CN104931690A - PD-1 antibody detection kit and application thereof - Google Patents

PD-1 antibody detection kit and application thereof Download PDF

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CN104931690A
CN104931690A CN201510267428.7A CN201510267428A CN104931690A CN 104931690 A CN104931690 A CN 104931690A CN 201510267428 A CN201510267428 A CN 201510267428A CN 104931690 A CN104931690 A CN 104931690A
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antibody
solid phase
phase carrier
assay kit
recombinant protein
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王琳
王征
李永奎
方敏
张剑
祁闪闪
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Tongji Medical College of Huazhong University of Science and Technology
Union Hospital Tongji Medical College Huazhong University of Science and Technology
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Union Hospital Tongji Medical College Huazhong University of Science and Technology
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

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Abstract

The invention provides a PD-1 antibody detection kit and application thereof. The PD-1 antibody detection kit comprises a solid phase carrier, an antibody trapping agent and a labeled antibody, wherein the antibody trapping agent is a recombinant protein of a PD-1 cytomembrane outer structure, and the amino acid sequence of the antibody trapping agent is SEQ ID NO.1. The PD-1 antibody detection kit can quickly detect a PD-1 antibody in a to-be-detected sample, such as in-vitro serum and tissues homogenate, the specificity of detection is high, the sensitivity of detection is high, and the application range is wide.

Description

A kind of PD-1 antibody assay kit and application thereof
Technical field
The present invention relates to a kind of antibody assay kit, particularly relate to a kind of PD-1 antibody assay kit and application thereof.
Background technology
Under normal circumstances, the specificity cell toxicity T lymphocyte (CTL) that organism immune response activation produces can kill the tumour cell in body.The immunosuppressive action of tumour is the important mechanisms that tumour cell escape body immune system kills and wounds.The current research in tumor immunology field finds, the immunosupress reaction that programmed death molecule-1 (PD-1) mediates is the major reason that apoptosis occurs CTL, unable and function is exhausted.The inhibitive ability of immunity cell surface expression B7-H1/4 molecule that tumour cell and tumour thereof are correlated with, they are known PD-1 parts, when CTL enters tumor microenvironment, the PD-1 of CTL cell surface can by its ligand activation, cause the phosphorylation of the SHP1/2 in born of the same parents, and then cause Apoptosis or function to be exhausted, thus cannot effective killing tumor cell.
At present, the correlative study of path blocking agent anti-PD-1 and B7-H1/4 monoclonal antibody has breakthrough progress, research finds that it can block the inhibiting effect of PD-1 to T cell, thus the immunocyte Tumor-cytotoxic efiect activated in tumor patient body, reduce inhibition regulatory T-cell effect, kill and wound the function of cancer cell with enhanced CT L, it has become the focus of immunization therapy in recent years.Multinomial tumor animal experimental result shows, blocks PD-1 path and can strengthen Graft Versus Tumor, extends life cycle, improve prognosis.The phase iii clinical trial that monoclonal antibody for the blocking-up of PD-1 path is used for progressive stage malignant mela noma completes, and its antitumous effect is remarkable, and being approved as by U.S. FDA can marketed drug.Due to the broad spectrum anticancer effect of PD-1 antibody, its to the lanqin oral solutions of the cancers such as kidney, cancer of the stomach, breast cancer, carcinoma of urinary bladder, leukaemia, head and neck cancer, intestinal cancer and brain tumor also second phase or phase iii clinical trial among.
Novel, safe as one, the efficient antineoplastic of PD-1 antibody, certainly will will be widely applied in clinical cancer therapy, scientific research field also can do deep research, therefore in the urgent need to a kind of method for detecting specificity for PD-1 antibody to the formulation of this medicine, pharmacokinetics etc.
