CN104072586B

CN104072586B - The detection reagent and kit of people's kidney injury molecule-1

Info

Publication number: CN104072586B
Application number: CN201310109245.3A
Authority: CN
Inventors: 蔡逸强; 闵洪中; 李锐
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-03-29
Filing date: 2013-03-29
Publication date: 2019-03-01
Anticipated expiration: 2033-03-29
Also published as: CN104072586A

Abstract

The present invention relates to novel injury of kidney detection reagent and kits.The present inventor identifies crucial antigenic determinant respectively from the Kidney injury molecule 1 (KIM-1) of overall length, and the short-peptide mixture to contain these critical antigen determinants obtains the polyclonal antibody of the anti-KIM-1 of specificity as immunogene.Antigen fragment and its polyclonal antibody provided by the invention, preparation method is simple, and potency is high, high specificity, high sensitivity.

Description

The detection reagent and kit of people's kidney injury molecule-1

Technical field

The invention belongs to biotechnologys and immunity field；More particularly it relates to people's kidney injury molecule-1 (KIM- 1) novel agent and kit.

Background technique

Human kidney injury molecule 1 (KIM-1) is a kind of transmembrane protein, is not expressed in normal kidney, and is damaged in kidney Expression obviously increases in the animal model of wound.Zooscopy show KIM-1 ectodomain fracture after product can by urine in Discharge, therefore KIM-1 level can evaluate kidney injury situation in detection urine.Existing research shows in all kidney troubles KIM-1 level obviously increases in the expression of nephridial tissue KIM-1 and urine.Therefore, KIM-1 has been used as a kind of mark of seizure of disease Remember that object is studied and applied by people.People KIM-1 includes 359 amino acid altogether, and wherein 1-20 are signal peptide, is removed when expression As maturity state；21-290 are extracellular portion；291-311 are one section of cross-film sequence with 21 amino acid；Finally 48 amino acid then constitute the Intracellular domain of the albumen.

Although KIM-1 has been understood by people as the marker of medical diagnosis on disease, due to their full-length proteins Immunogenicity itself is unsatisfactory, be immunized during obtaining antibody that there is also antibody titers poor, unstable defect.

Therefore, this field there is a need to advanced optimize the antibody for KIM-1 marker.Also, it there is a need to combine The antibody of the relevant marker of other injury of kidney carrys out combined application in the monitoring of injury of kidney, to improve the accurate of injury of kidney diagnosis Rate.

Summary of the invention

The purpose of the present invention is to provide a kind of novel agents and kit for monitoring injury of kidney.

In the first aspect of the present invention, a kind of isolated polypeptide is provided, it is such as SEQ ID NO:1 or SEQ ID NO:2 The polypeptide of shown amino acid sequence, the polypeptide derive from Kidney injury molecule 1 (KIM-1).

In another aspect of this invention, a kind of isolated polynucleotides are provided, it encodes the polypeptide.

In another aspect of this invention, a kind of mixtures of polypeptides is provided, by SEQ ID NO:1 and SEQ ID NO:2 institute Show that the polypeptide of amino acid sequence mixes.

In a preferred embodiment, the ratio of the polypeptide mixing of amino acid sequence shown in SEQ ID NO:1 and SEQ ID NO:2 It is 1:5~5:1 according to weight ratio.

Preferably, the ratio that the polypeptide of amino acid sequence shown in SEQ ID NO:1 and SEQ ID NO:2 mixes is according to weight Than for 1:3~3:1；It is more preferably 1:2~2:1 according to weight ratio.

In another aspect of this invention, the purposes of the mixtures of polypeptides is provided, the anti-kidney damage of specificity is used to prepare Hurt the antibody of molecule 1.

In another aspect of this invention, it provides a kind of for detecting the antibody of injury of kidney, is mixed by the polypeptide Object is immunized animal and obtains.

In a preferred embodiment, the antibody is polyclonal antibody.

In another aspect of this invention, a kind of method for preparing antibody is provided, which comprises mixed with the polypeptide It closes object and animal is immunized, the antibody of specific anti-human kidney injury molecule-1 (KIM-1) is isolated in the animal body after being immunized.

