CN111856032A - Method for rapidly detecting concentration of anti-PD-L1 monoclonal antibody - Google Patents
Method for rapidly detecting concentration of anti-PD-L1 monoclonal antibody Download PDFInfo
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Abstract
The invention discloses a method for rapidly detecting the concentration of an anti-PD-L1 monoclonal antibody, which adopts a full-automatic molecular luminescence immunoassay analyzer for detection and specifically comprises the following steps: a. preparing a standard solution; b. respectively adding a sample to be detected and a standard solution with series concentration into an AML instrument, mixing and reacting with nanometer magnetic beads coupled with PD-L1 protein and isoluminol derivative labeled Goat anti-Human IgG (tracer), adding a system liquid, washing, adding an excitation liquid, and determining a luminescence peak value; c. and (4) calculating the concentration of the anti-PD-L1 monoclonal antibody in the sample to be tested. According to the detection method, the magnetic beads are used as the solid phase carriers, and the isoluminol derivative is adopted to label the antibody, so that the reaction of the antigen and the antibody is accelerated, the concentration detection result of the anti-PD-L1 monoclonal antibody is accurate, the automation degree, the detection speed and the sensitivity are improved, and the detection method has very important significance.
Description
Technical Field
The invention particularly relates to a method for rapidly detecting the concentration of an anti-PD-L1 monoclonal antibody.
Background
The anti-PD-L1 monoclonal antibody medicine is a star medicine in the existing tumor treatment medicines, has obvious treatment effects on various tumors such as melanoma, lung cancer, urothelial cancer, head and neck squamous carcinoma and the like, is different from the traditional treatments such as chemotherapy, radiotherapy, targeted therapy and the like which directly kill tumor cells, and the anti-PD-L1 monoclonal antibody indirectly kills the tumor cells by acting on an immune system. Like other antitumor drugs, the anti-PD-L1 monoclonal antibody is also accompanied by adverse reactions related to the action mechanism thereof during the use process, and is called immune-related adverse reactions (iraE). irAE may be characterized by delayed onset, long duration and more organ involvement than adverse events resulting from chemotherapy. .
In recent years, biotechnological drugs, in particular antibody drugs, have been developed more and more rapidly. In the case of biotechnological drugs, in vivo drug analysis is mainly based on immunoassay. For the anti-PD-L1 monoclonal antibody drugs, the analytical methods we used are mainly ELISA and MSD. The two methods have the main defects of low automation degree, high labor cost and long time consumption, and the experiment can be completed in 5-8 hours except that the coating needs to be carried out overnight in advance and the subsequent experiment operation is completed. The development of a detection method for quantitatively detecting the anti-PD-L1 antibody with high automation degree and rapid detection has very important significance.
The full-automatic immunoassay method is detected by a full-automatic molecular luminescence immunoassay analyzer, wherein the chemiluminescence reaction is divided into two types: the first one is to use luminous agent as substrate of enzyme immunoassay to enhance the sensitivity of the assay by luminous reaction, the second one is to use luminous agent as antibody or antigen marker to directly detect the content of antigen or antibody in the sample by luminous reaction, the detection time by full-automatic molecular luminescence immunoassay analyzer is short and only 5-20 minutes is needed to obtain the result, but not any chemical reaction kinetic energy generates chemiluminescence reaction, therefore, if the measuring condition is proper, the chemiluminescence analysis has enough selectivity.
At present, no fully automatic analysis method for the anti-PD-L1 antibody is available.
Disclosure of Invention
In order to solve the problems, the invention provides a method for rapidly detecting the concentration of an anti-PD-L1 monoclonal antibody, which adopts a full-automatic molecular luminescence immunoassay analyzer for detection and specifically comprises the following steps:
a. preparation of standard solution: preparing a standard substance of the anti-PD-L1 monoclonal antibody into standard solutions with series concentrations by using a blank sample solution;
b. respectively adding a sample to be detected and a standard solution with a series of concentrations into an AML instrument, mixing and reacting the sample to be detected and the standard solution with a nano magnetic bead coupled PD-L1 protein solution and a tracer solution for 15-20 min, adding a system liquid for washing, removing the washed system liquid, adding an excitation liquid, mixing uniformly, and measuring a light emitting value;
c. and establishing a four-parameter fitting standard curve according to the concentration and the luminous value of the standard solution, and calculating the concentration of the anti-PD-L1 monoclonal antibody in the sample to be detected according to the standard curve and the luminous value of the sample to be detected.
