CN111190020B

CN111190020B - Method for optimizing universal biological analysis by adding monkey serum

Info

Publication number: CN111190020B
Application number: CN202010066176.2A
Authority: CN
Inventors: 盛稳稳; 刘玮博; 金晓莺; 冀群升; 金毅; 施婧; 张欣
Original assignee: Wuxi Apptec Suzhou Co Ltd
Current assignee: Wuxi Apptec Suzhou Co Ltd
Priority date: 2020-01-20
Filing date: 2020-01-20
Publication date: 2024-02-02
Anticipated expiration: 2040-01-20
Also published as: CN111190020A

Abstract

The invention discloses a method for optimizing universal bioanalytical by adding monkey serum, which comprises the following steps: preparing a universal coated antibody buffer solution, and incubating overnight; adding a sealing buffer solution for incubation, adding two sets of identical to-be-detected objects with certain gradients, and then respectively adding a detection antibody working solution containing monkey serum and a detection antibody working solution not containing monkey serum into the to-be-detected objects; color development and termination, background values, sensitivity of each set of data were compared after microplate reader treatment. The method can solve the problems that the general background value of the general method in monkey serum or monkey plasma is relatively high, thereby affecting the sensitivity of the method and being unfavorable for the detection of the exposure of animals in low-dose groups.

Description

Method for optimizing universal biological analysis by adding monkey serum

Technical Field

The invention relates to the field of biological analysis, in particular to a method for optimizing universal biological analysis by adding monkey serum aiming at reducing background signal value to optimize method and method sensitivity.

Background

The investigation of sensitivity in a macromolecular bioanalytical method is an important index and is also a parameter which must be verified for the bioanalytical method by the current FDA, NMPA, OECD proposal. Description of LLOQ in the FDA defines the sensitivity of a method, which should be determined during method development. And develop and verify the sensitivity so that it can meet the requirements of the intended biological sample detection. NMPA also describes sensitivity as the lowest concentration of analyte in a sample that can be reliably quantified with acceptable accuracy and precision. Sensitivity is the lowest point of the standard curve and should be suitable for the intended concentration and assay purposes. The background value, if high, generally shows that the method is greatly affected by the matrix. The accuracy and precision of the sensitivity in the method can also be affected laterally.

There are many ways to reduce the high background signal values, such as increasing the minimum dilution factor, changing the blocking solution, changing the reagents and even the changing method. However, increasing the minimum dilution factor to reduce the matrix content to achieve the interference effect of the reduced matrix may also result in reduced sensitivity, changing the blocking solution may increase the cost of the method, and changing reagents and methods may render the general method unsuitable for different drugs.

In order to ensure that the universal method can provide a detection way for each drug, the solution mode of effectively reducing the background value and optimizing the sensitivity is mastered, so that the development effectiveness of the universal biological analysis method can be ensured, and the subsequent method verification and effective sample analysis can be ensured.

Meanwhile, the method has very important significance for detecting the whole medicine in preclinical pharmacodynamics. Different medicines are detected, and the established general method is used, so that the method has the advantages of saving time and reagent cost. However, the current general method has the problems of high background value and relatively low sensitivity.

The current time cost and reagent cost of some preclinical sample analysis can be greatly reduced by using a general method, and the reagent selection link of the general method is particularly important. And the general background value of the general method in monkey serum or monkey plasma is relatively high, thereby affecting the sensitivity of the method and being unfavorable for detecting the exposure of animals in low dose groups.

Disclosure of Invention

The invention aims to provide a method for optimizing universal bioanalytical by adding monkey serum, which can effectively improve the background value and the optimized sensitivity of the universal bioanalytical method by adding monkey serum, and is effective no matter whether the coated antibody is a common anti-human Fc antibody or a polyclonal antibody of a specific fragment. The method can solve the problems that the general background value of the general method in monkey serum or monkey plasma is relatively high, thereby affecting the sensitivity of the method and being unfavorable for the detection of the exposure of animals in low-dose groups.