In another aspect of this invention, it provides a kind of for detecting the kit of injury of kidney, comprising:

Solid phase carrier, and the polyclonal antibody on solid phase carrier, the polyclonal antibody are mixed by the polypeptide Object is immunized animal and obtains；It or include: container, and above-mentioned mixtures of polypeptides in container is immunized animal and obtains Polyclonal antibody.

In a preferred embodiment, in the kit further include: solid phase carrier, and the antibody on solid phase carrier (such as polyclonal antibody), the antibody are the antibody of gelatinase-associated rouge fortune albumen (NGAL) of the anti-neutrophil cell of specificity； Or include: container, and gelatinase-associated rouge fortune albumen (NGAL) of the anti-neutrophil cell of specificity in container Antibody.

In a preferred embodiment, the solid phase carrier is coating plate, test paper and/or microballoon.

In another preferred example, the kit further include: the detection antibody of label, color developing agent, cleaning solution, terminate liquid And/or operation instructions.

Other aspects of the invention are apparent to those skilled in the art due to this disclosure 's.

Detailed description of the invention

After Fig. 1, ELISA reaction, the absorbance in each hole on ELISA Plate, reading obtained are surveyed in A405nm.Wherein A, B row SEQ ID NO:1 polypeptide is coated in micropore；SEQ ID NO:2 polypeptide is coated in C, D row micropore.1,2 it is classified as #YZ2861 rabbit The serum (how anti-) of sub- 1:1000 dilution；3,4 serum for being classified as #YZ2861 rabbit 1:10,000 dilution；5,6 it is classified as # YZ2861 rabbit 1:100,000 gradient dilution serum；7,8 serum for being classified as #YZ22862 rabbit 1:1000 dilution；3,4 It is classified as the serum of #YZ2862 rabbit 1:10,000 dilution；5,6 it is classified as #YZ2862 rabbit 1:100,000 gradient dilution blood Clearly.A, pre-immune serum is added in the hole C；B, the serum finally obtained is added in the hole D.

Different dilutions after #YZ2861 rabbit are immunized in Fig. 2, SEQ ID NO:1 (above), SEQ ID NO:2 (following figure) respectively Titre (potency) measurement result of the antibody of degree.

Different dilutions after #YZ2862 rabbit are immunized in Fig. 3, SEQ ID NO:1 (above), SEQ ID NO:2 (following figure) respectively Titre (potency) measurement result of the antibody of degree.

Polyclonal antibody of the invention can specifically combine the KIM-1 of overall length as the result is shown by Fig. 4, WESTERN BLOT Polypeptide fragment.

Specific embodiment

It is frequently found in the practice of those skilled in the art, since the antigenic determinant of albumen is easy to be embedded in protein steric The inside of structure, therefore, it is difficult to find the high antibody of a species specificity.Problem in view of the above technology, in order to efficiently be detected The specific antibody of KIM-1 injury of kidney marker, the present inventor pass through in-depth study, identify respectively from the KIM-1 of overall length Crucial antigenic determinant, the short-peptide mixture to contain these critical antigen determinants obtain respectively as immunogene out The polyclonal antibody of the anti-KIM-1 of specificity.Antigen fragment and its polyclonal antibody provided by the invention, preparation method is simple, effect Valence is high, high specificity, high sensitivity.

In the present invention, " isolated polypeptide " refers to polypeptide substantially free of natural relative other albumen, lipid, sugar Class or other materials, those skilled in the art can purify the polypeptide with the purified technology of protein of standard, and substantially pure is more Peptide generates single master tape in non-reducing polyacrylamide gel.

Any polypeptide of SEQ ID NO:1-2 of the invention can be recombinant polypeptide, natural polypeptides, synthesis polypeptide, Preferably synthetic polypeptide.Polypeptide of the present invention can be native purified product or chemically synthesized product, or use weight Group technology is generated from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).

The polynucleotides of the coding polypeptide can be DNA form or rna form.DNA form includes cDNA, genome DNA or artificial synthesized DNA.DNA can be single-stranded or double-strand.

