CN103074303A - Hybridoma cell strain generating anti-human NGAL specific monoclonal antibody, monoclonal antibody generated by same and application - Google Patents
Hybridoma cell strain generating anti-human NGAL specific monoclonal antibody, monoclonal antibody generated by same and application Download PDFInfo
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Abstract
The invention discloses a hybridoma cell strain generating an anti-human NGAL specific monoclonal antibody and a monoclonal antibody generated by the same. The hybridoma cell strain is generated in a way that after an amino acid sequence shown by SEQIDNO:1 immunizes a Balb/c rat, a spleen B lymph cell and a myeloma cell of the rat are fused for further cultivation. The antibody can be used for the immunity detection of the NGAL. Based on the invention, an immunity detection reagent for the NGAL can be developed and used for diagnosing and detecting acute kidney injury and other diseases.
Description
Technical field
The present invention relates to bio-pharmaceutical engineer technology domain, relate in particular to hybridoma cell strain and by the anti-human neutrophil gelatinase-associated lipocalin monoclonal antibody specific of its generation.
Background technology
Injury of the kidney is one of modal complication of various critical illness clinically.Although in recent decades injury of the kidney pathologic, physiologic and pathogenetic research and clinical treatment method have been had very much progress, its M ﹠ M is still high.Clear and definite, early treatment is very important to the reverse of injury of the kidney, and early diagnosis is the key that instructs clinical timely treatment.At present, the classical injury of the kidney biochemical markers of commonly using clinically mainly contains blood urea nitrogen, serum creatinine, blood bladder chalone C.Urea is important end last small molecule metabolite of human body protein metabolism, its concentration renal function that do not place one's entire reliance upon, when glomerular filtration rate(GFR drops to normal 50% when following, blood urine element concentration just raises rapidly, can only be as the index of guestimate renal function; Creatinine is the dead end product of creatine metabolism, compares with urea, and it is little that it is affected by the kidney other factor, can indicate preferably renal function.But the same with the blood urine element, serum creatinine is insensitive to the loss of kidney reserve function; There are some researches show that the blood bladder chalone C at injury of the kidney just rising in 12 hours occurs, this moment, renal function was subjected to irreversible damage, had increased the mortality ratio of injury of the kidney.Neutrophil gelatinase-associated lipocalin (neutrophil gelatinase-associated lipocalin, NGAL) in the extremely early stage just significantly rising of acute injury of kidney, its susceptibility is high, window phase is short, is subject to extensive concern as biology early warning and the diagnosis index of injury of the kidney.
NGAL is the small molecules secreted protein of a kind of 25Kd of finding in the neutrophil leucocyte specific granule.Be distributed widely in Adult Human Bone Marrow, uterus, prostate gland, sialisterium, stomach, appendix, colon and tracheae and Fetal Spleen and the lung tissue.The protein of this 25kDa is comprised of 198 amino acid, front 20 amino acid are leader sequence, NGAL is the newcomer of lipocalin protein (lipocalin) superfamily, can in conjunction with and transport hydrophobic small molecules, participate in the body anti-inflammatory action and may have close ties with tumour.Mishra J in 2003 etc. observe in the nephrotoxicity of mouse kidney behind the ischemical reperfusion injury and cisplatin induction, NGAL significantly increases in the mouse urine, Barasch research groups in 2011 have reported the experimental result that drives lower knock in mouse model with Luciferase-2 and the two fluorescent reporter genes of mCherry at NGAL promotor and its 5 ' UTR at Nature Medcine, the Henles of NGAL activity of gene expression in nephridial tissue manages and the epithelial cell of collecting tubule is the noticeable change of dose-dependently when having disclosed ischemia and cytotoxicity renal impairment.When injury of the kidney is described in the urine NGAL mainly from nephridial tissue, rather than when normal also low epithelial cells such as neutrophil leucocyte, liver cell, respiratory tract and enteron aisle of expressing NGAL.Illustrate that NGAL has important using value in the injury of the kidney diagnosis.
