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Chemiluminiscence kit for quantitatively detecting human chitinase 3-like protein 1

A chemiluminescence reagent and chemiluminescence technology, applied in the field of immunoassay, can solve the problems of high detection technology threshold, narrow detection linear range, difficulty in sample acquisition, etc., achieve semi-automatic or fully automatic batch operation, and improve sensitivity and accuracy , the effect of reducing blood sample volume requirements

Pending Publication Date: 2020-11-20
SHANGHAI CHANGZHENG HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the detection of chitinase 3-like protein 1, there are mainly enzyme-linked immunoassay (ELISA), immunohistochemistry (IHC), etc., and the detection of chitinase 3-like protein 1 by ELISA is a serological test. Limited by its methodology, the detection sensitivity is low, and the detection linear range is narrow; the detection samples of immunohistochemical method are derived from human tissues / cells, and it is difficult to obtain samples, the detection cost is high, and the detection technology threshold is relatively high

Method used

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  • Chemiluminiscence kit for quantitatively detecting human chitinase 3-like protein 1
  • Chemiluminiscence kit for quantitatively detecting human chitinase 3-like protein 1

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1: the preparation of kit of the present invention

[0046] 1. Preparation of human YKL-40 monoclonal antibody or polyclonal antibody coated chemiluminescence plate

[0047] Dilute the human YKL-40 monoclonal antibody or polyclonal antibody to 2-8ug / ml with phosphate buffer or carbonate buffer, add 100ul / well to the chemiluminescent plate, and place it at 4°C for 16-20 hours Finally, take out the chemiluminescent plate, dry the residual liquid, and use bovine serum albumin, casein, gelatin, animal or human serum, etc. to block, add samples at 120ul-300ul / well, and place at 37°C for 2 hours or at 4°C Leave it for 16-20 hours, shake off the residue, dry and store at 4°C for later use.

[0048] 2. Preparation of biotin-labeled human YKL-40 monoclonal antibody or human YKL-40 polyclonal antibody

[0049] Prepare 10 mM NHS-DPEG4-Biotin working solution with 0.1 M PBS pH7.2. Add the human YKL-40 monoclonal antibody or polyclonal antibody into an appropriate amou...

Embodiment 2

[0067] Embodiment 2: the method for detecting human YKL-40 using the kit of the present invention

[0068] The method for detecting human YKL-40 using the kit of the present invention comprises the following steps:

[0069] a) Dilute the concentrated washing solution 20 times to the working concentration;

[0070] b) Dilute the concentration of the standard substance according to the dilution gradient, and add it to the chemiluminescence plate, 50ul / well, and mark it;

[0071] c) Add serum / plasma / cell lysate according to 50ul / well to the corresponding plate wells, and make a mark;

[0072] d) Take the biotin-labeled human YKL-40 antibody working solution and add 50ul / well to the corresponding plate well;

[0073] e) Incubate at 37°C for 2 hours, take out, wash 4 times with 250ul washing solution per well, and pat dry;

[0074] f) Take streptavidin-labeled peroxidase working solution or streptavidin-labeled alkaline phosphatase working solution, add 50ul / well to the correspo...

Embodiment 3

[0078] Embodiment 3: the methodological evaluation of kit of the present invention

[0079] a) Standard curve

[0080] The test kit of the present invention uses the concentration value of the calibrator (0 pg / ml is eliminated, this value is considered as the background of the reaction plate, and is not included in the standard curve range) as the X value, and the luminescence value RLU corresponding to the concentration detection is used as the Y value, so as to make One-dimensional equation. like figure 1 As shown, the drawn standard curve regression equation is y=108.3x-15630, R 2 = 0.99634.

[0081] b) Minimum detection limit

[0082] The kit of the present invention detects the minimum detection limit reference product (62.5pg / ml) 10 times while detecting the standard curve, and calculates the average concentration value (x) and standard deviation (SD) of the minimum detection limit reference product through the standard curve . According to the formula: detection l...

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Abstract

The invention relates to the field of immunoassay, in particular to a chemiluminiscence kit for quantitatively detecting human chitinase 3-like protein 1 (YKL-40 / CHI3L1). The kit comprises a chemiluminescent plate coated with a human YKL-40 monoclonal antibody or a human YKL-40 polyclonal antibody, a biotin-labeled human YKL-40 monoclonal antibody or a human YKL-40 polyclonal antibody, a streptavidin-labeled enzyme substrate and an enzymatic luminescent substrate. An enzymatic chemiluminiscence method is adopted; a human YKL-40 monoclonal antibody or a human YKL-40 polyclonal antibody in a chemiluminiscence plate is used as a capture antibody; a biotin-labeled human YKL-40 monoclonal antibody or a human YKL-40 polyclonal antibody is used as a detection antibody, an enzyme substrate is usedas a catalytic enzyme to catalyze a luminous substrate to emit light, and a biotin amplification system is matched for detection. The detection sensitivity and accuracy of the kit are improved, and large-flux sample detection is facilitated.

Description

Technical field [0001] The invention relates to the technical field of immunoassay, specifically, it is a chemiluminescence kit for quantitative detection of human chitinase 3-like egg 1 (YKL-40 / CHI3L1). Background technique [0002] Chitinase-3-like-1protein (CHI3L1 for short), also known as cartilage glycoprotein 39 (YKL-40), is a member of the glycosyl hydrolase 18 family, but it lacks hydrolytic activity, CHI3L1 The protein is involved in cell proliferation and angiogenesis. Current academic research has found that chitinase 3-like protein 1 is related to asthma and allergic diseases as well as various tumors. [0003] At present, the detection of chitinase 3-like protein 1 is mainly used in scientific research, and clinical research is still in its infancy. For the detection of chitinase 3-like protein 1, currently there are mainly enzyme-linked immunoassay (ELISA), immunohistochemistry (IHC), etc. The detection of chitinase 3-like protein 1 by enzyme-linked immunoass...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/573G01N33/535G01N21/76
CPCG01N33/577G01N33/573G01N33/535G01N21/76
Inventor 唐昊缪瑜姗仲彩铭胡家勋王伟
Owner SHANGHAI CHANGZHENG HOSPITAL
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