The polynucleotides for encoding polypeptide of the invention can usually use PCR amplification method, recombination method or artificial synthesized method It obtains.For PCR amplification method, disclosed related nucleotide sequence, especially open reading frame sequence can come according to the present invention Design primer, and use the commercially available library cDNA or by the library cDNA prepared by conventional method well known by persons skilled in the art as mould Plate expands and obtains related sequence.Once obtaining related sequence, so that it may obtain related sequence in large quantity with recombination method Column.At present, it is already possible to obtain encoding the DNA sequence dna of polypeptide of the invention by chemical synthesis completely.

The present invention also provides a kind of mixtures of polypeptides, the amino acid sequence as shown in SEQ ID NO:1 and SEQ ID NO:2 The polypeptide of column mixes.

Polypeptide is prepared into mixture crosslinking and shares in carrying out animal immune as immunogene, can get the antibody of high-titer, exempts from The significant effect of epidemic disease is immune better than single polypeptide.

The present invention also provides can specific recognition KIM-1 antibody.Here, " specificity " refers to that antibody can be identified and be tied Together in KIM-1 albumen, but nonrecognition and the antibody for being incorporated into other non related antigen molecules.Antibody of the invention can pass through this In field prepared by various technologies known to technical staff.

The more preferable polyclonal antibody of the present invention, in the present invention, term used " polyclonal antibody " (more anti-) refer to one group with Antigen has the globulin of specific binding capacity, be by antigenic stimulus body, it is thin by the slurry of body after generating immunological response Born of the same parents' synthesis and secretion.Antigen is usually to be made of multiple antigenic determinants, body is stimulated by a kind of antigenic determinant, by one A bone-marrow-derived lymphocyte receives antibody caused by the antigen and is referred to as monoclonal antibody.Body, phase are stimulated by a variety of antigenic determinants Just generate various monoclonal antibodies with answering, these monoclonal antibodies mixed in together are exactly polyclonal antibody.It is polyclonal Antibody is advantageous in that their potency is high, and specificity is high, and affinity is strong, and sensitivity is good, is convenient for artificial processing and quality control, In addition, polyclonal antibody preparation is relatively easy, it is more economical.

Polyclonal antibody can be made with various methods well known to those skilled in the art.Mixtures of polypeptides of the invention, It can be applied to animal (such as rabbit, mouse, rat etc.；Preferably rabbit) to induce the generation of polyclonal antibody.Similar, The cell for expressing polypeptide of the invention can also be used to immune animal to produce antibody.Polyclonal antibody can use lymph node injection Method, subcutaneous multi-point injection method, the immunization methods such as Multiple modality injection method are made.Mixtures of polypeptides and Freund are used in embodiment After adjuvant mixing, new zealand rabbit is immunized in dorsal sc multi-point injection；And carry out booster immunization；Final more grams for obtaining high-titer Grand antibody.

Using mixtures of polypeptides of the invention, monoclonal antibody can also be produced.Such monoclonal antibody can use miscellaneous Tumor technology is handed over to prepare (see Kohler et al., Nature256；495,1975；Kohler et al., Eur.J.Immunol.6: 511,1976；Kohler et al., Eur.J.Immunol.6:292,1976；Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).A variety of adjuvants can be used for enhancing immune anti- It answers, including but not limited to Freund's adjuvant etc..

The present invention also provides the methods that whether there is KIM-1 in test sample, are prepared by the method anti-KIM-1 Specific polyclonal antibody detected, it includes: to contact sample with the specific polyclonal antibody of anti-KIM-1；Observation Whether antibody complex is formed, forms antibody complex and mean that there are KIM-1 in sample.More grams of the specificity of anti-KIM-1 Grand antibody, can be with well known to those skilled in the art various according to antigen-antibody In-vitro specificity for detecting KIM-1 albumen In conjunction with the method that designs of principle reach, such as protein immunoblot experiment, co-immunoprecipitation experiment etc..

The present invention also provides a kind of detection injury of kidney kits, wherein contain antibody of the invention, preferably more grams Grand antibody.Preferably, the detection kit includes: solid phase carrier, and the polyclonal antibody on solid phase carrier, it should Polyclonal antibody is immunized animal by the mixtures of polypeptides and is obtained.

The solid phase carrier can be coating plate, test paper and/or microballoon.To pass through immunity test strip, colloidal gold Etc. technologies detected.

