CN101339195A - Elisa kit for checking soluble PD-1 protein and checking method - Google Patents
Elisa kit for checking soluble PD-1 protein and checking method Download PDFInfo
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Abstract
The invention provides an enzyme-linked immune detection reagent kit for detecting soluble PD-1 protein and a detection method thereof. The reagent kit consists of a PD-1 fusion protein standard compounds, a monoclonal antibody coating plate, an enzyme labeled monoclonal antibody and an auxiliary reagent; wherein, the reagent kit applies two strains of mouse anti-human PD-1 hybridomas and monoclonal antibodies secreted by the hybridomas which are called 1F2 and 5F10 and that are developed independently by the inventor and establishes the enzyme-linked immune detection reagent kit for detecting the soluble PD-1 (sPD-1) protein and the detection method thereof according to different characteristics of recognition sites of the two single strains of monoclonal antibodies. The reagent kit can detect the sPD-1 protein accurately and sensitively. The method can be applied to the detection of scientific research as well as clinical autoimmune diseases and hematological diseases, and the like, and provides the evaluation for diagnosis, monitoring and prognosis of relevant clinical diseases.
Description
Technical field
The present invention relates to medical science and Biological Detection field, be specifically related to a kind of enzyme-linked immunologic detecting kit that is used to detect solubility PD-1 albumen, and use the method that this kit detects soluble protein.
Background technology
PD-1 is the immunoglobulin superfamily I type transmembrane glycoprotein of a 50～55kD, mainly is expressed in the moment of growing in T, B cell and the marrow of activation (referring to people such as Ishida Y, The EMBOJournal, 11:3887-3895 (1992); People such as Agata Y, Int.Immunol, 8:765-772 (1996)), initial studies show that, two tyrosine residues of PD-1 cytoplasmic domain and the signaling molecule effect in downstream, can bring into play immune negativity adjusting function (referring to people such as Latchman Y, Nat Immunol, 2 (3): 261-268 (2001); People such as Ravetch JV, Science, 290 (5489): 84-89 (2000).PD-1 can also promote the propagation of T cell, and this effect depends on CD28 costimulatory signal (referring to people such as Rio ML, Eur J Immunol, 35:3545-3560 (2005)).Other has zoopery to show that in the animal model of PD-1 knock out mice, the shortage of PD-1 can cause the generation of a series of immunity diseases.The PD-1 deficient mice can cause the expansion cardiomyopathy (referring to people such as Hiroyuki N, Science, 291:319-321 (2001)) of autonomous immunity.Research also shows graft-versus-host reaction that blocking-up PD-1 signal can quicken IFN-γ and rely on (referring to people such as Blazar BR, J Immunol, 171:1272-1277 (2003)).PD-1/PD-L signal pathway with EAE (EAE) relevant (referring to people such as Salama AD, J Exp Med, 198:71-78 (2003)).Studies show that of clinical diseases such as AIDS, the expression of PD-1 is relevant with the immunoregulation effect of T cell in the body, HIV infects the expression of the PD-1 of the infected cell of alternative downward modulation, and the early apoptosis that prevents cell is (referring to people such as Venkatachari NJ, Virology.; 376:140-153 (2008)).
The PD-1 gene is made up of 5 extrons and 4 intrones, the montage of different PD-1mRNA will cause PD-1 molecule born of the same parents inner segment, stride the disappearance of film section and extracellular fragment, experiment confirm PD-1 Δ ex3 (No. 3 Exon deletion) will form solubility PD-1 (soluble PD-1, sPD-1), and the method with western blot in cell proliferation test confirms, after the T cell is stimulated by CD3CD28, the sPD-1 expression does not increase (referring to people such as Nislsen C in the culture supernatant, Cell Immunol, 235:109-116 (2005)); Bing W etc. has confirmed the existence of sPD-1 in RA patient's peripheral blood and articular cavity, and with the method validation of RT-PCR the mRNA of this solubility PD-1 be that the product of No. 3 Exon deletion (Δ ex3) is (referring to Wan B, Nie H, people such as Zhang JW, J Immunol, 177:8844-8850 (2006)).
People such as Feng Zuohua use the characteristics of this path of PD-1/PD-L, cDNA with coding PD-1 extracellular fragment makes up carrier for expression of eukaryon, has expressed solubility PD-1, and proof sPD-1 can be with high-affinity in conjunction with PD-Ls, the interaction of blocking-up PD-Ls/PD-1, thereby strengthen the T cell and kill tumor effect (referring to Qiu Hui, Zhang Hui, Feng Zuohua, Geng Hui, Zhang Guimei, Chinese liver magazine, 14 (7): 505-509 (2006); Hunan, Helan, Zhang Guimei, He Yufei, Zhang Hui, Xiang Jinyi, Zhu Hangang, Feng Zuohua, Chinese microbiology and Journal of Immunology, 26 (5): 463-467 (2006)).
