Immunofluorescence reagent strip for determining urinary nuclear matrix protein as well as preparation method and application thereof

A nuclear matrix protein and immunofluorescence technology, applied in the field of quantitative detection of concentration, can solve problems such as low specificity, and achieve the effect of simple and convenient operation and reduced operation time.

Inactive Publication Date: 2018-11-02
ANIMAL AND PLANT & FOOD DETECTION CENTER JIANGSU ENTRY EXIT INSPECTION AND QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Studies have shown that the sensitivity (detection rate) of NMP22 combined with cystoscopy is over 99%; whether in early screening or recurrence monitoring, the sensitivity of NMP22 detection is much higher than that of urine exfoliated cells, but the specificity is slightly lower than that of urine cells

Method used

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  • Immunofluorescence reagent strip for determining urinary nuclear matrix protein as well as preparation method and application thereof
  • Immunofluorescence reagent strip for determining urinary nuclear matrix protein as well as preparation method and application thereof
  • Immunofluorescence reagent strip for determining urinary nuclear matrix protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Preparation of specific monoclonal antibody against nuclear matrix protein (NMP22)

[0032] 1. Animal immunity:

[0033]Female Balb / c mice aged 6-8 weeks were selected as immunization objects. Before the initial immunization, blood was collected from the tail vein of each mouse, and 200 μl of the emulsified immunogen (antigen plus complete adjuvant) was injected intraperitoneally during the initial immunization; after the initial immunization On the 14th day and the 28th day, the second and third immunizations (antigen plus incomplete adjuvant) were carried out in the same way. On the 5th day after the third immunization, blood was collected from the orbit of each mouse to collect serum to evaluate the antibody effect. On the seventh day after the third immunization, booster immunization was carried out, and 100 μl of immunogen was injected into the tail vein. On the third day after the booster immunization, spleen lymphocytes and SP2 / 0 (mixing ratio of 5:1) ...

Embodiment 2

[0041] An immunofluorescence reagent strip for measuring urinary nuclear matrix protein, characterized in that: in the immunofluorescence reagent strip, water-absorbing pads, chromatographic membranes, binding pads, and sample pads are arranged in sequence; the sample pad corresponds to the sample adding area, and the binding pad Corresponding to the binding area; the chromatographic membrane corresponds to the detection area; the chromatographic membrane is equipped with a detection line T line and a quality control line C line; the detection line T line is coated with NMP22 monoclonal antibody N-C2; the quality control line C line Goat anti-mouse IgG is coated on the top; antibody complex Cy3-N-C1 labeled with fluorescent substances is laid on the binding pad.

[0042] T-line and C-line diluent formula: 0.01M PBS, 5% sucrose, 1% BSA; binding pad treatment solution formula: 0.1MTris-HCl, 0.1% Tween-20, 0.5% PVA, 5% sucrose, 1% BSA; Cy3-N-C1 diluent formula: 0.01MPBS, 0.5% PVA...

Embodiment 3

[0054] Example 3: Standard Curve for Determination of Urinary Nuclear Matrix Protein Immunofluorescence Reagent Strip

[0055] The NMP22 antigen solution can be diluted with antigen diluent to a final concentration of 0ng / mL, 4ng / mL, 8ng / mL, 16ng / mL, 32ng / mL, 64ng / mL serial standard solution.

[0056] Antigen diluent is 0.05mol / L phosphate buffer containing BSA and sucrose, pH=7.4, containing 20gBSA, 50g sucrose, 8g NaCl, 0.2g KCl, 0.24g KH per liter 2 PO 4 , 1.44 g Na 2 HPO 4 of aqueous solution.

[0057] According to the detection steps of the urine nuclear matrix protein NMP22 immunofluorescence reagent strip assay method, the fluorescence detection intensity of the NMP22 series standard products was obtained. According to the fluorescence intensity value and concentration of the standard as the coordinate axis, select a suitable mathematical model to establish a standard curve, the standard curve is as follows figure 2 As shown, the correlation coefficient r=0.9982 w...

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Abstract

The invention discloses an immunofluorescence reagent strip for determining a urinary nuclear matrix protein as well as a preparation method and application thereof. The immunofluorescence reagent strip disclosed by the invention comprises three areas, namely a sample adding zone, a binding zone and a detection zone. The reagent strip respectively comprises an absorbent pad, a chromatography membrane, a conjugate pad and a sample pad from top to bottom. A fluorescent substance-labeled anti-NMP22 antibody complex is tiled on the conjugate pad, and an anti-nuclear matrix protein (NMP22) monoclonal antibody enveloped on a T line on the chromatography membrane can be used for detecting the NMP22 in urine. When the reagent strip is used with a fluorescence analyzer together, quantitative determination can be realized, and the operation is simple and convenient. The immunofluorescence reagent strip can be applied to laboratory detection and on-site quick testing, detection only needs about 10 minutes, the operation time is greatly shortened, and the kit can be applied to auxiliary diagnosis for early screening and recurrence monitoring of bladder cancer.

Description

technical field [0001] The invention discloses an immunofluorescent reagent strip for measuring urinary nuclear matrix protein and its preparation method and application, which are used for quantitative detection of the concentration of urinary nuclear matrix protein (NMP22) in urine. This kit can be used for the auxiliary diagnosis of early screening and recurrence monitoring of bladder cancer. It belongs to the field of immunological detection. technical background [0002] Bladder cancer is the most common malignant tumor of the urinary system in my country. The postoperative recurrence rate of bladder cancer can reach 70% to 80%, and 10% to 15% of patients die due to tumor progression. Early detection, regular postoperative follow-up, and detection of recurrence are the keys to the treatment of bladder tumors. [0003] The age-standardized incidence rate of bladder cancer in my country is 3.8 / 100,000 for males and 1.8 / 100,000 for females. In recent years, reports on ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/543G01N33/531
CPCG01N33/6803G01N33/531G01N33/54306
Inventor 王毅谦陈雷龙云凤蔡正清高玲丁涛姜珊方云涛厉蓉蓉祁专成
Owner ANIMAL AND PLANT & FOOD DETECTION CENTER JIANGSU ENTRY EXIT INSPECTION AND QUARANTINE BUREAU
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