The key issue that realizes the NGAL immunodetection is how to obtain its monoclonal antibody and polyclonal antibody and as the antigen standard substance of immunodetection.According to Bioinformatics Prediction, NGAL albumen may have glycosylation in 86 amino acids, for can be best near the structure and function of native protein, be applicable to the restructuring NGAL albumen of immunology detection working standard at 293 cells by genetically engineered, for the preparation of next step NGAL monoclonal antibody and polyclonal antibody provides antigen, and be used for the research of NGAL characteristic.NGAL has more specificity and susceptibility for the early diagnosis of injury of the kidney, is expected to become desirable injury of the kidney early diagnosis biochemical indicator.The principle that immunoassay technology is based on antigen-antibody response is carried out the detection method of quantitative and qualitative analysis to determinand (antigen/antibody), have high specificity, the advantage such as highly sensitive, convenient, be the important research means of modern life science, have a wide range of applications at the bioanalysis detection field.Structure all has great importance for kidney disease early diagnosis, human health etc. based on the NGAL detection system of immunoassay technology, has simultaneously larger social effect and economic worth.The enzyme linked immunological for detection of NGAL (ELISA) test kit of selling in the market all adopts external import NGAL antibody, and is expensive, and is the scientific research test kit, is difficult to big area and carries out clinical application.Therefore, the preparation high specific the NGAL monoclonal antibody specific become in the urgent need to.
Summary of the invention
The purpose of this invention is to provide the hybridoma cell strain that two strains produce anti-human NGAL monoclonal antibody specific.
The hybridoma cell strain of the monoclonal antibody of the anti-human NGAL of generation provided by the present invention all is preserved in Chinese Typical Representative culture collection center (CCTCC), address: Hubei China province Wuhan City Wuhan University, postcode: 430072, preservation date is on December 26th, 2012, preserving number is respectively CCTCC NO.C2012197 and CCTCC NO.C2012198, Classification And Nomenclature is respectively hybridoma cell strain NGAL (1-F2) and hybridoma cell strain NGAL (1-G2), distinguishes in this application called after 1-F2 and 1-G2.
Hybridoma cell strain 1-F2 and 1-G2 by the aminoacid sequence shown in SEQ ID NO:1 immunity Balb/c mouse after the mouse spleen bone-marrow-derived lymphocyte merge rear cultivation generation with the myeloma cell.
The present invention also provides a kind of monoclonal antibody that is produced by hybridoma cell strain 1-F2, and the subclass of this monoclonal antibody belongs to IgG1.
The present invention also provides a kind of monoclonal antibody that is produced by hybridoma cell strain 1-G2.The subclass of this monoclonal antibody belongs to IgG1.
The monoclonal antibody that the present invention also provides monoclonal antibody that hybridoma cell strain 1-F2 produces and/or hybridoma cell strain 1-G2 to produce prepares the injury of the kidney diagnosis or detects application in the preparation.
It is a kind of for diagnosis or detect the test kit of injury of the kidney that the present invention also provides, and it comprises the monoclonal antibody that monoclonal antibody that hybridoma cell strain 1-F2 produces and/or hybridoma cell strain 1-G2 produce.
The present invention specifically finishes purpose of the present invention by following technical proposals:
1.NGAL the preparation of recombinant protein: from GENEBANK, obtain the human neutrophil genatinase associated lipocalin encoding sequence, gene segment by amplification NGAL NGAL, goal gene is connected with carrier for expression of eukaryon, makes up Eukaryotic expression recombinant plasmid.Recombinant plasmid is stably transfected in 293 cells, expresses NGAL.Purification of recombinant proteins also carries out purity, concentration and activity identification, obtains highly purified NGAL recombinant protein.
2. pass through Bioinformatics Prediction, NGAL albumen 86 amino acids have obvious glycosylation, for obtaining the best monoclonal antibody specific that can identify native protein, with the NGAL recombinant protein that obtains in the scheme 1 as immunogen immune animal Balb/c mouse,, utilize indirect elisa method and cloning to filter out hybridoma cell strain that can the anti-NGAL monoclonal antibody of stably excreting and detect tiring of its cells and supernatant through cytogamy repeatedly by hybridoma technology.Carry out afterwards the specificity experiment of Subclass of antibody evaluation and antibody, confirm that the antibody that obtains is that specificity is for NGAL.
3. set up the ELISA detection system of NGAL: utilize the anti-NGAL antibody that has prepared as capture antibody and detection antibody, with the NGAL recombinant protein as detectable antigens, carry out the antibody pair test, through optimizing the ELISA reaction conditions, obtain to detect the pairing antibody of natural NGAL and successfully set up the double-antibodies sandwich ELISA that detects NGAL with this.