In order to solve the technical problems, the invention adopts the following technical scheme:

the invention provides a method for optimizing universal bioanalytical by adding monkey serum.

As a preferred technical scheme of the invention, the method comprises the following steps:

I. adding a universal coated antibody buffer solution into a high adsorption plate, and incubating overnight; washing the plate with washing liquid, adding a sealing buffer solution, and incubating at room temperature; washing the plate with washing liquid, adding two sets of identical objects to be detected, and incubating at room temperature;

II, respectively preparing a detection antibody working solution containing monkey serum and a detection antibody working solution without the monkey serum, and respectively adding the working solutions into an object to be detected for incubation at room temperature;

and III, developing and stopping, and comparing the background value and the sensitivity of each group of data after the treatment of the enzyme label instrument.

As a preferred technical scheme of the invention, the preparation method of the universal coated antibody buffer solution in the step I comprises the following steps: polyclonal antibodies against the human Fc segment and polyclonal antibodies against the human CH2 segment were prepared separately, and coated antibody buffers were prepared using carbonate buffers.

As a preferable technical scheme of the invention, the washing liquid in the step I contains 0.05% -0.1% of PBS; the blocking buffer solution is 3% -5% BSA; the test substance was formulated in monkey serum containing drug with a gradient of 5000-10ng/mL and was pre-diluted 50-fold with 1% BSA.

In the preferred technical scheme of the invention, in the step I, the high adsorption plate is a 96-well plate, 100 microlitres of general coated antibody buffer solution is added into each well, and the mixture is incubated overnight at 2-8 ℃.

In the step I, the high adsorption plate is a 96-well plate, the plate is washed by washing liquid for 3-5 times and then is patted dry, 300 microliters of sealing buffer solution is added into each well, and the plate is incubated for 1-3 hours at room temperature.

In the step I, the high adsorption plate is a 96-well plate, the plate is washed by washing liquid for 3-5 times and then is patted dry, 100 microliters of an object to be detected is added into each hole, and the plate is incubated for 2 hours at room temperature at 400-600 revolutions per second.

In a preferred embodiment of the present invention, in step II, the preparation method of the working solution for detecting antibodies containing monkey serum comprises: 1: 20000-fold dilutions in 1% bsa with 5% monkey serum; the preparation method of the working solution of the detection antibody without monkey serum comprises the following steps: 1: 20000-fold dilutions were in 1% bsa.

As a preferred embodiment of the present invention, 100. Mu.l of the detection antibody working solution is added to each well in step II, and the reaction is incubated at room temperature for 1 hour at 400-600 rpm.

As a preferable technical scheme of the invention, the following steps are added between the step I and the step II: washing the plate with washing liquid for 3-5 times, and then beating to dry.

As a preferable technical scheme of the invention, the washing liquid is PBS washing liquid containing 0.05% -0.1% Tween, 300 microliter of washing liquid is added into each hole, and the plate is washed for 3-5 times and then is dried by beating.

In step III, as a preferred embodiment of the present invention, the color development and termination are specifically: and adding 100 microliters of TMB into each corresponding hole, developing at room temperature in a dark place, and adding 100 microliters of 2N (equivalent concentration) H2SO4 into each hole when the maximum concentration value of the microplate reader reading at the wavelength of 370nm is 1.4-1.6.

In step III, the background value and sensitivity of each group of data are compared after the treatment of the microplate reader, specifically: and reading the final data at the wavelength of 450nm, processing the fitting curve processing data by setting a template according to test conditions on an enzyme label instrument, and comparing the background value and the sensitivity of each group of data.

As a preferable technical scheme of the invention, the following steps are added between the step II and the step III: washing the plate with washing liquid for 3-5 times, and then beating to dry.

