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A human urine albumin latex-enhanced secondary antibody competitive immunoturbidimetric detection kit and its production and use method

A detection kit and latex-enhanced technology, applied in the biological field, can solve the problems of multiple detections, difficult standardization of clinical results, and incompletely consistent preparation methods of latex-antibody conjugates.

Active Publication Date: 2020-12-29
长沙文瀚生物技术有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the above factors are affected, and due to the diversity of antibody proteins and latex particles, even if it is the same latex and antibody, no matter whether physical or chemical methods are used, different manufacturers have different preparation methods for different latex-antibody conjugates. Can not get exactly the same kit products
As a result, clinical results are difficult to standardize due to the influence of kit product factors, so that different hospitals cannot use the same test results universally, resulting in multiple tests and increasing the cost of testing for patients
[0007] 3. In the existing immunoturbidimetric method, for each analyte, it is necessary to prepare a corresponding latex-antibody conjugate. The development process of the kit requires research on the type of antibody, latex type, latex size and coupling method Obtain the best detection scheme and prepare the kit; biotechnology is changing with each passing day, clinically valuable new markers are constantly being discovered, and the number of new kits to be developed continues to increase, which adds a huge workload to kit development

Method used

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  • A human urine albumin latex-enhanced secondary antibody competitive immunoturbidimetric detection kit and its production and use method
  • A human urine albumin latex-enhanced secondary antibody competitive immunoturbidimetric detection kit and its production and use method
  • A human urine albumin latex-enhanced secondary antibody competitive immunoturbidimetric detection kit and its production and use method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Preparation of rabbit anti-mouse IgG secondary antibody (A) or goat anti-rabbit IgG (B)-latex conjugate

[0056] Commercially available particle size of 100nm, 0.2ml of carboxyl nano-latex particles with a concentration of 10%, add pH 4.6, 0.8ml of 0.02M phosphate buffer, add 20μl of dicyclohexylcarbodiimide with a concentration of 20mg / ml, and incubate on a shaking table at 37°C After 20 minutes of reaction and activation, add 0.5ml, pH 7.5, 0.02M phosphate buffer and 0.02ml purified rabbit anti-mouse IgG antibody or 0.05ml goat anti-rabbit IgG (secondary antibody), and then incubate at 37°C for 60°C. Minutes, after the ligation reaction is finished, add pH 7.0, 0.02M phosphate buffer to make the total solution volume to 10ml, then add 0.2M glycine 0.1ml, 1% Tween 20 0.1ml, proclin 3000.1ml, shake at 37°C Incubate and react for 30 minutes to block and terminate the reaction. Add proclin300 at 0.1% to prepare rabbit anti-mouse IgG (A) or goat anti-rabbit IgG secondary a...

Embodiment 2

[0058] Determination of mouse anti-human albumin monoclonal antibody or rabbit anti-human albumin polyclonal antibody titer:

[0059] Dilute mouse anti-human albumin monoclonal antibody or rabbit anti-human albumin polyclonal antibody with water ratio, use 0.02M, pH7.0 (containing NaCl0.15M) phosphate buffer as R1, use the above method to prepare rabbit antibody respectively The mouse IgG secondary antibody-latex conjugate is R2. On the Hitachi 7170S automatic biochemical analyzer, set the measurement program according to the following parameters, and measure the primary antibody with different dilutions; measurement wavelength: 546nm; R 1 :R 2 : Sample=100:100:20, that is, after adding 100μl R1, add 20μl sample, keep warm at 37°C for 5 minutes, then add 100μl R 2 , read the light absorption value at point 17 and point 34, and calculate the light absorption difference between point 34 and point 17: △A=A34-A17. The results of titer determination are shown in Table (1) and Fig...

Embodiment 3

[0063] 3.1 Preparation of Human Urine Albumin Latex Enhanced Secondary Antibody Competitive Immunoturbidimetric Detection Kit:

[0064] Prepare the secondary antibody competition immunoturbidimetric detection reagent according to the following formula

[0065] Kit A:

[0066] R1: pH7.0, 0.02M phosphate buffer

[0067] Rabbit anti-mouse IgG secondary antibody-latex (0.01-0.05%)

[0068] Glycine (0.002M)

[0069] Tween20 (0.01%)

[0070] Proclin 300 (0.1%)

[0071] R2: pH7.5, 0.02M phosphate buffer

[0072] Mouse anti-human albumin monoclonal antibody (1:200)

[0073] NaCl(0.15M)

[0074] Casein (0.01%)

[0075] Tween20 (0.001%)

[0076] Proclin 300 (0.1%)

[0077] Kit B

[0078] R1: pH7.0, 0.02M phosphate buffer

[0079] Goat anti-rabbit IgG secondary antibody-latex (0.01-0.05%)

[0080] Glycine (0.002M)

[0081] Tween20 (0.01%)

[0082] Proclin 300 (0.1%)

[0083] R2: pH7.5, 0.02M phosphate buffer

[0084] Rabbit anti-human albumin (1:500)

[0085] NaCl(0.15M) ...

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Abstract

The invention discloses a human urinary albumin latex enhanced secondary-antibody competitive immunity turbidity detection kit as well as a production method and a use method thereof. By utilizing a secondary-antibody competitive immunity turbidity method, the defects of existing enhanced immunity turbidity methods are overcome from immune-reaction principles; when measuring that no antigen existsin a reaction system, a secondary antibody linked to a latex particle reacts with a primary antibody in the system to generate agglutination reaction, and the agglutination effect reaches the maximum; and when measuring that the antigen exists in the reaction system, the antigen is bound with the primary antibody to form an antigen-antibody (primary antibody) immune compound, a binding site between the primary antibody and the secondary antibody is closed by through the binding, the competition is formed with immune agglutination reaction of the primary antibody and the secondary antibody, and if the amount of the primary antibody is limited, the agglutination effect of a primary antibody-secondary antibody immune compound in a sample is competitively inhibited by the antigen, so that theagglutination effect of the immune compound generated through the reaction between the primary antibody and secondary antibody-latex is reduced along the increase of free antibodies in the reaction system.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a human urine albumin latex-enhanced secondary antibody competitive immunoturbidimetric detection kit and a production and application method thereof. Background technique [0002] Immunoturbidimetric analysis methods have been widely used clinically, and dozens of kits have been used clinically. At the same time, the discovery of new biomarkers will continue to promote the production of this new kit. At present, there are mainly two types. One is to directly add the antibody into the reaction system. Under certain conditions, the antigen and antibody react to form an immune complex, which causes agglutination or precipitation to change the turbidity of the system. This turbidity change is related to the system. There is a positive correlation between the antigen concentration in the sample, and the antigen concentration in the sample can be determined accordingly. This m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
CPCG01N33/6827
Inventor 文建新刘株漆乐新
Owner 长沙文瀚生物技术有限责任公司
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