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Kit for testing early lung cancer specific autoantibody enzyme linked immunity and preparation method thereof

An enzyme-linked immunosorbent assay and autoantibody technology, applied in the field of medical detection reagents, can solve problems such as lack of sensitivity and specificity, patient suffering, and inability to diagnose early lung cancer.

Inactive Publication Date: 2010-01-13
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current methods for early diagnosis of lung cancer mainly rely on imaging examinations, such as X-ray examination, CT, bronchoscopy, etc., but these methods can only be completed when the lung cancer tissue is large enough and rely on corresponding equipment, which is costly and expensive. Causes suffering to patients and is difficult to use for census
The method of using serology to detect lung cancer mainly relies on carcinoembryonic antigen (CEA), NSE, C A 199,C A Single indicators such as 125 lack sensitivity and specificity, and cannot be used for early diagnosis of lung cancer of various histological types

Method used

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  • Kit for testing early lung cancer specific autoantibody enzyme linked immunity and preparation method thereof
  • Kit for testing early lung cancer specific autoantibody enzyme linked immunity and preparation method thereof
  • Kit for testing early lung cancer specific autoantibody enzyme linked immunity and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0049] The T7 tail fiber antibody was diluted 1:1000, each well was coated with 100 μl PBS (PH7.4) on a 96-well ELISA plate, and shaken overnight at 4°C on a shaker. 150 μl washing solution per well, wash four times, about 1 minute each time, and pat dry. 2% BSA / PBS 200ul at room temperature (24°C) to block for 2 hours. ) Add 150 μl washing solution to each well, wash four times, about 1 minute each time, and pat dry. A total of 12 wells were set up, repeated every two wells, and set as auxiliary wells. Add 1% BSA 1:5 diluted lung cancer antigen peptide I (SMMU-EPII) 100 μl to the first and second wells, and add 100 μl to the third and fourth wells 1% BSA 1:5 diluted lung cancer antigen peptide II (SMMU-EPIII) 100μl, lung cancer antigen peptide III (SMMU-EPIIII), lung cancer antigen peptide IV (SMMU-EPIIV), lung cancer antigen peptide V (SMMU-EPIV) and lung cancer Similarly, 100 μl of each antigenic peptide VI (SMMU-EPIVI) was added to the remaining 8 wells sequentially, and...

Embodiment 2

[0051] Coat the T7 tail fiber antibody at a dilution of 1:1000 with 100 μl PBS (PH7.4) per well on a 96-well ELISA plate, shake gently on a shaker at 4°C overnight, add 150 μl washing solution to each well, and wash four times , about 1 minute each time, pat dry. 2% BSA / PBS 200ul at room temperature (24°C) to block for 2 hours. )

[0052] Add 150 μl washing solution to each well, wash four times, about 1 minute each time, and pat dry. A total of 12 wells were set up, and every two wells were repeated, set as auxiliary wells, 1% BSA 1:4 diluted lung cancer antigen peptide I (SMMU-EPII) 100 μl was added to the first and second wells, and 100 μl of lung cancer antigen peptide I (SMMU-EPII) diluted in the third and fourth wells 1% BSA 1:5 diluted lung cancer antigen peptide II (SMMU-EPIII) 100μl, lung cancer antigen peptide III (SMMU-EPIIII), lung cancer antigen peptide IV (SMMU-EPIIV), lung cancer antigen peptide V (SMMU-EPIV) and lung cancer Similarly, 200 μl of each antigeni...

Embodiment 3

[0055] Add 100 μl of 1% BSA 1:500 diluted lung cancer patient plasma to each phage sample, and incubate at room temperature for 1 hour. Wash the plate with washing solution, 150 μl per well, wash four times, about 1 minute each time, and pat dry. Add 100 μl of 1:10000 diluted HRP-coupled goat anti-human IgG to each well and incubate at room temperature for 1 hour. Wash the plate with washing solution, 150 μL per well, wash four times, about 1 minute each time, and pat dry. Add 1 drop each of chromogenic reagents A and B at room temperature to each well, mix well, incubate at 37°C for 20 minutes, and add 25 μl of stop solution. Immediately detect with a microplate reader, take a wavelength of 450nm, first use a blank well to zero, then read the OD value of each well, and record the results.

[0056] Result judgment

[0057] Each ELISA is calculated and judged according to the following formula: after zeroing with a blank well, the sample OD value / the average OD value of the ...

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Abstract

The invention provides a kit for testing early lung cancer specific autoantibody enzyme linked immunity, consisting of a solid phase carrier and six recombined bacteriophage liquid which are packed at the surface of the solid phase carrier, contains lung cancer antigenic peptide I, lung cancer antigenic peptide II, lung cancer antigenic peptide III, lung cancer antigenic peptide IV, lung cancer antigenic peptide V and lung cancer antigenic peptide VI, and is equivalently respectively packaged into different enzyme marking holes on the solid phase carrier, wherein each of the six recombined bacteriophage liquid is 100-200mu l. The recombined lung cancer specific antigenic peptide uses T7 bacteriophage to display a lung cancer cDNA library onto the surface of the bacteriophage, is sieved out by means of affinity sieving and serologic analysis, has a capability for testing the autoantibody generated by stimulating tumor in blood serum of a patient, provides an effective testing effect for early lung cancer patients, and has greater clinic application value.

Description

technical field [0001] The invention relates to the technical field of medical detection reagents, in particular to an ELISA detection kit for early stage specific autoantibodies of lung cancer and a preparation method thereof. Background technique [0002] Lung cancer is a common malignant tumor, and it is also the most serious malignant tumor that threatens human life and health. The incidence rate of different races is different. According to the data from the Health Information Center of the National Health Bureau of China, the mortality rate of lung cancer from 2004 to 2005 was 30.61 / 100,000 , ranking first among male and female malignant tumors. Early diagnosis and timely treatment of lung cancer are the key to prolonging the 5-year survival period. However, due to the lack of obvious early symptoms, more than 50% of lung cancers are already in the middle and advanced stages when they are clearly diagnosed, and even metastasized, losing the opportunity for surgery. The...

Claims

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Application Information

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IPC IPC(8): G01N33/574G01N33/531C07K14/435C07K5/113C07K7/08
Inventor 曹广文吴玲玲赵晋丰常文军
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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