CN103074303A - Hybridoma cell strain generating anti-human NGAL specific monoclonal antibody, monoclonal antibody generated by same and application - Google Patents

Abstract

The invention discloses a hybridoma cell strain producing specific monoclonal antibody against human neutrophil gelatinase-associated lipocalin and the monoclonal antibody produced therefrom. The hybridoma cell strain is produced by immunizing Balb/c mice with the amino acid sequence shown in SEQ ID NO: 1, then fused mouse spleen B lymphocytes with myeloma cells. The antibody can be used for immunodetection of human neutrophil gelatinase-associated lipocarrier. In this way, an immunoassay reagent for human neutrophil gelatinase-associated lipocarcin can be developed, which can be used for diagnosing or detecting diseases such as acute kidney injury.

Description

Hybridoma cell line producing anti-human NGAL specific monoclonal antibody and the monoclonal antibody produced and application thereof

technical field

The invention relates to the technical field of biomedical engineering, in particular to a hybridoma cell line and an anti-human neutrophil gelatinase-associated lipocalin specific monoclonal antibody produced therefrom. the

Background technique

Renal injury is one of the most common clinical complications of various critical illnesses. Although great progress has been made in research on the pathophysiology and pathogenesis of renal injury and clinical treatment methods in recent decades, its morbidity and mortality are still high. It has been clear that early treatment is very important for the reversal of renal injury, and early diagnosis is the key to guide clinical and timely treatment. At present, the classic biochemical markers of renal injury commonly used in clinical practice mainly include blood urea nitrogen, blood creatinine, and blood cystatin C. Urea is an important final small molecule metabolite of protein metabolism in the human body, and its concentration does not completely depend on renal function. When the glomerular filtration rate drops below 50% of the normal level, the blood urea concentration rises rapidly. As an indicator for roughly estimating renal function; creatinine is the end product of creatine metabolism. Compared with urea, it is less affected by extrarenal factors and can better indicate renal function. However, like blood urea, serum creatinine is not sensitive to the loss of renal reserve function; some studies have shown that blood cystatin C does not rise until 12 hours after renal injury occurs, at this time renal function has been irreversibly damaged, which increases the mortality rate of renal injury . Neutrophil gelatinase-associated lipocalin (NGAL) is significantly elevated in the very early stage of acute kidney injury, with high sensitivity and short window period, and it can be used as a biological early warning and diagnostic indicator of kidney injury Widespread concern. the

NGAL is a 25Kd small molecule secreted protein found in neutrophil-specific granules. Widely distributed in adult bone marrow, uterus, prostate, salivary glands, stomach, appendix, colon and trachea, as well as fetal spleen and lung tissues. This 25kDa protein is composed of 198 amino acids, and the first 20 amino acids are the leader sequence. NGAL is a new member of the lipocalin superfamily, which can bind and transport small hydrophobic molecules, participate in anti-inflammatory effects in vivo and may There is a close relationship with tumors. In 2003, Mishra J et al. observed that NGAL in mouse urine was significantly increased in mouse kidneys after ischemia-reperfusion injury and cisplatin-induced toxic kidney damage. -2 and mCherry dual fluorescent reporter gene in the knock in mouse model driven by the NGAL promoter and its 5'UTR revealed the role of NGAL gene expression activity in kidney tissue during ischemic and cytotoxic renal damage The epithelial cells of Henles' duct and collecting duct showed significant changes in a dose-dependent manner. It shows that NGAL in urine mainly comes from kidney tissue during renal injury, not from neutrophils, hepatocytes, respiratory tract and intestinal epithelial cells, which also have low expression of NGAL in normal conditions. It shows that NGAL has important application value in the diagnosis of renal injury. the

