CN102967692A - In-vitro detection method for evaluating CD20 target site treatment medicine and means - Google Patents
In-vitro detection method for evaluating CD20 target site treatment medicine and means Download PDFInfo
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Abstract
The invention belongs to the field of biological medicine, and particularly relates to an in-vitro detection method for evaluating a CD20 target site treatment medicine and a means. The in-vitro detection method disclosed by the invention can be used for rapidly, accurately and efficiently detecting the cell killing bioactivity of the CD20 target site treatment medicine and means, and has good specialization, strong specificity and high accuracy, and the determination range of activity reaches up to 50-150 percent; the precision is high, and the repeatability, the inter-day difference and personnel operating deviation RSD (relative standard deviation) are smaller than 10 percent; and the CV (variable coefficient) percent of triple multiple pores is smaller than 20 percent, and the fitting constants R2 of four parameter fitting are larger than 0.98. In addition, the method does not need costly instruments, has no complicated operation, is convenient for popularization and high-flux operation, and can be used for completely meeting the requirements of screening treatment medicines and means with inhibitory against CD20 target site tumor cell growth activity and evaluating the stability.
Description
Technical field
The invention belongs to the biological medicine technology field, be specifically related to the external detection method of a kind of CD20 of evaluation target position medicine and means.
Background technology
Lymthoma is one group and originates from lymph node or other adenoid malignant tumours.The incidence of disease in China rises year by year in recent years, and mortality ratio is about 1.5/10 ten thousand, accounts for the 11st～13 of all mortality of malignant tumors.Lymthoma can be divided into Hodgkin lymphoma (being called for short HD) and non-Hodgkin lymphoma (being called for short NHL) two large classes, and wherein NHL is modal Malignancy, also is one of fastest-rising malignant tumour of the incidence of disease.In NHL patient, about 85%～90%, B cell lymphoma, the serious harm human health, and namely use the treatment meanss such as a large amount of chemicotherapies or hematopoietic stem cell transplantation still can not solve this difficult problem with present medical level.Studies show that CD20 is the proprietary film surface protein in bone-marrow-derived lymphocyte and B lymphoma cell surface, is expressed in early stage B cell and mature B cell stage, be divided into behind the thick liquid cell CD20 and express and disappear.The expression of CD20 molecule in the diseases such as non-Hodgkin lymphoma and Huppert's disease increases unusually.On the oncocyte film of B cell lymphoma patient above 90% the CD20 developed by molecule is arranged, and superficial density is very high.This shows that the CD20 molecule is the desirable target molecule for the treatment of non-Hodgkin lymphoma (NHL).Development has novel therapeutic mechanism and the lower anti-CD-20 monoclonal antibody of toxic and side effect just may kill and wound the B lymphoma cell in specific manner, reaches preferably result for the treatment of.
In the performance history of biotherapeutics and means, for the ease of having in a large number the screening of biologically active treatment medicine and means, the researchist need to set up biologically active, effect and the stability thereof that a kind of fast and efficiently detection means is accurately investigated medicine to be selected and means.This method must satisfy following several condition: at first, the method is experiment in vitro preferably, based on cellular level, such as ADCC (Antibody-Dependent Cell-Mediated Cytotoxicity), CDC (Complement Dependent Cytotoxicity), direct effect etc., or based on enzymatic reaction; Secondly, the experiment mechanism of the method should be consistent as far as possible with this medicine and the means mechanism of action in vivo, can react really the curative effect that this medicine and means act in vivo by experiment in vitro, be included in the selection of clone and also should approach with true sufferer situation as far as possible; The 3rd, the method must be passed through method validation, satisfy the requirement of the aspects such as method specificity, accuracy, precision and durability, such as the repeatability that detects for same sample, the result's such as the person's of dispatching a person operate miss RSD should satisfy less than 10% in the daytime, on this basis, guarantee biologically active testing result reliable, and be used for guiding treatment medicine and means screening operation; At last, under identical experiment condition, the method is simpler, operation steps is more few better, because such method is more flexible, more is conducive to promote, and is applicable to high flux work.