Double antibody sandwich method Enzyme-multiplied immune technique carries out accurate quantification effective method the most to protein substance at present, its principle is by solid phase carrier (such as ELISA Plate) by a kind of antibody bag of testing protein, catch testing protein for specificity, then adopt the another kind of labelled antibody of testing protein (such as two of coupling HRP anti-etc.) to carry out demarcating and detecting.But, PD-1 antibody is a kind of IgG antibody inherently, it has the identical structure of the IgG that to originate with same species, major part region (about 2/3 sequence) is constant region, the amino acid sequence of the IgG constant region of same species is identical with structure, and the IgG constant region of different plant species is according to the different homology had in various degree of evolution degree; Variable region is binding specificity antigenic domains mainly, owing to being difficult to obtain the anti-igg antibody for variable region, the epi-position in this district therefore generally can not being adopted to prepare two and resist.If adopt two kinds of anti-igg antibody for constant region epi-position, obviously cannot get rid of the interference of the IgG contained in test serum, tissue fluid equal samples itself, therefore traditional double antibody sandwich method cannot realize the specific detection to PD-1 antibody.At present, there is no the effective method accurately measuring PD-1 antibody concentration, the concentration especially how measuring PD-1 antibody in animal or human's serum becomes problem demanding prompt solution.
Summary of the invention
The invention provides a kind of PD-1 antibody assay kit and application thereof, cannot realize technological deficiencies such as the specific detection of PD-1 antibody for solving double antibody sandwich method of the prior art.
The invention provides a kind of PD-1 antibody assay kit, comprise solid phase carrier, antibody capture agent and labelled antibody, described antibody capture agent is the recombinant protein of PD-1 cell membrane extracellular portion, and its amino acid sequence is as SEQ ID NO.1.
In PD-1 antibody assay kit of the present invention, the recombinant protein of PD-1 cell membrane extracellular portion specificity can catch PD-1 antibody in sample to be tested, it can by common commercially available acquisition, and the article No. that ACRO Biosystems company such as can be adopted to produce is the recombinant protein of PD1-M5228; Labelled antibody can be combined with PD-1 antibody specifically and coupling has the material (such as horseradish peroxidase, streptomysin etc.) producing detection signal.
The principle of work of this kit is: after sample to be tested being joined the solid phase carrier of pre-coated antibody capture agent, antibody capture agent specificity catches the PD-1 antibody in sample to be tested, the labelled antibody added subsequently is combined with PD-1 antibody specifically, detected by the detection signal produced coupling material on labelled antibody, thus the concentration of PD-1 antibody in sample to be tested can be obtained.
Be understandable that, in PD-1 antibody assay kit of the present invention, solid phase carrier and antibody capture agent can independently be packed, and also can form pre-coated solid phase carrier by pre-coated for antibody capture agent on solid phase carrier in advance, thus pack pre-coated solid phase carrier.
In the present invention, described labelled antibody can be the IgG antibody of horseradish peroxidase or streptomysin mark; Particularly, this labelled antibody is identical with the source species of the recombinant protein of PD-1 cell membrane extracellular portion, in one embodiment, the recombinant protein of PD-1 cell membrane extracellular portion derives from mouse, and labelled antibody can be the mountain goat anti rat IgG antibody of horseradish peroxidase (HRP) coupling.Described solid phase carrier can adopt this area conventional carrier, such as ELISA Plate.
In addition, PD-1 antibody assay kit of the present invention can also comprise with reference to product, is conducive to the accuracy ensureing to detect with reference to product, such as, can be one or more in PD-1 antibody standard substance, positive reference substance and negative controls.Wherein, the PD-1 antibody that PD-1 antibody standard substance can be clone number that Bioxcell company of the U.S. produces is RMP1-14, it blocks the most frequently used treatment antibody for PD-1 in Murine Malignant tumor model Experiment on therapy, and antitumous effect is remarkable; Positive reference substance can be positive serum, assaypositive tissue homogenate etc.; Negative controls can be negative serum, negative tissue homogenate etc.
Further, PD-1 antibody assay kit of the present invention can also comprise the conventional reagent needed for mensuration, such as can comprise be buffered in liquid, lavation buffer solution, sample diluting liquid, confining liquid, nitrite ion and stop buffer one or more.
In one embodiment, bag is buffered the carbonate buffer solution that liquid is pH9.6, containing Na in often liter of solution 2cO 31.59g and NaHCO 32.93g; Lavation buffer solution is the phosphate buffer of pH7.4, containing NaCl 8.0g, KCl 0.2g, KH in often liter of solution 2pO 40.24g, Na 2hPO 412H 2o 3.628g, can contain Tween-20 0.5mL further; Confining liquid is the above-mentioned phosphate buffer of the pH7.4 containing 1mg/mL bovine serum albumin(BSA); Stop buffer is the sulfuric acid of 2mol/L.