The key issue in realizing NGAL immunoassay is how to obtain its monoclonal antibody and polyclonal antibody as well as antigen standards for immunoassay. According to bioinformatics prediction, NGAL protein may have glycosylation at the 86th amino acid. In order to best approach the structure and function of natural protein, recombinant NGAL suitable for immunological detection standard was expressed in 293 cells through genetic engineering. The protein provides antigens for the preparation of NGAL monoclonal antibody and polyclonal antibody in the next step, and is used for the research of NGAL characteristics. NGAL is more specific and sensitive for the early diagnosis of renal injury, and is expected to become an ideal biochemical indicator for early diagnosis of renal injury. Immunoassay technology is a detection method based on the principle of antigen-antibody reaction for quantitative and qualitative analysis of the target object (antigen/antibody). It has the advantages of strong specificity, high sensitivity, and convenience. It is an important research method in modern life sciences. The field of analysis and detection has broad application prospects. The construction of an NGAL detection system based on immunoassay technology is of great significance for the early diagnosis of kidney disease and human health, as well as of great social and economic value. The enzyme-linked immunosorbent immunoassay (ELISA) kits currently on the market for the detection of NGAL all use imported NGAL antibodies, which are expensive and are for scientific research, making it difficult for large-scale clinical applications. Therefore, the preparation of highly specific NGAL-specific monoclonal antibodies has become an urgent need. the

Contents of the invention

The purpose of the present invention is to provide two hybridoma cell lines producing anti-human NGAL specific monoclonal antibody. the

The hybridoma cell lines producing anti-human NGAL monoclonal antibody provided by the present invention are all preserved in the China Center for Type Culture Collection (CCTCC), address: Wuhan University, Wuhan City, Hubei Province, China, postcode: 430072, and the preservation date is 2012 On December 26, the preservation numbers were CCTCC NO.C2012197 and CCTCC NO.C2012198, and the classification and designation were hybridoma cell line NGAL (1-F2) and hybridoma cell line NGAL (1-G2), respectively. Named 1-F2 and 1-G2. the

Both hybridoma cell lines 1-F2 and 1-G2 were produced by fusion of mouse spleen B lymphocytes and myeloma cells after immunizing Balb/c mice with the amino acid sequence shown in SEQ ID NO:1. the

The present invention also provides a monoclonal antibody produced by the hybridoma cell line 1-F2, the subclass of the monoclonal antibody belongs to IgG1. the

The invention also provides a monoclonal antibody produced by the hybridoma cell line 1-G2. The subclass of this monoclonal antibody is IgG1. the

The present invention also provides the use of the monoclonal antibody produced by the hybridoma cell line 1-F2 and/or the monoclonal antibody produced by the hybridoma cell line 1-G2 in preparing preparations for diagnosing or detecting kidney injury. the

The present invention also provides a kit for diagnosing or detecting renal injury, which comprises the monoclonal antibody produced by the hybridoma cell line 1-F2 and/or the monoclonal antibody produced by the hybridoma cell line 1-G2. the

The present invention specifically completes the object of the present invention through the following technical solutions:

1. Preparation of NGAL recombinant protein: Obtain human NGAL coding sequence from GENEBANK, amplify the gene fragment of human neutrophil gelatinase-associated lipocalin NGAL, connect the target gene with eukaryotic expression vector, and construct eukaryotic Expression of recombinant plasmids. The recombinant plasmid was stably transfected into 293 cells to express human neutrophil gelatinase-associated lipocalin. Purify the recombinant protein and identify its purity, concentration and activity to obtain high-purity NGAL recombinant protein. the

2. According to bioinformatics prediction, the 86th amino acid of NGAL protein has obvious glycosylation. In order to obtain the best specific monoclonal antibody that can recognize the natural protein, the NGAL recombinant protein obtained in scheme 1 was used as the immunogen to immunize animals Balb/c mice, through multiple cell fusions by hybridoma technology, use indirect ELISA method and cloning to screen out hybridoma cell lines that can stably secrete anti-NGAL monoclonal antibody and detect the titer of the cell culture supernatant. Afterwards, antibody subtype identification and antibody specificity experiments were carried out, and it was confirmed that the obtained antibody was specific for NGAL. the

3. Establish an ELISA detection system for NGAL: use the prepared anti-NGAL antibody as the capture antibody and detection antibody, and use the NGAL recombinant protein as the detection antigen to conduct antibody pairing experiments. After optimizing the ELISA reaction conditions, obtain a paired antibody that can detect natural NGAL Based on this, a double-antibody sandwich ELISA method for the detection of NGAL was successfully established. the

