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Immunoassay for chromogranin A, antibodies and kit

A kit and a technology for detecting antibodies, applied in the direction of anti-animal/human immunoglobulin, biological testing, immunoglobulin, etc., can solve problems such as assay methods, false negatives, inferior polyclonal antibodies, etc.

Active Publication Date: 2015-04-08
DIASOURCE IMMUNOASSAY SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The methods known in the art have the disadvantage of false positive or false negative results, mainly due to cross-reactivity with CGA-derived peptides which are usually also present close to the native CGA
Assays using polyclonal antibodies in particular suffer from the disadvantage of
The use of monoclonal antibodies has been suggested in order to overcome this problem, however, the monoclonal antibody-based CGA assays described in the prior art actually perform even worse than polyclonal antibody-based assays

Method used

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  • Immunoassay for chromogranin A, antibodies and kit
  • Immunoassay for chromogranin A, antibodies and kit
  • Immunoassay for chromogranin A, antibodies and kit

Examples

Experimental program
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Effect test

Embodiment 1

[0057] Monoclonal antibodies

[0058] has basically passed and Milstein's method in 1975 prepared monoclonal antibodies (also expressed as antibody 2E8) that specifically reacted with the polypeptides represented by the 236th to 251st amino acid sequences of the human CGA amino acid sequence, and with the 264th to 279th A monoclonal antibody (also denoted herein as antibody 1H6) specifically reacts with the polypeptide indicated by the amino acid sequence.

[0059] Antibody 2E8 was used as a capture antibody in the Elisa; antibody 1H6 was used as a detection antibody bound to HRP.

Embodiment 2

[0061] Calibration curve of CGA ELISA

[0062] A set of 6 calibrators are provided, the values ​​of which are calibrator 1: 0nmol / L, calibrator 2: 1nmol / L, calibrator 3: 5nmol / L, calibrator 4: 15nmol / L, calibrator 5: 30nmol / L, calibrator 6: 50nmol / L.

[0063] The calibrator in the kit is a synthetic peptide corresponding to CGA. Peptide calibrators set up to produce responses equivalent to purified native CGA fragments (Stridsberg, M et al 1993, Stridsberg et al 1995)

[0064] The OD of the six calibrators was plotted against the nmol / L value to construct a calibration curve.

[0065] The results of calibrator measurements are in Table 1 and figure 1 described in.

[0066] Table 1

[0067]

Embodiment 3

[0069] Correlation between the test system of embodiment 1 and commercial test kit (Dako)

[0070] For this comparison, the example of the present invention was used alongside the assay kit of the Chromogranin A ELISA kit (code K0025) commercially available from Dako (Dako Denmark A / S; Produktionsvej 42; DK-2600 Glostrup; Denmark). The testing system of 1 tests a batch of patient samples. The commercially available assay kit is widely used and is based on a simplified double polyclonal antibody sandwich assay in which the sample is simultaneously treated with a peroxidase-conjugated anti-chromogranin A antibody on a surface coated with anti-chromogranin Antibody A was incubated in the microwells. All antibodies in this test kit are reported to be rabbit polyclonal antibodies. Use the Dako assay kit according to the manufacturer's instructions. In the assay, the OD is read at 450nm and 650nm.

[0071] The results of this comparison are shown in the figure 2 middle. Her...

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Abstract

The invention relates to monoclonal antibodies which are reactive with an epitope in the polypeptide represented by amino acid sequence 236 to 251 or 264 to 279 of the human CGA amino acid sequence. The invention further relates to the use of these monoclonal antibodies in an immunoassay for CGA, to immunoreagents comprising any of these two antibodies, and to test kits for the determination of CGA containing immunoreagents based on both of the monoclonal antibodies.

Description

field of invention [0001] The present invention relates to a method for the determination of chromogranin A (CGA). It also relates to monoclonal antibodies against CGA and immunoreagents based thereon which can be used in the assay of the present invention, and test kits for use in the assay of CGA. Background of the invention [0002] Chromogranin (CG) and secretogranins constitute a family of acidic proteins that are co-stored with neurotransmitters and peptide hormones in the brain and diffuse neuroendocrine system (Winkler, H. & Fischer-Colbrie, R., 1992). Although these proteins are the products of different genes, they share some general structural properties, such as an abundance of acidic amino acid residues and several pairs of basic amino acids that serve as potential sites for post-translational cleavage. CG is co-stored and co-released with neuropeptides and hormones in neuroendocrine cells throughout the body. A role for CG in hormone granule production and ho...

Claims

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Application Information

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IPC IPC(8): C07K16/18G01N33/53G01N33/577G01N33/68
CPCG01N33/74C07K2317/34G01N33/577G01N33/68C07K16/18C07K16/26C07K16/30G01N33/53
Inventor 麦茨·斯特里斯贝格英韦·索马林
Owner DIASOURCE IMMUNOASSAY SA
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