The present invention also provides the above-mentioned arbitrary described application of PD-1 antibody assay kit in PD-1 antibody test.Particularly, this application can be embodied in scientific experiment the pharmacokinetics research etc. in the mensuration of PD-1 antibody concentration, the application of PD-1 antibody clinical.
The present invention also provides a kind of PD-1 antibody detection method, adopts above-mentioned PD-1 antibody assay kit to carry out, and described PD-1 antibody detection method comprises the step that following order is carried out:
1) by pre-coated on described solid phase carrier for the recombinant protein of described PD-1 cell membrane extracellular portion, obtained pre-coated solid phase carrier;
2) in described pre-coated solid phase carrier, add sample to be tested, hatch, wash subsequently;
3) in described pre-coated solid phase carrier, add described labelled antibody, hatch, wash subsequently;
4) in described pre-coated solid phase carrier, add nitrite ion, hatch, add stop buffer subsequently, and measure OD 450.
Be understandable that, as antibody capture agent is pre-coated in the kit that adopts on solid phase carrier, form pre-coated solid phase carrier, then detection method can omit above-mentioned steps 1).
Further, described PD-1 antibody assay kit also comprises PD-1 antibody standard substance, and described PD-1 antibody detection method also comprises:
Gradient dilution is carried out to described PD-1 antibody standard substance, and adopts step 1) to step 4) measure the OD of each gradient dilution liquid 450, obtain PD-1 antibody standard substance concentration and OD 450linear relationship;
According to the OD of measured sample to be tested 450with described linear relationship, obtain the concentration of PD-1 antibody in described sample to be tested.
Further, can adopt and prepare described pre-coated solid phase carrier with the following method:
Adopt bag to be buffered the recombinant protein of liquid to described PD-1 cell membrane extracellular portion to dilute, the concentration controlling the recombinant protein solution after dilution is not less than 15.625ng/mL, is joined on described solid phase carrier by the recombinant protein solution after dilution and carries out bag quilt;
After bag is moved to end, employing lavation buffer solution washs solid phase carrier, closes subsequently to adding confining liquid in solid phase carrier, closes washing after terminating also dry, obtained described pre-coated solid phase carrier.
Enforcement of the present invention, at least has following advantage:
1, kit of the present invention adopts Ag-Ab sandwich method, by adopt PD-1 antibody for antigen, namely the recombinant protein of PD-1 cell membrane extracellular portion realizes catching the specificity of PD-1 antibody, thus overcome this technical barrier of interference that traditional double antibody sandwich cannot get rid of the IgG itself contained in sample to be tested, thus achieve the specific detection to PD-1 antibody.
2, in kit of the present invention, the recombinant protein of PD-1 cell membrane extracellular portion and labelled antibody all can be combined with PD-1 antibody specifically, thus ensure that high specific and the high sensitivity of detection, and detectability can be low to moderate 15.625ng/mL.
3, kit using method of the present invention is simple and easy, quick, only need 2 hours namely can complete pattern detection, and usable range is extensive, PD-1 antibody in damping fluid, serum, tissue homogenate equal samples can be commonly used to laboratory to detect, and can be used for the pharmacokinetics research in clinical practice.
Accompanying drawing explanation
Fig. 1 is concentration and the OD of each gradient PD-1 antibody diluent in physiological saline 450linear relationship chart;
Fig. 2 is concentration and the OD of each gradient PD-1 antibody diluent in serum 450linear relationship chart;
Fig. 3 is concentration and the OD of each gradient PD-1 antibody diluent in tissue homogenate 450linear relationship chart;
Fig. 4 is PD-1 antibody concentration curve in different time serum after mouse peritoneal injection PD-1 antibody.
Embodiment
For making the object, technical solutions and advantages of the present invention clearly, below in conjunction with drawings and Examples of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, obviously, described embodiment is the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1PD-1 antibody assay kit
1, material and source thereof:
Antibody capture agent: the recombinant protein of mouse PD-1 cell membrane extracellular portion, ACRO Biosystems company produces, and article No. is PD1-M5228, and its amino acid sequence is as SEQ ID NO.1;
Solid phase carrier: ELISA Plate, Coster company produces;
Labelled antibody: the mountain goat anti rat IgG antibody of HRP coupling, KPL company produces, and article No. is 141612.