Further, the concentration of the anti-PD-L1 monoclonal antibody in the series of concentration standard solutions in the step a is 0.2-51.2 ng/mL.
Further, the volume ratio of the sample to be detected or the standard solution with the series of concentrations to the nano magnetic bead coupled PD-L1 protein solution, the tracer solution, the system liquid, the excitation liquid A and the excitation liquid B in the step B is 4-6: 1-3: 10-20: 60-70: 10-30, preferably 5: 2: 15: 65: 20.
Still further, the test sample includes, but is not limited to, serum, plasma, or a sample that requires formulation with a protein buffer.
Furthermore, the nano magnetic bead coupled PD-L1 protein solution is a solution prepared by diluting a nano magnetic bead coupled PD-L1 protein stock solution by 10 times with a 1% bovine serum albumin diluent; the tracer solution is prepared by diluting a Goat Anti-Human IgG stock solution marked by isoluminol derivative by 2000 times with a 1% bovine serum albumin diluent.
Further, the exciting liquid in the step B is composed of an exciting liquid (A) NaOH solution and an exciting liquid (B) carbamide peroxide solution, wherein the ratio of the exciting liquid (A) to the exciting liquid (B) is 1: 1.
further, the concentration of the NaOH solution is 0.5mol/L, and the concentration of the carbamide peroxide solution is 0.4 g/L.
Further, the blank sample solution is protein buffer solution, blank serum or blank plasma; the protein buffer solution is bovine serum albumin diluent, and the concentration is 0.5-1.5%, preferably 1%.
Further, the mixture of step b is reacted for 20 min.
Further, the system liquid in the step b is boric acid buffer liquid; the pH value of the boric acid buffer solution is 8.0.
The invention "blank serum or blank plasma" refers to serum or plasma without anti-PD-L1 monoclonal antibody, and the source is animal or human;
According to the method for rapidly detecting the concentration of the anti-PD-L1 monoclonal antibody, magnetic beads are used as solid phase carriers, and the isoluminol derivative is used for marking the antibody, so that the reaction of an antigen and an antibody is accelerated, the concentration detection result of the anti-PD-L1 monoclonal antibody is accurate, the automation degree, the detection speed and the sensitivity are improved, and the method has very important significance.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Detailed Description
The raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products.
Example 1 detection of the concentration of monoclonal antibody against PD-L1 in the serum of the invention
First, preparation of solution
a. Preparation of standard solution: taking a standard substance of the anti-PD-L1 monoclonal antibody, adding blank serum to prepare standard solutions of which the concentrations of the anti-PD-L1 monoclonal antibody are respectively 0.2 ng/mL, 0.4 ng/mL, 0.8 ng/mL, 1.6 ng/mL, 3.2 ng/mL, 6.4 ng/mL, 12.8 ng/mL, 25.6 ng/mL and 51.2 ng/mL;
b. preparation of nano magnetic bead coupled PD-L1 protein solution: taking nano magnetic bead coupled PD-L1 protein stock solution, and diluting by 10 times with 1% bovine serum albumin diluent to obtain the nano magnetic bead coupled PD-L1 protein stock solution;
c. preparing a tracer solution: diluting the isoluminol derivative labeled Goat anti-Human IgG stock solution by 2000 times with 1% bovine serum albumin diluent to obtain the isoluminol derivative labeled Goat anti-Human IgG stock solution;
second, detecting
d. Putting a serum sample to be detected and a standard solution with a series of concentrations on a sample frame of an AML instrument, putting a nano magnetic bead coupled PD-L1 protein solution and a tracer solution in a reagent disc of the AML instrument, then respectively sucking the series of standard solutions and the sample to be detected by using a sample adding needle, respectively sucking the nano magnetic bead coupled PD-L1 protein solution and the tracer solution by using a reagent needle, respectively mixing and reacting 50 mu L of the series of standard solutions or the serum sample to be detected with 20 mu L of the nano magnetic bead coupled PD-L1 protein solution and 150 mu L of the tracer solution for 20min, automatically adding 650 mu L of system liquid (pH value 8.0 boric acid buffer solution) for washing, automatically removing the washed system liquid, and finally automatically adding 200 mu L of the standard solution with the volume ratio of 1: 1, uniformly mixing an excitation liquid A (NaOH solution) and an excitation liquid B (carbamide peroxide solution), and measuring a luminous value;
e. Fitting the measured values of the standard solutions with the series of concentrations and the theoretical concentration by adopting four parameters to obtain a four-parameter equation, and substituting the measured values of the sample to be detected into the four-parameter equation for back calculation to obtain the concentration of the anti-PD-L1 monoclonal antibody in the sample to be detected.