Compared with the prior art, the invention has the beneficial effects that:

the addition of monkey serum in the method can effectively improve the background value and optimize the sensitivity of the universal bioanalytical method, and the method is effective no matter the coated antibody is a common anti-human Fc antibody or a polyclonal antibody of a specific fragment. The method can solve the problems that the general background value of the general method in monkey serum or monkey plasma is relatively high, thereby affecting the sensitivity of the method and being unfavorable for the detection of the exposure of animals in low-dose groups. Experiments prove that the detection antibody working solution is an anti-human F (ab) 2 polyclonal antibody, and after the monkey serum is added into the detection antibody, the components homologous to the monkey IgG in the detection antibody can be neutralized, so that the sensitivity and the background value are obviously improved (see table 1), the background value is greatly reduced, and the function of an optimization method is achieved. Meanwhile, the difference between the capture antibody which is a common anti-human Fc polyclonal antibody and a polyclonal antibody of a specific anti-human CH2 fragment is compared, and the change between reagents is proved to be effective in adding monkey serum into the detection antibody.

Detailed Description

The above-described aspects are further described below in conjunction with specific embodiments. It should be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

Embodiment one:

1. experimental procedure

Mainly comprises the following steps:

1) Polyclonal antibodies against the human Fc fragment and polyclonal antibodies against the human CH2 fragment were prepared separately, and 2. Mu.g/mL of the coating buffer was prepared using a carbonate buffer. After adding 100 mu L of the solution to each well, the solution is placed at 2-8 ℃ and incubated overnight.

2) 300 microliters of PBS wash containing 0.5% Tween was added to each well, and the plate was washed 3 times and then dried. 300 microliters of 3% BSA blocking buffer was added to each well and incubated for 1-3 hours at room temperature.

3) 300 microliters of PBS wash containing 0.5% Tween was added to each well, and the plate was washed 3 times and then dried. Simultaneously, drugs with a gradient (5000, 2500, 1600, 800, 400, 200, 100, 50, 25, 10 ng/mL) were formulated in monkey serum and 50-fold pre-dilution was performed using 1% bsa. Then 100 microliters of sample was added per well and incubated at room temperature for 2 hours at 400-600 revolutions per second.

4) 300 microliters of PBS wash containing 0.5% Tween was added to each well, and the plate was washed 3 times and then dried. Meanwhile, preparing a detection antibody working solution. The detection antibody working solution comprises the following components in parts by weight: 20000-fold dilutions in 1% bsa, one portion is 1: 20000-fold dilutions were made in 1% BSA with 5% Monkey Serum. 100 microliters per well was added to the corresponding well and incubated at room temperature for 1 hour at 400-600 revolutions per second.

5) 300 microliters of PBS wash containing 0.5% Tween was added to each well, and the plate was washed 3 times and then dried. When 100 microliter TMB is added to each corresponding hole and color development is carried out at room temperature in a dark place, and the maximum concentration value of the microplate reader at the wavelength of 370nm is 1.4-1.6, 100 microliter 2N H2SO4 is added to each hole to terminate the reaction. And the final data were read at a wavelength of 450nm (see Table 2 for results).

6) And setting the concentration of each hole on an enzyme label instrument according to a test plate bitmap, fitting each curve according to the corresponding relation between the concentration and an OD value, and setting a curve weight factor to be 1. Blank wells for each condition did not participate in the curve fit. (the results are shown in tables 3 to 6).

2. Experimental results

1) The current general method is that the coating antibody is a polyclonal antibody of an anti-human Fc segment, the detection antibody is another polyclonal antibody of an anti-human Fc segment, the established method has the general disadvantage of high background value, the result of STD1 shown in the table 1 is that the background value is as high as 1.475, the sensitivity is 200ng/mL (the acceptance standard is less than or equal to 20% according to CV%, bias% is less than or equal to 20% according to CV%, and the upper limit and the lower limit of quantification are 25% according to the acceptance standard).