Take the research and development of carrying out biotherapeutics and means based on target molecule CD20 as example, B lymphoma cell overexpression CD20, take CD20 as target antigen, the effect that kills and wounds in vivo the B lymphoma cell has three, be respectively ADCC effect, CDC effect and Apoptosis, wherein one of main mechanism is the CDC effect, namely is combined with the surface of cell membrane corresponding antigens by specific antibody, form compound and the activating complement classical pathway, formed membrane attack complex is to target cell performance cracking effect.According to the experimental design mechanism of action consistency principle, the researchist utilizes this medicine and means that the killing effect in vitro of high expressed CD20 cell is estimated its biologically active and effect usually.The clone of high expressed CD20 commonly used mainly is Human B lymphoma cell Daudi.From bibliographical information, the cytology activity methods that this kills and wounds does not gain public acceptance or standardization yet up to the present,, the for example selection of clone, cell bed board density, medicine and means action time, developing time and colour developing mode etc. are all different.The more important thing is, in the killing activity method of having reported, also lack the relevant evaluation to aspects such as detection system specificity, accuracy, repeatability, comprise that also there is difference in the evaluation method to experimental result, be difficult to guarantee the Stability and dependability of testing result.
Summary of the invention
Technical matters to be solved by this invention is the problems and shortcomings for above existence, provide a kind of in estimating CD20 target position medicine and means process, biologically active treatment medicine and means are screened and the cell killing biologically active detection method of estimation of stability to having.The method can satisfy in the method validation process specificity, accuracy, precision are comprised repeatability, poor, personnel operation error in the daytime, and the requirement of the aspect such as durability, can carry out quick, accurate, high-throughout screening to large number of biological active treatment medicine and means, satisfy medicine and the means demand in R﹠D process.
For this reason, the invention discloses the external detection method of a kind of CD20 of evaluation target position medicine and means, comprise the following steps:
A takes the logarithm and expresses the cell of CD20 growth period, makes cell suspension with basal medium, and counting also calculates cell density and motility rate, and being adjusted to suitable viable cell density is n * 10
5Individual/ml, n=6-10 adds the cell suspension equal-volume in the Tissue Culture Plate hole, sets up simultaneously the blank hole, and the blank hole adds the equal-volume basal medium;
B gets reference substance and testing sample, carries out serial gradient dilution with basal medium, obtains reference substance and the testing sample of serial dilution concentration;
C adds reference substance and the testing sample of serial dilution concentration in the described Tissue Culture Plate of the step a hole successively, the blank hole then adds isopyknic basal medium, then Tissue Culture Plate is positioned in 37 ℃, 5% CO2gas incubator and cultivates 10-20min;
D gets human serum, dilutes with basal medium, adds successively in step c described Tissue Culture Plate hole and the blank hole, and the concentration that makes human serum is 3%-7%, then Tissue Culture Plate is positioned in 37 ℃, 5% CO2gas incubator and cultivates 45-75min;
E adopts the detection system that detects living cells or dead cell amount that Tissue Culture Plate is carried out chromogenic reaction;
After the f chromogenic reaction is finished, measure the result of chromogenic reaction, and calculate the cell killing biologically active of testing sample according to measurement result.
Term of the present invention " reference substance " refers to that known its shows compound or the composition of killing functions of immunocytes activity to the cell of expressing CD20.
In certain embodiments, the CD20 target position medicine of addressing is one of in monoclonal antibody, inhibitors of kinases, antisense oligonucleotides, compound, Chinese medical extract or the Chinese medicinal compound extract or their any combination, wherein is preferably monoclonal antibody.
In certain embodiments, the cell of described exponential phase expression CD20 is Human B lymphoma cell Daudi.
In certain embodiments, described basal medium is the RPMI1640 nutrient culture media.
In certain embodiments, described serial gradient dilution is 2 times of serial gradient dilutions.
In certain embodiments, in step e, the detection system of described detection living cells or dead cell amount comprises tetrazole compound redox detection system, ATP total amount detection system or LDH burst size detection system etc.
In certain embodiments, described tetrazole compound is MTT, MTS, CCK-8, wherein is preferably CCK-8.With respect to other tetrazole compounds such as MTT, MTS, therefore the formed reduction substance that show color of CCK-8 can directly be soluble in the aqueous phase, and need not to add the DMSO hydrotropy, uses conveniently, and detection sensitivity is also higher.