Therefore, the detection of the expression of sPD-1 is significant to the monitoring of the generation of diseases such as various autoimmune disease and blood disease development in the serum, yet domestic now also do not have a sPD-1 enzyme linked immunological kit.
Show one's high ideals monarch's PhD dissertation " mouse-anti people PD-1 Study of Monoclonal Antibodies and biological function research " thereof of inventor discloses the technical scheme for preparing the different mouse-anti people PD-1 molecule monoclonal antibody of two strain recognition sites, independent research the monoclonal antibody of two strain mouse-anti people PD-1 hybridomas and secretion thereof, called after 1F2 and 5F10.Still be not in the news at present about the enzyme linked immunological kit that utilizes these two kinds of monoclonal antibodies to detect sPD-1.
Summary of the invention
The present invention seeks to utilize monoclonal antibody 1F2 and 5F10 preparation to detect the enzyme-linked immunologic detecting kit of sPD-1 albumen, and detection method is provided, thereby realize sPD-1 albumen is carried out accurate and sensitive detection.
For achieving the above object, the concrete technical scheme of the present invention is, a kind of enzyme-linked immunologic detecting kit that is used to detect sPD-1 albumen, comprise: PD-1 fusion standard items, PD-1 monoclonal antibody 1F2 bag is by plate, biotin labeled PD-1 monoclonal antibody 5F10, horseradish peroxidase and auxiliary reagent.
The preparation method of above-mentioned PD-1 monoclonal antibody 1F2 and 5F10 is open at show one's high ideals monarch's PhD dissertation " mouse-anti people PD-1 Study of Monoclonal Antibodies and biological function research " thereof of inventor, this paper has been embodied in incomparably in the database, and applicant University Of Suzhou guarantees to provide in from the applying date 20 years this two kinds of monoclonal antibodies: PD-1 monoclonal antibody 1F2 and 5F10.
The preparation process of described PD-1 fusion standard items may further comprise the steps:
The extracellular fragment sequence of PCR method human cloning PD-1 gene, RT-PCR amplifies human IgG1 Fc constant region gene from people's spleen cell, both splice the back and insert among the retroviral vector pEGZ-Term, recombinant vector and two helper viral vector liposome method cotransfection 293T incasing cellss, infect the L929 cell with the culture supernatant that contains virion, the Zeocin screening obtains the gene transfecting cell that the energy stably excreting is expressed PD-1Ig albumen, cultivate through serum free medium, the supernatant of collecting detects with dot blot, after ELISA is quantitative, concentrate and go up Protein G post purifying, further western blot identifies.
Described monoclonal antibody 1F2 bag is got with PD-1 monoclonal antibody 1F2 coated elisa plate by plate, and ELISA Plate can be selected the removable batten in 12 * 8 holes for use, and its making step is as follows:
(1) preparation monoclonal antibody 1F2: clone's mouse-anti people PD-1 gene, the hybridoma cell strain 1F2 of the anti-people PD-1 of preparation stably excreting antibody places to contain hyclone and the conventional cultivation of DMEM nutrient culture media; Collect well-grown hybridoma 1F2, inject, collect ascites after 7～10 days, Protein G immunoaffinity chromatography purified monoclonal antibody through pristane (Pristane) the Balb/c mouse peritoneal in 2 weeks of sensitization;
Its detailed preparation method is referring to the monarch's that shows one's high ideals PhD dissertation " mouse-anti people PD-1 Study of Monoclonal Antibodies and biological function research thereof ";
(2) bag quilt: said monoclonal antibody 1F2 is diluted to 2 μ g/ml with coating buffer, adds in each hole of ELISA ELISA Plate, every hole 100 μ l spend the night under 4 ℃; Second day, with the confining liquid sealing, spent the night in 4 ℃ in 200 μ l/ holes; The 3rd day, wash with cleaning fluid; Promptly obtain monoclonal antibody 1F2 coated elisa plate.
Described coating buffer is made up of the Tirs-HCL of 1 parts by volume and the water of 9 parts by volume; Described confining liquid is: massfraction is the PBS solution of 1% BSA; Described cleaning fluid is the phosphate buffered solution (pH=7.4) that contains solution gross mass 1% Tween-20, and its collocation method is a prior art.
