CN116990528B - Analysis method for rapidly determining anti-CD40 monoclonal antibody based on Gyrolab platform - Google Patents

Analysis method for rapidly determining anti-CD40 monoclonal antibody based on Gyrolab platform Download PDF

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CN116990528B
CN116990528B CN202311262970.4A CN202311262970A CN116990528B CN 116990528 B CN116990528 B CN 116990528B CN 202311262970 A CN202311262970 A CN 202311262970A CN 116990528 B CN116990528 B CN 116990528B
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扈正桃
薛爱琴
张楠
赖力
贺艳琳
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Chengdu Huaxi Haiqi Medical Technology Co ltd
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Abstract

The invention belongs to the technical field of biological detection, and particularly relates to an analysis method for rapidly determining anti-CD40 monoclonal antibody based on a Gyrolab platform. The invention adopts a Gyrolab platform to detect the concentration of the anti-CD40 monoclonal antibody, in the detection process, the capture antibody is diluted to 50-100 mu g/ml by PBS-T, and the detection antibody is Rexxip TM F, diluting to 10-150 mug/ml. According to the invention, through optimizing sample processing conditions and preparation conditions, the detection accuracy can be improved, the purpose of detecting the anti-CD40 monoclonal antibody by using the Gyrolab platform is further realized, and the method has a good application prospect.

Description

Analysis method for rapidly determining anti-CD40 monoclonal antibody based on Gyrolab platform
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to an analysis method for rapidly determining anti-CD40 monoclonal antibody based on a Gyrolab platform.
Background
anti-CD40 monoclonal antibody medicine has unique effect on treating solid tumor, lymphoma and multiple myeloma. In recent years, with the rapid development of antibody drug application, the development of bioanalytical technology faces greater challenges and opportunities. Currently, quantitative analysis methods of anti-CD40 monoclonal antibodies are mainly ELISA and MSD, but the methods need large sample size, are difficult to automate, and are time-consuming and labor-consuming. The development of a detection method for quantitatively analyzing the anti-CD40 monoclonal antibody with high automation degree and high detection speed has very important significance.
The Gyrolab platform (Gyrolab xPlore) is an analysis device developed by Gyros of sweden enterprises, and can replace the traditional method to carry out immune experiments of various links of biomacromolecule drug research and development, preclinical, clinical and production. By adopting the Gyrolab xPlore system in an automatic sample injection mode, the traditional double-antibody sandwich macromolecule detection method and the micro-fluidic CD technology of the patent are perfectly combined together, so that experimental effects which cannot be achieved by other experimental methods requiring manual operation can be achieved, and the experimental effects comprise perfect data repeatability, extremely high detection sensitivity, extremely wide detection range, strong matrix resistance, extremely small sample injection quantity and the like. Therefore, it would be of great interest if gyrolab xplore could be applied to screening of anti-CD40 mab drugs.
However, in the prior art, there is still a difficulty in applying Gyrolab xPlore to the analysis of anti-CD40 mab drugs. The most important problem is that the prior setting of the analysis conditions of the Gyrolab xPlore applied to the anti-CD40 monoclonal antibody lacks relevant research, so that the linearity of a standard curve obtained in analysis is poor, and an accurate detection result is difficult to obtain.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an analysis method for rapidly determining anti-CD40 monoclonal antibody based on a Gyrolab platform.
An analytical method for rapidly determining anti-CD40 monoclonal antibody based on Gyrolab platform adopts GyThe method comprises the steps of detecting the concentration of anti-CD40 monoclonal antibody by a rolab platform, diluting a capture antibody to 50-100 mug/ml by PBS-T in the detection process, and detecting the antibody by Rexxip TM F, diluting to 10-150 mug/ml.
Preferably, the capture antibody is diluted to 50 μg/ml with PBS-T and/or the detection antibody is Rexxip TM F is diluted to 40 mug/ml.
Preferably, the capture antibodies are labeled with the EZ-Link ™ Micro Sulfo-NHS-LC-Biotinylation Kit kit and/or the detection antibodies are labeled with the Alexa Fluor ™ 647 antibody labeling kit.
Preferably, in the detection process, the preparation method of the standard curve sample is as follows: standard solutions of serial concentrations were prepared with rhesus monkey blank serum using anti-CD40 mab standard.
