind of test kit for detecting injury of the kidney being provided, comprising:
Solid phase carrier, and be positioned at the polyclonal antibody on solid phase carrier, this polyclonal antibody is obtained by described polypeptide mixture immune animal.
In a preference, described solid phase carrier is that bag is by plate, test paper and/or microballoon.
In another preference, described detection kit also comprises: detection antibody, developer, washings, stop buffer and/or the working instructions of mark.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
After Fig. 1, ELISA reaction, survey the absorbancy in each hole on enzyme plate at A405nm, the reading obtained.Wherein wrap by SEQIDNO:1 polypeptide in the capable micropore of A, B; Wrap by SEQIDNO:2 polypeptide in the capable micropore of C, D.1,2 be classified as the dilution serum (resisting) of #YZ2391 rabbit 1: 1000 more; 3,4 the dilution serum of #YZ2391 rabbit 1: 10,000 is classified as; 5,6 #YZ2391 rabbit 1: 100,000 gradient dilution serum is classified as; 7,8 the dilution serum of #YZ2392 rabbit 1: 1000 is classified as; 3,4 the dilution serum of #YZ2392 rabbit 1: 10,000 is classified as; 5,6 #YZ2392 rabbit 1: 100,000 gradient dilution serum is classified as.A, C hole adds pre-immune serum; B, D hole adds the final serum obtained.
Titre (tiring) measurement result of different dilution antibody after Fig. 2, SEQIDNO:1 (upper figure), the immune #YZ2392 rabbit of SEQIDNO:2 (figure below) difference.
Embodiment
Often find in the practice of those skilled in the art, the antigenic determinant due to albumen is easy to the inside being embedded in protein steric structural, is therefore difficult to the antibody finding a species specificity high.For above-mentioned technical barrier, in order to efficiently obtain the specific antibody detecting NGAL injury of the kidney mark, the present inventor is through deep research, crucial antigenic determinant is identified respectively from the NGAL of total length, using the short-peptide mixture containing these critical antigen determinants as immunogen, obtain the polyclonal antibody of the anti-NGAL of specificity respectively.Antigen fragment provided by the invention and polyclonal antibody thereof, preparation method is simple, height of tiring, and high specificity is highly sensitive.
In the present invention, " isolated polypeptide " refers to that polypeptide is substantially free of natural other albumen relative, lipid, carbohydrate or other material, those skilled in the art can purify this polypeptide with the purified technology of protein of standard, and substantially pure polypeptide produces single master tape on non-reducing polyacrylamide gel.
The arbitrary described polypeptide of SEQIDNO:1-2 of the present invention can be recombinant polypeptide, natural polypeptides, improvement on synthesis, preferred improvement on synthesis.Polypeptide of the present invention can be native purified product, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (such as, bacterium, yeast, higher plant, insect and mammalian cell).
The polynucleotide of the polypeptide described in coding can be DNA form or rna form.DNA form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-strand.
The polynucleotide of polypeptide of the present invention of encoding can obtain by the method for pcr amplification method, recombination method or synthetic usually.For pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the cDNA storehouse prepared by ordinary method well known by persons skilled in the art as template, amplification and relevant sequence.Once obtain relevant sequence, just relevant sequence can be obtained in large quantity with recombination method.At present, can obtain encoding by chemosynthesis the DNA sequence dna of polypeptide of the present invention completely.
Present invention also offers a kind of polypeptide mixture, it is mixed by the polypeptide of aminoacid sequence shown in SEQIDNO:1 and SEQIDNO:2.
Polypeptide preparation resulting mixture is combined for carrying out animal immune as immunogen, can obtain the antibody of high-titer, and the Be very effective of immunity is better than the immunity of wall scroll polypeptide.
Present invention also offers can the antibody of specific recognition NGAL.Here, " specificity " refers to antibody capable identification and is incorporated into NGAL albumen, but nonrecognition and be incorporated into the antibody of other non related antigen molecule.Antibody of the present invention can be prepared by the various technology that those skilled in that art are known.
The present invention is polyclonal antibody more preferably, in the present invention, term " polyclonal antibody " (resisting) used refers to that one group has the sphaeroprotein of specific binding capacity with antigen more, and it is by antigenic stimulation body, to produce after immunological response, synthesized by the plasmocyte of body and secrete.Antigen is normally made up of multiple antigenic determinant, stimulates body by a kind of antigenic determinant, accepts the antibody that this antigen produces be referred to as monoclonal antibody by a bone-marrow-derived lymphocyte.Stimulate body by plurality of antigens determinant, correspondingly just produce various monoclonal antibody, these monoclonal antibodies are mixed in together is exactly polyclonal antibody.The benefit of polyclonal antibody is their height of tiring, and specificity is high, and avidity is strong, and sensitivity is good, is convenient to artificial process and quality control, and in addition, polyclonal antibody preparation is relatively easy, more economical.