Biotin labeled PD-1 monoclonal antibody 5F10 in the kit of the present invention is the PD-1 monoclonal antibody 5F10 of biotin N-maloyl imines ester mark, and its preparation process is as follows:
(1) preparation monoclonal antibody 5F10: clone's mouse-anti people PD-1 gene prepares the hybridoma cell strain 5F10 of the anti-people PD-1 of stably excreting antibody, places to contain 15% hyclone and the conventional cultivation of DMEM nutrient culture media; Collect well-grown hybridoma 5F10, inject, collect ascites after 7～10 days, through Protein G immunoaffinity chromatography purified monoclonal antibody 5F10 through pristane (Pristane) the BALB/c mouse abdominal cavity in 2 weeks of sensitization;
(2) prepare 1mg monoclonal antibody 5F10, add carbonic acid buffer (pH 9～9.5) to 1ml, tap water flushing bag filter is then with Coating Buffer flushing bag filter; Under 4 ℃, diafiltration in the carbonic acid buffer of 0.01mol/L pH=9.5; Adding concentration is the BNHS-DMSO solution 40 μ l of 1mg/ml, uses shaking table, lucifuge, room temperature concussion 4h; With PBS dialysis 7 times, change two not good liquors every day; Use the PD-1/L929 gene transfecting cell, detect the expression rate of PD-1 on PD-1/L929 with the fluidic cell method, with the identification marking effect, this biotin labeled 5F10 monoclonal antibody detects the expression rate of PD-1 on PD-1/L929 and reaches 99%.
Described horseradish peroxidase is made up of the Avidin-HRP of 1 parts by volume and the BSA of 16667 parts by volume.
Described auxiliary reagent comprises: cleaning fluid, and colour developing liquid and stop buffer, they consist of:
Cleaning fluid: the phosphate buffer (pH=7.4) that contains the Tween-20 of massfraction 1%;
Colour developing liquid: the aqueous solution of tetramethyl benzidine (TMB), wherein the volume ratio of tetramethyl benzidine and water is 1: 2;
Stop buffer: 2M sulfuric acid.
The method of testing of kit of the present invention may further comprise the steps:
(1) antigen-antibody reaction: in the micropore of the antibody sandwich plate that kit provides, add the gradient dilution liquid of standard items PD-1 fusion or the gradient dilution liquid of test serum sample respectively, 37 ℃ of insulation 1.5h; Wash plate 3 times with cleaning fluid then;
The gradient dilution liquid of described standard items PD-1Ig albumen is that maximum concentration is 50ng/ml, by 0.4 times of dilution gradient dilution liquid that is 7 concentration: 50ng/ml, 20ng/ml, 8ng/ml, 3.2ng/ml, 1.28ng/ml, 0.512ng/ml, 0.2048ng/ml; The gradient dilution liquid of described testing sample is 7 concentration by 0.4 times of dilution equally;
(2) adding concentration again is the biotin labeled PD-1 monoclonal antibody of 2 μ g/ml 5F10,100 μ l/ holes, 37 ℃ of insulation 1h; After washing plate 4 times with cleaning fluid, every hole adds Avdin-HRP 100 μ l, 0 ℃ of insulation 20min; Wash plate 6 times with cleaning fluid then;
(3) every hole adds developer TMB100 μ l, 37 ℃ of insulation 15min; Last every hole adds 50 μ l stop buffer cessation reactions;
(4) light absorption value with the blank hole is zero, records the OD value with microplate reader in 450nm;
(5) result calculates:
A. production standard working curve: the concentration with standard items sPD-1 is horizontal ordinate, and the OD value is an ordinate, draws the standard working curve of sPD-1.Basis of calculation curvilinear regression coefficients R is worked as R
2Greater than 0.98 this mensuration effectively;
B. calculate the content of the sPD-1 of test serum sample from typical curve according to the OD value of testing sample.
Because the technique scheme utilization, the present invention compared with prior art has following advantage:
1. because the application of PD-1 monoclonal antibody 1F2 and 5F10, the binding site difference of these two kinds of antibody, and have specificity, therefore utilize these two kinds of antibody can realize detecting the purpose of solubility PD-1 albumen;
2. in the process of production standard working curve, general method adopts the two-fold dilution, and the standard items gradient dilution liquid that the present invention adds has adopted 0.4 times of dilution, and the working curve linearity of acquisition is good, and regression coefficient has reached 0.9997;
3. the invention provides the enzyme-linked immunologic detecting kit that is applied in detection solubility PD-1 albumen and have stability, detect accurately sensitive advantage.
Description of drawings
Make kit and the process flow diagram that obtains standard working curve among Fig. 1 embodiment one;
Use the process flow diagram that kit detects sPD-1 albumen among Fig. 2 embodiment two;
The standard working curve figure that obtains among Fig. 3 embodiment one and two;
Respectively organize sPD-1 content distribution figure in the tested object serum among Fig. 4 embodiment two.