The present invention passes through hybridoma technology, the hybridoma cell strain 1-F2 and the 1-G2 that have prepared anti-NGAL monoclonal antibody, can secrete corresponding NGAL monoclonal antibody specific, can successfully match and detect natural sera, tentatively set up the double-antibodies sandwich ELISA that detects NGAL.
For above and other objects of the present invention, feature and advantage can be become apparent, preferred embodiment cited below particularly, and cooperation accompanying drawing are described in detail below.
Description of drawings
Fig. 1 is the analysis of NGAL gene reverse transcription PCR amplified production agarose gel electrophoresis.Wherein, M is DNA Marker; 1-5 is respectively 60 ℃ of five annealing temperatures, 62 ℃, 64 ℃, 66 ℃, 68 ℃.
Fig. 2 is the agarose gel electrophoresis interpretation of result figure that pcDNA3.1-NGAL uses EcoR I and Acc65 I double digestion product.Wherein, M is DNA marker; Swimming lane 1 is pcDNA3.1, and swimming lane 2 is pcDNA3.1-NGAL.
Fig. 3 identifies figure for restructuring NGAL protein expression Western blot; Wherein, 1 is cell extraction albumen; 2 is cells and supernatant.
Fig. 4 is restructuring NGAL protein purification figure.Wherein 1 is ultraviolet absorption curve, and 2 is solution conductivity, and 3 is antibody purification concentration, F1, F2, is respectively the absorption peak that refers to that the different time protein purification occurs.
Fig. 5 is that the SDS-PAGE of restructuring NGAL albumen behind the purifying identifies.Wherein, M is molecular weight standard; Swimming lane 1 is NGAL behind the purifying.
Fig. 6 is that ELISA identifies that monoclonal antibody 1-F2 detects the NGAL recombinant protein.Wherein, the negative contrast in left 1 hole, left 2～12 holes are respectively antibody dilution multiple 1:1000,1:2000,1:4000,1:8000,1:16000,1:32000,1:64000,1:128000,1:256000,1:512000,1:1024000.
Fig. 7 is that ELISA identifies that monoclonal antibody 1-G2 detects the NGAL recombinant protein.Wherein, the negative contrast in left 1-3 hole, left 4～12 holes are respectively antibody dilution multiple 1:1000,1:2000,1:4000,1:8000,1:16000,1:32000,1:64000,1:128000,1:256000.
Fig. 8 is that Western blot identifies that monoclonal antibody 1-F2 is in conjunction with the NGAL recombinant protein; Wherein, swimming lane 1 is recombinant protein NGAL; Swimming lane 2 is irrelevant albumen PCT.
Fig. 9 is that Western blot identifies that monoclonal antibody 1-G2 is in conjunction with the NGAL recombinant protein.Wherein, swimming lane 1 is recombinant protein NGAL; Swimming lane 2 is irrelevant albumen PCT.
Figure 10 is the typical curve that standard ELISA detects recombinant protein.
Figure 11 is that standard ELISA detects NGAL result in the clinical samples (normal people and nephrotic are relatively).
Embodiment
The present invention is connected goal gene by the gene segment of RT-PCR amplification NGAL NGAL with carrier for expression of eukaryon pcDNA3.1, make up Eukaryotic expression recombinant plasmid pcDNA3.1-NGAL.Be stably transfected into 293 cells with recombinant plasmid, express NGAL.The NGAL eukaryon expression plasmid that utilizes pcDNA3.1 to make up has been realized secreting, expressing at 293 cells, shows through the SDS-PAGE electrophoretic analysis, and the recombinant protein molecular mass of expression is about 25kD.Adopt albumen affinity chromatography column purification NGAL recombinant protein, by obtaining high purity N GAL recombinant protein behind the ni-sepharose purification, content is about 0.54mg/mL.
The present invention learns that by Bioinformatics Prediction NGAL albumen 86 amino acids may have glycosylation, for obtaining the best monoclonal antibody specific that can identify native protein, with the NGAL recombinant protein of 293 cell expressings as immunogen immune animal Balb/c mouse, by hybridoma technology through cytogamy repeatedly, utilize indirect elisa method and cloning to filter out the hybridoma cell strain of the anti-NGAL monoclonal antibody specific of energy stably excreting, ascites is carried out titration and avidity mensuration through HiTrap IgG Purification HP affinity chromatography column purification to the monoclonal antibody of gained.Obtain hybridoma cell strain 1-F2 and the 1-G2 of 2 strain stably excreting NGAL monoclonal antibody specifics through screening, be IgG1 through subgroup identification, 2 strain monoclonal antibodies are tired all more than 1:256000, and avidity is respectively 6.39 * 10
10M
-1With 3.65 * 10
9M
-1, Western blot identifies and shows that 2 strain monoclonal antibodies all have higher specificity.