Preferably, before the rhesus monkey blank serum is used, the rhesus monkey blank serum is centrifuged for 5-10 min at 12000-14000 Xg and 2-8 ℃ and an intermediate clarified serum matrix is selected for use.
Preferably, the concentration range of the standard solution is 19.5-80000 ng/mL.
Preferably, in the detection process, the sample to be detected is subjected to Rexxip TM HX is diluted 10-20 times and then loaded.
Preferably, in the analysis method, the solution prepared by the quality control sample comprises a solution containing the anti-CD40 monoclonal antibody standard with the following concentration:
500000 ng/mL of A solution,
HQC solution 6400 ng/mL,
4000 ng/mL of solution B,
MQC solution 1000 ng/mL,
LQC solution 100 ng/mL.
Preferably, the Gyrolab Bioaffy 200 CD of the Gyrolab platform is equilibrated to room temperature prior to use.
Preferably, in the detection process, the solution prepared by the capture antibody, the solution prepared by the detection antibody, the solution prepared by the sample to be detected, the standard solution and the solution prepared by the quality control sample are respectively subjected to the following operations before use: mixing uniformly, centrifuging and using;
wherein, the centrifugal conditions of the solution prepared by the capture antibody and the solution prepared by the detection antibody are as follows: 3000-5000 Xg, 2-8deg.C, centrifuging for 3-5min;
the centrifugal conditions of the solution prepared by the sample to be detected, the standard solution and the solution prepared by the quality control sample are as follows: 12000-14000 Xg, 2-8deg.C, and centrifuging for 5-10 min.
The invention optimizes the detection method of the Gyrolab platform, and can obtain the following beneficial effects by optimizing sample preparation conditions (such as dilution ratio of capture antibody and detection antibody):
1. the standard curve with better linearity can be obtained, and the detection accuracy is further improved.
2. In a preferred embodiment, the invention also optimizes other processing conditions (e.g., centrifugation conditions) to further improve the accuracy of the detection.
Based on the condition optimization, the invention realizes the purpose of detecting the anti-CD40 monoclonal antibody by using the Gyrolab platform for the first time, and has good application prospect.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 is a standard curve obtained during the optimization of the optimum conditions in Experimental example 2; wherein, curve 1: the concentration of the capture antibody (Biotin-Human CD 40) is 100 mug/mL, and the concentration of the detection antibody (Alex 647-Human IgG h+l) is 37.5 mug/mL; curve 2: the concentration of the capture antibody (Biotin-Human CD 40) is 100 mug/mL, and the concentration of the detection antibody (Alex 647-Human IgG h+l) is 75 mug/mL; curve 3: the concentration of the capture antibody (Biotin-Human CD 40) is 100 mug/mL, and the concentration of the detection antibody (Alex 647-Human IgG h+l) is 150 mug/mL;
FIG. 2 is a standard curve obtained during the optimization of the optimum conditions in Experimental example 2; wherein, curve 1: the concentration of the capture antibody (Biotin-Human CD 40) is 50 mug/mL, and the concentration of the detection antibody (Alex 647-Human IgG h+l) is respectively 10 mug/mL; curve 2: the concentration of the capture antibody (Biotin-Human CD 40) is 50 mug/mL, and the concentration of the detection antibody (Alex 647-Human IgG h+l) is 40 mug/mL respectively; curve 3: when the concentration of the capture antibody (Biotin-Human CD 40) is 100 mug/mL, the concentration of the detection antibody (Alex 647-Human IgG h+l) is 10 mug/mL respectively; curve 4: when the concentration of the capture antibody (Biotin-Human CD 40) is 100 mug/mL, the concentration of the detection antibody (Alex 647-Human IgG h+l) is 40 mug/mL respectively;
FIG. 3 is a standard curve obtained during the optimization of the optimum conditions in Experimental example 2; wherein, curve 1: when the concentration of the capture antibody (Biotin-Human CD 40) is 50 mug/mL, and the concentration of the detection antibody (Alex 647-Human IgG h+l, self-labeled antibody) is 40 mug/mL respectively, the corresponding standard curve is obtained; curve 2: when the concentration of the capture antibody (Biotin-Human CD 40) is 50 mug/mL, the concentration of the detection antibody (Goatti-Human IgG, monkey ads-Alexa Fluor 647, purchased commercial antibody) is 40 mug/mL respectively;
FIG. 4 is a standard curve obtained in the preferred embodiment of Experimental example 2.