EXAMPLE 2 detection of the concentration of the anti-PD-L1 monoclonal antibody in plasma according to the invention
First, preparation of solution
a. Preparation of standard solution: preparing a standard substance of the anti-PD-L1 monoclonal antibody into standard solutions with the concentrations of the anti-PD-L1 monoclonal antibody of 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6 and 51.2ng/mL respectively by using blank plasma;
b. preparation of nano magnetic bead coupled PD-L1 protein solution: taking nano magnetic bead coupled PD-L1 protein stock solution, and diluting by 10 times with 1% bovine serum albumin diluent to obtain the nano magnetic bead coupled PD-L1 protein stock solution;
c. preparing a tracer solution: diluting the isoluminol derivative labeled Goat anti-Human IgG stock solution by 2000 times with 1% bovine serum albumin diluent to obtain the isoluminol derivative labeled Goat anti-Human IgG stock solution;
second, detecting
d. Putting a blood plasma sample to be detected and a standard solution with a series of concentrations on a sample frame of an AML instrument, putting a nano magnetic bead coupled PD-L1 protein solution and a tracer solution into a reagent disc of the AML instrument, then respectively sucking the series of standard solutions and the sample to be detected by using a sample adding needle, respectively sucking the nano magnetic bead coupled PD-L1 protein solution and the tracer solution by using a reagent needle, respectively mixing and reacting 50 mu L of the series of standard solutions or the blood plasma sample to be detected with 20 mu L of the nano magnetic bead coupled PD-L1 protein solution and 150 mu L of the tracer solution for 20min, automatically adding 650 mu L of system liquid (pH value 8.0 boric acid buffer solution) for washing, automatically removing the washed system liquid, and finally automatically adding 200 mu L of the standard solution with the volume ratio of 1: 1, uniformly mixing an excitation liquid A (NaOH solution) and an excitation liquid B (carbamide peroxide solution), and measuring a luminous value;
e. Fitting the measured values of the standard solutions with the series of concentrations and the theoretical concentration by adopting four parameters to obtain a four-parameter equation, and substituting the measured values of the sample to be detected into the four-parameter equation for back calculation to obtain the concentration of the anti-PD-L1 monoclonal antibody in the sample to be detected.
Example 3 detection of the concentration of anti-PD-L1 monoclonal antibody in the protein buffer solution of the invention
First, preparation of solution
a. Preparation of standard solution: preparing a standard solution with the concentrations of the anti-PD-L1 monoclonal antibodies of 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6 and 51.2ng/mL by taking the anti-PD-L1 monoclonal antibody standard substance and using a 1% bovine serum albumin diluent;
b. preparation of nano magnetic bead coupled PD-L1 protein solution: taking nano magnetic bead coupled PD-L1 protein stock solution, and diluting by 10 times with 1% bovine serum albumin diluent to obtain the nano magnetic bead coupled PD-L1 protein stock solution;
c. preparing a tracer solution: diluting the isoluminol derivative labeled Goat anti-Human IgG stock solution by 2000 times with 1% bovine serum albumin diluent to obtain the isoluminol derivative labeled Goat anti-Human IgG stock solution;
second, detecting
d. Putting a protein buffer solution and a standard solution with a series of concentrations of a sample to be detected on a sample frame of an AML instrument, putting a nano magnetic bead coupled PD-L1 protein solution and a tracer solution into a reagent disc of the AML instrument, then respectively sucking the series of standard solutions and the sample to be detected by using a sample adding needle, respectively sucking the nano magnetic bead coupled PD-L1 protein solution and the tracer solution by using a reagent needle, respectively mixing and reacting 50 mu L of the series of standard solutions or the sample to be detected with 20 mu L of the nano magnetic bead coupled PD-L1 protein solution and 150 mu L of the tracer solution for 20min, automatically adding 650 mu L of system liquid (pH value 8.0 boric acid buffer solution) for washing, automatically removing the washed system liquid, and finally automatically adding 200 mu L of the standard solution with the volume ratio of 1: 1, uniformly mixing an excitation liquid A (NaOH solution) and an excitation liquid B (carbamide peroxide solution), and measuring a luminous value;
e. Fitting the measured values of the standard solutions with the series of concentrations and the theoretical concentration by adopting four parameters to obtain a four-parameter equation, and substituting the measured values of the sample to be detected into the four-parameter equation for back calculation to obtain the concentration of the anti-PD-L1 monoclonal antibody in the sample to be detected.