The present invention has set up the ELISA detection system of NGAL: using anti-NGAL antibody 1-F2 is capture antibody, anti-NGAL antibody 1-G2 is for detecting antibody, the double-antibody sandwich elisa detection method of Criterion, can detect the natural NGAL in the urine, and successfully set up the double-antibodies sandwich ELISA that detects NGAL with this.
Material and source
Laboratory animal: the Balb/c mouse is available from Third Military Medical University's Experimental Animal Center (Chongqing in China).
Cell strain and plasmid: (CCTCC) bought at 293 cell strain Chinese Typical Representative culture collection centers, and the pcDNA3.1 plasmid is available from Novagen company.
Cell culture medium and foetal calf serum are available from Gibco company, and Lipo2000 is available from Novagen company.
Enzyme and test kit: restriction enzyme EcoR I and Acc65 I and plasmid extraction test kit are available from OMEGA company, and sepharose reclaims test kit available from sky root company.
Other reagent: the HRP-goat anti-mouse IgG is available from middle mountain company.
Embodiment 1The preparation of NGAL recombinant protein
1.NGAL the full gene of encoding sequence obtains
According to the NGAL gene order (NM_005564.3) that GeneBank provides, determine NGAL gene plan cloned sequence, specifically shown in SEQ ID NO:2:
Design NGAL total length primer: respectively shown in SEQ ID NO:3 and 4
SEQ?ID?NO:3：NGAL-1：5’-TAAGGTACCCATGCCCCTAGGTCTCCTG-3’
SEQ?ID?NO::4：NGAL-2：5’-GGTGGAATTTTAGCCGTCGATACACTG-3’
Primer contains respectively restriction enzyme site: NGAL-1:Acc651; The NGAL-2:EcoR I, synthetic by Invitrogen company.
Extract the total RNA of white corpuscle by Trizol Reagent method, obtain white corpuscle cDNA by reverse transcription, obtain human neutrophil genatinase associated lipocalin by pcr amplification, set up the PCR system, under different annealing temperatures, carry out PCR.Get 25 μ L PCR product 1%TAE electrophoresis and analyze (Fig. 1).Use the sepharose DNA of sky, Beijing root to reclaim test kit (common centrifugal column type) recovery PCR product.2.NGAL construction of recombinant plasmid and enzyme are cut evaluation
PcDNA3.1 carrier and NGAL PCR are reclaimed product respectively with behind EcoR I and the Acc65 I double digestion digestion 1h, carry out the dna gel electrophoresis, reclaim the purpose band, with the connection of spending the night of 4 ℃ of T4DNA ligase enzymes.To connect product transformed competence colibacillus bacillus coli DH 5 alpha, to be applied to behind the bacterium liquid mixing on the LB culture plate that contains penbritin, 37 ℃ of constant incubator incubated overnight, the single colony inoculation of picking next day spends the night in the LB liquid nutrient medium enlarged culturing that contains penbritin.
From culturing bacterium, extract plasmid with plasmid extraction kit, identify with row 1% agarose gel electrophoresis behind EcoR I and the Acc65 I double digestion, at the about visible specific band in 600bp place, show and inserted the fragment (Fig. 2) that size is about 600bp in the pcDNA3.1 carrier.
3.NGAL recombinant plasmid purpose segment order-checking
The pcDNA3.1-NGAL recombinant plasmid is sent to the order-checking of Shanghai Ying Jun company, and uses the Vector5.0 instrument NGAL sequence of sequencing result and GeneBank is carried out homology analysis.Result's demonstration of order-checking, Insert Fragment length is 597bp, and is in full accord with the NGAL sequence of reporting among the GeneBank.
4.NGAL the expression of recombinant protein and evaluation
1) recovery of 293 cells and cultivation
293 frozen cells are changed in 37 ℃ of water-baths by liquid nitrogen rapidly, DMEM substratum and 37 ℃ of preheatings of serum are also configured the DMEM perfect medium that contains 10% calf serum.Cell suspension is moved into the perfect medium of 37 ℃ of preheatings, the centrifugal 5min of 1000rmp/min abandons supernatant, adds 3-4mL and contains the perfect medium of 10% foetal calf serum and blow gently evenly, changes in the culturing bottle, places 5%CO
2, 37 ℃ of incubators cultivate, and change liquid every day.
