Quantitative determination method for specific IgG antibody in plasma

Abstract

The invention discloses a quantitative determination method for a specific IgG antibody in plasma, which belongs to the field of antibody detection. The method comprises the following steps of 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 contrast plasma, and neutralizing to-be-detected antibodies in the to-be-treated contrast plasma to obtain contrast plasma, 3) taking to-be-detected plasma and contrast 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 compound with IgG antibodies and signal molecules to generate quantifiable data, and subtracting two groups of quantifiable data to obtain detection data, 4) diluting thousands of mixed plasma serving 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 to-be-detected sample into the standard curve to obtain the content of the specific IgG 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 common central nervous system degenerative disease with the main characteristics of memory cognitive function, which mainly clinically manifested the deletion of memory capabilities and the decline in cognitive function. The main pathological features are extracellular β-amylin, AmyiD β-Protein, Aβ) precipitation, Neurofibrillary Tangles, NFTS and a large number of apoptosis and loss of neuronal neurons. According to these pathological features, a variety of pathogenic hypothesis, in the explanation of many of the pathogenesis of AD, is currently "A [beta] cascaded holiday", which believes that the occurrence of AD is due to the formation of Aβ in vivo The metabolic imbalance was eliminated, so that it was deposited in a particular brain to form an elderly spot, and further triggered Tau protein to form NFTS, which eventually led to sudden l...

Examples

Embodiment 1

[0040] Example 1 Special anti-Aβ in plasma 42 Quantitative measurement method of IgG antibody of oligomer

[0041] The method of the present invention includes the following steps:

[0042] 1.a [beta] oligomer preparation: Aβ 42 Polypeptide, processed (to A] 42 Acid is added to the polypeptide, and naturally volatilized in the fume hood is naturally volatilized for 5-6 hours) makes it become average A [beta] 42 After the monomer, the polymer solution (0.01 M PBS, 0.085% mass volume ratio of NaCl, 0.05% mass volume ratio SDS, water, pH 7.2) 42 The concentration to 100 m was polymerized at 4 ° C (450 μg / ml).

[0043] 2. Package: Will prepare a beta 42 The oligomer was diluted with 0.01 M phosphate buffer (pH 6.0) and added to the enzyme board, and the package was overnight at 4 ° C.

[0044]3. Close: Turn off the microplate liquid and wash the plate 5 times with wash. The addition of 200 μl / well enclosed liquid, closed 37 ° C, 3H.

[0045] 4. Standard plasma, preparation for pla...

experiment example 1

[0054] Experimental case 1 Aβ 42 Optimize the conditions for incubation with plasma

[0055] Hereinafter, the concentration is divided into two parts by mixing plasma, and one of the standard plasma is divided into standard plasma. 42 After incubation, as the control plasma, to Aβ 42 Optimize with plasma common incubation conditions (Aβ, which is not mentioned 42 The antibody detection steps are evaluated by the exception of the optimization effect, and the optimization effect is evaluated by the standard curve made by the standard plasma final detection value.

[0056] Condition 1: Different Aβ 42 Types and plasma incubation: dilute 10 times, 20 times, 40 times, 80-fold plasma, respectively and Aβ, respectively 42 Oligomer and Aβ 42 The monomer was incubated at pH 7.2 buffer and incubated under conditions of 4 ° C. Taking it as a control plasma, detecting Aβ in plasma 42 The antibody content is subtracted from the absorbance measured by different dilution plasma. 42 The absorbanc...

experiment example 2

[0076] Experiment 2 Methodology Verification

[0077] In this first experiment, methodatic evaluation of the method of Example 1, as follows:

[0078] 1. Method detection limit: The blank control was measured 8 times, and the OD values ​​were 0.129, 0.127, 0.117, 0.121, 0.126, 0.127, 0.126, 0.132, and calculated the standard deviation 6 was 0.00466, and the formula (LOD = 3.3 * 8) / Standard curve slope) The calculation of the LOD value is 0.0167. Then the brought the marking line is calculated to obtain method detection is: 6.73%.

[0079] 2. Method Repeat: Three different concentrations to be tested by the plasma samples: 50 times dilution, 100 times dilution, 200 times dilution, each concentration three repetitions, calculate the CV value. The results were 92.02%, 90.50%, 93.04%, 39.74%, 41.04%, 47.36%, 23.80%, 19.32%, 21.63%, respectively. After the dilution multiple, its value was 115.03%, 113.12%, 116.30%, 99.35%, 102.76%, 118.41%, 119.00%, 96.62%, 108.16%, average value of ...