2, PD-1 antibody assay kit
The PD-1 antibody assay kit of the present embodiment is made up of above-mentioned solid phase carrier, antibody capture agent and labelled antibody, independently packs and is positioned in box body.
Embodiment 2PD-1 antibody assay kit and preparation method thereof
The PD-1 antibody assay kit of the present embodiment is the improvement carried out on the basis of the PD-1 antibody assay kit of embodiment 1, it forms pre-coated solid phase carrier by pre-coated for antibody capture agent on solid phase carrier, and this kit is made up of above-mentioned pre-coated solid phase carrier, labelled antibody and following component:
PD-1 antibody standard substance: Bioxcell company of the U.S. produces, clone number is RMP1-14;
Bag is buffered liquid: often liter containing Na 2cO 31.59g, NaHCO 32.93g, pH value is 9.6;
Lavation buffer solution (PBS): often liter containing NaCl 8.0g, KCl 0.2g, KH 2pO 40.24g, Na 2hPO 412H 2o 3.628g, pH value is 7.4; Tween-20 0.5mL (PBST) can be added further;
Confining liquid: add 1mg/mL bovine serum albumin(BSA) (production of BSA, Sigma company) in above-mentioned PBS;
Nitrite ion: KPL company produces, and article No. is 520001, comprising A liquid and B liquid;
The sulfuric acid of stop buffer: 2mol/L;
Above-mentioned each component is independently packed and is positioned in box body.
Wherein, the preparation method of above-mentioned pre-coated solid phase carrier is:
1, adopt above-mentioned bag to be buffered liquid and the recombinant protein of mouse PD-1 cell membrane extracellular portion is diluted to the recombinant protein solution that concentration is 0.01mg/mL, add this recombinant protein solution of 100 μ L in the every hole of ELISA Plate after, shrouding, with being placed on 4 DEG C of refrigerator overnight.
2, the solution in ELISA Plate is outwelled, above-mentioned lavation buffer solution PBS is adopted to wash plate twice, subsequently to adding the above-mentioned confining liquid of 200 μ L in the every hole of ELISA Plate, after hatching 1 hour in 37 DEG C, outwell the solution in ELISA Plate, and after adopting above-mentioned lavation buffer solution PBS to wash plate twice, clappers, normal temperature dries, and namely obtains pre-coated elisa plate, be placed in 4 DEG C of preservations after shrouding.
The detection of PD-1 antibody concentration in embodiment 3 physiological saline
Adopt the PD-1 antibody assay kit of embodiment 2 to detect, method is specially:
1, be the gradient dilution liquid that concentration is respectively 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL and 15.625ng/mL by above-mentioned PD-1 antibody standard substance physiological saline gradient dilution; In addition, the mountain goat anti rat IgG antibody adopting above-mentioned PBST to add 0.2 μ L HRP coupling by every mL prepares labelled antibody working fluid.
2, get each gradient dilution liquid 100 μ L, add in the plate hole of above-mentioned pre-coated elisa plate respectively, shrouding, hatch 1 hour in 37 DEG C.
3, outwell the solution in ELISA Plate, adopt above-mentioned lavation buffer solution PBST to wash plate 5 times, after clappers, add the above-mentioned labelled antibody working fluid of 100 μ L to every hole, shrouding, hatches 30 minutes in 37 DEG C.
4, outwell the solution in ELISA Plate, adopt above-mentioned lavation buffer solution PBST to wash plate 5 times, after clappers, add A liquid and each 50 μ L of B liquid of above-mentioned nitrite ion to every hole, shrouding, hatches 15 minutes for 37 DEG C.
5, take out ELISA Plate, add the above-mentioned stop buffer of 30 μ L to every hole, be placed in microplate reader and read OD 450, the results are shown in Table 1.
The OD of each gradient PD-1 antibody diluent in table 1 physiological saline 450value
PD-1 antibody concentration (ng/mL) OD 450Value
1000 1.4263
500 0.7509
250 0.4227
125 0.2051
62.5 0.1251
31.25 0.0834
15.625 0.0515
According to above-mentioned table 1 data creating linear relationship chart, as shown in Figure 1, and obtain linear equation, wherein R 2=0.999, PD-1 antibody concentration and OD in physiological saline are described 450between in extremely strong linear relationship.Illustrate thus, kit of the present invention effectively can measure the PD-1 antibody concentration in physiological saline, and can realize accurate detection when sample to be tested PD-1 antibody concentration is not less than the scope of 15.625ng/mL, and detection sensitivity is high.