Embodiment
Below in conjunction with drawings and Examples the present invention is further described, unless otherwise indicated, the percentage that relates among the embodiment is all mass percent:
Embodiment one, makes the elisa kit for detecting that detects solubility PD-1 albumen
For convenience of description, at first list main agents and the instrument that uses among the embodiment:
Biotin: N-maloyl imines ester ((BNHS) Sigma company)
Coating buffer: volume ratio Tirs-HCL: H
2O=1: 9
The solution that contains massfraction 1% polysorbas20 of cleaning fluid: PB preparation (pH=7.4)
Confining liquid: the PBS solution of massfraction 1%BSA (Sigma company)
Horseradish peroxidase: volume ratio Avidin-HRP (Sigma company): BSA=1: 16667
Colour developing liquid: volume ratio tetramethyl benzidine (TMB) (Sigma company): H
2O=1: 2
Stop buffer: 2M sulfuric acid
The fixed version of enzyme mapping (12 * 8 holes, Nunc company);
Enzyme mark analyzer (Bio-Rad company)
A kind of elisa kit for detecting that detects solubility PD-1 albumen comprises:
1 bottle of PD-1Ig fusion standard items, 50ng/ml;
1 of the ELISA ELISA Plate (96 hole) of PD-1 monoclonal antibody 1F2 bag quilt;
The PD-1 monoclonal antibody 5F10 of biotin N-maloyl imines ester mark, 2 μ g/ml;
Horseradish peroxidase, Avidin-HRP (Sigma company): BSA=1: 16667;
Colour developing liquid (TMB) 1 bottle;
The solution that contains 1% polysorbas20 of 1 bottle of cleaning fluid: PB preparation (pH=7.4);
1 bottle of stop buffer: the sulfuric acid of 2mol/L;
The concrete operations step of elisa kit for detecting for preparing above-mentioned detection solubility PD-1 albumen is as follows, and flow process can be referring to Fig. 1:
1. the preparation process of preparation PD-1Ig fusion standard items may further comprise the steps:
The extracellular fragment sequence of PCR method human cloning PD-1 gene, RT-PCR amplifies human IgG1 Fc constant region gene from people's spleen cell, both splice the back and insert among the retroviral vector pEGZ-Term, recombinant vector and two helper viral vector liposome method cotransfection 293T incasing cellss, infect the L929 cell with the culture supernatant that contains virion, the Zeocin screening obtains the gene transfecting cell that the energy stably excreting is expressed PD-1Ig albumen, cultivate through serum free medium, the supernatant of collecting detects with dot blot, after ELISA is quantitative, concentrate and go up Protein G post purifying, further western blot identifies, obtains standard items PD-1Ig fusion.
2. make PD-1 monoclonal antibody 1F2 and 5F10:
2.1 the cultivation of cell line
Employing contains the RPMI1640 nutrient culture media of 10%FCS, and the gene transfecting cell strain regularly uses resistance screening medicine Zeocin (500 μ g/ml) to cultivate, and to keep the stably express of genes of interest product, the Zeocin with 50 μ g/ml is kept cultivation usually.PD-1/L929 cell and empty retrovirus change the L929 cell line and are the growth of adherent property, when going down to posterity with 0.25% trypsinization, interval 3～4d cultivation of going down to posterity.SP2/0 cultivates with the DMEM nutrient culture media that contains 10%FCS is conventional, and interval 2～3d changes liquid and goes down to posterity.
2.2 the immunity of animal
Collect well-grown PD-1/L929 cell, (behind twice of the 1200rpm * 5min), add mitomycin solution (50 μ g/1 * 10 with aseptic PBS washing is centrifugal
7Individual cell), mixing places 37 ℃ of reaction 45min, and PBS washing, centrifugal (behind the 1200rpm * 5min) three times, the NS with 0.3～0.4ml suspends again again, employing lumbar injection (1 * 10
7Individual cell/only) 6～8 age in week the Balb/c mouse.Carried out immunity once more respectively in the 21st day and the 35th day behind initial immunity, cell quantity is about 8 * 10
6Cell/only (the 21st day) and 5 * 10
6Cell/only (the 35th day), the pre-service of cell and the position of injection are with for the first time.In merging preceding 4～5d, lumbar injection 3 * 10 once more
6Cell/only, carry out booster immunization.
2.3 myeloma cell's preparation
Merge preceding 10～14d, recovery murine myeloma cell SP2/0 is with the DMEM nutrient culture media that the contains 10%FCS cultivation of going down to posterity, treat that the cell growth is vigorous, form is good (round and mellow, bright, big or small homogeneous), and cell viability can be in order to merge greater than 95% (tongue is expected blue dyeing).Merge preceding 24～36h and cell concentration is adjusted into 3 * 10 with the DMEM nutrient culture media of the fresh 10%FCS of containing
5/ ml.
