Turbidimetric immunoassay for assessing human cysteine proteinase inhibitor C

A technology of cysteine ​​protease and immunoassay, which is applied to measuring devices, instruments, scientific instruments, etc., to achieve the effects of reducing measurement time, improving linearity, and reducing interference

Active Publication Date: 2009-01-28
根田股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0034] Surprisingly, the above-mentioned problems associated with the prior art nephelometric immunoassays for the measurement of cystatin C can be solved by Explained turbidimetric method and solved

Method used

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  • Turbidimetric immunoassay for assessing human cysteine proteinase inhibitor C
  • Turbidimetric immunoassay for assessing human cysteine proteinase inhibitor C
  • Turbidimetric immunoassay for assessing human cysteine proteinase inhibitor C

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0263] Synthesis Example 1: Preparation of polyclonal poultry serum and its affinity purification

[0264] a) Preparation of poultry serum

[0265] Polyclonal IgY antibodies to cystatin C can be produced using the following immunization methods:

[0266] Use 2-4 hens per immunization experiment. Before immunization begins, a pair of eggs is collected. IgY purified from these eggs will serve as control IgY. 100 μg of highly purified human cystatin C (purified from the urine of patients with renal tubular proteinuria) in phosphate buffer was emulsified with Freund's complete adjuvant and injected into the breast muscle of hens middle. Injections were repeated every 4 weeks. 10 weeks after initiation of injections, eggs were collected. Egg yolk was isolated from the egg, and the IgY fraction from the egg yolk was isolated by ammonium chloride precipitation in a conventional manner following prior art methods of egg antibody isolation (see, e.g., Larsson A, Baaloew R-M, Lind...

Embodiment 2

[0270] Synthesis Example 2: Preparation of Monoclonal Anti-Human Cystatin C Antibody

[0271] Preparation of monoclonal anti-human cystatin C antibodies can be carried out as follows, by utilizing methods well known in the art, for example, from Harlow et al., 1988, "Antibodies: a Laboratory Manual" "Section 6, Cold Spring Harbor Press, New York, USA. Human PSA was isolated from human semen plasma as described by Sensabaugh et al., 1990, J. Urology 144, 1523.

[0272] Mice were immunized with 4 injections of 50 μg human Cystatin C in RAS (RIBI Adjuvant System) at regular intervals. Four months after the first injection, spleens from immunized mice were isolated using the polyethylene glycol method described by G. Galfre et al., 1981, Methods in Enzymology, 73, 3-46. Lymphocytes were fused with the myeloma cell line SP2 / 0-Ag14.

[0273] Hybridomas secreting antibodies against cystatin C were identified by the following screening ELISA: microtiter plates were coated with rabb...

Embodiment 3

[0277] Synthesis Example 3: Preparation of Anti-cystatin C Immune Particles

[0278] a) Coupling method - standard method:

[0279] (1) Buffers and reagents

[0280]

[0281] In order to have more signal / mg antibody from the polymer particles, the pH of the coupling buffer is adapted in a special way to the pi of the antibody to be coupled. Additionally, the antibodies were diluted with albumin / transferrin prior to conjugation (see below). For conjugation, when using monoclonal antibodies, choose a pH that is half a unit above the pi of the antibody. Polyclonal antibodies have a very variable pi, and therefore a pH 8.8 polyclonal antibody was used. The selected pH was obtained by mixing the above mentioned PBS and borate buffer.

[0282] The following standard methods outline two simple one-step methods for attachment by physical adsorption of antibodies to chloromethyl groups. The method was used for a 1 μm chloromethyl latex at 1% solids. This reaction can be easi...

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Abstract

There is a demand for improved turbidimetric immunoassays for human Cystatin C in biological samples, especially in human clinical samples of body fluids. The present invention provides a turbidimetric immunoassay method and reagent set enabling measurement of human Cystatin C by turbidimetric methods, resulting in a surprisingly stronger and faster turbidimetric signal than in the present state of the art. The increased and faster signal is accomplished by the use of new reagents and compositions, and enables shorter assay times and kinetic reading with a stronger signal, improving overall assay speed and quality. Improved robustness to lipid interference and improved linearity is achieved.

Description

[0001] The present invention relates to an improved turbidimetric immunoassay for the assessment of human cystatin C in samples of human body fluids by utilizing specific nanoparticle-antibody conjugates; by utilizing said assay to assess Methods of glomerular filtration rate of patients; corresponding reaction kits; improved nanoparticle-antibody conjugates and their use for the above mentioned medical purposes. Background of the invention: [0002] Cystatin C was discovered and characterized in 1981 by A. Grubb and H. Lovberg [Grubb AO, et al: Proc. Natl. Acad. Sci. USA 1982; 79]. Cystatin C was the first protein discovered in the superfamily of proteins later named Cystatin. The cystatin protein superfamily is a group of proteins that inhibit cysteine ​​protease enzymes. Other biological functions of cystatin C are currently being investigated. In contrast to other members of the cystatin superfamily, cystatin C is present in all body fluids, and the overall composition o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N
Inventor 卡特林·森德汤姆·尼尔森
Owner 根田股份有限公司
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