The present invention prepares anti-NGAL monoclonal antibody hybridoma cell lines 1-F2 and 1-G2 through hybridoma technology, can secrete corresponding NGAL-specific monoclonal antibodies, can successfully match and detect natural serum, and initially establishes Double-antibody sandwich ELISA method for detecting NGAL. the

In order to make the above and other objects, features and advantages of the present invention more comprehensible, preferred embodiments are described below in detail with accompanying drawings. the

Description of drawings

Figure 1 is the agarose gel electrophoresis analysis of the NGAL gene reverse transcription PCR amplification product. Among them, M is DNA Marker; 1-5 are five annealing temperatures of 60°C, 62°C, 64°C, 66°C, and 68°C, respectively. the

Fig. 2 is an analysis diagram of agarose gel electrophoresis results of pcDNA3.1-NGAL double-digested products with EcoRI and Acc65I. Among them, M is DNA marker; lane 1 is pcDNA3.1, and lane 2 is pcDNA3.1-NGAL. the

Figure 3 is a Western blot identification diagram of recombinant NGAL protein expression; wherein, 1 is the cell extracted protein; 2 is the cell culture supernatant. the

Fig. 4 is a purification diagram of recombinant NGAL protein. Among them, 1 is the ultraviolet absorption curve, 2 is the conductivity of the solution, 3 is the concentration of the purified antibody, and F1 and F2 respectively refer to the absorption peaks that appear at different times of protein purification. the

Figure 5 is the SDS-PAGE identification of the purified recombinant NGAL protein. Among them, M is the molecular weight standard; lane 1 is NGAL after purification. the

Figure 6 shows the detection of NGAL recombinant protein by ELISA identification monoclonal antibody 1-F2. Among them, the left 1 well is the negative control, and the left 2 to 12 wells are antibody dilution ratios of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000 , 1:256000, 1:512000, 1:1024000. the

Figure 7 shows the ELISA identification of monoclonal antibody 1-G2 to detect NGAL recombinant protein. Among them, the left 1-3 wells are the negative control, and the left 4-12 wells are the antibody dilution ratios of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1 :128000, 1:256000. the

Figure 8 is the Western blot identification of monoclonal antibody 1-F2 binding to NGAL recombinant protein; wherein, lane 1 is the recombinant protein NGAL; lane 2 is the irrelevant protein PCT. the

Figure 9 is the Western blot identification of monoclonal antibody 1-G2 binding to NGAL recombinant protein. Among them, lane 1 is the recombinant protein NGAL; lane 2 is the irrelevant protein PCT. the

Fig. 10 is a standard curve for detection of recombinant protein by standard ELISA. the

Figure 11 shows the results of standard ELISA detection of NGAL in clinical specimens (comparison between normal people and patients with kidney disease). the

Detailed ways

The invention amplifies the gene fragment of human neutrophil gelatinase-associated lipocalin NGAL by RT-PCR, connects the target gene with the eukaryotic expression vector pcDNA3.1, and constructs the eukaryotic expression recombinant plasmid pcDNA3.1-NGAL. The recombinant plasmid was stably transfected into 293 cells to express human neutrophil gelatinase-associated lipocalin. The NGAL eukaryotic expression plasmid constructed by pcDNA3.1 was secreted and expressed in 293 cells, and the molecular weight of the expressed recombinant protein was about 25kD as shown by SDS-PAGE electrophoresis analysis. The NGAL recombinant protein was purified by a protein affinity chromatography column, and the high-purity NGAL recombinant protein was obtained after purification by a nickel column, with a content of about 0.54 mg/mL. the

In the present invention, the 86th amino acid of NGAL protein may be glycosylated through bioinformatics prediction. In order to obtain the best specific monoclonal antibody capable of recognizing natural protein, NGAL recombinant protein expressed by 293 cells is used as an immunogen to immunize animals. Balb/c mice, through multiple cell fusions using hybridoma technology, using indirect ELISA method and cloning to screen out hybridoma cell lines that can stably secrete anti-NGAL specific monoclonal antibodies, ascitic fluid was passed through HiTrap IgG Purification HP affinity layer The monoclonal antibody was purified by column analysis, and the titer and affinity were determined for the obtained monoclonal antibody. After screening, two hybridoma cell lines 1-F2 and 1-G2 that stably secrete NGAL-specific monoclonal antibodies were obtained, both of which were identified as IgG1 by subtype identification. 6.39×10 ¹⁰ M ^-1 and 3.65×10 ⁹ M ^-1 , Western blot identification showed that both monoclonal antibodies had high specificity.

