CN114994331B - Kit for detecting protein expression level and preparation method and application thereof - Google Patents
Kit for detecting protein expression level and preparation method and application thereof Download PDFInfo
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- CN114994331B CN114994331B CN202210541972.6A CN202210541972A CN114994331B CN 114994331 B CN114994331 B CN 114994331B CN 202210541972 A CN202210541972 A CN 202210541972A CN 114994331 B CN114994331 B CN 114994331B
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6869—Interleukin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/244—Interleukins [IL]
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- 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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/54—Interleukins [IL]
- G01N2333/5434—IL-12
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Abstract
The invention discloses a kit for detecting protein expression level, wherein the protein is human IL-12B protein, the kit comprises effective dose of human IL-12B protein and two strains of effective dose of human IL-12B protein monoclonal antibodies, the two strains of human IL-12B protein monoclonal antibodies are monoclonal antibody 2 and monoclonal antibody 4, the sequences of heavy chain variable region and light chain variable region of monoclonal antibody 2 are shown as SEQ ID NO.2 and SEQ ID NO.3, and the sequences of heavy chain variable region and light chain variable region of monoclonal antibody 4 are shown as SEQ ID NO.4 and SEQ ID NO. 5. The kit has the advantages of high sensitivity, wide linear range and high detection speed.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a kit for detecting protein expression level, and a preparation method and application thereof.
Background
Interleukin, abbreviated as IL, is involved in the expression and regulation of a functional immune response by a number of factors derived from lymphocytes or macrophages. Lymphokines derived from lymphocytes and generally called monokines derived from macrophages have different biological activities of factors (e.g., macrophage activation, T cell proliferation promotion, etc.), and many physical and chemical properties of the factors themselves are unknown. Interleukins are a very important family of cytokines, and as of 2013, there were up to 29 members recognized; they play an important role in a series of processes such as maturation, activation, proliferation and immunoregulation of immune cells, and in addition, they are involved in various physiological and pathological reactions of the body.
Interleukin (IL) 12, antigen presenting cells and BCells produce, and secrete extracellular in this form, a pro-inflammatory cytokine in the form of a heterodimer consisting of two subunits with molecular weights of 40kDa (IL-12B) and 35kDa (IL-12A). IL-12 can induce IFN-gamma production, in vivo immune response (especially in bacterial or parasitic infection) it is also IFN-gamma production required. It has been reported that (Ek WE, karlsson T,
J,Rask-Andersen M,Johansson
practical effects of fluorescent proteins biomarkers on fluorescent devices Sci adv.2021Dec 10;7 (50): eabl 4359.), IL-12B has a protective effect on psoriasis and psoriatic arthropathy, therefore, monitoring of IL-12B is necessary, and therefore, the development of a kit for detecting human IL-12B protein is urgently needed.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention aims to provide a detection kit for human IL-12B protein and a preparation method thereof.
Therefore, on one hand, the invention provides a kit for detecting the expression level of a protein, wherein the protein is human IL-12B protein, the kit consists of an effective amount of human IL-12B protein and two effective amounts of human IL-12B protein resistant monoclonal antibodies, the two human IL-12B protein resistant monoclonal antibodies are monoclonal antibody 2 and monoclonal antibody 4, the sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 2 are shown as SEQ ID NO.2 and SEQ ID NO.3, and the sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 4 are shown as SEQ ID NO.4 and SEQ ID NO. 5.
Preferably, the effective amount of the human IL-12B protein is a standard, and the gradient of the standard is 3000pg/ml, 500pg/ml, 100pg/ml, 25pg/ml, 5pg/ml, 2.5pg/ml, 0pg/ml.
Preferably, the epitope sequence of monoclonal antibody 2 of the present invention is LLLHKKEDGIWSTDILKDQKEPKNK.
Preferably, the epitope sequence of monoclonal antibody 4 of the present invention is hklkyenytssffirdhikpdpkn.
Preferably, the monoclonal antibody 2 of the present invention is coupled to magnetic particles, and the concentration of the magnetic particles coupled to the monoclonal antibody 2 is 0.25mg/ml.
Preferably, the monoclonal antibody 4 provided by the invention is marked with acridinium ester, and the concentration of the acridinium ester marked monoclonal antibody 4 is 40ng/ml.
In still another aspect, the present invention also provides a method for detecting human IL-12B using the kit, the method comprising the steps of: 1) 10 μ l sample +50 μ l reagent R1+50 μ l reagent R2+90 μ l wash buffer; 2) Mixing and incubating at 37 ℃ for 15min; 3) wash buffer 3 times, 500 mul each time; 4) Adding 200 mul of substrate, and immediately detecting the RLU value by a chemiluminescence apparatus; the wash buffer is TBST buffer solution.