The advantageous effects of the present invention are described below by way of test examples.
Test example 1 precision, accuracy and linearity verification of the detection method of the present invention
1. Primary reagent information
1) And (3) standard substance:
chinese name: programmed cell death-ligand 1 monoclonal antibody
Batch number: 2018120501
Packaging: nalgene bottle
Concentration/content: 30mg/mL
Physical and chemical properties: colorless clear solution
The validity period is as follows: 2019.12.4
Production unit: NP scientific inc
Providing a unit: beijing Xinai advanced Biotechnology Co Ltd
And (3) storage: and (5) preserving at 2-8 ℃ in a dark place.
2) Other reagent information
2. Sample preparation
1) Standard curve sample
mu.L of anti-PD-L1 monoclonal antibody was added to 45. mu.L of 1% BSA diluent to prepare a solution Stock A at 3000. mu.g/mL.
5 μ L of LStock A was added to 45 μ L of 1% BSA dilution to make up 300 μ g/mL solution Stock B.
mu.L of Stock B was added to 45. mu.L of 1% BSA dilution to prepare 30. mu.g/mL of solution Stock C.
mu.L of Stock C was added to 180. mu.L of 1% BSA dilution to prepare 3. mu.g/mL of solution Stock D.
Standard curve samples were prepared by dilution as follows.
The solution Stock A was diluted as in the following table to prepare a standard curve sample.
2) Quality control sample
The solution Stock D was diluted according to the following table to prepare quality control samples:
remarking: and respectively taking out the prepared standard curve sample and quality control sample, subpackaging 12 parts for later use, storing each part in a refrigerator at-70 ℃ of 150ul, and using after completely re-dissolving during detection.
3) Precision and accuracy sample
Using a fully automated molecular luminescence bioanalyzer, following the detection method of example 3, 3 days were measured, and 6 assay batches were examined, 2 assay batches were run each day, standard curve, quality control and Blank samples were run in each assay batch, each batch containing 9 concentrations of standard curve samples (cal.1, cal.2, cal.3, cal.4, cal.5, cal.6, cal.7, cal.8, AP1), each concentration level standard curve sample being measured twice each; each batch contained 5 concentrations (ULOQ, QC-H, QC-M, QC-L, and LLOQ) of quality control samples, each measured 5 times per concentration level of quality control samples. The results were recorded and the CV% values were calculated.
4) Autodilutability-verifying sample
Programmed cell death-ligand 1 was first prepared as 3000. mu.g/mL solution Stock A with 1% BSA dilution.
Then diluting 150 times with 1% BSA diluent, setting an instrument to automatically dilute 10 times, wherein the specific dilution steps are as shown in the following table, and the theoretical concentration of the instrument before dilution is 20000 ng/mL. And comparing the actual concentration value obtained by multiplying the detection result by the dilution factor with a theoretical value, and calculating the% RE of the diluted sample.
Remarking: the instrument was used to pre-dilute 10 fold (Step3) to a final dilution of 1500.
5) Validating requirements
At least 6 points (including LLOQ and ULOQ) are constructed, and the measured values of the points on the standard curve (without ULOQ and LLOQ) should be between 80-120% of the theoretical values, and the measured values of LLOQ and ULOQ should be between 75-125% of the theoretical values. The measured value of autodilutability verification should be between 80-120% of its theoretical value.
