A kit for magnetic particle chemiluminescence immunodetection of CA125 surface TN antigen and its preparation method

A chemiluminescence immunity, CA125 technology, applied in chemiluminescence/bioluminescence, analysis and fermentation through chemical reaction of materials, can solve problems such as long reaction time, affecting detection sensitivity and linear range, and Fab segment influence, etc. Achieve the effect of avoiding non-specific adsorption, avoiding false positive phenomenon, and improving the linear range

Active Publication Date: 2022-03-25
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 reaction time is long. The test process is eluted 12 times, and it takes nearly 4 hours to complete from the beginning of the test to the end of the test, and it is difficult to realize integration and full automation;
[0010] 5) This patent uses the OD value tested by the microplate reader as the final data for ROC curve analysis, and does not involve calibrator fitting. This processing method has certain limitations, and the final result fluctuates due to the influence of different manufacturers or models of microplate readers larger;
[0011] 6) The patent does not treat the sialic acid on the surface of the CA125 antigen, which can cover the recognition site of the lectin VVA, thereby affecting the detection sensitivity and linear range

Method used

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  • A kit for magnetic particle chemiluminescence immunodetection of CA125 surface TN antigen and its preparation method
  • A kit for magnetic particle chemiluminescence immunodetection of CA125 surface TN antigen and its preparation method

Examples

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Effect test

Embodiment 1

[0076] The kit for magnetic particle chemiluminescence immunodetection of CA125 surface Tn antigen in this example includes magnetic separation reagent coupled with Fab fragment of 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 magnetic particle chemiluminescence immunodetection of Tn antigen on the surface of CA125 in the present embodiment includes the following operation steps:

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

[0079] 1. Preparation of immobilized papain:

[0080] Ⅰ. Accurately measure 50mg Fe 3 O 4 Nanomagnetic particles were placed in a 10ml polystyrene plastic test tube, placed on a magnetic rack, and the supernatant was removed after precipitation, and washed twice with 5ml of pure water. Na 2 HPO 4 , 2mM K...

Embodiment 2

[0146] Unlike Example 1,

[0147] Step Ⅰ is to accurately measure 50mg Fe 3 O 4 Nanomagnetic 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 after washing was completed, 10ml of 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 a magnetic rack after mixing, remove the supernatant after precipitation, add 7.5 ml of 0.2 mM hydrochloric acid buffer, and mix at room temperature for 10 minutes;

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

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

[0151] Step 5: After mixing, place the magn...

Embodiment 3

[0167] Unlike Example 1,

[0168] Step Ⅰ is to accurately measure 50mg Fe 3 O 4 Nanomagnetic 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 25ml of pure water, and 25ml of 0.01M PBS buffer (0.14MNaCl, 3mM KCl, 10mM PBS) was added after washing. 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 a magnetic rack after mixing, remove the supernatant after precipitation, add 15 ml of 0.2 mM hydrochloric acid buffer, and mix at room temperature for 10 minutes;

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

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

[0172] Step V: After mixing, place the ma...

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Abstract

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. The kit includes a magnetic separation reagent coupled with the Fab fragment of the CA125 antibody, a biotin-lectin reagent containing sialidase that can recognize the Tn antigen, and a streptavidin-biotin-alkaline phosphatase coupling reagent; The magnetic separation reagent specifically binds the CA125 antigen, the biotin-lectin can specifically recognize the Tn antigen on the surface of the CA125 antigen, and the streptavidin-biotin-alkaline phosphatase coupling reagent can specifically bind the biotin-lectin The reagent realizes the amplification of the detection signal, and the sialidase hydrolyzes the glycosidic bond connected with sialic acid on the surface of the Tn antigen, releases the sialic acid to expose the Tn antigen, and improves the detection accuracy, sensitivity and linear range. The kit of the invention has high specificity and good sensitivity, and can realize fully automatic testing.

Description

technical field [0001] The invention relates to the technical field of immunodetection, in particular to a magnetic particle chemiluminescence immunodetection kit for Tn antigen on the surface of CA125 and a preparation method thereof. Background technique [0002] Ovarian cancer ranks third in the incidence of gynecological malignant tumors, second only to cervical cancer and endometrial cancer, but its mortality rate ranks first. In recent decades, although a large number of 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 (cancer antigen 125, CA125) was originally discovered and named by Bast et al. in 1981. It is an antigenic epitope recognized by the monoclonal antibody OC125, and was first quantified by radioimmunoassay technology in 1984 by Klug et al. Detection, and set the normal range of CA125 <35U / ml. On the one hand, CA125 is a commonly us...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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