Mouse anti-human neutrophil gelatinase-associated lipocalin (NGAL) monoclonal antibodies and hybridoma cell strains and application thereof

A hybridoma cell line and monoclonal antibody technology, applied in the direction of anti-animal/human immunoglobulin, biochemical equipment and methods, instruments, etc., can solve the problems of high color background, difficult promotion, and difficult quality control, etc. Achieve the effect of important application prospects

Inactive Publication Date: 2012-11-14
GETEIN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The specificity and consistency of the NGAL antibody prepared by this method are limited, the color background is high when used in immunohistochemistry, the quality is not easy to control, and it is difficult to promote in clinical application
There are NGAL monoclonal antibodies on the market, but these antibodies are mainly used for laboratory research and cannot be used for clinical detection of NGAL in human cells.

Method used

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  • Mouse anti-human neutrophil gelatinase-associated lipocalin (NGAL) monoclonal antibodies and hybridoma cell strains and application thereof
  • Mouse anti-human neutrophil gelatinase-associated lipocalin (NGAL) monoclonal antibodies and hybridoma cell strains and application thereof
  • Mouse anti-human neutrophil gelatinase-associated lipocalin (NGAL) monoclonal antibodies and hybridoma cell strains and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: Preparation of NGAL recombinant protein

[0024] 1. Construction of NGAL prokaryotic expression vector

[0025] Synthesize the primer sequences in Table 1, use the nested (Gene SOEing) PCR technique to add all the primers for PCR amplification, then use the primary PCR product as a template, and use primer 1 and primer 24 as upstream and downstream primers to perform secondary PCR amplification. The target fragment of NGAL (as shown in the gene sequence of SEQ ID No.2) was obtained. Link it into the prokaryotic expression plasmid pET32a, through CaCl 2 Transform Escherichia coli Top10 competent cells by method, pick colonies and inoculate them in LB medium with 50 μg / mL ampicillin, culture overnight at 37°C with shaking at 220 r / min, use the bacterial solution for PCR identification, and send positive clones for sequencing verification.

[0026] Table 1 Primer sequences

[0027]

[0028] 2. Expression of NGAL-Trx fusion protein

[0029] Sequence the...

Embodiment 2

[0038] Example 2: Animal immunization

[0039] Using purified human NGAL recombinant protein as antigen, immunize 6-week-old BALB / c mice by conventional methods, and inject 200 μg of antigen subcutaneously for the first basic immunization, using complete Freund’s adjuvant; immunize once every 2 weeks, a total of three times, 100 μg / One mouse / time, two weeks after the last basic immunization, intraperitoneal injection of booster immunization, 50 μg / mouse, tail-docked blood sampling to detect the serum antibody titer of the mice reached 1:10 4 Above, immunization is completed.

Embodiment 3

[0040] Embodiment 3: the preparation of hybridoma cell

[0041] 1. Preparation of feeder cells

[0042] The peritoneal macrophages of BALB / c mice were used as feeder cells. The preparation method was as follows: take a BALB / c mouse, kill the mouse by pulling the neck, disinfect the body surface with 75% alcohol, and remove the Lift the abdominal skin to expose the peritoneum. Wipe the peritoneum with 75% alcohol cotton ball for disinfection. Inject 10ml of DMEM culture solution into the peritoneal cavity with a syringe, rinse repeatedly, recover the washing solution, centrifuge at 1000r / min for 10 minutes, discard the supernatant, and obtain feeder cells. The pellet was resuspended in DMEM medium containing 15% calf serum, and the cell concentration was adjusted to 2×10 5 pieces / ml. Add the above cell suspension into a 96-well plate, 0.1ml per well, set at 37°C, 5% CO 2 overnight in an incubator for use the next day.

[0043] 2. Preparation of immune splenocytes

[0044...

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Abstract

The invention discloses a hybridoma cell strain 3B1 and a hybridoma cell strain 4C2 and a mouse anti-human NGAL monoclonal antibody 3B1 and a mouse anti-human NGAL monoclonal antibody4C2 produced respectively. The two kinds of monoclonal antibodies belong to a same immunoglobulin G (IgG) 2a subclass, but identify various epitopes and can perform specific binding to NGAL recombinant protein with amino acid sequences represented by SEQ ID NO.1. The two monoclonal antibodies can be applied to detect the concentration of the NGAL in human body fluids and have significant application prospect.

Description

technical field [0001] The invention belongs to the field of monoclonal antibodies, and relates to hybridoma cell line 3B1 and hybridoma cell line 4C2, and the mouse anti-human NGAL monoclonal antibody 3B1 and mouse anti-human NGAL monoclonal antibody 4C2 produced respectively, which can be used to detect human body fluid Concentration of NGAL. Background technique [0002] In 1993, neutrophil gelatinase-associated lipocalin (NGAL) was first discovered in neutrophils. Studies at home and abroad have shown that NGAL is related to inflammation, embryonic development, immune response, signal transduction, and the occurrence and development of various tumors. The expression of NGAL was significantly enhanced in ovarian cancer cell lines, colon tumors and breast cancer. Immunohistochemistry demonstrated that NGAL increased from negative to strongly positive in pancreatic cancer, NGAL was strongly positive in lung cancer, and the staining was strongest in bronchoalveolar cell ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/18G01N33/577C12R1/91
Inventor 苏恩本陈玲杨艳沈小娟孔婷婷
Owner GETEIN BIOTECH
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