Technical field

[0001] The invention belongs to the field of antibody detection. Background technique

[0002] Alzheimer's Disease (AD) is a common central nervous system degenerative disease with the main characteristics of memory cognitive function, which mainly clinically manifested the deletion of memory capabilities and the decline in cognitive function. The main pathological features are extracellular β-amylin, AmyiD β-Protein, Aβ) precipitation, Neurofibrillary Tangles, NFTS and a large number of apoptosis and loss of neuronal neurons. According to these pathological features, a variety of pathogenic hypothesis, in the explanation of many of the pathogenesis of AD, is currently "A [beta] cascaded holiday", which believes that the occurrence of AD is due to the formation of Aβ in vivo The metabolic imbalance was eliminated, so that it was deposited in a particular brain to form an elderly spot, and further triggered Tau protein to form NFTS, which eventually led to sudden l...

Examples

Embodiment 1

[0040] Example 1 Special anti-Aβ in plasma 42 Quantitative measurement method of IgG antibody of oligomer

[0041] The method of the present invention includes the following steps:

[0042] 1.a [beta] oligomer preparation: Aβ 42 Polypeptide, processed (to A] 42 Acid is added to the polypeptide, and naturally volatilized in the fume hood is naturally volatilized for 5-6 hours) makes it become average A [beta] 42 After the monomer, the polymer solution (0.01 M PBS, 0.085% mass volume ratio of NaCl, 0.05% mass volume ratio SDS, water, pH 7.2) 42 The concentration to 100 m was polymerized at 4 ° C (450 μg / ml).

[0043] 2. Package: Will prepare a beta 42 The oligomer was diluted with 0.01 M phosphate buffer (pH 6.0) and added to the enzyme board, and the package was overnight at 4 ° C.

[0044]3. Close: Turn off the microplate liquid and wash the plate 5 times with wash. The addition of 200 μl / well enclosed liquid, closed 37 ° C, 3H.

[0045] 4. Standard plasma, preparation for pla...

experiment example 1

[0054] Experimental case 1 Aβ 42 Optimize the conditions for incubation with plasma

[0055] Hereinafter, the concentration is divided into two parts by mixing plasma, and one of the standard plasma is divided into standard plasma. 42 After incubation, as the control plasma, to Aβ 42 Optimize with plasma common incubation conditions (Aβ, which is not mentioned 42 The antibody detection steps are evaluated by the exception of the optimization effect, and the optimization effect is evaluated by the standard curve made by the standard plasma final detection value.

[0056] Condition 1: Different Aβ 42 Types and plasma incubation: dilute 10 times, 20 times, 40 times, 80-fold plasma, respectively and Aβ, respectively 42 Oligomer and Aβ 42 The monomer was incubated at pH 7.2 buffer and incubated under conditions of 4 ° C. Taking it as a control plasma, detecting Aβ in plasma 42 The antibody content is subtracted from the absorbance measured by different dilution plasma. 42 The absorbanc...

experiment example 2

[0076] Experiment 2 Methodology Verification

[0077] In this first experiment, methodatic evaluation of the method of Example 1, as follows:

[0078] 1. Method detection limit: The blank control was measured 8 times, and the OD values ​​were 0.129, 0.127, 0.117, 0.121, 0.126, 0.127, 0.126, 0.132, and calculated the standard deviation 6 was 0.00466, and the formula (LOD = 3.3 * 8) / Standard curve slope) The calculation of the LOD value is 0.0167. Then the brought the marking line is calculated to obtain method detection is: 6.73%.

[0079] 2. Method Repeat: Three different concentrations to be tested by the plasma samples: 50 times dilution, 100 times dilution, 200 times dilution, each concentration three repetitions, calculate the CV value. The results were 92.02%, 90.50%, 93.04%, 39.74%, 41.04%, 47.36%, 23.80%, 19.32%, 21.63%, respectively. After the dilution multiple, its value was 115.03%, 113.12%, 116.30%, 99.35%, 102.76%, 118.41%, 119.00%, 96.62%, 108.16%, average value of ...

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