In addition, the detection kit further include: the detection antibody of label, color developing agent, cleaning solution, terminate liquid and/or Operation instructions.These reagents can be adjusted according to the difference of detection method.

Main advantages of the present invention are:

(1) first identified of the present invention has arrived the critical antigen determinant in KIM-1 protein sequence, with anti-containing these keys For the short-peptide mixture of former determinant as immunogene, the antibody titer of acquisition is high, identifies that the specificity of antigen is good.

(2) polyclonal antibody is prepared using small peptide, small peptide synthetic method is simple.

(3) antigen fragment and its polyclonal antibody provided by the invention, preparation method is simple, and potency is high, high specificity, spirit Sensitivity is high.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part such as J. Pehanorm Brooker etc. is write, Molecular Cloning:A Laboratory guide, Science Press, condition described in 2002, or according to system Make condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are calculated by weight.

Unless otherwise defined, it anticipates known to all professional and scientific terms as used herein and one skilled in the art Justice is identical.In addition, any method similar to or equal to what is recorded and material all can be applied in the present invention.It is described in text Preferred implement methods and materials be for illustrative purposes only.

In order to efficiently obtain the specific antibody of detection two kinds of injury of kidney markers of KIM-1, the present inventor is by deep Research identifies crucial antigenic determinant, respectively from the KIM-1 of overall length with the small peptide containing these critical antigen determinants Mixture obtains the polyclonal antibody that specificity resists anti-KIM-1 as immunogene.Antigen fragment provided by the invention and its more Clonal antibody, preparation method is simple, and potency is high, high specificity, high sensitivity.

Polypeptide is prepared into mixture crosslinking and shares in carrying out animal immune as immunogene, can get the antibody of high-titer, exempts from The significant effect of epidemic disease is immune due to single polypeptide.

Polyclonal antibody can be made with various methods well known to those skilled in the art.Mixtures of polypeptides of the invention, It can be applied to animal (such as rabbit, mouse, rat etc.；Preferably rabbit) to induce the generation of polyclonal antibody.Similar, The cell for expressing polypeptide of the invention can also be used to immune animal to produce antibody.Polyclonal antibody can use lymph node injection Method, subcutaneous multi-point injection method, the immunization methods such as Multiple modality injection method are made.Mixtures of polypeptides and Freund are used in embodiment After adjuvant mixing, new zealand rabbit is immunized in dorsal sc multi-point injection；And carry out booster immunization；Final acquisition high-titer Polyclonal antibody.

Using mixtures of polypeptides of the invention, monoclonal antibody can also be produced.Such monoclonal antibody can use miscellaneous Tumor technology is handed over to prepare (see Kohler et al., Nature256;495,1975;Kohler et al., Eur.J.Immunol.6: 511,1976;Kohler et al., Eur.J.Immunol.6:292,1976;Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas,Elsevier,N.Y.,1981)。

A variety of adjuvants can be used for enhancing immune response, including but not limited to Freund's adjuvant etc..

It is more grams of the specificity using anti-KIM-1 the present invention also provides the method that whether there is KIM-1 in test sample Grand antibody is detected, it includes: to contact sample with the specific polyclonal antibody of anti-KIM-1；It sees whether to form antibody Compound forms antibody complex and means that there are KIM-1 in sample.The specific polyclonal antibody of anti-KIM-1 is resisted to be used for KIM-1 albumen is detected, the various principles combined according to antigen-antibody In-vitro specificity well known to those skilled in the art can be used The method of design reaches, such as protein immunoblot experiment, co-immunoprecipitation experiment etc..

The present invention also provides a kind of detection injury of kidney kits, wherein contain antibody of the invention, preferably more grams Grand antibody.Preferably, the detection kit includes:

Container 1 or coating plate 1, and in container 1 or coating plate 1 on polyclonal antibody, the polyclonal antibody by The mixtures of polypeptides is immunized animal and obtains.

In order to further increase the sensibility and accuracy of injury of kidney detection, it may also include in the kit and detect other The detection reagent of injury of kidney marker.As of the invention one preferred aspect, it may also include specificity in the kit Detect the reagent of NGAL.

For the ease of those skilled in the art's use, it may also include the use for illustrating each reagent or tool in the kit The operation instructions of method dosage.