3 results
1) Result of precision and accuracy verification
TABLE 1 Standard Curve verification results (unit: ng/mL) of PD-L1 detected by full-automatic molecular luminescence biological immunity analyzer
TABLE 2 precision and accuracy (unit: ng/mL) of PD-L1 detected by full-automatic molecular luminescence biological immunity analyzer
TABLE 3 automatic dilution verification result (unit: ng/mL) of PD-L1 detected by full-automatic molecular luminescence bioanalyzer
The results in tables 1-3 show that the precision and accuracy between batches and in batches are good, the requirements of biological analysis guiding principles of Chinese pharmacopoeia are met, and the result is accurately calculated after automatic dilution, so that the method is stable and reliable. After general biological analysis methods in the field are verified, the result meets the requirements, the method is considered to be reliable, if serum, plasma or other samples needing to be prepared by protein buffer solution are required to detect the concentration of the anti-PD-L1 monoclonal antibody, and the anti-PD-L1 monoclonal antibody standard is replaced by corresponding blank serum or blank plasma for 1% bovine serum albumin diluent.
In conclusion, the method for detecting the concentration of the anti-PD-L1 monoclonal antibody accelerates the reaction of the antigen and the antibody, ensures that the detection result of the concentration of the anti-PD-L1 monoclonal antibody is accurate, improves the automation degree, the detection speed and the sensitivity, and has very important significance.
Claims (10)
1. A method for rapidly detecting the concentration of an anti-PD-L1 monoclonal antibody is characterized by comprising the following steps: the method adopts a full-automatic molecular luminescence immunoassay analyzer for detection, and specifically comprises the following steps:
a. preparation of standard solution: taking a standard substance of the anti-PD-L1 monoclonal antibody, and adding a blank sample solution to prepare a standard solution with a series of concentrations;
b. respectively adding a sample to be detected and a standard solution with a series of concentrations into an AML instrument, mixing and reacting the sample to be detected and the standard solution with a nano magnetic bead coupled PD-L1 protein solution and a tracer solution for 15-20 min, adding a system liquid for washing, removing the washed system liquid, adding an excitation liquid, mixing uniformly, and measuring a light emitting value;
c. and establishing a four-parameter fitting standard curve according to the concentration and the luminous value of the standard solution, and calculating the concentration of the anti-PD-L1 monoclonal antibody in the sample to be detected according to the standard curve and the luminous value of the sample to be detected.
2. The detection method according to claim 1, characterized in that: the concentration of the anti-PD-L1 monoclonal antibody in the series of concentration standard solutions in the step a is 0.2-51.2 ng/mL.
3. The detection method according to claim 1, characterized in that: and b, coupling the standard solution with the sample to be detected or the series of concentrations with the nano magnetic beads to obtain a PD-L1 protein solution, a tracer solution, a system liquid and an excitation liquid in a volume ratio of 4-6: 1-3: 10-20: 60-70: 10-30, preferably 5: 2: 15: 65: 20.
4. the detection method according to claim 1 or 3, characterized in that: the sample to be tested includes but is not limited to serum, plasma or a sample which needs to be prepared by protein buffer.
5. The detection method according to claim 1 or 3, characterized in that: the nano magnetic bead coupled PD-L1 protein solution is prepared by diluting a nano magnetic bead coupled PD-L1 protein stock solution by 10 times with a 1% bovine serum albumin diluent.
6. The detection method according to claim 1 or 3, characterized in that: the tracer solution is prepared by diluting a Goat Anti-Human IgG stock solution marked by isoluminol derivative by 2000 times with a 1% bovine serum albumin diluent.
7. The detection method according to claim 1 or 3, characterized in that: and step b, the exciting liquid is prepared from the following components in a volume ratio of 1: 1 NaOH solution and carbamide peroxide solution; the concentration of the NaOH solution is 0.5mol/L, and the concentration of the carbamide peroxide solution is 0.4 g/L.
8. The detection method according to claim 1, characterized in that: the blank sample solution is protein buffer solution, blank serum or blank plasma; the protein buffer solution is bovine serum albumin solution, and the concentration is 0.5-1.5%, preferably 1%.
9. The detection method according to claim 1, characterized in that: mixing and reacting for 20min in the step b.
10. The detection method according to claim 1, characterized in that: the system liquid in the step b is boric acid buffer liquid; the pH value of the boric acid buffer solution is 8.0.
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