In certain embodiments, described Tissue Culture Plate is 96 porocyte culture plates, so that high flux ground carries out drug evaluation.
The present invention can be fast, accurately and efficiently the cell killing biologically active for CD20 target position medicine and means is detected.The method specificity is good, high specificity, accuracy high, and the determination of activity scope reaches 50%-150%; Precision is high, and repeatability, poor and personnel operation error RSD is all less than 10% in the daytime, and the CV% in two multiple holes is less than the fitting constant R of 20%, 4 parameter fitting
2All greater than 0.98.In addition, the present invention does not need very expensive instrument and loaded down with trivial details operation, be convenient to promote and the high flux operation, can satisfy it fully and have the target position for CD20, by CDC effect killer cell, have the screening of biologically active treatment medicine and means and the needs of estimation of stability thereof.
Description of drawings
Fig. 1 Rituximab and reference material 4 parameter fitting figure.
Fig. 2 Infliximab 4 parameter fitting figure.
Fig. 3 adds 1% and 5% complement and keeps 100% reference material and 75% reference material, 4 parameter fitting figure under the normal human serum condition.
Fig. 4 adds 20% and 5% complement and keeps 100% reference material and 75% reference material, 4 parameter fitting figure under the normal human serum condition.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are not used in for explanation the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is usually according to normal condition or the condition of advising according to manufacturer.Unless otherwise defined, employed all specialties are identical with the meaning that scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The usefulness that better implementation method described in the literary composition and material only present a demonstration.
One, material and reagent
Material: Rituximab(originates in Roche for CD20 target position monoclonal antibody drug, lot number: B6109), for CD20 target position monoclonal antibody drug reference material (good and biological medicine company preparation), for CD20 target position monoclonal antibody drug testing sample (good and biological medicine company preparation), the monoclonal antibody drug Infliximab(for the CD20 target position does not originate in Johnson ﹠ Johnson, lot number: AAM05014).
Reagent: dyeing liquor CCK-8(Dojindo, Cat. No. CK04-20), RPMI1640 culture medium powder (GIBCO, Cat. No. 31800-022), streptomysin/penicillin two anti-(GIBCO, Cat. No.15070-063), FBS(GIBCO, Cat. No. 10099-141).
Solution preparation:
RPMI1640 nutrient culture media: get the 1000ml beaker, pour 1 bag of RPMI1640 culture medium powder into, sodium bicarbonate 2.0g, add about 900ml ultrapure water, the lucifuge magnetic agitation is until watery hydrochloric acid adjust pH to 7.00 ± 0.05 is used in dissolving, pour solution into graduated cylinder, be settled to 1000ml, through 0.22 μ m filter membrane aseptic filtration and get final product, 4 ℃ keep in Dark Place after the packing.
Cell line: the Daudi cell line (is purchased from typical case's culture collection council of Chinese Academy of Sciences cell bank, catalog number (Cat.No.): TCHu140).
Two, detection method step
1) cell bed board: the growth period Daudi cell of taking the logarithm is made single cell suspension with the RPMI1640 basal medium.Counting also calculates cell density and motility rate, adjusts viable cell density to 6.0-10.0 * 10
5Individual/ml, add in the 96 porocyte culture plates with 100 μ l/ holes.The blank hole then adds the RPMI1640 basal medium with 100 μ l/ holes.
2) reference material and testing sample dilution: get reference substance and testing sample, according to indicating concentration, be diluted to suitable concn with the RPMI1640 basal medium, in 96 new porocyte culture plates, do 2 times of serial gradient dilutions, obtain 11 dilutabilitys.
3) application of sample: get the Tissue Culture Plate in the step 1), reference substance and the testing sample of the serial dilution concentration that dilution is good add in the Tissue Culture Plate hole of completing cell successively with 50 μ l/ holes, the blank hole then adds basal medium with 50 μ l/ holes, the multiple hole of 2-3.After finishing Tissue Culture Plate is positioned in 37 ℃, 5% CO2gas incubator and cultivates 10-20min.
4) get human serum, be diluted to suitable concn with basal medium, then with in the Tissue Culture Plate hole and blank hole in the 50 μ l/ holes adding step 3), so that the concentration of human serum is 3%-7%.
