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Quantitative determination method for specific IgM antibody in plasma

A determination method and specific technology, which can be used in the measurement of color/spectral properties, biological testing, measuring devices, etc., can solve the problems of affecting accuracy, inconvenient detection, unavoidable non-specific binding effects, etc., and achieves low detection limit, good The effect of repeatability, excellent detection effect

Pending Publication Date: 2021-07-02
BLOOD TRASFUSION INST CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has great limitations: first, the plasma components are complex, and plasma components other than Aβ antibodies will bind to the coated Aβ non-specifically, which will eventually affect the accuracy of detection; secondly, mouse-derived monoclonal antibodies and human-derived multiple There are obvious differences in antibodies. Aβ has non-uniform forms such as monomers and multimers. The antibodies produced by it contain many polyclonal antibodies. There are problems in using mouse monoclonal antibodies to calibrate human polyclonal antibodies.
However, these methods can only reduce non-specific binding or increase specific binding to a certain extent, but none of them can avoid the impact of non-specific binding on the detection results.
In addition, for the detection of specific anti-Aβ IgM antibodies, neither the mouse-derived IgM monoclonal antibody nor the human-derived anti-Aβ IgM antibody has been commercialized. caused great inconvenience

Method used

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  • Quantitative determination method for specific IgM antibody in plasma
  • Quantitative determination method for specific IgM antibody in plasma
  • Quantitative determination method for specific IgM antibody in plasma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Specific anti-Aβ in the plasma of embodiment 1 42 Quantitative determination method of oligomer IgM antibody

[0039] Method of the present invention comprises the steps:

[0040] 1. Aβ oligomer preparation: Aβ 42 Peptides, processed (to Aβ 42 Hexafluoroisopropanol (HFIP) was added to the peptide, and it was naturally volatilized in a fume hood to dry for 5-6 hours) to make it into a uniform Aβ 42 After monomer, add polymerization liquid (0.01M PBS, the NaCl of 0.085% mass volume ratio, the SDS of 0.05% mass volume ratio, medium is water, pH 7.2) makes Aβ 42 Concentration to 100M, polymerized at 4°C for 48-72h (polymerization concentration 450μg / ml).

[0041] 2. Coating: the prepared Aβ 42 The oligomers were diluted with 0.01M phosphate buffer (pH6.0), added to the microtiter plate, and coated overnight at 4°C.

[0042]3. Sealing: Pour off the microplate liquid and wash the plate 5 times with the plate washing solution. Add 200 μl / well blocking solution, block at...

experiment example 1

[0052] Experimental Example 1Aβ 42 Optimization of co-incubation conditions with plasma

[0053] After diluting the mixed plasma of thousands of people to different concentrations, each concentration is divided into 2 parts, one of which is the standard plasma, and the other is treated with Aβ 42 As a control plasma after incubation, for Aβ 42 Co-incubation conditions with plasma were optimized (unmentioned Aβ 42 The antibody detection steps are the same as in Example 1), and the optimization effect is judged by the standard curve obtained from the final detection value of the standard plasma.

[0054] Condition 1: different Aβ 42 Co-incubation of species and plasma: Dilute 20 times, 40 times, 80 times, 160 times, 320 times, 640 times plasma respectively with Aβ 42 Oligomers and Aβ 42 Monomers were incubated in pH 8.8 buffer at 37°C for 3h. Use it as a control plasma to detect Aβ in plasma 42 IgM antibody content, the absorbance measured with different dilutions of pla...

experiment example 2

[0068] Experimental example 2 methodological verification of the present invention

[0069] This experimental example carries out methodological evaluation to the method of embodiment 1, specifically as follows:

[0070] 1. Method detection limit: The blank control was measured 8 times, and the OD values ​​were 0.097, 0.102, 0.099, 0.095, 0.102, 0.097, 0.102, 0.097, and the calculated standard deviation δ was 0.0028, which was brought into the formula (LOD=3.3*δ / standard curve slope) the calculated LOD value is 0.0146. Then bring it into the marking line to calculate the detection limit of the method: 2.07%.

[0071] 2. Method repeatability: Dilute a plasma sample to be tested to three different concentrations: 50-fold dilution, 100-fold dilution, and 200-fold dilution. Each concentration is tested in three repetitions, and the CV value is calculated. The measured results were 91.14%, 95.68%, 99.029%, 42.46%, 46.54%, 42.15%, 23.20%, 23.98%, 23.98%, respectively. After conv...

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Abstract

The invention discloses a quantitative determination method for a specific IgM antibody in plasma, and belongs to the field of antibody detection. The method comprises the following steps: 1) dividing one plasma sample into two groups as to-be-detected plasma and to-be-treated contrast plasma; (2) incubating excessive antigens and to-be-treated control plasma, and neutralizing to-be-detected antibodies in the to-be-treated control plasma to obtain control plasma; 3) taking to-be-detected plasma and the control plasma as samples, combining the to-be-detected plasma and the contrast plasma with corresponding antigens connected to the solid-phase carrier, successively combining the antigen-antibody complex with the IgM antibody and the signal molecule to generate quantifiable data, and subtracting two groups of quantifiable data to obtain detection data; (4) diluting the mixed plasma of thousands of persons as a standard substance into different concentrations according to the steps (1)-(3) to make a standard curve; and 5) substituting detection data of a sample to be detected into the standard curve to obtain the content of the specific IgM antibody in the plasma. According to the determination method, the influence of non-specific binding can be eliminated, and the detection result is reliable.

Description

technical field [0001] The invention belongs to the field of antibody detection. Background technique [0002] Alzheimer's disease (AD) is a neurodegenerative disease with insidious onset and the most common cause of dementia. Most AD patients develop after the age of 65, and the incidence and severity of AD are positively correlated with the age of the patients. There is currently no cure for AD, and few drugs can slow its progression. With the aging of the global population, the prevalence of AD is increasing year by year, which will also generate huge social public expenditures. my country is already the country with the largest elderly population in the world. With the increasing degree of aging in the next few decades, AD patients will continue to increase. There are two main reasons why there are rare drugs for AD that can slow down the course of the disease: (1) AD has an insidious onset and is difficult to detect in the early stage, thus losing the "golden period"...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N21/31
CPCG01N33/6854G01N33/6896G01N21/31G01N2333/4709G01N2800/2821
Inventor 曹海军杜晞李长清张容马莉王宗奎刘凤娟叶生亮
Owner BLOOD TRASFUSION INST CHINESE ACAD OF MEDICAL SCI
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