Detailed Description
In the following examples and experimental examples, reagents and raw materials not specifically described are commercially available.
Example 1 analytical method for rapidly determining anti-CD40 monoclonal antibody based on Gyrolab platform
The embodiment provides a method for measuring anti-CD40 monoclonal antibody by using a Gyrolab platform, which is specifically as follows:
1. principle of detection
Adding a standard curve sample, a quality control sample, a sample to be tested and a reagent into a PCR plate according to a typesetting schematic diagram (MP), preparing a mechanical arm in a Gyrolab xPLand, accurately transferring the sample and the reagent from the PCR plate to each microstructure in a CD by using a pipetting needle during operation, wherein the CD microstructure is provided with a microbead affinity column wrapped by 15 nL streptavidin, and the CD accurately rotates at a proper speed and time to ensure that the sample and the reagent flow through the affinity column in parallel for reaction. The microbeads are combined with biotin capture reagent, the capture reagent is combined with the sample to be detected in a specific way, and finally, a secondary antibody detection reagent with fluorophores is connected to form a composite of streptavidin microbeads, biotin capture reagent, sample and secondary antibody fluorescence detection reagent. Fluorescence readings were excited with a laser, the magnitude of the fluorescence value correlated positively with the anti-CD40 mab concentration.
2. Method of
And preparing a standard curve sample with the concentration range of 19.5-80000 ng/mL by using the anti-CD40 humanized monoclonal antibody as a target drug and using rhesus monkey blank serum. For the antibody drug, biotin-labeled Human CD40 is used as a capture reagent (capture antibody), fluorescent-labeled Human IgG h+l is used as a detection reagent (detection antibody), a mechanical arm is arranged in the Gyrolab xPand, a pipetting needle is used for accurately transferring samples and reagents from a PCR plate to each microstructure in the CD in operation, a 15 nL streptavidin-coated microsphere affinity column is arranged in the CD microstructure, and the CD precisely rotates to ensure that the samples and the reagents flow through the affinity column in parallel for reaction. The microbeads are combined with a capture reagent, the capture reagent is combined with a sample to be detected in a specific way, and finally a detection reagent is connected to form a composite of streptavidin microbeads, biotin capture reagent, sample and secondary anti-fluorescence detection reagent. The fluorescence reading is excited by laser, and the fitting analysis is carried out on the fluorescence value by adopting analysis software of the instrument in a 5-parameter method. The whole process is fully automatically completed by the Gyrolab xPLore.
3. The specific operation steps are as follows
a. Preparation of standard curve samples: anti-CD40 monoclonal antibody standard (recombinant Anti-CD40 antibody, abcam, ab 280207) was prepared into standard solutions of a series of concentrations with pooled rhesus monkey blank serum. Specifically, a rhesus monkey blank serum with corresponding volumes is added into a centrifuge tube, and anti-CD40 monoclonal antibody standard substances (concentration: 1.055 mg/mL) with corresponding volumes and A, STD-STD 7 are respectively taken into the centrifuge tube (see Table 1) and are uniformly mixed by vortex for later use. Wherein, because the rhesus monkey blank serum matrix components are complex, before use, the medium clarified serum matrix is selected for use at 14000 Xg and 2-8 ℃ for 5min, otherwise the% CV of the compound hole of the sample is larger than 20%.
Table 1 preparation of standard curve samples
b. Preparation of quality control samples: according to the following table, quality control samples are prepared, firstly, rhesus monkey blank serum with corresponding volumes is added into a centrifuge tube, and respectively, anti-CD40 monoclonal antibody standard substances (concentration: 1.055 mg/mL), A, high Quality Control (HQC), B, medium Quality Control (MQC) and Low Quality Control (LQC) with corresponding volumes are taken into the centrifuge tube, and vortex mixing is carried out for standby.