Main advantages of the present invention are:

(1) first identified of the present invention has arrived the antigenic determinant of KIM-1 key, to contain these critical antigen determinants For short-peptide mixture as immunogene, the antibody titer of acquisition is high, identifies that the specificity of antigen is good.

(2) antigen fragment and its polyclonal antibody provided by the invention, preparation method is simple, and potency is high, high specificity, spirit Sensitivity is high.

The mixture of embodiment 1, special KIM-1 amino acid fragment sequence and its product is as immunogene

Full length amino acid (source of people, the 359 amino acid) sequence (SEQ ID NO:3) of KIM-1 albumen is as follows:

MHPQVVILSLILHLADSVAGSVKVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTCQN

GIVWTNGTHVTYRKDTRYKLLGDLSRRDVSLTIENTAVSDSGVYCCRVEHRGWFNDMKI

TVSLEIVPPKVTTTPIVTTVPTVTTVRTSTTVPTTTTVPTTTVPTTMSIPTTTTVLTTMTVS

TTTSVPTTTSIPTTTSVPVTTTVSTFVPPMPLPRQNHEPVATSPSSPQPAETHPTTLQGA

IRREPTSSPLYSYTTDGNDTVTESSDGLWNNNQTQLFLEHSLLTANTTKGIYAGVCISVL

VLLALLGVIIAKKYFFKKEVQQLSVSFSSLQIKALQNAVEKEVQAEDNIYIENSLYATD

Since full length sequence haves the shortcomings that synthesis is difficult and immunogenicity is not strong as immunogene, the present inventor is according to above Full length sequence, using segment as immunogene, is tested various repeatedly using the sequence fragment of the artificial synthesized various length of conventional method The immune effect of immunogene.Finally, suitable immunogene is obtained comprising two polypeptide fragments from KIM-1, sequence (N → C-terminal) as follows:

CRREPTSSPLYSYTTD(SEQ ID NO:1)；

CTHVTYRKDTRYKLLGDLSRRD(SEQ ID NO:2)。

Two polypeptide fragments are mixed with mass ratio 1:1, obtain mixtures of polypeptides.It is special using the mixture as production Property Anti-TNF-α KIM-1 antibody specific immune it is former.After animal is immunized with the mixture of the two segments, can get has efficiently The specific polyclonal antibody of valence, high immunogenicity (i.e. high antigenic).

The generation of the anti-KIM-1 antibody of embodiment 2, specific polyclonal

The mixture of the specific polypeptide prepared respectively using above-described embodiment 1 is even as immunogene and hemocyanin (KLH) Connection is immunized new zealand white rabbit and generates immune serum.The methods and procedures of animal immune is as follows:

1) two new zealand male rabbit (#YZ2861 are immunized；#YZ2862), it acquires preimmune serum and stores in -20 DEG C.

2) first immunisation takes the immunogene being coupled with hemocyanin (KLH) and equivalent Freund's complete adjuvant to emulsify 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 mixtures.

3) after 14 days, back is repeated with Freund's complete adjuvant emulsification antigen (antigen: Freund's complete adjuvant=1:1 (v/v)) Multi-point injection booster immunization.The injection volume of booster immunization is 400 μ g polypeptide mixtures.

4) it after 28 days, is mixed using same dosage polypeptide with isometric incomplete Freund's adjuvant, back multi-point injection adds It is strong immune.

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, arteria carotis blood sampling takes serum, -70 DEG C of storages.Obtain specific antibody after, carry out ELISA and WESTERN BLOT verifies the potency of antibody and identifies the performance of antigen.

ELISA method is as follows:

Using direct method, synthesis polypeptide is coated in 96 hole elisa Plates, Post-immunisation serum is added, compareing is immune preceding blood Clearly.

Material:

1, envelope antigen: polypeptide antigen is coated in 96 hole elisa Plates, 0.1 hole μ g/100 μ l/；

SEQ ID NO:1 polypeptide is wherein coated in A, B row micropore of ELISA Plate；SEQ ID is coated in C, D row micropore NO:2 polypeptide.