In addition, be understandable that, in sample to be tested, the scope of application of PD-1 antibody concentration does not have the upper limit, when sample to be tested concentration detects higher than being diluted to by a certain percentage in the above-mentioned range of linearity during 1000ng/mL, and the actual concentrations of sample to be tested PD-1 antibody can be calculated by dilution ratio after sensing.
The detection of PD-1 antibody concentration in embodiment 4 serum
Adopt the PD-1 antibody assay kit of embodiment 2 to detect, method is specially:
1, be the gradient dilution liquid that concentration is respectively 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL and 15.625ng/mL by above-mentioned PD-1 antibody standard substance mice serum gradient dilution; In addition, the mountain goat anti rat IgG antibody adopting above-mentioned PBST to add 0.2 μ L HRP coupling by every mL prepares labelled antibody working fluid.
2, the physiological saline adding 3 times of volumes in each gradient dilution liquid carries out diluting, mixing, and respectively gets 100 μ L subsequently, adds in the plate hole of above-mentioned pre-coated elisa plate respectively, shrouding, hatch 1 hour in 37 DEG C.
3, outwell the solution in ELISA Plate, adopt above-mentioned lavation buffer solution PBST to wash plate 5 times, after clappers, add the above-mentioned labelled antibody working fluid of 100 μ L to every hole, shrouding, hatches 30 minutes in 37 DEG C.
4, outwell the solution in ELISA Plate, adopt above-mentioned lavation buffer solution PBST to wash plate 5 times, after clappers, add A liquid and each 50 μ L of B liquid of above-mentioned nitrite ion to every hole, shrouding, hatches 15 minutes for 37 DEG C.
5, take out ELISA Plate, add the above-mentioned stop buffer of 30 μ L to every hole, be placed in microplate reader and read OD 450, the results are shown in Table 2.
The OD of each gradient PD-1 antibody diluent in table 2 serum 450value
PD-1 antibody concentration (ng/mL) OD 450Value
1000 0.9609
500 0.5727
250 0.3651
125 0.2265
62.5 0.1855
31.25 0.1603
According to above-mentioned table 2 data creating linear relationship chart, as shown in Figure 2, and obtain linear equation, wherein R 2=0.9978, PD-1 antibody concentration and OD in serum are described 450between in extremely strong linear relationship.Illustrate thus: kit of the present invention effectively can measure the PD-1 antibody concentration in serum, and can realize accurate detection when sample to be tested PD-1 antibody concentration is not less than the scope of 31.25ng/mL, and detection sensitivity is high.
The detection of PD-1 antibody concentration in embodiment 5 tumor tissues homogenate liquid
Adopt the PD-1 antibody assay kit of embodiment 2 to carry out, method is specially:
1, be the gradient dilution liquid that concentration is respectively 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL and 15.625ng/mL by above-mentioned PD-1 antibody standard substance melanoma homogenate gradient dilution; In addition, the mountain goat anti rat IgG antibody adopting above-mentioned PBST to add 0.2 μ L HRP coupling by every mL prepares labelled antibody working fluid.
2, in each gradient dilution liquid, add isopyknic physiological saline to carry out diluting, mixing, respectively get 100 μ L subsequently, add in the plate hole of above-mentioned pre-coated elisa plate respectively, shrouding, hatch 1 hour in 37 DEG C.
3, outwell the solution in ELISA Plate, adopt above-mentioned lavation buffer solution PBST to wash plate 5 times, after clappers, add the above-mentioned labelled antibody working fluid of 100 μ L to every hole, shrouding, hatches 30 minutes in 37 DEG C.
4, outwell the solution in ELISA Plate, adopt above-mentioned lavation buffer solution PBST to wash plate 5 times, after clappers, add A liquid and each 50 μ L of B liquid of above-mentioned nitrite ion to every hole, shrouding, hatches 15 minutes for 37 DEG C.
5, take out ELISA Plate, add the above-mentioned stop buffer of 30 μ L to every hole, be placed in microplate reader and read OD 450, the results are shown in Table 3.