The present invention establishes an ELISA detection system for NGAL: using anti-NGAL antibody 1-F2 as a capture antibody and anti-NGAL antibody 1-G2 as a detection antibody, a standard double-antibody sandwich ELISA detection method is established, which can detect natural NGAL in urine, Based on this, a double-antibody sandwich ELISA method for detecting NGAL was successfully established. the

Materials and Sources

Experimental animals: Balb/c mice were purchased from the Experimental Animal Center of the Third Military Medical University (Chongqing, China). the

Cell lines and plasmids: 293 cell lines were purchased from China Center for Type Culture Collection (CCTCC), and pcDNA3.1 plasmid was purchased from Novagen. the

Cell culture medium and fetal bovine serum were purchased from Gibco, and Lipo2000 was purchased from Novagen. the

Enzymes and kits: Restriction enzymes EcoRI and Acc65I and plasmid extraction kits were purchased from OMEGA Company, and agarose gel recovery kits were purchased from Tiangen Company. the

Other reagents: HRP-goat anti-mouse IgG was purchased from Zhongshan Company. the

Example 1 Preparation of NGAL recombinant protein

1. Acquisition of the whole gene of NGAL coding sequence

According to the NGAL gene sequence (NM_005564.3) provided by GeneBank, the pseudo-cloned sequence of the human neutrophil gelatinase-associated lipocalin gene was determined, as shown in SEQ ID NO:2:

Design NGAL full-length primers: shown in SEQ ID NO:3 and 4 respectively

SEQ ID NO: 3: NGAL-1: 5'-TAAGGTACCCATGCCCCTAGGTCTCCTG-3'

SEQ ID NO::4:NGAL-2:5'-GGTGGAATTTTAGCCGTCGATACACTG-3'

The primers contain enzyme cutting sites: NGAL-1: Acc651; NGAL-2: EcoRI, synthesized by Invitrogen. the

The total RNA of leukocytes was extracted by Trizol Reagent method, cDNA of leukocytes was obtained by reverse transcription, and human NGAL was obtained by PCR amplification. A PCR system was established and PCR was performed at different annealing temperatures. Take 25 μL of the PCR product and analyze it by 1% TAE electrophoresis (Figure 1). The PCR products were recovered using Beijing Tiangen's agarose gel DNA recovery kit (ordinary spin column type). 2. NGAL recombinant plasmid construction and enzyme digestion identification

The pcDNA3.1 vector and the NGAL PCR recovery product were digested with EcoRI and Acc65I for 1 hour, respectively, and then subjected to DNA gel electrophoresis to recover the target band, which was ligated overnight at 4°C with T4 DNA ligase. Transform the ligation product into competent Escherichia coli DH5α, mix the bacterial solution evenly and spread it on the LB culture plate containing ampicillin, cultivate overnight in a constant temperature incubator at 37°C, pick a single colony and inoculate it on the LB liquid culture containing ampicillin the next day Base expansion overnight. the

The plasmid was extracted from the cultured bacteria with a plasmid extraction kit, and identified by 1% agarose gel electrophoresis after double digestion with EcoRI and Acc65Ⅰ. A specific band was seen at about 600 bp, indicating that the pcDNA3.1 vector had been inserted with a size of about It is a 600bp fragment (Figure 2). the

3. Sequencing of target fragments of NGAL recombinant plasmids

The pcDNA3.1-NGAL recombinant plasmid was sent to Shanghai Yingjun Company for sequencing, and the vector5.0 tool was used to analyze the homology between the sequencing results and the NGAL sequence in GeneBank. Sequencing results showed that the length of the inserted fragment was 597bp, which was completely consistent with the sequence of NGAL reported in GeneBank. the