2.4 the preparation of trophocyte and immune mouse spleen cell
1 of Balb/c mouse, 1d extracts the eyeball bloodletting before merging, and disconnected neck is put to death, place 75% alcohol 10min, be fixed on the dissection plate of aseptic super-clean bench, open the mouse thoracic cavity and take out thymus gland, open the mouse thoracic cavity and take out spleen with method along breastbone, both are placed 120 purpose woven wires, the screen cloth immigration is filled in the plate of 20ml basal medium, grind thymus gland and spleen, make it become individual cells and be suspended from the nutrient culture media, centrifugal (1000rpm * 5min), resuspension cell and counting; Adjusting trophocyte concentration with complete medium or HAT nutrient culture media is 3～4 * 10
5/ ml, with above-mentioned cell point plate in 96 well culture plates (totally 10 96 well culture plates), every hole adds i.e. (3～4) * 10 of 0.1ml cell suspension
4/ hole is cultivated standby in the CO2 incubator.
Get the Balb/c mouse behind the booster immunization, handle mouse as stated above, and take out spleen, obtain single spleen cell through grinding at left back belly.Expect that with tongue blue liquid has nuclear counting as living cells, centrifugal (1000rpm * 5min), DMEM is suspended from sedimentation cell in the 10ml DMEM basal medium after washing twice, places incubator standby.
2.5 the fusion of cell and selectivity are cultivated
DMEM basal medium, PEG solution are placed the pre-temperature of 37 ℃ of water-baths.Collect 2 * 10
7Individual well-grown SP2/0 cell is with 1 * 10
8Individual spleen cell is mixed in the 50ml transparent plastic centrifuge tube, with serum-free RPMI1640 washing twice, behind the centrifugal 5min of 1000rpm, abandons supernatant, at the bottom of the light finger bomb tube, makes two kinds of immunoprecipitation cells fully be mixed into pasty state.Plastic tube is placed 37 ℃ of thermos cups, draw 50% the PEG solution of 0.7ml through 37 ℃ of pre-temperature, suction pipe is inserted the cell bottom gently, in 1min, at the uniform velocity add, and the limit edged stirs gently, leaves standstill 90s then in water-bath, adds the DMEM basal medium of 37 ℃ of pre-temperature of 40ml again, centrifugal behind the static 5min of room temperature (1000rpm * 10min), supernatant discarded.Sedimentation cell is contained gently the DMEM cultivation suspension of 1%HAT, 15%FCS with 100ml.Drip behind the mixing in above-mentioned 96 well culture plates that contain trophocyte, 100 μ l/ holes place 37 ℃, 5%CO
2Cultivate in the incubator, the later half amount of 3～4d is changed liquid, uses the HT medium culture behind 10～12d instead, the DMEM medium culture of migrating after 2 weeks and containing 15%FCS.
2.6 the screening of the positive hybridoma cell strain of secretion PD-1 monoclonal antibody
(it is yellow that the nutrient culture media color is) draws supernatant when treating that cell clone grows to 1/3～1/4 culture hole, screens with the indirect immunofluorescence labelling method.Behind well-grown PD-1/L929 cell dissociation, with PBS washing and adjust its concentration, divide in the streaming pipe (5 * 10
5Cell/pipe), adds the culture supernatant (60 μ l/ pipe) of hybridoma, simultaneously with the positive contrast of PE-PD-1mAb.In 4 ℃ of reaction 45min, the PBS washing twice with containing 5% calf serum adds sheep anti mouse PE labelled antibody; In 4 ℃ of reaction 45min, washing back is analyzed with FCM again, simultaneously with empty retrovirus change the L929 cell as negative control cell strain screen.
2.7 the cloning of positive hybridoma cell is cultivated
Adopt limiting dilution assay that positive colony is carried out subclone.Draw the hybridoma in the positive hole of PD-1 antibody response, the counting back is 50/ml and 10/ml/ penicillin bottle with HAT selective medium gradient dilution to cell, add people 100 μ l to 96 well culture plates that contain trophocyte, make every hole on average contain 5 and 1 cell, 37 ℃, 5%CO
2Cultivate the observation of cell upgrowth situation.When treating 1/5 hole at the bottom of clone cell grows to the hole, carry out multiple sieve by the screening technique of the positive colony of having set up.Select the antibody titer height, be single clonal growth and the form good cell continues subclone, until the antibody-secreting positive rate greater than 95%; Enlarged culture and timely liquid nitrogen cryopreservation.