The OD of each gradient PD-1 antibody diluent in table 3 tumor tissues homogenate liquid 450value
PD-1 antibody concentration (ng/mL) OD 450Value
1000 1.6969
500 0.9673
250 0.5537
125 0.4055
62.5 0.2231
According to above-mentioned table 3 data creating linear relationship chart, as shown in Figure 3, and obtain linear equation, wherein R 2=0.9967, PD-1 antibody concentration and OD in tissue homogenate are described 450between in extremely strong linear relationship.Illustrate thus: kit of the present invention effectively can measure the PD-1 antibody concentration in tissue homogenate, and can realize accurate detection when sample to be tested PD-1 antibody concentration is not less than the scope of 62.5ng/mL, and detection sensitivity is high.
Embodiment 6 mice serum pharmacokinetic study
Test mice is C57BL/6 mouse, totally 5, is numbered 1# respectively, 2#, 3#, 4#, 5#; Adopt the PD-1 antibody assay kit of embodiment 2 to detect, method is specially:
1, adopt physiological saline that PD-1 antibody standard substance is mixed with 1mg/mL, to 2#, 3#, 4#, 5# mouse lumbar injection 0.1mL respectively, 2# after 1 day, 3# after 3 days, 4# after 7 days, 5# respectively eye socket blood sampling after 14 days, in 4 DEG C of cold postpone 2000g centrifuging and taking serum, be denoted as 2#, 3#, 4#, 5# serum respectively.
2, to 1# mouse orbit blood sampling, in 4 DEG C of cold postpone 2000g centrifuging and taking serum, adopt this serum using PD-1 antibody standard substance gradient dilution be 125ng/mL as standard items, be denoted as 1# serum.
3, in above-mentioned 1# to 5# serum, add normal saline dilution, the mixing of 9 times of volumes respectively, the parallel sampling of each serum three times, respectively gets 100 μ L at every turn, is added in the plate hole of above-mentioned pre-coated elisa plate respectively, after shrouding, hatches 1 hour for 37 DEG C.
4, outwell the solution in ELISA Plate, adopt above-mentioned lavation buffer solution PBST to wash plate 5 times, after clappers, every hole adds the above-mentioned labelled antibody working fluid of 100 μ L, and shrouding, hatches 30 minutes for 37 DEG C.
5, outwell the solution in ELISA Plate, adopt above-mentioned lavation buffer solution PBST to wash plate 5 times, after clappers, every hole adds A liquid and each 50 μ L of B liquid of above-mentioned nitrite ion, and shrouding, hatches 15 minutes for 37 DEG C.
6, take out ELISA Plate, every hole adds 30 μ L stop buffers, is placed in microplate reader and reads OD 450value, the linear equation obtained according to embodiment 4 calculates PD-1 antibody concentration, the results are shown in Table 4.
PD-1 antibody concentration in different time serum after table 4 mouse peritoneal injection PD-1 antibody
According to table 4 data creating curve map, as shown in Figure 4.Result shows: during mice serum three the parallel samplings carried out in different time points after injection PD-1 antibody detect, the PD-1 antibody concentration testing result of each serum is basically identical, relative error≤2.07%.Illustrate thus, kit of the present invention and detection method highly sensitive, and can be used for the pharmacokinetics research of PD-1 antibody.
Last it is noted that above each embodiment is only in order to illustrate technical scheme of the present invention, be not intended to limit; Although with reference to foregoing embodiments to invention has been detailed description, those of ordinary skill in the art is to be understood that: it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein some or all of technical characteristic; And these amendments or replacement, do not make the essence of appropriate technical solution depart from the scope of various embodiments of the present invention technical scheme.

Claims (10)

1. a PD-1 antibody assay kit, is characterized in that, comprises solid phase carrier, antibody capture agent and labelled antibody, and described antibody capture agent is the recombinant protein of PD-1 cell membrane extracellular portion, and its amino acid sequence is as SEQ ID NO.1.
2. PD-1 antibody assay kit according to claim 1, is characterized in that, the recombinant protein of described PD-1 cell membrane extracellular portion is pre-coated on described solid phase carrier.