4. Expression and identification of NGAL recombinant protein

1) Recovery and culture of 293 cells

The frozen 293 cells were quickly transferred from liquid nitrogen to a 37°C water bath, DMEM medium and serum were preheated at 37°C, and DMEM complete medium containing 10% calf serum was prepared. Transfer the cell suspension into the preheated complete medium at 37°C, centrifuge at 1000rmp/min for 5min, discard the supernatant, add 3-4mL complete medium containing 10% fetal bovine serum and blow it evenly, transfer it to a culture bottle, Place in a 5% CO ₂ , 37°C incubator for culture, and change the medium every day.

2) Transfection of 293 cells

The cultured 293 cells were inoculated into a six-well plate, and the next day, the cells were observed to cover the plate to a density of 90-95%, and the recombinant plasmid pcDNA3.1 was transfected into the 293 cells with the transfection reagent Lipofectamin2000 (Invitrogen Company). After 48 hours of culture, G418 (800 μg/ml) was added, cultured for 3 days, and the supernatant and cells were collected for Western blot identification (Figure 3). It was confirmed that the transfection was successfully cloned, and a stable expression cell line was obtained and expanded for culture. the

3) Expression and purification of NGAL recombinant protein

Collect the culture supernatant, and use a protein affinity chromatography column (Ni ²⁺ 22DA2Sepharose Fast Flow) to purify the recombinant protein (Figure 4). The finished protein was collected, and then samples were taken for SDS-PAGE electrophoresis (Figure 5). The purity was 95% as detected by HPLC.

5. NGAL recombinant protein concentration and purity identification

Determination of recombinant protein concentration by Lowry method: prepare a standard curve for analysis according to conventional methods and determine the concentration of the sample, calculate the average value of multiple tubes, and the measured concentration is 0.54 mg/ml. the

Example 2 Bioinformatics Analysis and Determination of Immunogen

1. Find the amino acid sequence of NGAL protein from GENEBANK, which consists of 198 amino acid residues. the

2. Online bioinformatics glycosylation prediction analysis 86 amino acids in NGAL protein may be glycosylated. In order to obtain the best specific monoclonal antibody that can recognize natural protein, select the full-length NGAL protein expressed by 293 cells As an immunogen, its amino acid sequence is specifically the same as SEQ ID NO:1. the

Embodiment 3 Preparation of anti-NGAL monoclonal antibody

1. Preparation of NGAL monoclonal antibody

The recombinant full-length NGAL protein was emulsified with an equal amount of Freund's complete adjuvant, and the antigen emulsification was carried out by double syringes. After the emulsification was completed, five Balb/c mice aged about 8 weeks were given basic immunization (100 μg/mouse) by multi-point subcutaneous injection in limbs and intraperitoneal injection. Two weeks later, NGAL whole antigen was emulsified with the same amount of Freund's incomplete adjuvant, and 5 Balb/c mice were boosted by multi-point injection in the limbs and intraperitoneal injection (100 μg/mouse); 2 One week later, the same method was used for booster immunization once again, and the mouse spleen was taken out after 7 days, and the splenocytes were subjected to cell fusion. the

The fusion process is to mix spleen cells and myeloma cells (Sp2/0) at a ratio of 8:1, and use polyethylene glycol as a fusion agent. Fused cells were suspended in HAT medium containing calf serum and cultured at 37°C in 6% CO ₂ . Screening was performed by ELISA. The detected positive clone wells were subcloned by the limiting dilution method, and cultured at 37°C in 5% CO ₂ until the culture medium of all cell growth wells were positive, then the expansion culture of the monoclonal antibody could be carried out .

The method of inducing monoclonal antibody in the peritoneal cavity of mice was used to expand the culture. Adult Balb/c mice were taken, and 0.5 mL of liquid paraffin was injected intraperitoneally, and hybridoma cells were injected intraperitoneally one week later. The hybridoma cells were suspended and mixed with physiological saline, and the number of cells was adjusted to 4×10 ⁵ cells/mL, and 0.5 mL of hybridoma cells were injected intraperitoneally into each Balb/c mouse. Collect ascites after 10-14 days.