2) rotaring redyeing 293 cell
293 cells of cultivating are seeded in six orifice plates, observation of cell covered with flat board and reached 90-95% density next day, with transfection reagent Lipofectamin2000(Invitrogen company) recombinant plasmid pcDNA3.1 transfection is transfected in 293 cells, add G418(800 μ g/ml after cultivating 48h), cultivated 3 days, and collected cleer and peaceful cell and carry out Wester nblot evaluation (Fig. 3).Determine that transfection successfully carries out cloning, obtain stable expression cell strain and enlarged culturing.
3) expression of NGAL recombinant protein and purifying
Collect culture supernatant, use albumen affinity column (Ni
2+22DA2Sepharose Fast Flow) purification of recombinant proteins (Fig. 4).Collection obtains finished product albumen, the row SDS-PAGE electrophoresis (Fig. 5) of then taking a sample respectively, and detecting its purity through HPLC is 95%.
5.NGAL recombinant protein concentration, Purity
The Lowry method is measured recombinant protein concentration: according to ordinary method for the preparation of the typical curve of analyzing and measure sample concentration, the multitube calculating mean value, recording concentration is 0.54mg/ml.
Embodiment 2Bioinformatic analysis and immunogenic definite
1. search the aminoacid sequence of NGAL albumen from GENEBANK, this albumen is comprised of 198 amino-acid residues.
2. 86 amino acids may be by glycosylation in the online information biology glycosylation forecast analysis NGAL albumen, for obtaining the best monoclonal antibody specific that can identify native protein, choose the total length NGAL albumen of 293 cell expressings as immunogen, its aminoacid sequence is specifically with SEQ ID NO:1.
Embodiment 3The preparation of anti-NGAL monoclonal antibody
1.NGAL the preparation of monoclonal antibody
With the Freund's complete adjuvant emulsification of recombinant full-lenght NGAL albumen and equivalent, the mutual pushing manipulation of double syringe is adopted in antigen emulsification.After emulsification is finished, add the abdominal injection approach to the Balb/c mouse of 58 weeks about age with the subcutaneous multi-point injection of four limbs, carry out fundamental immunity (100 μ g/ mouse).After 2 weeks, with the Freund's incomplete adjuvant emulsification of NGAL holoantigen and equivalent, add the abdominal injection approach to 5 Balb/c mouse with the subcutaneous multi-point injection of four limbs, carry out supplementary immunization (100 μ g/ mouse); After 2 weeks, adopt again the same manner supplementary immunization once, put to death afterwards the taking-up mouse spleen, splenocyte is carried out cytogamy in 7 days.
Fusion process is that splenocyte is mixed with 8:1 with myeloma cell (Sp2/0), take polyoxyethylene glycol as fusogen.Fused cell is suspended from the HAT nutrient solution that contains calf serum, and places 6%CO
2In 37 ℃ of cultivations.Screen with the ELISA method.Adopt limiting dilution assay to carry out subclone to the positive colony hole that detects, and place 5%CO
2In in 37 ℃ of cultivations, until till the nutrient solution in all cells growth hole all is positive, can carry out the enlarged culturing of monoclonal antibody.
Adopt the method that induces monoclonal antibody in the mouse peritoneal to carry out enlarged culturing.Get the Balb/c mouse that has grown up, inject liquid paraffin 0.5mL in intraperitoneal, 1 Zhou Houzai injects hybridoma in intraperitoneal.With the hybridoma mixing that suspends, and cell count transferred to 4 * 10 with physiological saline
5Individual/mL, every Balb/c mouse peritoneal injection 0.5mL hybridoma.Collect ascites after 10-14 days.