5) colour developing: adopt the CCK-8 color development system respectively at adding CCK-8 with 20 μ l/ holes in reference substance hole, testing sample hole, the blank hole in the Tissue Culture Plate, after finishing Tissue Culture Plate is positioned in 37 ℃, 5% CO2gas incubator and continues to hatch 3.5-4.5h.
6) reading: take out the Tissue Culture Plate mixing, put into microplate reader, take 650nm as reference wavelength, under 450nm, measure the OD in reference substance hole, testing sample hole and blank hole
450-650, the record measurement result.
7) interpretation of result: carry out 4 parameter fittings with the dose-effect relationship of reference substance hole and testing sample hole OD value and adding consistency respectively, determine the EC of reference substance and testing sample
50Value, curve constant R
2, multiple hole CV%.Calculate the cell killing biologically active of testing sample according to following formula:
Take reference material and Rituximab as material, Rituximab is product in contrast, be diluted to suitable concn with the RPMI1640 basal medium, in 96 new porocyte culture plates, do 2 times of serial gradient dilutions, obtain 11 dilutabilitys, adopt above-mentioned detection method step to investigate the biologic activity of the relative Rituximab of reference material.
The result as shown in Figure 1, Rituximab and reference material 4 parameter fittings are all right, curve constant R
2All satisfy＞0.98, the CV% of 3 multiple holes detection OD values all satisfies＜20% under the different drug-diluting concentration.By with RituximabEC
50The comparison of value, the cell killing biologically active of reference material is 95%.
Take Infliximab as material, be diluted to suitable concn with the RPMI1640 basal medium, in 96 new porocyte culture plates, do 2 times of serial gradient dilutions, obtain 11 dilutabilitys, adopt above-mentioned detection method step to detect its killing activity.
Result such as Fig. 2, when the method is applied to Infliximab, although the CV% of 3 multiple holes detection OD values all satisfies＜20% under the different drug-diluting concentration, can't carry out 4 parameter fittings to the result, this medicine can not produce lethal effect to the Human B lymphoma cell Daudi of high expressed CD20.Relative, the testing result of reference material then can normally be carried out 4 parameter fittings among Fig. 1, satisfies simultaneously fitting constant R
2The CV% in＞0.98,3 multiple holes＜20%.This experimental result shows that the method for the invention has specificity.
The test of embodiment 3 evaluation of the accuracy
Take reference material (with embodiment 1) as material, with the RPMI1640 basal medium it is diluted to the level of tiring and is respectively 50%, 75%, 100%, 125%, 150% testing sample, 3 experimenters (Analyst1,2,3) independently adopt separately above-mentioned detection method step that these 5 testing samples are carried out the cell killing biologically active and detect.
Result such as table 1 are respectively 5 testing samples of 50%, 75%, 100%, 125%, 150% to the level of tiring, and 3 different experiments personnel utilize the inventive method the testing result of these samples all to be satisfied the fitting constant R of 4 parameter fitting curves of reference material
2The CV% in＞0.98,3 multiple holes all satisfy＜20%, and different personnel all satisfy＜10% to the tire measured value of level of difference with respect to the mean deviation of theoretical value.This explanation is when the cell killing biologically active that detects for CD20 target position biotherapeutics and means, and the inventive method can reach accuracy requirement to field of activity testing sample in 50%-150%.
Take reference material (with embodiment 1) and testing sample as material, the precision of the method for the invention is estimated.At first adopt above-mentioned detection method step respectively to choose 1 testing sample by 3 experimenters and carry out the detection of cell killing biologically active, parallel laboratory test 3 times, the repeatability of investigation method, then adopting above-mentioned detection method step that same testing sample is carried out the cell killing biologically active in different 3 days by 1 experimenter detects, investigation method poor in the daytime, adopt above-mentioned detection method step that same testing sample is carried out the cell killing biologically active by 3 experimenters (Analyst1,2,3) at last and detect the personnel operation error of investigation method.