2. Purification of NGAL monoclonal antibody

Purify the ascites after collecting it. The specific plan is: configure the buffers required for antibody purification: Binding Buffer (solution A, 20mmol/L sodium phosphate, 0.8mol/L (NH4) ₂ SO ₄ , pH7.5); Elution Buffer ( Solution B, 20mmol/L sodium phosphate, pH7.5); Regeneration buffer (solution C, 20mmol/L sodium phosphate, pH7.5, and 30% isopropanol by volume). Add ammonium sulfate to the monoclonal antibody ascitic fluid to make the final solubility consistent with that of ammonium sulfate in solution A, filter through a 0.45 μm filter membrane and wait for sample loading; select HiTrap IgG Purification HP column to connect to AKTA prime protein purification instrument, use A , B solution and C solution to fully wash the column, and after fully equilibrating with A solution, load the prepared sample from A tube, equilibrate the column with A solution after loading, remove impurity proteins, and then use B solution to elute and purify Column, collect the elution peak; adjust the pH of the eluted protein to 7.0-8.0, freeze the antibody in aliquots and store at -20°C; use C solution to regenerate the filler, and then use A solution to balance.

3. Identification of anti-NGAL monoclonal antibody subclasses

Mouse Monoclonal Antibody Isotyping Reagents (ELISA/Ouchterlony Double Diffusion, Stock No.ISO-2LOT114k4817) from Sigma Company of the United States was used, and the operation was carried out according to the instructions. the

1) Coating microtiter plate: Dilute NGAL recombinant protein with coating solution to the optimal working concentration of 5 μg/mL, add 100 μL antigen solution to each well, add 12 wells to each plant, overnight at 4°C, wash 5 times, and air-dry . the

2) Blocking: Add 350 μL of blocking solution to each well, incubate at 37°C for 1h to 1.5h, wash 5 times, and air dry. the

3) Add 100 μl of fresh hybridoma cell culture supernatant or diluted monoclonal antibody to each well, let stand at room temperature (20°C-25°C) for 1 hour, and shake for 3 minutes, 5 times in total. the

4) Dilute IgM, IgG2a, IgG2b, IgG3, IgG, IgA with antibody diluent at 1:2000. the

5) Add 100 μL of diluted antibody to each well, add each antibody to 2 wells, let stand at room temperature for 30 minutes, shake and wash for 3 minutes, a total of 5 times. the

6) Add 100 μL 1:1000 diluted rabbit anti-goat IgG antibody to each well, let stand at room temperature for 15 minutes, shake and wash for 3 minutes, a total of 5 times. the

7) Add 100 μL of substrate chromogenic solution to each well, and react at room temperature for 10 minutes to 15 minutes in the dark. h. Add 50 μL of stop solution to each well, observe the results, and determine the Ig subclass. the

4. Monoclonal antibody affinity identification

The affinity constant was determined by the Friguent method, and the NGAL antigen was dissolved in 0.05mol/L carbonic acid buffer (pH9.6), with a final concentration of 1 μg/ml, and the solution was added to a 96-well plate in an amount of 100 μl per well and kept at 4°C. After overnight, the plate was blocked with PBS solution containing 1% BSA, and the plate was dried and stored at 4°C for future use. the

Establishment of the reaction system: the initial concentration of the anti-NGAL monoclonal antibody in the reaction system was 20 ng/mL. The initial concentration of NGAL antigen was diluted downward from 1000ng/ml (1250x l0mol/L) to form 8 reaction systems, and reacted at 37°C for 1 hour; Wash the plate 5 times after incubating at 37°C for 1 hour; add 100 μl of goat anti-mouse secondary antibody to each conjugate solution, react for 1 hour at 37°C, wash the plate 5 times, add the substrate for 15 minutes of color development, and then add the stop solution. The OD450 value was calculated to calculate the antigen binding rate of each reaction system to calculate the affinity constant, and the affinity of 1-F2 was 6.39×10 ¹⁰ M ^-1 , and that of 1-G2 was 3.65×10 ⁹ M ^-1 .