3. PD-1 antibody assay kit according to claim 1, is characterized in that, described labelled antibody is the IgG antibody of horseradish peroxidase or streptomysin mark.
4. PD-1 antibody assay kit according to claim 1, is characterized in that, described solid phase carrier is ELISA Plate.
5. PD-1 antibody assay kit according to claim 1, is characterized in that, also comprises one or more in PD-1 antibody standard substance, positive reference substance and negative controls.
6. PD-1 antibody assay kit according to claim 1, is characterized in that, also comprise be buffered in liquid, lavation buffer solution, sample diluting liquid, confining liquid, nitrite ion and stop buffer one or more.
7. the arbitrary described application of PD-1 antibody assay kit in PD-1 antibody test of claim 1 to 6.
8. a PD-1 antibody detection method, is characterized in that, adopts PD-1 antibody assay kit according to claim 1 to carry out, and described PD-1 antibody detection method comprises the step that following order is carried out:
1) by pre-coated on described solid phase carrier for the recombinant protein of described PD-1 cell membrane extracellular portion, obtained pre-coated solid phase carrier;
2) in described pre-coated solid phase carrier, add sample to be tested, hatch, wash subsequently;
3) in described pre-coated solid phase carrier, add described labelled antibody, hatch, wash subsequently;
4) in described pre-coated solid phase carrier, add nitrite ion, hatch, add stop buffer subsequently, and measure OD 450.
9. PD-1 antibody detection method according to claim 8, is characterized in that, described PD-1 antibody assay kit also comprises PD-1 antibody standard substance, and described PD-1 antibody detection method also comprises:
Gradient dilution is carried out to described PD-1 antibody standard substance, and adopts step 1) to step 4) measure the OD of each gradient dilution liquid 450, obtain PD-1 antibody standard substance concentration and OD 450linear relationship;
According to the OD of measured sample to be tested 450with described linear relationship, obtain the concentration of PD-1 antibody in described sample to be tested.
10. PD-1 antibody detection method according to claim 8 or claim 9, is characterized in that, adopt and prepare described pre-coated solid phase carrier with the following method:
Adopt bag to be buffered the recombinant protein of liquid to described PD-1 cell membrane extracellular portion to dilute, the concentration controlling the recombinant protein solution after dilution is not less than 15.625ng/mL, is joined on described solid phase carrier by the recombinant protein solution after dilution and carries out bag quilt;
After bag is moved to end, employing lavation buffer solution washs solid phase carrier, closes subsequently to adding confining liquid in solid phase carrier, closes washing after terminating also dry, obtained described pre-coated solid phase carrier.
CN201510267428.7A 2015-05-22 2015-05-22 PD-1 antibody detection kit and application thereof Pending CN104931690A (en)

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CN109425736A (en) * 2017-08-25 2019-03-05 北京百普赛斯生物科技有限公司 A kind of method and kit detecting PD-1 antibody blood concentration
CN110687282A (en) * 2019-08-26 2020-01-14 中国医学科学院肿瘤医院 PD-1 and/or p53 autoantibodies as markers for tumor efficacy prediction or prognosis evaluation
CN110887961A (en) * 2018-09-10 2020-03-17 中国科学院苏州纳米技术与纳米仿生研究所 Solid phase carrier and kit for antibody screening
TWI718528B (en) * 2019-05-03 2021-02-11 國立高雄大學 Method for manufacturing immune checkpoint blockade particles and use of the immune checkpoint blockade particles

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109425736A (en) * 2017-08-25 2019-03-05 北京百普赛斯生物科技有限公司 A kind of method and kit detecting PD-1 antibody blood concentration
CN109425736B (en) * 2017-08-25 2021-04-09 北京百普赛斯生物科技股份有限公司 Method and kit for detecting blood concentration of PD-1antibody
CN110887961A (en) * 2018-09-10 2020-03-17 中国科学院苏州纳米技术与纳米仿生研究所 Solid phase carrier and kit for antibody screening
TWI718528B (en) * 2019-05-03 2021-02-11 國立高雄大學 Method for manufacturing immune checkpoint blockade particles and use of the immune checkpoint blockade particles
CN110687282A (en) * 2019-08-26 2020-01-14 中国医学科学院肿瘤医院 PD-1 and/or p53 autoantibodies as markers for tumor efficacy prediction or prognosis evaluation

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