Such as table 2, utilize above-mentioned detection method, respectively choose 1 testing sample by 3 experimenters and carry out the detection of cell killing biologically active, the result all satisfies the fitting constant R of 4 parameter fitting curves of reference material and testing sample
2＞0.98, the CV% in 3 multiple holes all satisfies＜20%, and the RSD of each testing sample testing result satisfies＜10%, illustrate that in detection the repeatability of the inventive method can reach precision requirement during for the cell killing biologically active of CD20 target position biotherapeutics and means.
Such as table 3, in different 3 days, same testing sample is carried out Parallel testing by 1 experimenter, the result all satisfies the fitting constant R of 4 parameter fitting curves of reference material and testing sample
2The CV% in＞0.98,3 multiple holes all satisfy＜20%, and the RSD of 3 testing results satisfies＜10%, illustrates that in detection the poor in the daytime of the inventive method can reach precision requirement during for the cell killing biologically active of CD20 target position biotherapeutics and means.
Such as table 4, by 3 experimenters same testing sample is carried out Parallel testing, the result all satisfies the fitting constant R of 4 parameter fitting curves of reference material and testing sample
2＞0.98, the CV% in 3 multiple holes all satisfies＜20%, and the RSD of 3 testing results satisfies＜10%, illustrate that in detection the personnel operation error of the inventive method can reach precision requirement during for the cell killing biologically active of CD20 target position biotherapeutics and means.
Take reference material (with embodiment 1) and testing sample as material, select the Daudi cell of different cell generations (10,20,28,38), adopt above-mentioned detection method step that same testing sample is carried out the cell killing biologically active and detect.
Such as table 5, use the Daudi cell of different cell generations, utilize this detection method that same testing sample is detected, the result all satisfies the fitting constant R of 4 parameter fitting curves of reference material and testing sample
2＞0.98, the CV% in 3 multiple holes all satisfies＜20%, and under the different cell generation conditions (in 40 generations), the RSD of same testing sample testing result satisfies＜10%, explanation is when the cell killing biologically active that detects for CD20 target position biotherapeutics and means, and the inventive method can reach durability to the requirement of cell generation.
Embodiment 6 durability evaluation tests (complement keep normal human serum batch)
Take reference material (with embodiment 1) and Rituximab as material, select the complement of different batches to keep normal human serum, adopt above-mentioned detection method step that same testing sample is carried out the cell killing biologically active and detect.
Such as table 6, use the complement of different batches to keep normal human serum, utilize this detection method that same testing sample is detected, the result all satisfies the fitting constant R of 4 parameter fitting curves of reference material and testing sample
2＞0.98, the CV% in 3 multiple holes all satisfies＜20%, and under the different serum batch conditions, the RSD of same testing sample testing result satisfies＜10%, explanation is when the cell killing biologically active that detects for CD20 target position biotherapeutics and means, and the inventive method can reach durability to the requirement of serum batch.
The impact of different cell incubation times after embodiment 7 dosings
Take reference material (with embodiment 1) as material, with the RPMI1640 basal medium it being diluted to the level of tiring is 75% and 100% testing sample, select the complement of variable concentrations to keep normal human serum (1%, 5%, 20%), testing sample is carried out the cell killing biologically active detect.
The complement of 1% and 5% concentration keeps the contrast of normal human serum testing result and sees Fig. 3.After can finding to add 5% complement and keeping normal human serum, can effectively bring into play the CDC effect, killer cell, actual testing result and notional result are basically identical; And add after 1% complement keeps normal human serum, because serum-concentration is excessively low, can not effectively bring into play the CDC effect cell is killed, and actual testing result and notional result deviation are large, can't accurately judge the biologic activity of sample.
The complement of 20% and 5% concentration keeps the contrast of normal human serum testing result and sees Fig. 4.After can finding to add 5% complement and keeping normal human serum, can effectively bring into play the CDC effect, killer cell, actual testing result and notional result are basically identical; And add after 20% complement keeps normal human serum, because serum-concentration is too high, produce cytotoxic effect, cause can not obtaining killing and wounding curve, actual testing result and notional result deviation are large, can't accurately judge the biologic activity of sample.
In sum, the present invention has set up a kind of cell killing biologically active detection method for CD20 target position biotherapeutics and means, the method is quick, efficiently, easily promote, be fit to the high flux operation, by to the method specificity, accuracy, it is strong that the checking of the many-side such as precision and durability and investigation have confirmed that this inventive method has stability, the characteristics such as applicability is good can satisfy in the R﹠D process for CD20 target position biotherapeutics and means, and screening and stability 12 with cell killing bioactivator are estimated.