5. ELISA identification of anti-NGAL monoclonal antibody

Dilute the purified NGAL antigen to 5 μg/ml with coating diluent, add 100 μl to each well of the ELISA strip plate, and coat overnight at 4°C. The next day, take out the plate, discard the antigen, and wash the plate. Add monoclonal antibodies to be identified at 1:1000, 1:2000, 1:4000, 1:8000, 1:16000..., add 100μl/well to the corresponding wells, and make blank and positive and negative control wells . Incubate at 37°C for 1 hour, wash the plate, add secondary antibody, dilute HRP-labeled goat anti-mouse IgG at 1:3000, add 100 μl/well to the corresponding wells, and incubate at 37°C for 40 minutes. Discard the solution, wash the plate, pat dry, add 100 μl/well of substrate solution, develop color for 10 minutes in the dark, and add 50 μl/well of stop solution. The results showed that the anti-NGAL monoclonal antibodies 1-F2 and 1-G2 presented a concentration gradient visible to the naked eye (Figure 6, Figure 7). the

6. Western blot identification of anti-NGAL monoclonal antibody

1) Electrophoresis of 3 μl of Marker, 20 μl of NGAL antigen, and 20 μl of irrelevant protein procalcitonin (PCT). the

2) After electrophoresis, remove the gel and place it in transfer buffer to equilibrate for 10 minutes. the

3) The 0.22μm PVDF membrane was first treated with anhydrous methanol for 20s, and then washed with ddH2O for 5min. Then soak in Transfer Buffer for more than 5 minutes. At the same time, soak the filter paper in Transfer Buffer. the

4) Membrane transfer: Arrange from bottom to top, filter paper-PVDF membrane-glue-filter paper, discharge air bubbles, put into membrane transfer apparatus, 18V constant voltage electrotransfer for 1.5h. the

5) After the membrane transfer is completed, clear protein markers can be seen on the membrane, wash twice with ddH2O, and wash with TBST for 5 minutes. Place the transfer membrane in blocking solution and block at room temperature for 2 h. the

6) Discard the blocking solution, rinse the membrane 3 times with 1×TBST, 15min each time. the

7) Add purified antibody diluted 1:1000 and incubate overnight at 4°C. the

8) Rinse the membrane 3 times with 1×TBST, 15min each time. the

9) Add HRP-goat anti-mouse IgG antibody diluted to 1:10000 and incubate at 37°C for 3h. the

10) Wash 3 times with 1×TBST, 15min each time. the

11) Use chemiluminescence to develop color, after developing and fixing, observe the analysis results and take pictures. Clear bands of anti-NGAL monoclonal antibody binding can be seen at 25KD of the recombinant NGAL protein (Figure 8, Figure 9). the

NGAL in the detection standard of the double antibody sandwich ELISA method of embodiment 4

1. HRP-labeled antibody

This method uses _NaIO4 to oxidize the sugar molecules on the surface of HRP into aldehyde groups, and then combine with the amino groups of the antibody protein. The yield of the enzyme-labeled antibody is high. The specific steps are as follows: Weigh 10mg of HRP enzyme, add 2mL Prepare pure water as 5mg/ml HRP liquid, add 34μL per mg of HRP enzyme, add 340μL of NaIO ₄ , place in the dark at 4°C, after 1h, add 250μL of ethylene glycol according to the amount of 250μL per mg of HRP enzyme, keep away from light Place it at 4°C for 30 minutes, then transfer it to a dialysis bag, and dialyze overnight with 1mM acetate buffer (pH 4.0-4.4). Antibody preparation: 0.01M PBS buffer was dialyzed overnight at 4°C, and the protein concentration was determined. Labeling: Mix antibody and HRP enzyme at 1:1 (mass ratio), add 1mol/L Na ₂ CO ₃ buffer (1:80), adjust the pH of the reaction to 9.5, and react at 25°C for 2-3h. Termination: freshly prepare 0.1mol/L NaH ₄ B (4mg/mL), add 47μl per mg of HRP enzyme. After standing at 4°C for 2 hours, transfer to a dialysis bag. Dialyze with 0.01mol/L PBS buffer (pH7.0-7.2) overnight, change the solution at least twice during the period, and avoid light. Aliquot storage: the labeled antibody is divided into light-proof EP tubes, and the antibody titer is determined. Store at -20°C.