2) Under the same conditions as in 1), 5% monkey serum was added to the detection antibody, the background value was greatly improved to 0.268, and the sensitivity reading was also relatively improved to 50 ng/mL. But the background value is still high (the general method suggests that the background value is less than 0.2). See table 1 for STD2 results.

3) Under otherwise identical conditions of 1), the coated antibodies were polyclonal antibodies specific for CH2 at the same working concentration, see the results for STD3 in Table 1, with a background value of 0.321, a sensitivity of about 50ng/mL, and a much improved background value and sensitivity over 1), but the background value was still high.

4) Under the other conditions of 3), 5% monkey serum is added into the detection antibody, the background value is greatly improved to be 0.057, the sensitivity reading is relatively improved to be 25ng/mL, and CV% and Bias% are relatively low and stable. See table 1 for STD4 results.

Table 1 plate position meter

In Table 1, STD1: coating: 2. Mu.g/mL polyclonal antibody against human Fc fragment; detection: 1:20000 fold dilution in 1% bsa;

STD2 Coating 2. Mu.g/mL polyclonal antibody against human Fc fragment; detection 1:20000 fold dilutions in 1% BSA with 5% monkey serum;

STD3 Coating 2 mug/mL anti-human CH2 segment polyclonal antibody; detection: 1:20000 fold dilution in 1% bsa;

STD4 Coating 2 mug/mL anti-human CH2 segment polyclonal antibody; detection 1:20000 fold dilutions were made in 1% BSA with 5% monkey serum.

The concentrations of all standard curves were 5000, 2500, 1600, 800, 400, 200, 100, 50, 25, 10ng/mL. OD signal values were read by a 450nm wavelength microplate reader, and the results are shown in Table 2.

Table 2 original data of OD signal values read by 450nm wavelength microplate reader

Tables 3-6 show the data obtained after the OD signal values were read by a 450nm wavelength microplate reader.

TABLE 3 data after processing of reading OD signal values by 450nm wavelength ELISA

As shown in Table 3, coating 2. Mu.g/mL of polyclonal antibody against human Fc fragment; detection 1:20000 fold dilutions in 1% BSA. The concentration variation coefficient CV% of the curve should be 20% or less (the lowest quantitative limit and the highest quantitative limit are 25%), and the deviation Bias% should be within + -20% (the lowest quantitative limit and the highest quantitative limit are 25%). In combination with the above data, the sensitivity satisfying the acceptance criteria was 200ng/mL, and the background signal value was as high as 1.475.

TABLE 4 data after processing of reading OD signal values by 450nm wavelength ELISA

As shown in Table 4, coating 2. Mu.g/mL of polyclonal antibody against human Fc fragment; detection 1:20000 fold dilutions were made in 1% BSA with 5% monkey serum. The concentration variation coefficient CV% of the curve should be 20% or less (the lowest quantitative limit and the highest quantitative limit are 25%), and the deviation Bias% should be within + -20% (the lowest quantitative limit and the highest quantitative limit are 25%). In combination with the above data, the sensitivity that meets the acceptance criteria is 50ng/mL, and the background signal value drops to 0.268.

TABLE 5 data after processing of reading OD signal values by 450nm wavelength ELISA

As shown in Table 5, coating 2. Mu.g/mL of polyclonal antibody against the human CH2 segment; detection 1:20000 fold dilutions in 1% BSA. The coefficient of variation CV% of the concentration of the curve should be 20% or less (the lowest quantitative limit and the highest quantitative limit are 25%),

the Bias% should be within + -20% (the lowest limit and the highest limit are 25%). In combination with the above data, the sensitivity satisfying the acceptance criteria was 50ng/mL, and the background signal value was 0.321.

