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Novel reagent and kit for renal injury monitoring

A mixture, antibody technology, applied in the field of biotechnology and immunity, can solve the problems of antibody titer difference, unsatisfactory immunogenicity, instability and so on

Active Publication Date: 2012-11-14
SHANGHAI SUNFORY BIOPHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although NGAL has been known as a marker for disease diagnosis, due to the unsatisfactory immunogenicity of its full-length protein itself, there are still defects in antibody titer difference and instability in the process of obtaining antibodies by immunization

Method used

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  • Novel reagent and kit for renal injury monitoring
  • Novel reagent and kit for renal injury monitoring
  • Novel reagent and kit for renal injury monitoring

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1, the mixture of specific NGAL amino acid fragment sequence and its products as immunogen

[0046] The full-length amino acid sequence of NGAL protein is as follows:

[0047] The full-length amino acid of NGAL protein (human source, 198aa), the sequence is as follows (SEQ ID NO: 3):

[0048] MPLGLLWLGL ALLGALHAQA QDSTSDLIPA PPLSKVPLQQ NFQDNQFQGK

[0049] WYVVGLAGNA ILREDKDPQK MYATIYELKE DKSYNVTSVL FRKKKCDYWI

[0050] RTFVPGCQPG EFTLGNIKSY PGLTSYLVRV VSTNYNQHAM VFFKKVSQNR

[0051] EYFKITLYGR TKELTSELKE NFIRFSKSLG LPENHIVFPV PIDQCIDG

[0052] Due to the disadvantages of difficult synthesis and low immunogenicity of the full-length sequence as an immunogen, the inventors used conventional methods to artificially synthesize sequence fragments of various lengths based on the above full-length sequence, and used the fragments as immunogens to repeatedly test various The immune effect of the immunogen. Finally, a suitable immunogen was obtained, which included...

Embodiment 2

[0056] Example 2, Production of specific polyclonal anti-NGAL antibodies

[0057] The immune sera produced by immunizing New Zealand white rabbits were prepared by using the mixture of specific polypeptides prepared in Example 1 above as immunogens coupled with hemocyanin (KLH). The methods and procedures of animal immunization are as follows:

[0058] 1) Two New Zealand male rabbits (#YZ2391; #YZ2392) were immunized, and the pre-immune serum was collected and stored at -20°C.

[0059] 2) For the first immunization, take the immunogen coupled with hemocyanin (KLH) and the equivalent amount of Freund's complete adjuvant emulsified antigen (antigen: Freund's complete adjuvant = 1:1 (v / v)), multiple points on the back injection. The injection volume for the first immunization was 400 μg of the polypeptide mixture.

[0060] 3) After 14 days, the antigen was emulsified with Freund's complete adjuvant (antigen: Freund's complete adjuvant = 1:1 (v / v)) and repeated back multi-point...

Embodiment 3

[0085] Embodiment 3, test kit

[0086] Described test kit comprises:

[0087] Container 1, and the polyclonal antibody prepared in the aforementioned Example 2 contained in the container;

[0088] Container 2, and the horseradish peroxidase-labeled goat anti-human IgG polyclonal antibody contained in the container.

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Abstract

The invention relates to a novel reagent and a kit for renal injury monitoring. The inventor identifies critical antigenic determinants from full-length NGAL (neutrophil gelatinase-associated lipocalin). And a short peptide mixture containing the critical antigenic determinants is adopted as an immunogen to obtain a specific polyclonal antibody against NGAL. The antigen fragments and its polyclonal antibody provided in the invention have the advantages of simple preparation method, high titer, strong specificity, and high sensitivity.

Description

technical field [0001] The present invention belongs to the fields of biotechnology and immunology; more specifically, the present invention relates to novel reagents and kits for monitoring renal injury. Background technique [0002] Neutrophil gelatinase-associated lipotransportin (NGAL), previously known as a marker of neutrophil activation, is released when invading microorganisms cause neutrophil degranulation and exocytosis of granule proteins into the blood. Later, NGAL was reported as a urinary biomarker to detect the early onset of renal tubular cell injury. US patent application 2005 / 0272101 discloses the use of NGAL in serum for the same purpose. PCT application WO2006 / 066587 discloses how to use NGAL in urine or plasma or serum for detection. Therefore, NGAL has been studied and applied as a marker of disease onset. Human NGAL comprises a total of 198 amino acids. [0003] Although NGAL has been known as a marker for disease diagnosis, due to the unsatisfact...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C07K19/00C07K16/18C12N15/12G01N33/53G01N33/543
Inventor 蔡逸强闵洪中李锐
Owner SHANGHAI SUNFORY BIOPHARM INC
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