Scope of the present invention is not subjected to the restriction of described specific embodiments, and described embodiment also comprises method and the component of functional equivalent only as the single example of illustrating various aspects of the present invention in the scope of the invention.In fact, except content as herein described, those skilled in the art can easily grasp multiple improvement of the present invention with reference to above description and accompanying drawing.Described improvement also falls within the scope of appended claims.Every piece of list of references mentioned above is listed this paper in as a reference all in full.
Claims (10)
1. an external detection method of estimating CD20 target position medicine and means comprises the following steps:
A takes the logarithm and expresses the cell of CD20 growth period, makes cell suspension with basal medium, and counting also calculates cell density and motility rate, and the adjustment viable cell density is n * 10
5Individual/ml, the cell suspension equal-volume is added in the Tissue Culture Plate hole, set up simultaneously the blank hole, the blank hole adds the equal-volume basal medium;
B gets reference substance and testing sample, carries out serial gradient dilution with basal medium, obtains reference substance and the testing sample of serial dilution concentration;
C adds reference substance and the testing sample of serial dilution concentration in the described Tissue Culture Plate of the step a hole successively, the blank hole then adds isopyknic basal medium, then Tissue Culture Plate is positioned in 37 ℃, 5% CO2gas incubator and cultivates 10-20min;
D gets human serum, dilutes with basal medium, adds successively in step c described Tissue Culture Plate hole and the blank hole, and the concentration that makes human serum is 3%-7%, then Tissue Culture Plate is positioned in 37 ℃, 5% CO2gas incubator and cultivates 45-75min;
E adopts the detection system that detects living cells or dead cell amount that Tissue Culture Plate is carried out chromogenic reaction;
After the f chromogenic reaction is finished, measure the result of chromogenic reaction, and calculate the cell killing biologically active of testing sample according to measurement result.
2. external detection method as claimed in claim 1 is characterized in that described CD20 target position medicine is one of in monoclonal antibody, inhibitors of kinases, antisense oligonucleotides, compound, Chinese medical extract or the Chinese medicinal compound extract or their any combination.
3. external detection method as claimed in claim 1 is characterized in that described n=6-10.
4. external detection method as claimed in claim 1 is characterized in that it is Human B lymphoma cell Daudi that described exponential phase is expressed the cell of CD20.
5. external detection method as claimed in claim 1 is characterized in that described basal medium is the RPMI1640 basal medium.
6. external detection method as claimed in claim 1 is characterized in that described serial gradient dilution is 2 times of serial gradient dilutions.
7. external detection method as claimed in claim 1 is characterized in that described human serum is that complement keeps normal human serum.
8. external detection method as claimed in claim 1 is characterized in that in step e, and the detection system of described detection living cells or dead cell amount comprises tetrazole compound redox detection system, ATP total amount detection system or LDH burst size detection system etc.
9. external detection method as claimed in claim 8 is characterized in that described tetrazole compound is MTT, MTS or CCK-8.
10. external detection method as claimed in claim 1 is characterized in that described Tissue Culture Plate is 96 porocyte culture plates.
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| CN107870232A (en) * | 2016-09-28 | 2018-04-03 | 三生国健药业（上海）股份有限公司 | A kind of biological activity detection method for RANKL target therapeutic agents |
| CN110455868A (en) * | 2019-08-19 | 2019-11-15 | 昆明理工大学 | A method of concrete construction site early moist curing effect is detected using surface resistivity |
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| CN104502254A (en) * | 2014-12-09 | 2015-04-08 | 中国科学院合肥物质科学研究院 | Automatic cell cloning survival rate detection device and detection method thereof |
| CN107870232A (en) * | 2016-09-28 | 2018-04-03 | 三生国健药业（上海）股份有限公司 | A kind of biological activity detection method for RANKL target therapeutic agents |
| CN110455868A (en) * | 2019-08-19 | 2019-11-15 | 昆明理工大学 | A method of concrete construction site early moist curing effect is detected using surface resistivity |
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