Table 6 data after reading OD signal value by 450nm wavelength enzyme labelling instrument

As shown in Table 6, coating 2. Mu.g/mL of polyclonal antibody against the human CH2 segment; detection 1:20000 fold dilutions were made in 1% BSA with 5% monkey serum. The concentration variation coefficient CV% of the curve should be 20% or less (the lowest quantitative limit and the highest quantitative limit are 25%), and the deviation Bias% should be within + -20% (the lowest quantitative limit and the highest quantitative limit are 25%). In combination with the above data, the sensitivity satisfying the acceptance criteria was 25ng/mL, and the background signal value was reduced to 0.057.

In summary, the improvement of sensitivity and background value is very remarkable after 5% monkey serum is added to the detection antibody, and the method is effective whether the coated antibody is a general anti-human Fc antibody or a polyclonal antibody against a specific fragment.

Claims

1. A method for optimizing a universal bioassay by adding monkey serum, comprising the steps of:

III, developing and stopping, and comparing the background value and the sensitivity of each group of data after the treatment of the enzyme label instrument;

the preparation method of the universal coated antibody buffer solution in the step I comprises the following steps: preparing polyclonal antibody of the anti-human CH2 segment, and preparing a coated antibody buffer solution by using a carbonate buffer solution; the washing liquid is PBS containing 0.05-0.1% Tween; the blocking buffer solution is 3% -5% BSA; the test substance is prepared from monkey serum, contains a drug with a gradient of 5000-10ng/mL, and is subjected to 50-time pre-dilution by using 1% BSA;

the preparation method of the working solution containing the monkey serum for detecting antibodies in the step I I comprises the following steps: 1: 20000-fold dilutions in 1% bsa with 5% monkey serum; the preparation method of the working solution of the detection antibody without monkey serum comprises the following steps: 1: 20000-fold dilutions were in 1% bsa.

2. The method of claim 1, wherein in step I, the high adsorption plate is a 96-well plate, 100 μl of universal coated antibody buffer is added to each well, and incubated overnight at 2-8deg.C.

3. The method according to claim 1, wherein in step I, the high adsorption plate is a 96-well plate, the plate is washed with a washing liquid 3-5 times and then is patted dry, 300 μl of a blocking buffer is added to each well, and the plate is incubated at room temperature for 1-3 hours.

4. The method according to claim 1, wherein in step I, the high adsorption plate is a 96-well plate, the plate is washed with a washing liquid 3 to 5 times and then is patted dry, 100 microliters of the test substance is added to each well, and the plate is incubated at room temperature for 2 hours at 400 to 600 revolutions per second.

5. The method according to claim 1, wherein 100 microliters of detection antibody working solution is added to each well in step II, and incubated at room temperature for 1 hour at 400-600 revolutions per second.

6. The method of claim 1 or 5, wherein the following steps are added between step I and step I I: washing the plate with washing liquid for 3-5 times, and then beating to dry.

7. The method of claim 6, wherein the wash solution is a PBS wash solution containing 0.05% to 0.1% tween, 300 μl of wash solution is added per well, and the plate is washed 3-5 times and then dried.

8. The method according to claim 1, wherein in step III, the color development and termination is specifically: adding 100 microliter TMB into each corresponding hole, developing at room temperature in dark, and adding 100 microliter 2N H into each hole when the maximum concentration value of the microplate reader is 1.4-1.6 under 370nm wavelength ₂ SO ₄ And (5) terminating.

9. The method according to claim 1, wherein in step III, the background value and the sensitivity of each set of data are compared after the microplate reader processing, specifically: and reading the final data at the wavelength of 450nm, processing the fitting curve processing data by setting a template according to test conditions on an enzyme label instrument, and comparing the background value and the sensitivity of each group of data.

10. The method according to claim 1 or 7 or 8, characterized in that the following steps are added between step II and step III: washing the plate with washing liquid for 3-5 times, and then beating to dry.

11. The method of claim 10, wherein the wash solution is a PBS wash solution containing 0.05% to 0.1% tween, 300 μl of wash solution is added per well, and the plate is washed 3-5 times and then dried.