TABLE 2 preparation of quality control samples
c. Rexxip TM HX、Rexxip TM F. Gyrolab Bioaffy 200 CD was equilibrated to room temperature for use prior to testing.
d. After the preparation of all blank samples, standard curve samples and quality control samples with serum is completed, 14000 Xg is centrifuged for 5min at 2-8 ℃.
e. Diluting a sample to be detected: all samples were run with Rexxip TM After 10-fold dilution of HX, 14000 Xg, 2-8℃and centrifugation for 5min.
f. The capture antibody (Biotin-Human CD40, the reagent is self-labeling reagent by adopting a finished product kit, the kit is EZ-Link ™ Micro Sulfo-NHS-LC-Biotinylation Kit, thermo, 21935) is diluted to 50 mug/ml by PBS-T, the detection antibody (Alex 647-Human IgG h+l, the kit is Alexa Fluor ™ 647 antibody labeling kit, thermo, A20186) is self-labeling reagent by adopting a finished product kit, and the kit is Rexxip TM F is diluted to 40 mug/ml. Wherein, biotin-Human CD40 and Alex647-Human IgG h+l are mixed uniformly before use, and centrifuged for 3min at 2-8 ℃ and 3000 Xg.
g. The plate was loaded with 3000 Xg, 2-8℃and centrifuged for 3min (removing air bubbles) according to MP loading.
h. The PCR plate and CD were loaded and the instrument was run. And detecting the fluorescence value of the sample by using a Gyrolab xPand full-automatic nano-upgrading immunoassay workstation, fitting a standard curve equation by using analysis software of the instrument by a 5-parameter method, and calculating the concentration result of each sample.
The technical scheme of the invention is further described through experiments.
Experimental example 1 methodological verification
In the detection method of this experimental example, the procedure not specifically described was performed as in example 1.
1. Validating content
a. Standard curve and quantitative range
Standard curve each concentration point was set with 2 multiplex wells and at least 3 days for at least 3 independent analytical batches of verification. The% RE of the concentration of each standard curve sample and the theoretical concentration in each analytical batch and the average% RE and average% CV of each concentration among all analytical batches were counted.
Batch acceptance criteria: the standard curve sample concentration for each analytical batch should be 75% to meet that its% RE is within + -20.0% (ULOQ and LLOQ% RE is within + -25.0%).
Batch-to-batch acceptance criteria: the standard curve sample concentrations between each analytical batch met that their average% RE was within.+ -. 20.0% (ULOQ and LLOQ% RE was within.+ -. 25.0%) and average% CV was within..ltoreq.20.0% (ULOQ and LLOQ% CV. Ltoreq.25.0%).
b. Accuracy and precision
Serum samples of 5 concentrations of ULOQ, HQC, MQC, LQC and LLOQ were prepared in 3 sets each, each set being provided with 2 wells, for at least 3 days for 3 batch validations. Average% RE, average% CV, and total allowable error were counted for each concentration quality control sample within and between batches.
Acceptance criteria: average% RE within and between batches at each concentration is within + -20.0% (average% RE of ULOQ & LLOQ is within + -25.0%); average precision (% CV) is less than or equal to 20.0% (average% CV of ULOQ & LLOQ is less than or equal to 25.0%); the Total allowable Error (% Total Error) is less than or equal to 30.0% (the Total allowable Error of ULOQ & LLOQ is less than or equal to 40.0%).
c. Selectivity of
10 rhesus monkey Blank serum matrixes with different sources are respectively taken, 2 compound holes are arranged on each Blank sample (SE-Blank), the fluorescence value of the Blank serum sample matrixes is obtained through detection, and no obvious endogenous interfering substances need to influence the detection of the to-be-detected object.
Acceptance criteria: the fluorescence value of the blank serum sample matrix < LLOQ fluorescence value indicates that no significant endogenous interfering substances affect the assay of the test substance.
10 rhesus monkey blank serum matrices of different origins were taken separately, and samples of lower quantitative limit (SE-LLOQ) and high (SE-HQC) concentration were prepared separately, each sample being provided with 2 wells. The% RE of each concentration and theoretical concentration was counted.
Acceptance criteria: compared to the theoretical values, the% RE of more than 80% samples is within + -20.0% (SE-LLOQ% RE is within + -25.0%).
d. Dilution linearity and "HOOK" effect
Preparing a serum sample with a certain concentration from a rhesus monkey blank serum, further preparing 5 sets (2 complex holes) of parallel prepared rhesus monkey blank serum after serial dilution, using the prepared partial concentration sample for dilution linear investigation, comparing the actual concentration value obtained by multiplying the detection result by the dilution multiple with a theoretical value, and calculating average% RE and average% CV of the diluted sample; 2 samples with final concentrations higher than the upper limit of quantification were used for "HOOK" effect investigation, and judged according to the sample detection result (fluorescence value).