Polyclonal antibody can obtain by various method well known to those skilled in the art.Polypeptide mixture of the present invention, can be applied to animal (as rabbit, mouse, rat etc.; Be preferably rabbit) to induce the generation of polyclonal antibody.Similarly, the cell of expressing polypeptide of the present invention also can be used to immune animal to produce antibody.Polyclonal antibody can use lymph node injection method, subcutaneous multi-point injection method, and the immunization methods such as Multiple modality injection obtain.After adopting polypeptide mixture to mix with freund's adjuvant in embodiment, dorsal sc multi-point injection, immunize New Zealand rabbit; And carry out booster immunization; The polyclonal antibody of final acquisition high-titer.
Utilize polypeptide mixture of the present invention, also can manufacture order clonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare (see people such as Kohler, Nature256; 495,1975; The people such as Kohler, Eur.J.Immunol.6:511,1976; The people such as Kohler, Eur.J.Immunol.6:292,1976; The people such as Hammerling, InMonoclonalAntibodiesandTCellHybridomas, Elsevier, N.Y., 1981).
Multiple adjuvant can be used for strengthening immune response, includes but not limited to freund's adjuvant etc.
Present invention also offers the method detecting and whether there is NGAL in sample, be that the specific polyclonal antibody of the anti-NGAL utilizing the present invention to prepare detects, it comprises: contacted by the specific polyclonal antibody of sample with anti-NGAL; Observe and whether form antibody complex, define antibody complex and just represent in sample to there is NGAL.The specific polyclonal antibody of anti-NGAL, for detecting NGAL albumen, can reach by the method for the various principle design according to the combination of antigen-antibody In-vitro specificity well known to those skilled in the art, such as protein immunoblot experiment, co-immunoprecipitation experiment etc.
Present invention also offers a kind of detection injury of the kidney test kit, wherein containing antibody of the present invention, be preferably polyclonal antibody.Preferably, described detection kit comprises:
Solid phase carrier, and be positioned at the polyclonal antibody on solid phase carrier, this polyclonal antibody is obtained by described polypeptide mixture immune animal.
Described solid phase carrier can be that bag is by plate, test paper and/or microballoon.Thus by immunity test strip, the technology such as Radioactive colloidal gold detect.
In addition, described detection kit also comprises: detection antibody, developer, washings, stop buffer and/or the working instructions of mark.These reagent can be adjusted according to the difference of detection method.
Major advantage of the present invention is:
(1) first identified of the present invention has arrived the critical antigen determinant in NGAL protein sequence, and using the short-peptide mixture containing these critical antigen determinants as immunogen, the antibody titer of acquisition is high, identifies that the specificity of antigen is good.
(2) utilize small peptide to prepare polyclonal antibody, small peptide synthetic method is simple.
(3) antigen fragment provided by the invention and polyclonal antibody thereof, preparation method is simple, height of tiring, and high specificity is highly sensitive.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, conveniently condition such as J. Pehanorm Brooker etc. is write usually, Molecular Cloning: A Laboratory guide, Science Press, the condition described in 2002, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.
Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.
The miscellany of embodiment 1, special NGAL amino acid fragment sequence and product thereof is as immunogen
The full length amino acid sequence of NGAL albumen is as follows:
The full length amino acid (people source, 198aa) of NGAL albumen, sequence following (SEQIDNO:3):
MPLGLLWLGLALLGALHAQAQDSTSDLIPAPPLSKVPLQQNFQDNQFQGK
WYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWI
RTFVPGCQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNR
EYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG
Because full length sequence synthesizes difficult and that immunogenicity is not strong shortcoming as immunogen existence, the present inventor is according to above full length sequence, adopt the sequence fragment of ordinary method synthetic all lengths, using fragment as immunogen, repeatedly test various immunogenic immune effect.Finally, obtain applicable immunogen, it comprises two polypeptide fragments from NGAL, and sequence (N → C end) is as follows:
CREDKDPQKMYATIYELKEDKS(SEQIDNO：1)；
CYNQHAMVFFKKVSQNREY(SEQIDNO：2)。
Article two, polypeptide fragment mixes with mass ratio 1: 1, obtains polypeptide mixture.Former as the specific immune producing specific polyclonal anti-NGAL antibody using this mixture.After the miscellany immune animal of these two fragments, the specific polyclonal antibody of high-titer, high immunogenicity (i.e. high antigenic) can be obtained.
The generation of the anti-NGAL antibody of embodiment 2, specific polyclonal
The miscellany of specific polypeptide prepared using above-described embodiment 1 respectively as immunogen and hemocyanin (KLH) coupling, immunize New Zealand White Rabbit and the immune serum that produces.Method and the program of animal immune are as follows:
1) immunity two new zealand male rabbit (#YZ2391; #YZ2392), preimmune serum also-20 DEG C of storages are gathered.
2) first immunisation gets the immunogen good with hemocyanin (KLH) coupling and equivalent Freund's complete adjuvant emulsification antigen (antigen: Freund's complete adjuvant=1: 1 (v/v)), back multi-point injection.The injection volume of first immunisation is 400 μ g polypeptide miscellanys.
3), after 14 days, back multi-point injection booster immunization is repeated with Freund's complete adjuvant emulsification antigen (antigen: Freund's complete adjuvant=1: 1 (v/v)).The injection volume of booster immunization is 400 μ g polypeptide miscellanys.