2, the goat antirabbit polyclonal antibody of horseradish peroxidase-labeled；

3, the dilution of antibody or enzyme conjugate: 1.0% (v/v) is prepared with 1 × PBS solution containing 0.05% (v/v) polysorbas20 BSA solution, wherein containing normal goats IgG, concentration 0.1mg/ml；

4, eluent: contain the PBS solution of 0.05% (v/v) polysorbas20；

5, ABTS substrate.

Method:

1, the polyclonal antibody of the above-mentioned preparation of the present embodiment of 100 μ l10 doubling dilutions is added in the every hole of ELISA Plate, 37 It is incubated for 30 minutes at DEG C；

2, it elutes: being added in 96 hole elisa Plates with buffer, the hole 300-360 μ l/, after 3min, washing lotion is gently thrown away, It is placed on paper and pats dry, repeat secondary；

3, the goat anti-rabbit antibodies (1:2000) of 100 μ l horseradish peroxidase-labeleds are added in every hole, are incubated at 37 DEG C 30 minutes；

4, it elutes, eluent is added in 96 hole elisa Plates, the hole 300-360 μ l/, after 3min, eluent is gently thrown away, It is inverted on paper and pats dry, repeat secondary；

5,100 μ l ABTS substrate solutions are added in every hole, are incubated at room temperature 10-20 minutes；

6, microplate reader is analyzed: being read under 405nm wavelength.

Fig. 1 is the reading in each hole on ELISA Plate.

ELISA is the results show that polyclonal antibody obtained can specifically identify two polypeptide fragments of KIM-1. Fig. 2 is the antibody that different dilutions after #YZ2861 rabbit are immunized in SEQ ID NO:1 (above), SEQ ID NO:2 (following figure) respectively Titre (potency) experimental result.Fig. 3 is that #YZ2862 is immunized in SEQ ID NO:1 (above), SEQ ID NO:2 (following figure) respectively Titre (potency) experimental result of the antibody of different dilutions after rabbit.The results show that antibody titer is non-in Post-immunisation serum Chang Gao.That is, the polyclonal antibody potency can reach 1:100,000.

The ability of embodiment 3, polyclonal antibody combination overall length KIM-1 polypeptide of the invention or segment

WESTERN BLOT detection is carried out, WESTERN BLOT is one of the important method with antibody detection protein. WESTERN BLOT is operated as follows:

1. collecting protein sample

The preparation of mouse kidney after ischemic-reperfusion injury homogenate: male C57BL/6 mouse, after closing Bilateral Renal base of a fruit 30min using folder The method of open 30min makes acute kidney injury (AKI) mouse model, and exoculation takes blood, takes tie boat anlongshore side Renal Cortex at once, after weighing Cortex renis is shredded in super-clean bench, is homogenate agent with PBS, and homogenate, final concentration of 20g/L, homogenate operation are made in glass paste device It is carried out in ice bath.Manufactured homogenate is centrifuged 15min, Aspirate supernatant, with 30 μm of apertures with 20000r/min low-temperature and high-speed Disposable sterilized filter filtering, gained filtrate packing after, -70 DEG C of freezen protectives are spare.

2. electrophoresis (Electrophoresis)

(1) PAGE gel is prepared

PAGE gel is formulated as 5% concentration.

(2) sample treatment

The SDS-PAGE albumen sample-loading buffer being concentrated in right amount is added in the protein sample of collection.

(3) loading and electrophoresis

Protein sample is directly loaded in SDS-PAGE glue well, SDS-PAGE can use common electrophoresis Instrument can be met the requirements.

3. transferring film (Transfer)

Select pvdf membrane.Using the standard wet type membrane-transferring device of Bio-Rad, transferring film electric current can be set as 300-400mA, The transferring film time is 30-60 minutes.

4. closing (Blocking)

After transferring film, protein film is placed into preprepared Western cleaning solution immediately, is rinsed 1-2 minutes, To wash away the transferring film liquid on film.The all steps after the transferring film, it is certain it is noted that film moisturizing, avoid the drying of film, it is no Then easily generate higher background.

Cleaning solution is exhausted with mini desktop vacuum pump, Western confining liquid is added, is slowly shaken on shaking table, room temperature envelope It closes 60 minutes.

5. primary antibody is incubated for

According to proper proportion dilution primary antibody (that is: of the invention is how anti-).