2.8 the chromosomal karyotyping of hybridoma
Get well-grown cell, add colchicine, making its final concentration is 0.04～0.08 μ l/ml.After cultivating 2h, collecting cell, centrifugal 1000rpm, 10min dropwise is incorporated in 37 ℃ of pre-warm 0.075mol/LKCl solution 0.5ml, adds 5～10ml immediately again, blows and beats gently evenly with suction pipe, hatches 20min for 37 ℃.Add the immobile liquid 1ml (facing the time spent preparation) of freshly prepared 3 parts of methyl alcohol+1 part glacial acetic acid, centrifugal 1000rpm * 10min abandons supernatant.Add 8～10ml immobile liquid again, with suction pipe piping and druming evenly, fix 15～20min, centrifugal, abandon supernatant.Add the 5ml immobile liquid again, fixedly 30min.The centrifugal supernatant of abandoning.Add the 1.5ml immobile liquid again, piping and druming evenly.Get-10 ℃ of freezing microslides, drip 1～2 cell suspension,, dry after the flowing water flushing with fresh Giemsa solution (1 part of Giemsa stoste adds 9 parts of 0.075mol/L, PH6.8 phosphate buffer) dyeing 10～20min.Transparent three times of dimethylbenzene, the neutral resins mounting.Microscopically selective staining body good dispersion, not overlapping, do not have the sample scatter, oily mirror is observed down, writes down and carries out cinephotomicrography.
2.9 the preparation of monoclonal antibody ascites
Get the Balb/c mouse (about 15) in 8～10 ages in week, the abdominal cavity only injects pristane (being called P) 0.5ml/, and hybridoma 5 * 10 is directly injected in the abdominal cavity in 1 all backs
5Cell/only, massage mouse web portion gently makes the abundant mixing of hybridoma, and diffuses to whole abdominal cavity.Collect ascites about 7～12d, centrifugal, get supernatant ,-80 ℃ are standby.
2.10 Purification of Monoclonal Antibodies (Protein G immune-affinity chromatography)
With the centrifugal Declotting of ascites, every 10ml ascites adds 1ml 1mol/L CaCl
2, 0.1ml 5%Sulfatedextran solution, stirring at room 15min, 4 ℃ centrifugal, and (12000rpm 20min) removes fibrins.Get supernatant and add the saturated (NH of equal-volume by 50% saturation degree
4)
2SO
4Solution, mixing rearmounted 4 ℃ 4～6 hours, the centrifugal supernatant of abandoning.Get the PBS dissolving that precipitation adds 0.01mol/L, PH7.4.(NH is removed in dialysis
4)
2SO
4, according to the monoclonal antibody purification scheme that Pharmacia company provides, sample is Protein G affinity column in the FPLC system, PH2.8 glycocoll wash-out.Collect the protein peak effluent, in time adjust PH to 7.0 with the Tris solution of PH9.0.Measure the content of antibody protein after the PBS dialysis with 751 ultraviolet spectrophotometers, the computing formula of its concentration is: antibody protein content (mg/ml)=OD
280* 1.55-OD
260* 0.76.After the antibody filtration sterilization, be sub-packed in-80 ℃ of preservations.
3. making PD-1 monoclonal antibody 1F2 wraps by plate:
ELISA Plate can be selected homemade plate or import plate for use, and specification is the removable batten in 96 orifice plates or 12 * 8,6 * 8 holes.The present invention adopts a kind of import ELISA Plate (Nunc company product).With the dilution of above-mentioned 1F2 monoclonal anti body and function coating buffer is to add ELISA Plate behind the 2 μ g/ml, every hole 100 μ l, and coating buffer is by the Tirs-HCL of 1 parts by volume and the H of 9 parts by volume
2O forms, and spends the night under 4 ℃; Second day, with the confining liquid 1%BSA sealing that Sigma company produces, spent the night in 4 ℃ in 200 μ l/ holes; The 3rd day,, promptly obtain monoclonal antibody 1F2 coated elisa plate with the cleaning fluid washing.
4. the PD-1 monoclonal antibody 5F10 for preparing biotin N-maloyl imines ester mark:
Prepare 1mg monoclonal antibody 5F10, the carbonic acid buffer that adds pH 9～9.5 is to 1ml, and tap water flushing bag filter is then with Coating Buffer flushing bag filter; Diafiltration in the carbonic acid buffer of 0.01mol/L pH 9.5 (4 ℃); Adding concentration is the BNHS-DMSO 40 μ l of 1mg/ml, uses shaking table, lucifuge, room temperature concussion 4h; With PBS dialysis 7 times, change two not good liquors every day; Use the PD-1/L929 gene transfecting cell, detect the expression rate of PD-1 on PD-1/L929 with the fluidic cell method, with the identification marking effect, the expression rate of this biotin labeled 5F10 monoclonal antibody on PD-1/L929 reaches 99%.
5. preparation cleaning fluid: PB prepares the solution that contains 1% polysorbas20 of (pH=7.4);
6. prepare horseradish peroxidase solution: Avidin-HRP (Sigma company): BSA=1: 16667;
7. prepare colour developing liquid: tetramethyl benzidine (TMB) (Sigma company): H
2O=1: 2;
8. preparation stop buffer: 2M sulfuric acid.