2.NGAL the purifying of monoclonal antibody
After collecting ascites it is carried out purifying, concrete scheme is: the required buffer:Binding Buffer(A of configuration antibody purification liquid, 20mmol/L sodium phosphate, 0.8mol/L (NH4)
2SO
4, pH7.5); Elution Buffer(B liquid, the 20mmol/L sodium phosphate, pH7.5); Regeneration buffer(C liquid, the 20mmol/L sodium phosphate, pH7.5, and 30% add Virahol by volume).Add ammonium sulfate in odd contradictive hydroperitoneum, make its whole solubility consistent with the solubility of ammonium sulfate in the A liquid, 0.45 μ m membrane filtration is waited for loading; Select HiTrap IgG Purification HP post access AKTA prime protein purification instrument, with A, B liquid and C liquid pillar is fully washed, after carrying out abundant balance with A liquid, the sample of preparing is managed loading from A, use A liquid balance pillar after the loading, remove foreign protein, use again B liquid wash-out purification column, collect elution peak; Regulate the pH to 7.0-8.0 of eluted protein, the antibody packing is frozen in-20 ℃ of preservations; With C liquid filler is regenerated, then carry out balance with A liquid and get final product.
3. the evaluation of anti-NGAL monoclonal antibody subclass
Adopt the Mouse Monoclonal Antibody Isotyping Reagents(ELISA/Ouchterlony Double Diffusion of U.S. Sigma company, Stock No.ISO-2LOT114k4817), the operation by specification carries out.
1) coated elisa plate: be best effort concentration 5 μ g/mL with coating buffer with NGAL recombinant protein dilution, every hole adds 100 μ L antigen liquids, and every strain adds 12 holes, spends the night in 4 ℃, wash 5 times, and is empty dried.
2) sealing: every hole adds confining liquid 350 μ L, hatches 1h～1.5h for 37 ℃, washs 5 times, empty doing.
3) every hole adds the fresh Hybridoma Cell Culture supernatant of 100 μ l or dilution monoclonal antibody, and room temperature (20 ℃～25 ℃) leaves standstill 1h, shakes and washes 3min, totally 5 times.
4) dilute IgM, IgG2a, IgG2b, IgG3, IgG, IgA with antibody diluent with 1:2000.
5) every hole adds the antibody that 100 μ L have diluted, and every kind of antibody all adds 2 holes, and room temperature leaves standstill 30min, shakes and washes 3min, totally 5 times.
6) every hole adds the anti-sheep IgG of the rabbit antibody of 100 μ L1:1000 dilution, and room temperature leaves standstill 15min, shakes and washes 3min, totally 5 times.
7) every hole adds 100 μ L substrate nitrite ions, room temperature lucifuge reaction 10min～15min.H. every hole adds 50 μ L stop buffers, and observations is determined the Ig subclass.
4. monoclonal antibody avidity is identified
Measure affinity constant with the Friguent method, NGAL antigen is dissolved in the 0.05mol/L carbonic acid buffer (pH9.6), final concentration is l μ g/ml, amount with every hole 100 μ l is added to 4 ℃ of coated spending the night in 96 orifice plates with solution, with the PBS solution that contains 1%BSA plate is sealed, will save backup at 4 ℃ after the plate drying.
The foundation of reactive system: the starting point concentration of anti-NGAL monoclonal antibody is 20ng/mL in the reactive system.The starting point concentration of NGAL antigen is from 1000ng/ml(1250x l0mol/L) doubling dilution down, form 8 reactive systems, at 37 ℃ of reaction lh; With every hole 100 μ l reaction solution is joined the immune response plate that has been coated with NGAL antigen with diplopore, wash plate 5 times after hatching lh for 37 ℃; Add 37 ℃ of reactions of people's 100 μ 1 mountain sheep anti mouse, two anti-every binding substances solution lh, wash plate 5 times, add people's stop buffer after adding people's substrate colour developing 15min, measure the antigen combination rate reckoning affinity constant that the OD450 value is calculated each reactive system, drawing 1-F2 avidity is 6.39 * 10
10M
-1, 1-G2 is 3.65 * 10
9M
-1
5.ELISA identify anti-NGAL monoclonal antibody
With coated diluted to the 5 μ g/ml of the NGAL antigen of purifying, the every hole of ELISA batten adds 100 μ l, 4 ℃ of coated spending the night.Take out plank and abandon antigen next day, washes plate.Monoclonal antibody to be identified is made 1:1000,1:2000,1:4000,1:8000,1:16000...... adds in the corresponding cylindrical void by 100 μ l/ holes, makes in addition blank and negative and positive control wells.Hatch 1h for 37 ℃, wash plate, add two and resist, 1:3000 dilution HRP mark goat anti-mouse IgG adds in the corresponding cylindrical void by 100 μ l/ holes, hatches 40min for 37 ℃.Abandon liquid, wash plate, pat dry, add substrate solution 100 μ l/ holes, lucifuge colour developing 10min adds stop buffer 50 μ l/ holes.The result as seen, anti-NGAL monoclonal antibody 1-F2 and 1-G2 present macroscopic concentration gradient (Fig. 6, Fig. 7).