Acceptance criteria: average% RE of each diluted linear sample and average% CV of all samples are less than or equal to 20.0%, and the maximum dilution factor meeting the requirement is the acceptable maximum dilution factor of the samples. The HOOK effect is judged according to a sample detection result (fluorescence value), and if the concentration reaction curve is in an ascending or platform shape and infinitely extends, the HOOK effect is not shown; if the concentration response curve is in a downward-curved state, it is indicated that the "HOOK" effect exists. If the HOOK effect exists, the sample needs to be diluted and detected after the proper dilution multiple is determined.
2. Verification result
a. Standard curve and quantitative range
Gyrolab platform: quantitative range for detection of anti-CD40 mab standard curve: 39.063-80000 ng/mL, the lower limit of quantification is: 39.063 ng/mL, setting an anchor point as follows: 19.531 ng/mL. The results of the validation of each concentration point on the standard curve of the received analytical batch are shown in Table 3.
TABLE 3 standard curve validation results of Gyrolab platform detection of anti-CD40 mab concentration in rhesus serum
Remarks: 1. the concentration point is an anchor point, participates in standard curve fitting, and can not follow the acceptance standard of a standard curve;
2. ", indicates that the whole point concentration that does not meet the acceptance criterion is deleted;
3. "/" indicates no calculated data.
b. Accuracy and precision
In the Gyrolab platform, anti-CD40 monoclonal antibody standard substances are respectively prepared into 3 sets of 5 concentration quality control samples of ULOQ, HQC, MQC, LQC and LLOQ in the same analysis batch, and 2 compound holes are arranged in each set. The verification results of the effective analysis batches of the 2 platforms all meet the following criteria: within and among the batches, the average% RE is within + -20.0% (average% RE of ULOQ & LLOQ is within + -25.0%); average precision (% CV) is less than or equal to 20.0% (average% CV of ULOQ & LLOQ is less than or equal to 25.0%); the Total allowable Error (% Total Error) is less than or equal to 30.0% (the Total allowable Error of ULOQ & LLOQ is less than or equal to 40.0%). The specific results of the Gyrolab platform receiving the analytical batches are shown (Table 4);
table 4 accuracy and precision of control samples of Gyrolab platform
Remarks: "/" indicates no calculated data.
c. Selectivity of
The Gyrolab platform results show: the instrument response values of the matrix SE-Blank of the rhesus monkey Blank serum samples of 10 different sources are smaller than the instrument response value of LLOQ, which shows that no obvious endogenous interfering substances affect the measurement of the to-be-measured substances, and the concrete results of the Gyrolab platform are shown in Table 5. Gyrolab platform: the% RE of SE-LLOQ samples (80% samples) is within 25.0%; the% RE of SE-HQC samples (90% samples) is within +/-20.0%, and the requirement of selectivity accuracy is met, so that the method is good in selectivity. The concrete results of the Gyrolab platform are shown in Table 6.
Table 5 results of the selective assay of anti-CD40 monoclonal antibody concentration in rhesus serum by Gyrolab platform (I)
Table 6 results of the selective validation of the Gyrolab platform for detection of anti-CD40 mab concentration in rhesus serum (two)
Remarks: "×" indicates that the% RE of the sample does not meet the acceptance criteria.
d. Dilution linearity and "HOOK" effect
The Gyrolab platform results indicate that: the measured signal value of the sample with the final concentration of 1 mg/mL and 0.1 mg/mL is higher than the upper limit of quantification (ALQ), and each dilution factor (100, 2000) sample with the final concentration within the standard curve meets the requirements that the average percent RE is within +/-20.0 percent and the average percent CV is less than or equal to 20.0 percent. The order of the instrument response values for each concentration point of anti-CD40 mab was substantially consistent with the order of the concentration, indicating that no HOOK effect was observed within this range, and specific results are shown in Table 7.
TABLE 7 Gyrolab platform detection of dilution linearity and "HOOK" Effect of anti-CD40 mab concentration in rhesus serum
In conclusion, the verification items meet the requirements of the biological analysis guiding principle of Chinese pharmacopoeia, and the automatic dilution back calculation result is accurate, so that the method is stable and reliable. After the verification of the common biological analysis method in the field, the result meets the requirements, and the method can be considered to be stable and reliable.