Confining liquid is exhausted with mini desktop vacuum pump, is added the primary antibody that has diluted immediately, room temperature or 4 DEG C are on the side shaker Slowly shake incubation one hour.If primary antibody incubation 1 hour ineffective, overnight incubation can be slowly shaken at 4 DEG C.

Recycle primary antibody.Western cleaning solution is added, slowly shakes washing 5-10 minutes on the side shaker.Exhaust washing After liquid, cleaning solution is added, is washed 5-10 minutes.It washs 3 times altogether.

6. secondary antibody is incubated for

According to the secondary antibody (goat antirabbit polyclonal antibody) of proper proportion dilution horseradish peroxidase (HRP) label.

Cleaning solution is exhausted with mini desktop vacuum pump, is added the secondary antibody that has diluted immediately, room temperature or 4 DEG C are on the side shaker Slowly shake incubation one hour.

Recycle secondary antibody.Western cleaning solution is added, slowly shakes washing 5-10 minutes on the side shaker.Exhaust washing After liquid, cleaning solution is added, is washed 5-10 minutes.It washs 3 times altogether.

7. Protein Detection

Albumen is detected using the ECL class reagent such as Western luciferase assay reagent.

As the result is shown (Fig. 4), polyclonal antibody obtained can be specifically in conjunction with overall length by WESTERN BLOT KIM-1 polypeptide, acute kidney injury (AKI) mouse model can be distinguished most clearly with normal mice is compareed.

Embodiment 4, kit

(1) kit 1

The kit 1 includes:

Container 1, and polyclonal antibody prepared by the previous embodiment 2 loaded on container；

Container 2, and the goat antirabbit polyclonal antibody of the horseradish peroxidase-labeled loaded on container.

(2) kit 2

The kit 2 includes:

The kit 2 further includes the detection reagent for detecting NGAL, as follows:

Container 3, and the anti-NGAL antibody loaded on container；

Container 4, and goat anti-human igg's polyclonal antibody of the horseradish peroxidase-labeled loaded on container.

The anti-NGAL preparation method for antibody is as follows:

According to the full length sequence of NGAL, two polypeptide fragments from NGAL are obtained, sequence (N → C-terminal) is as follows: CREDKDPQKMYATIYELKEDKS (SEQ ID NO:4) and CYNQHAMVFFKKVSQNREY (SEQ ID NO:5).More than two Peptide fragment is mixed with mass ratio 1:1.It is coupled, is exempted from hemocyanin (KLH) using the polypeptide fragment mixture as immunogene Epidemic disease new zealand white rabbit and the immune serum generated.The methods and procedures of animal immune is as follows:

1) two new zealand male rabbits, simultaneously -20 DEG C of storages of acquisition preimmune serum is immunized.

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, arteria carotis blood sampling takes serum, -70 DEG C of storages.

Obtain the anti-NGAL antibody of specificity.WESTERN BLOT is the results show that anti-NGAL polyclonal antibody obtained can Specifically to combine the NGAL polypeptide of overall length.

Gelatinase-associated rouge fortune albumen (NGAL) of neutrophil cell includes 198 amino acid altogether, previous reported It can be used as urine biomarker, for detecting the early onset thereof of renal tubular cell injury.It detects KIM-1 polypeptide and detection NGAL is more The antibody combined application of peptide, is used for clinical diagnosis kidney injury situation, and the sensibility and accuracy of diagnosis are significantly increased.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, 15 those skilled in the art The present invention can be made various changes or modifications, such equivalent forms are equally fallen within defined by the application the appended claims Range.

Claims

1. a kind of mixtures of polypeptides, which is characterized in that its amino acid sequence as shown in SEQ ID NO:1 and SEQ ID NO:2 Polypeptide mixes.

2. mixtures of polypeptides as described in claim 1, which is characterized in that amino shown in SEQ ID NO:1 and SEQ ID NO:2 The ratio of the polypeptide mixing of acid sequence is 1:5~5:1 according to weight ratio.

3. the purposes of mixtures of polypeptides as claimed in claim 1 or 2 is used to prepare the anti-of the anti-Kidney injury molecule 1 of specificity Body.