6. the Western blot of anti-NGAL monoclonal antibody identifies
1) with Marker3 μ l, NGAL antigen 20 μ l, irrelevant albumen procalcitonin(PCT) 20 μ l electrophoresis.
2) after electrophoresis finishes, take off gel, place transfer printing damping fluid balance 10min.
3) 0.22 μ m pvdf membrane is processed 20s with anhydrous methanol first, wash 5min with ddH2O again.And then immerse Transfer Buffer above 5min.Simultaneously filter paper is immersed among the Transfer Buffer.
4) transferring film: be arranged in order from bottom to up, filter paper-pvdf membrane-glue-filter paper is discharged bubble, puts into the transferring film instrument, 18V constant voltage electrotransfer 1.5h.
5) after transferring film is finished, visible clear albumen marker on film, ddH2O cleans twice, TBST and washes 5min.Transfer film is placed confining liquid, room temperature sealing 2h.
6) discard confining liquid, with 1 * TBST rinsing film 3 times, each 15min.
7) add the antibody purification that dilutes with 1:1000,4 ℃ of overnight incubation.
8) 1 * TBST rinsing film is 3 times, each 15min.
9) add the HRP-goat anti-mouse igg antibody that has been diluted to 1:10000, hatch 3h for 37 ℃.
10) 1 * TBST washing is 3 times, each 15min.
11) with the chemoluminescence colour developing, behind the developing fixing, observation analysis result and shooting.At restructuring NGAL protein 25 KD place all visible anti-NGAL monoclonal antibody in conjunction with clear band (Fig. 8, Fig. 9).
Embodiment 4NGAL in the double antibody sandwich ELISA examination criteria
1.HRP traget antibody
This law is with NaIO
4Glycan molecule with the HRP surface is oxidized to aldehyde radical first, and then combine with the amino of antibody protein, the productive rate of the enzymic-labelled antibody that obtains is high, concrete steps are as follows: take by weighing 10mg HRP enzyme, add the HRP liquid that the 2mL pure water is configured to 5mg/ml, add 34 μ L according to every mg HRP enzyme and calculate, add 340 μ LNaIO
4, 4 ℃ of lucifuges are placed, behind the 1h, the amount that adds 250 μ L according to every mg HRP enzyme adds ethylene glycol 250 μ L, and lucifuge is placed 4 ℃, changes in the dialysis tubing behind the 30min, with 1mM acetate buffer solution (pH4.0-4.4) dialysed overnight, during change at least liquid 2 times, all require lucifuge.The PBS damping fluid of the preparation of antibody: 0.01M carries out 4 ℃ of dialysed overnight, and measures protein concentration.Mark: antibody and HRP enzyme are by the 1:1(mass ratio) mix, add 1mol/LNa
2CO
3Damping fluid (1:80), the pH that reconciles reaction are 9.5,25 ℃ of reaction 2-3h.Stop: fresh preparation 0.1mol/LNaH
4B(4mg/mL), add 47 μ l according to every mg HRP enzyme.Behind 4 ℃ of placement 2h, change dialysis tubing over to.With 0.01mol/L PBS damping fluid (pH7.0-7.2) dialysed overnight, during change at least liquid twice, lucifuge.Packing is preserved: the antibody that mark is good is managed packing with lucifuge EP, and measures antibody titer.-20 ℃ of preservations.
2. the detection of antibody pairing and clinical sample
1) specimen collection:
Collect the clear and definite nephrotic's of clinical diagnosis urine specimen from the first and second affiliated hospitals of Third Military Medical University, fetch in the process and store with ice bag, to keep sample fresh, carrying out mark behind the centrifuging and taking supernatant adding preservative agent is sub-packed in the 1.5ml Ep pipe,-80 ℃ of preservations, and with patient's essential information typing computer, carry out numbering.
2) Samples detection:
Choose monoclonal antibody 1-F2 as capture antibody, monoclonal antibody linked with peroxidase 1-G2 is as detecting antibody, and recombinant protein N GAL is standard substance, and sample testing method is as follows:
A. coated elisa plate: be 5 μ g/ml with coating buffer with antibody dilution, every hole adds 100 μ l, 4 ℃ of coated spending the night.