Experimental example 2 dilution condition screening experiment
1. Experimental method
In this experimental example, dilution factors of the capture antibody and the detection antibody are preferably used, and in each experimental step, other detection steps and conditions are performed as described in example 1, except for the experimental conditions specifically described.
2. Standard curve range and detection antibody concentration are searched
1. Preparation of standard curve samples: taking anti-CD40 monoclonal antibody standard substance, and using Rexxip TM HX was formulated as standard solutions of a range of concentrations. Adding a corresponding volume of Rexxip into a centrifuge tube TM HX, and respectively taking corresponding volumes of anti-CD40 monoclonal antibody standard (concentration: 1.055 mg/mL) and A, B, C, STD-STD 6 into a centrifuge tube (see Table 8), and uniformly mixing by vortex for later use.
Table 8 preparation of standard curve samples
2. The capture antibody (Biotin-Human CD 40) was set at 1 concentration: 100. and (4) diluting the solution with the volume [ mu ] g/mL to 100 [ mu ] g/mL by using PBS-T.
3. Detection antibody (Alex 647-Human IgG h+l) set 3 concentrations: 150. mu g/mL, 75 mu g/mL and 37.5 mu g/mL, and Rexxip is used for TM F, respectively diluting to 150 mug/mL, 75 mug/mL and 37.5 mug/mL.
4. Results:
table 9 summary of standard curve fitting parameters
Remarks: standard curve fitting formula: y= (a-B)/(1+ (x/C) D ) +B, where y is the instrument response value and x is the sample concentration value. "E" is a symbol of scientific counting, for example: 1.7257e+0.5= 1.7257 ×10 0.5 。
TABLE 10 Standard Curve results
As shown in the results of fig. 1 and table 10, when the concentration of the capture antibody (Biotin-Human CD 40) is 100 μg/mL, the concentration of the detection antibody (Alex 647-Human IgG h+l) is 150 μg/mL, 75 μg/mL, and 37.5 μg/mL, respectively, only 3, 2, and 3 concentration points of the standard curve satisfy% RE within ±20.0%, and do not satisfy the acceptance criteria of the standard curve. And when the theoretical concentration point is lower than 250.000pg/mL, BLQ appears, which indicates that the detection sensitivity is not high, and the lower limit concentration needs to be adjusted.
3. Adjusting standard curve range, exploring capture antibody and detecting antibody concentration
1. Preparation of standard curve samples: taking anti-CD40 monoclonal antibody standard substance, and using Rexxip TM HX was formulated as standard solutions of a range of concentrations. Adding a corresponding volume of Rexxip into a centrifuge tube TM HX, and respectively taking corresponding volumes of anti-CD40 monoclonal antibody standard (concentration: 1.055 mg/mL) and A, B, STD-STD 6 into a centrifuge tube (see Table 11), and uniformly mixing by vortex for later use.
Table 11 preparation of standard curve samples
2. Preparation of quality control samples: preparing a quality control sample according to the following table, adding a corresponding volume of Rexxip into a centrifuge tube TM HX, respectively taking corresponding volumes of anti-CD40 monoclonal antibody standard (concentration: 1.055 mg/mL), A, B, high Quality Control (HQC), C, medium Quality Control (MQC) and D into a centrifuge tubeSee table 12), vortex mixing for later use.
Table 12 preparation of quality control samples
3. The capture antibody (Biotin-Human CD 40) was diluted to 100 μg/mL, 50 μg/mL with PBS-T.
4. The detection antibody (Alex 647-Human IgG h+l) was diluted to 40. Mu.g/mL, 10. Mu.g/mL with Rexxip F.
5. Results:
table 13 summary of standard curve fitting parameters
Remarks: standard curve fitting formula: y= (a-B)/(1+ (x/C) D ) +B, where y is the instrument response value and x is the sample concentration value.