The quality testing of the elisa kit for detecting of the solubility PD-1 albumen of application the technology of the present invention preparation:
(1) stability test: bag is put 4 ℃ of preservations after 0,10,20,30 day by good ELISA Plate, respectively coefficient of variation CV＜5.17% of part measuring point numerical value in the analytical standard curve.
Concrete outcome is referring to table 1:
The stability analysis of table 1 sPD-1ELISA kit (x ± s, CV%)
(2) accuracy testing: adopt addition method, in the serum specimen dilution of 5 parts of known sPD-1 concentration, add the standard items 5.00ng of sPD-1 respectively.Measure the recovery, on average be about 100%.
Concrete outcome is referring to form 2:
The analysis of the accuracy of table 2sPD-1ELISA kit
| Original bulk (ng/ml) | Addition (ng) | Record content (ng/ml) | Accuracy rate (%) |
| 12.07 | 25.00 | 42.73 | 115.4 |
| 4.08 | 10.00 | 14.37 | 102.0 |
| 2.77 | 10.00 | 13.72 | 107.4 |
| 140 | 4.00 | 5.31 | 98.4 |
| 1.35 | 4.00 | 5.10 | 95.4 |
(3) precision experiment: use 15 box different batches kits at random, carry out replication, calculate each measurement result, obtain average, SD and the coefficient of variation, CV＜12% with same sample.
(4) range of linearity: add standard items (PD-1Ig), maximum concentration is 50ng/ml, by 0.4 times of dilution is 7 concentration: 50ng/ml, 20ng/ml, 8ng/ml, 3.2ng/ml, 1.28ng/ml, 0.512ng/ml, the gradient dilution liquid of 0.2048ng/ml, step is to specifications carried out time-and-motion study.
Concentration with sPD-1 is horizontal ordinate, and the OD value is an ordinate, curve plotting, and the range of linearity of kit is 0.2-50ng/ml.
(5) detection sensitivity: according to above-mentioned range of linearity measurement result, the detection sensitivity of this kit is 0.2ng/ml.
Embodiment two: the use-case of the elisa kit for detecting of using soluble PD-1 albumen, its process flow diagram can be referring to Fig. 2
(1) collect the normal person, alpastic anemia (AA) patient, acute lymphoblastic leukaemia (ALL) patient, acute myeloid leukaemia (AML) patient, non-Ke's lymphomas (NHL) patient, the liver transfer operation patient, the serum of hyperthyroid patient, subzero 20 ℃ frozen;
(2) with standard items PD-1Ig (concentration 50ng/ml), dilute 7 gradient concentrations with redistilled water by 0.4 times, typical curve in the kit is made up of the standard items of 7 variable concentrations, the concentration of typical curve each point is respectively 50ng/ml, 20ng/ml, 8ng/ml, 3.2ng/ml, 1.28ng/ml, 0.512ng/ml, 0.2048ng/ml; Equally sample to be tested serum is diluted 7 gradient concentrations by 0.4 times;
(3) antigen-antibody reaction: in the micropore of the antibody sandwich plate that kit provides, add the gradient dilution liquid of standard items and sample to be tested serum respectively, 37 ℃ of insulation 1.5h wash plate three times with cleaning fluid then;
(4) integrated enzyme reaction: every hole adds the biotin labeled PD-1 monoclonal antibody Bio-5F10 that 100 μ l concentration are 2 μ g/ml, 37 ℃ of insulation 1h; After cleaning fluid was washed plate four times, every hole added the Avdin-HRP of 100 μ l, 0 ℃ of insulation 20min;
(5) wash plate 6 times with cleaning fluid at last, every hole adds developer TMB100 μ l, 37 ℃ of insulation 15min; The H that adds every hole 2M then
2SO
4Solution 50 μ l cessation reactions;
(6) colorimetric: the light absorption value with the blank hole is zero, records the OD value with microplate reader in 450nm;
(7) result calculates:
Concentration with sPD-1 is horizontal ordinate, and the OD value is an ordinate, draws the standard working curve of sPD-1, and referring to Fig. 3, application software curve expert 1.3 tries to achieve sample sPD-1 content.