2. Antibody pairing and detection of clinical samples

1) Specimen collection:

Urine samples from patients with clinically diagnosed kidney disease were collected from the First and Second Affiliated Hospitals of the Third Military Medical University. During the retrieval process, they were stored in ice packs to keep the samples fresh. The supernatant was collected by centrifugation and preservatives, and then labeled and packaged in In a 1.5ml Ep tube, store at -80°C, and enter the basic information of the patient into the computer and make a number. the

2) Specimen testing:

Select monoclonal antibody 1-F2 as the capture antibody, enzyme-labeled monoclonal antibody 1-G2 as the detection antibody, recombinant protein NGAL as the standard, and the sample detection method is as follows:

a. Coating microtiter plate: Dilute the antibody to 5 μg/ml with coating solution, add 100 μl to each well, and coat overnight at 4°C. the

b. Wash the ELISA plate coated overnight 3 times and shake dry. the

c. Blocking: Add 350 μl of blocking solution to each well, incubate at 37°C for 1 hour, wash 3 times, and shake dry. the

d. Dilute the NGAL recombinant protein starting from 500ng/ml with antibody diluent, add 100μl to each well, and use the first well as a blank control to make a standard curve. 33 cases of renal injury specimens and 24 cases of normal specimens were detected, 100 μl was added to each well, and incubated at 37°C for 1 hour. the

e. Wash the plate 3 times and shake dry. the

f. Dilute the corresponding enzyme-labeled secondary antibody at 1:5000, add 100 μl to each well, and incubate at 37°C for 30 minutes. the

g. Wash the plate 5 times and shake dry. the

h. Add 100 μl of substrate chromogenic solution to each well, and develop color at room temperature for 5 minutes in the dark. the

i. Add 50 μl of stop solution to each well, read the plate, and determine the content of NGAL in clinical samples according to the standard curve. the

3) Statistical analysis

Using SPSS 13.0 software, the results of NGAL measured by ELISA in the myocardial infarction group and the normal control group were statistically analyzed. the

The results showed that the standard curve was successfully drawn using the paired antibody 1-F2-1-G2 and the NGAL recombinant protein standard (Figure 10), and it was found that 33 Cases of kidney disease patients (285.854ng/ml) were significantly higher than 24 cases of normal serum (6.155ng/ml; P<0.01) (Figure 11), indicating that paired antibodies can be successfully applied to the detection of NGAL in natural serum. the

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make some modifications and improvements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims. the

sequence listing

the

Claims (10)

1. A hybridoma cell line producing anti-human neutrophil gelatinase-associated lipocalin specific monoclonal antibody, the preservation number of which is CCTCC NO.C2012197. 2. A hybridoma cell line producing a specific monoclonal antibody against human neutrophil gelatinase-associated lipocalin, the preservation number of which is CCTCC NO.C2012198. 3. the hybridoma cell strain as claimed in claim 1 or 2, is characterized in that, after the aminoacid sequence shown in SEQ ID NO:1 immunizes Balb/c mouse, mouse spleen B lymphocyte fuses with myeloma cell produced after cultivation. 4. the hybridoma cell strain as claimed in claim 1 or 2, is characterized in that, the gene sequence of described human neutrophil gelatinase-associated lipocalin is as shown in SEQ ID NO:2. 5. A monoclonal antibody produced by the hybridoma cell line of claim 1. 6. The monoclonal antibody according to claim 5, wherein the subclass of the monoclonal antibody belongs to IgG1. 7. A monoclonal antibody produced by the hybridoma cell line of claim 2. 8. A monoclonal antibody according to claim 7, wherein the subclass of said monoclonal antibody belongs to IgG1. 9. The application of the monoclonal antibody as claimed in claim 5 and/or 7 in the preparation of human neutrophil gelatinase-associated lipocalin immunoassay preparation. 10. A kit for diagnosing or detecting renal injury, characterized in that it comprises the monoclonal antibody as claimed in claim 5 and/or 7.

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