Kit for preparing CA125 surface Tn antigen by magnetic particle chemiluminescence immunoassay and preparation method thereof

A chemiluminescence immunoassay, CA125 technology, applied in chemiluminescence/bioluminescence, analysis, fermentation and other directions by making materials undergo chemical reactions, which can solve the problem of difficult integration and full automation, complicated operation steps, and large fluctuations in final results. And other issues

Active Publication Date: 2019-05-21
THE OBSTETRICS & GYNECOLOGY HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] 2) This patent oxidizes and derivatizes the coated plate, intending to solve the non-specific adsorption phenomenon of lectin VVA and antibody. Although the sugar chain of the Fc segment is blocked, it is unavoidable regardless of the oxidant or the derivatization reagent. The impact on the Fab segment, thereby affecting the activity of the antibody;
[0008] 3) This patent achieves signal amplification through the specific combination of biotin-lectin VVA and streptavidin-horseradish peroxidase. This method has uncertainty and cannot guarantee that each streptavidin Molecule binds only 1 biotin molecule, thereby impairing the final test result value;
[0009] 4) The operation steps of this patent are complicated, and the react

Method used

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  • Kit for preparing CA125 surface Tn antigen by magnetic particle chemiluminescence immunoassay and preparation method thereof
  • Kit for preparing CA125 surface Tn antigen by magnetic particle chemiluminescence immunoassay and preparation method thereof

Examples

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Embodiment 1

[0076] The kit for the magnetic particle chemiluminescence immunoassay of CA125 surface Tn antigen in this embodiment includes a magnetic separation reagent coupled with the Fab fragment of the Ov185 monoclonal antibody, Biotin-VVA reagent containing sialidase, SA-Biotin-ALP reagent, chemical Luminescence substrate solution, washing solution and calibrator working solution.

[0077] The preparation method of the kit for the magnetic particle chemiluminescence immunodetection of CA125 surface Tn antigen in this embodiment comprises the following steps:

[0078] 1. Preparation of magnetic separation reagent coupled with Ov185 monoclonal antibody Fab fragment:

[0079] 1. Preparation of immobilized papain:

[0080] Ⅰ. Accurately measure 50mg Fe 3 o 4 Nano-magnetic particles were placed in a 10ml polystyrene plastic test tube, placed on a magnetic rack, removed from the supernatant after precipitation, washed twice with 5ml pure water, and added 5ml 0.01M PBS buffer (0.14M NaCl...

Embodiment 2

[0146] Different from Example 1,

[0147] Step Ⅰ is to accurately measure 50mg Fe 3 o 4 Nano-magnetic particles were placed in a 10ml polystyrene plastic test tube, placed on a magnetic rack, and the supernatant was removed after precipitation, washed twice with 10ml pure water, and 10ml 0.01M PBS buffer (0.14MNaCl, 3mM KCl, 10mM Na 2 HPO 4 , 2mM KH 2 PO 4, pH=7.2±0.05), then add papain with 0.05 times the mass of magnetic beads, and mix at room temperature for 1.5 hours;

[0148] Step II is to place the magnetic beads on the magnetic rack after mixing, remove the supernatant after precipitation, add 7.5ml 0.2mM hydrochloric acid buffer, and mix at room temperature for 10 minutes;

[0149] Step III is to add 0.3mg of 0.5M HEPES buffer solution (pH=7.4±0.1) after mixing, adjust the pH to 7.3±0.1, and mix at 37°C for 2.5 hours;

[0150] Step IV is to add 225 mg of glycine after mixing, and mix for 1 hour at 37°C;

[0151] Step Ⅴ is to place the magnetic bead solution on ...

Embodiment 3

[0167] Different from Example 1,

[0168] Step Ⅰ is to accurately measure 50mg Fe 3 o 4 Nano-magnetic particles were placed in a 10ml polystyrene plastic test tube, placed on a magnetic rack, removed from the supernatant after precipitation, washed twice with 25ml pure water, and added 25ml 0.01M PBS buffer (0.14MNaCl, 3mM KCl, 10mM Na 2 HPO 4 , 2mM KH 2 PO 4 , pH=7.2±0.05), then add papain with 0.04 times the mass of magnetic beads, and mix at room temperature for 1 hour;

[0169] Step II is to place the magnetic beads on the magnetic rack after mixing, remove the supernatant after precipitation, add 15ml 0.2mM hydrochloric acid buffer, and mix at room temperature for 10 minutes;

[0170] Step III is to add 0.125mg of 0.5M HEPES buffer solution (PH=7.4±0.1) after the mixing, adjust the pH to 7.3±0.1, and mix at 37°C for 2.5 hours;

[0171] Step IV is to add 75 mg of glycine after mixing, and mix for 1 hour at 37°C;

[0172] Step Ⅴ is to place the magnetic bead solutio...

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Abstract

The invention relates to the technical field of immunoassay, in particular to a kit for preparing a CA125 surface Tn antigen by magnetic particle chemiluminescence immunoassay and a preparation methodthereof. The kit comprises a magnetic separation reagent coupled with a Fab fragment of a CA125 antibody, a biotin-lectin reagent, and a streptavidin-biotin-alkaline phosphatase coupling reagent, wherein the biotin-lectin reagent containing sialidase is capable of identifying the Tn antigen. The magnetic separation reagent is specifically bound to the CA125 antigen; the biotin-lectin specificallyrecognizes the Tn antigen of the CA125 antigen surface; the streptavidin-biotin-alkaline phosphatase coupling reagent is specifically bound to the biotin-lectin reagent to realize amplification of the detection signal; the sialidase hydrolyzes a glucosidic bond connected with the sialic acid on the Tn antigen surface and the sialic acid is released to expose the Tn antigen. Therefore, the detection accuracy, sensitivity and linear range are improved. The kit has advantages of high specificity and high sensitivity and is capable of realizing fully automated testing.

Description

technical field [0001] The invention relates to the technical field of immunodetection, in particular to a kit for magnetic particle chemiluminescence immunodetection of Tn antigen on the surface of CA125 and a preparation method thereof. Background technique [0002] Ovarian cancer ranks third in the incidence of gynecological malignancies, second only to cervical cancer and uterine body cancer, but its case fatality rate ranks first. In recent decades, although a large number of scientific researchers have devoted themselves to the research of ovarian cancer, the survival rate of this type of patients has not been significantly improved. Cancer antigen 125 (CA125) was first discovered and named by Bast et al. in 1981. It is an epitope recognized by the monoclonal antibody OC125. It was first quantified by Klug et al. in 1984 using radioimmunoassay techniques. Detect and set the normal range of CA125 <35U / ml. On the one hand, CA125 is a commonly used serum tumor marker...

Claims

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Application Information

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IPC IPC(8): G01N33/574G01N33/535G01N33/543G01N21/76C12P21/06C12N11/14
Inventor 徐丛剑王宜生路晟霍萌萌胡春颖张晓燕顾建新任士芳
Owner THE OBSTETRICS & GYNECOLOGY HOSPITAL OF FUDAN UNIV
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