Testing result is seen Fig. 4, and results of statistical analysis sees Table 3:
Table 3 is respectively organized the statistical analysis of human peripheral supernatant sPD-1 content
| Group name | The example number | (x±s) | t(95％) |
| Normal group | 28 | 1.103±0.240 | |
| AA patient's group | 29 | 3.542±3.065 | P＜0.01 |
| ALL patient's |
10 | 2.736±1.408 | P＜0.05 |
| AML patient's group | 5 | 3.864±4.184 | P＜0.05 |
| NHL patient's group | 3 | 3.145±1.159 | P＞0.05 |
| The |
8 | 0.904±0.140 | P＞0.05 |
| Liver transfer |
8 | 1.180±0.00633 | P＞0.05 |
SPD-1 Determination on content result in the human peripheral supernatant:
Shown in Fig. 4 and table 3, normal person's's (28 example) content is between 0.775～1.825, and mean value is 1.103, and median is 1.033; AA patient's (29 example) content is between 1.062～14.000, and mean value is 3.542, and median is 2.002; ALL (10 example) patient's content is between 1.242～5.604, and mean value is 2.736, and median is 2.187; AML (5 example) patient's content is 0.91～10.127, and average out to 3.864, median are 1.74; NHL (3 example) patient's content is between 2.056～3.946, and mean value is 3.145, and median is 2.325; Each 8 example of hyperthyroidism and organ transplant patient more all do not have significant difference with the normal person.SPD-1 content is apparently higher than normal person (P＜0.01, table 3) among the AA patients serum, and the blood disease patient of other types also has minority to be higher than normal value, and hyperthyroidism and organ transplant patient's sPD-1 content does not relatively have significant difference in the normal person.
This method can supply the detection of diseases such as scientific research, clinical autoimmune disease and blood disease, provides assessment to diagnosis, monitoring and the prognosis of clinical relevant disease.
Claims (6)
1. enzyme-linked immunologic detecting kit that is used to detect sPD-1 albumen, it is characterized in that comprising: PD-1Ig fusion standard items, PD-1 monoclonal antibody 1F2 wraps by plate, biotin labeled PD-1 monoclonal antibody 5F10, horseradish peroxidase and auxiliary reagent.
2. the enzyme-linked immunologic detecting kit that is used to detect sPD-1 albumen according to claim 1, it is characterized in that: described monoclonal antibody 1F2 bag is got with PD-1 monoclonal antibody 1F2 coated elisa plate by plate, and the concentration of monoclonal antibody 1F2 is 2 μ g/ml in the hole of ELISA Plate.
3. the enzyme-linked immunologic detecting kit that is used to detect sPD-1 albumen according to claim 1 is characterized in that: described biotin labeled PD-1 monoclonal antibody 5F10 is the PD-1 monoclonal antibody 5F10 of biotin N-maloyl imines ester mark.
4. the enzyme-linked immunologic detecting kit that is used to detect sPD-1 albumen according to claim 1 is characterized in that: described horseradish peroxidase is made up of the Avidin-HRP of 1 parts by volume and the BSA of 16667 parts by volume.
5. the enzyme-linked immunologic detecting kit that is used to detect sPD-1 albumen according to claim 1 is characterized in that: described auxiliary reagent comprises: cleaning fluid, colour developing liquid and stop buffer.
6. a method of testing sPD-1 albumen comprises antigen-antibody reaction, integrated enzyme reaction, chromogenic reaction, cessation reaction step, it is characterized in that: adopt the described kit of claim 1, comprise following concrete steps,
(1) antigen-antibody reaction: in the micropore of the antibody sandwich plate that kit provides, add the gradient dilution liquid of standard items PD-1Ig fusion or the gradient dilution liquid of test serum sample respectively, 37 ℃ of insulation 1.5h; Wash plate 3 times with cleaning fluid then;
The gradient dilution liquid of described standard items PD-1Ig is that maximum concentration is 50ng/ml, with the phosphate buffered solution of massfraction 1%BSA gradient dilution liquid: the 50ng/ml that is 7 concentration by 0.4 times of dilution, 20ng/ml, 8ng/ml, 3.2ng/ml, 1.28ng/ml, 0.512ng/ml, 0.2048ng/ml; The gradient dilution liquid of described testing sample is 7 concentration by 0.4 times of dilution equally;
(2) adding concentration again is the biotin labeled PD-1 monoclonal antibody of 2 μ g/ml 5F10,100 μ l/ holes, 37 ℃ of insulation 1h; After washing plate 4 times with cleaning fluid, every hole adds Avdin-HRP 100 μ l, 0 ℃ of insulation 20min; Wash plate 6 times with cleaning fluid then;
(3) every hole adds developer TMB100 μ l, 37 ℃ of insulation 15min; Last every hole adds 50 μ l stop buffer cessation reactions;
(4) light absorption value with the blank hole is zero, records the OD value with microplate reader in 450nm;
(5) result calculates:
A. production standard working curve: the concentration with standard items sPD-1 is horizontal ordinate, and the OD value is an ordinate, draws the standard working curve of sPD-1.Basis of calculation curvilinear regression coefficients R is worked as R
2Greater than 0.98 this mensuration effectively;
B. calculate the content of the sPD-1 of test serum sample from typical curve according to the OD value of testing sample.
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