4), after 28 days, same dosage polypeptide is used to mix with isopyknic incomplete Freund's adjuvant, back multi-point injection booster immunization.
5) after 42 days, with step 4) back multi-point injection booster immunization.
6) after 70 days, with step 4) back multi-point injection booster immunization.
7), after 77 days, serum is got in carotid artery blood sampling, deposits for-70 DEG C.
After obtaining specific antibody, carry out ELISA and WESTERNBLOT and verify tiring and identifying the performance of antigen of antibody.
ELISA method is as follows:
Adopt direct method, improvement on synthesis is coated in 96 hole enzyme plates, adds Post-immunisation serum, contrast as preimmune serum.According to.
Material:
1, envelope antigen: polypeptide antigen is coated in 96 hole enzyme plates, 0.1 μ g/100 μ l/ hole;
Wherein wrap by SEQIDNO:1 polypeptide in the capable micropore of A, B of enzyme plate; Wrap by SEQIDNO:2 polypeptide in the capable micropore of C, D.
2, the goat antirabbit polyclonal antibody of horseradish peroxidase-labeled;
3, the dilution of antibody or enzyme conjugate: with 1 × PBS solution preparation, 1.0% (v/v) BSA solution containing 0.05% (v/v) polysorbas20, wherein containing normal goats IgG, concentration is 0.1mg/ml;
4, elutriant: containing the PBS solution of 0.05% (v/v) polysorbas20;
5, ABTS substrate.
Method:
1, add the polyclonal antibody of the above-mentioned preparation of the present embodiment of 100 μ l10 doubling dilutions in the every hole of enzyme plate, at 37 DEG C, hatch 30 minutes;
2, wash-out: add in 96 hole enzyme plates with damping fluid, 300-360 μ l/ hole, after 3min, throws away gently by washing lotion, is inverted on paper and pats dry, repeats secondary;
3, every hole adds the goat anti-rabbit antibodies (1: 2000) of 100 μ l horseradish peroxidase-labeled, hatches 30 minutes at 37 DEG C;
4, wash-out, added by elutriant in 96 hole enzyme plates, 300-360 μ l/ hole, after 3min, throws away gently by elutriant, is inverted on paper and pats dry, and repeats secondary;
5, every hole adds 100 μ lABTS substrate solutions, at room temperature hatches 10-20 minute;
6, microplate reader analysis: reading under 405nm wavelength.
Fig. 1 is the reading in each hole on enzyme plate.
ELISA result shows, and the polyclonal antibody obtained can identify two polypeptide fragments of NGAL specifically.Fig. 2 is titre (tiring) experimental result of different dilution antibody after the immune #YZ2392 rabbit of SEQIDNO:1 (upper figure), SEQIDNO:2 (figure below) difference.Result shows, and in Post-immunisation serum, antibody titers is all very high.Also namely, this polyclonal antibody is tired and can be reached 1: 100,000.
WESTERNBLOT result shows, and the polyclonal antibody obtained can specifically in conjunction with two polypeptide fragments of NGAL and NGAL of total length.
Embodiment 3, test kit
Described test kit comprises:
Container 1, and polyclonal antibody prepared by the previous embodiment 2 being loaded on container;
Container 2, and the goat anti-human igg's polyclonal antibody of horseradish peroxidase-labeled being loaded on container.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (3)
1. a polypeptide mixture, is characterized in that, it is mixed by the polypeptide of aminoacid sequence shown in SEQIDNO:1 and SEQIDNO:2; The ratio of the polypeptide mixing of aminoacid sequence shown in SEQIDNO:1 and SEQIDNO:2 is 1: 5 ~ 5: 1 according to weight ratio.
2. the purposes of polypeptide mixture according to claim 1, for the preparation of the antibody of the anti-neutrophilic granulocyte of specificity gelatinase-associated fat fortune albumen.
3. a method for Dispersal risk, is characterized in that, described method comprises:
With polypeptide mixture immune animal according to claim 1, in the animal body after immunity, isolate the antibody of the anti-neutrophilic granulocyte of specificity gelatinase-associated fat fortune albumen.
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| CN104072586B (en) * | 2013-03-29 | 2019-03-01 | 李锐 | The detection reagent and kit of people's kidney injury molecule-1 |
| CN104792997B (en) * | 2014-01-22 | 2017-07-11 | 天津汇滨生物科技有限公司 | A kind of HCT's original immunity detection reagent and preparation method and application |
| CN105021829B (en) * | 2015-07-21 | 2017-09-29 | 河北特温特生物科技发展有限公司 | A kind of kit of quantitatively detection BNP and the detection method of BNP |
| CN111153996B (en) * | 2020-01-10 | 2021-12-14 | 苏州睿瀛生物技术有限公司 | Antibody of G protein coupled receptor, preparation method thereof and G protein coupled receptor kit |
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Effective date of registration: 20160321 Address after: 200030 Shanghai city tianyaoqiaolu Gateway International Plaza, building A room 1915 Patentee after: Sheng Fu source, Shanghai biological medicine company limited Address before: 200030, room 2, building 111, No. 707 Middle Road, Shanghai, Xuhui District Patentee before: Li Rui |