TABLE 14 Standard Curve results
TABLE 15 quality control results
As shown in fig. 2 and the results of tables 14 to 15, when the concentration of the capture antibody (Biotin-Human CD 40) was 50 μg/mL and the concentration of the detection antibody (Alex 647-Human IgG h+l) was 10 μg/mL, each of the 7 concentration points of the standard curve satisfied that% RE was within ±20.0%, but that% RE of HQC was 22.76%, more than 20%; when the concentration of the capture antibody (Biotin-Human CD 40) is 50 mug/mL and the concentration of the detection antibody (Alex 647-Human IgG h+l) is 40 mug/mL, 7 concentration points of the standard curve all meet the requirement that the percent RE is within +/-20.0 percent, and the RE percent of 3 quality control products is within +/-20.0 percent, so that the test requirement is met; when the concentration of the capture antibody (Biotin-Human CD 40) is 100 mug/mL and the concentration of the detection antibody (Alex 647-Human IgG h+l) is 10 mug/mL, 7 concentration points of the standard curve all meet the condition that the% RE is within +/-20.0%, but the RE% of the MQC and the LQC are 56.51% and 49.70% respectively and are more than 20%; when the concentration of the capture antibody (Biotin-Human CD 40) is 100 mug/mL and the concentration of the detection antibody (Alex 647-Human IgG h+l) is 40 mug/mL, 7 concentration points of the standard curve all meet that the percentage RE is within +/-20.0%, but the percentage RE of the MQC and the percentage RE of the LQC are 23.16%, 24.40% and exceed +/-20.0%.
The results show that the concentration re% of the standard curve is less than 20%, and the antibody concentration combination with the quality control concentration re% meeting the requirement of less than 20% is the capture antibody: 50 mug/mL, detection antibody: 40. and [ mu ] g/mL. The concentration combination is the optimal concentration.
4. Adding a matrix to compare the effect of the self-labeled detection antibody with that of a commercially available detection antibody
The self-labeled detection antibody is Anti-Human IgG-heavy and light chain monkey-adsorbed antibody (Bethyl, A80-319A) labeled with Alexa Fluor ™ 647 antibody labeling kit.
The commercial detection antibody is a purchased antibody marked with fluorescence, and specific information is as follows: goat Anti-Human IgG, monkey ads-Alexa Fluor cube 647,Southern Biotech,2049-31.
1. Preparation of standard curve samples: taking an anti-CD40 monoclonal antibody standard substance, and preparing standard solutions with serial concentrations by using rhesus monkey blank serum. Firstly, adding rhesus monkey blank serum with corresponding volumes into a centrifuge tube, respectively taking anti-CD40 monoclonal antibody standard substances (concentration: 1.055 mg/mL) with corresponding volumes and A, STD-STD 6 into the centrifuge tube (see Table 16), and uniformly mixing by vortex for later use.
Table 16 preparation of standard curve samples
2. Preparation of quality control samples: according to the following table, quality control samples were prepared, first, rhesus monkey blank serum of the corresponding volume was added to the centrifuge tube, and then, anti-CD40 monoclonal antibody standard (concentration: 1.055 mg/mL), A, B, high Quality Control (HQC), medium Quality Control (MQC), low Quality Control (LQC) solutions of the corresponding volumes were respectively taken and vortexed into the centrifuge tube (Table 17), and mixed well for use.
Table 17 preparation of quality control samples
3. Sample MRD dilution: all samples were run with Rexxip TM HX was diluted 20-fold and loaded.
4. The capture antibody (Biotin-Human CD 40) was diluted to 50 μg/ml with PBS-T.
5. Rexxip for detection of antibodies (Alex 647-Human IgG h+l) TM F was diluted to 40. Mu.g/mL while aligning commercially available antibodies (GoatAnti-Huamp IgG, monkey ads-AF 647), also with Rexxip TM F is diluted to 40 mug/mL.
6. Results:
table 18 summary of standard curve fitting parameters
Remarks: standard curve fitting formula: y= (a-B)/(1+ (x/C) D ) +B, where y is the instrument response value and x is the sample concentration value.
TABLE 19 Standard Curve results
TABLE 20 quality control results
As shown in fig. 3 and tables 19-20, the% RE of each concentration point of the self-labeled detection antibody, the standard curve and the quality control product is within ±20.0%, which satisfies the acceptance criteria; and the commercial detection antibody is purchased, the lower limit of the standard curve is not shown, 2 quality control products in 3 quality control products are not shown, the RE% of the MQC is 32.25%, and the RE% is more than 20%, and the acceptance standard is not met.
The results show that the detection performance of the self-labeled detection antibody is obviously superior to that of the commercial detection antibody.