B. will be coated with the enzyme plate that spends the night and wash 3 times, dry.
C. sealing: every hole adds confining liquid 350 μ l, hatches 1h for 37 ℃, washs 3 times, dries.
D. with antibody diluent the NGAL recombinant protein is begun doubling dilution from 500ng/ml, every hole adds 100 μ l, and the first hole is blank, does typical curve.Detect 33 routine injury of the kidney samples, 24 routine normal specimen, every hole adds 100 μ l, hatches 1h for 37 ℃.
F. corresponding ELIAS secondary antibody is diluted with 1:5000, every hole adds 100 μ l, hatches 30min for 37 ℃.
H. every hole adds 100 μ l substrate nitrite ions, room temperature lucifuge colour developing 5min.
I. every hole adds 50 μ l stop buffers, reads plate, determines the content of NGAL in the clinical sample according to typical curve.
3) statistical analysis
Use SPSS13.0 software that heart stalk combination Normal group is carried out statistical analysis with NGAL result that ELISA surveys.
The result shows, utilize pairing antibody 1-F2-1-G2 and NGAL recombinant protein standard substance successfully to draw typical curve (Figure 10), and find to the normal urine of 24 examples and 33 routine injury of the kidney Urine in Patients detection the time, 33 routine nephrotics (285.854ng/ml) are significantly higher than 24 routine normal serum (6.155ng/ml; P＜0.01) (Figure 11), illustrate that pairing antibody can be successfully applied to the detection of NGAL in the natural sera.
Although the present invention discloses as above with preferred embodiment; so it is not to limit the present invention; any person of ordinary skill in the field; without departing from the spirit and scope of the invention; when can doing a little change and improvement, so the present invention's protection domain is as the criterion when looking the claim person of defining.
Sequence table
Claims (10)
1. hybridoma cell strain that produces anti-human neutrophil gelatinase-associated lipocalin monoclonal antibody specific, its preserving number is CCTCC NO.C2012197.
2. hybridoma cell strain that produces anti-human neutrophil gelatinase-associated lipocalin monoclonal antibody specific, its preserving number is CCTCC NO.C2012198.
3. hybridoma cell strain as claimed in claim 1 or 2 is characterized in that, behind the immunity of the aminoacid sequence shown in SEQ ID NO:1 Balb/c mouse, mouse spleen bone-marrow-derived lymphocyte and myeloma cell are merged rear cultivation and produced.
4. hybridoma cell strain as claimed in claim 1 or 2 is characterized in that, the gene order of described NGAL is shown in SEQ ID NO:2.
5. monoclonal antibody that is produced by hybridoma cell strain claimed in claim 1.
6. monoclonal antibody as claimed in claim 5 is characterized in that, the subclass of described monoclonal antibody belongs to IgG1.
7. monoclonal antibody that is produced by hybridoma cell strain claimed in claim 2.
8. a monoclonal antibody as claimed in claim 7 is characterized in that, the subclass of described monoclonal antibody belongs to IgG1.
9. the application in preparation NGAL immunodetection preparation such as claim 5 and/or 7 described monoclonal antibodies.
10. a test kit that is used for diagnosis or detects injury of the kidney is characterized in that, comprises such as claim 5 and/or 7 described monoclonal antibodies.
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| CN107045062A (en) * | 2017-03-28 | 2017-08-15 | 广州瑞博奥生物科技有限公司 | Detect colloidal gold immuno-chromatography test paper strip, kit of human neutrophil genatinase associated lipocalin and preparation method thereof |
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| CN107045062A (en) * | 2017-03-28 | 2017-08-15 | 广州瑞博奥生物科技有限公司 | Detect colloidal gold immuno-chromatography test paper strip, kit of human neutrophil genatinase associated lipocalin and preparation method thereof |
| CN107045062B (en) * | 2017-03-28 | 2019-01-29 | 广州瑞博奥生物科技有限公司 | Detect colloidal gold immuno-chromatography test paper strip, the kit and preparation method thereof of human neutrophil genatinase associated lipocalin |
| CN113402606A (en) * | 2020-03-17 | 2021-09-17 | 江苏众红生物工程创药研究院有限公司 | Neutrophil gelatinase-associated lipocalin detection kit and clinical application thereof |
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| CN115991768B (en) * | 2022-09-16 | 2023-07-07 | 北京中楷健康科技有限公司 | Neutrophil gelatinase-associated lipocalin (NGAL) detection kit |
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