5. Final scheme
Through the above experiments, the preferred sample formulation protocol of the present invention is as follows:
1. preparation of standard curve samples: taking an anti-CD40 monoclonal antibody standard substance, and preparing standard solutions with serial concentrations by using mixed rhesus monkey blank serum. Firstly, adding rhesus monkey blank serum with corresponding volumes into a centrifuge tube, respectively taking anti-CD40 monoclonal antibody standard substances (concentration: 1.055 mg/mL) with corresponding volumes and A, STD-STD 7 into the centrifuge tube (see Table 21), and uniformly mixing by vortex for later use.
Table 21 preparation of standard curve samples
2. Preparation of quality control samples: according to the following table, quality control samples are prepared, firstly, rhesus monkey blank serum with corresponding volumes is added into a centrifuge tube, and then anti-CD40 monoclonal antibody standard substances (concentration: 1.055 mg/mL), A, B, high Quality Control (HQC), medium Quality Control (MQC) and Low Quality Control (LQC) with corresponding volumes are respectively taken and mixed into the centrifuge tube by vortex for standby.
Table 22 preparation of quality control samples
3. Sample dilution: all samples were run with Rexxip TM HX was diluted 10-fold and loaded.
4. The capture antibody (Biotin-Human CD 40) was diluted to 50 μg/ml with PBS-T.
5. Rexxip for detection of antibodies (Alex 647-Human IgG h+l) TM F is diluted to 40 mug/mL.
The standard curve results are shown in fig. 4 and table 23.
TABLE 23 Standard Curve results
It can be seen from the above examples and experimental examples that the detection method of the Gyrolab platform is optimized for the purpose of quantitative analysis of anti-CD40 monoclonal antibodies, and the accuracy of detection can be improved by optimizing sample processing conditions and preparation conditions. Therefore, the invention realizes the purpose of detecting the anti-CD40 monoclonal antibody by using the Gyrolab platform for the first time and has good application prospect.

Claims (9)

1. An analytical method for rapidly determining anti-CD40 monoclonal antibody based on a Gyrolab platform is characterized by comprising the following steps of: detecting the concentration of the anti-CD40 monoclonal antibody by adopting a Gyrolab platform, wherein in the detection process, a capture reagent is diluted to 50 mug/ml by using PBS-T, and a detection antibody is Rexxip TM F was diluted to 40 μg/ml and the capture reagent was biotin labelled Human CD40.
2. The method of analysis according to claim 1, wherein: the capture reagent was labeled with EZ-Link ™ Micro Sulfo-NHS-LC-Biotinylation Kit kit and/or the detection antibody was labeled with Alexa Fluor ™ 647 antibody labeling kit.
3. The method of analysis according to claim 1, wherein: in the detection process, the preparation method of the standard curve sample comprises the following steps: standard solutions of serial concentrations were prepared with rhesus monkey blank serum using anti-CD40 mab standard.
4. An assay method according to claim 3, wherein: before the rhesus monkey blank serum is used, centrifuging for 5-10 min at 12000-14000 Xg and 2-8 ℃ to select a medium clarified serum matrix for use.
5. An assay method according to claim 3, wherein: the concentration range of the standard solution is 19.5-80000 ng/mL.
6. The method of analysis according to claim 1, wherein: in the detection process, the sample to be detected uses Rexxip TM HX is diluted 10-20 times and then loaded.
7. The method of analysis according to claim 1, wherein: in the analysis method, the solution prepared by the quality control sample comprises a solution containing the following anti-CD40 monoclonal antibody standard with the concentration:
500000 ng/mL of A solution,
HQC solution 6400 ng/mL,
4000 ng/mL of solution B,
MQC solution 1000 ng/mL
LQC solution 100 ng/mL.
8. The method of analysis according to claim 1, wherein: the Gyrolab biolab Bioaffy 200 CD of the Gyrolab platform was equilibrated to room temperature prior to use.
9. The method of analysis according to claim 1, wherein: in the detection process, the solution prepared by the capture reagent, the solution prepared by the detection antibody, the solution prepared by the sample to be detected, the standard solution and the solution prepared by the quality control sample are respectively subjected to the following operations before use: mixing uniformly, centrifuging and using;
wherein, the centrifugal conditions of the solution prepared by the capture reagent and the solution prepared by the detection antibody are as follows: 3000-5000 Xg, 2-8deg.C, centrifuging for 3-5min;
the centrifugal conditions of the solution prepared by the sample to be detected, the standard solution and the solution prepared by the quality control sample are as follows: 12000-14000 Xg, 2-8deg.C, and centrifuging for 5-10 min.
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