Preferably, the sample according to the present invention is serum, whole blood, urine, interstitial fluid.
In still another aspect, the invention also provides an application of the kit in detecting human IL-12B protein.
The human IL-12B protein detection kit prepared by the invention is a chemiluminescence detection kit, has the advantages of high sensitivity, good specificity and wide linear range, and has the prospect of large-scale and automatic application (the detection kit is integrated into the existing chemiluminescence detection instrument to realize automatic detection). In addition, the monoclonal antibody is prepared by designing the antigen polypeptide and immunizing a mouse, so that the epitope recognized by the monoclonal antibody can be directly determined, and the monoclonal antibody has the advantages of strong targeting property and good specificity; the antigenic polypeptide disclosed by the invention has a good reference effect on the subsequent study of the human IL-12B protein.
Drawings
FIG. 1IL-12B protein signal peptide analysis.
FIG. 2 removal of signal peptide IL-12B protein transmembrane region analysis.
FIG. 3 shows the calibration results of the standards.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1: analysis and design of IL-12B proteins
Human IL-12B protein (NCBI Reference Sequence: NP-002178.2) was selected and its amino acid Sequence was systematically analyzed. The signal peptide was first analyzed and the results showed (FIG. 1) that the sequence had a signal peptide probability of 78.878%, the signal peptide type was SP (Sec/SPI), the cleavage sites were aa22-aa23, and the signal peptide sequence was MCHQLVISWFSLVFLASPLVA. Next, the sequence from which the signal peptide was removed was analyzed for the transmembrane region, and the results showed that the remaining sequences were extracellular regions (FIG. 2). Therefore, the amino acid sequence of the recombinant IL-12B protein is determined to be shown in SEQ ID NO.1 (22 amino acids are removed from the N terminal).
Designing antigen polypeptide of SEQ ID NO.1, designing and synthesizing 5 polypeptides in total, wherein the specific sequence is as follows:
antigenic polypeptide 1 (aa 11-aa 35): VELDWYPDAPGEMVVLTCTPEEDG;
antigenic polypeptide 2 (aa 80-aa 104): LLLHKKEDGIWSTDILKDQKEPKNK;
antigenic polypeptide 3 (aa 150-aa 174): aatlsaervvrgdnkeystqed;
antigenic polypeptide 4 (aa 194-aa 218): HKLKYENYTSSSFFIRDIIKPDPPKN;
antigenic polypeptide 5 (aa 256-aa 280): QGKSKREKKDRVFTDKSATKICKKICRK.
Example 2: preparation and test of monoclonal antibody for specifically recognizing antigen polypeptide
Mice were immunized with 5 antigen polypeptides synthesized in example 1, and monoclonal antibodies were prepared according to conventional hybridoma screening (see example 2 of CN 106226512B for details). Through screening and preparation, each antigen polypeptide screens a hybridoma cell and prepares a batch of monoclonal antibodies, the serial numbers of the hybridoma cells are 1-5, the serial numbers of the monoclonal antibodies are 1-5, and the monoclonal antibodies correspond to 1-5 of antigen epitopes (antigen polypeptides). As shown in table 1.
TABLE 1 hybridoma cells, monoclonal antibodies and epitope mapping Table
| Hybridoma cell | Monoclonal antibodies | Antigenic epitope |
| Hybridoma |
|
Epitope 1: VELDWYPDAPGEMVVLTCTPEEDG |
| Hybridoma cell 2 | Monoclonal antibody 2 | Epitope 2: LLLHKKEDGIWSTDILKDQKEPKNK |
| Hybridoma cell 3 | Monoclonal antibody 3 | Epitope 3: AATLSAERVRGDNKEYEYSVECQED |
| Hybridoma cell 4 | Monoclonal antibody 4 | Epitope 4: HKLKYENYTSSSFFIRDIIKPDPPKN |
| Hybridoma cell 5 | Monoclonal antibody 5 | Epitope 5: QGKSKREKKDRVFTDKSATVSKICRK |
Screening and detecting the prepared monoclonal antibody by using ELISA, wherein the specific method is briefly described as follows: an enzyme-labeled plate (0.5. Mu.g/ml) was coated with IL-12B protein (purchased from GmbH, PCK 172) at 0.1ml per well and overnight at 4 degrees; after washing for 3 times, sealing by using 5% skimmed milk, wherein each hole is 0.2ml and the temperature is 37 ℃ for 2 hours; after washing for 3 times, adding 10 times of serially diluted monoclonal antibodies (diluted after adjusting the concentration to be 1 mg/ml), wherein each hole is 0.1ml and the temperature is 37 ℃ for 1h; after washing for 3 times, adding a secondary goat anti-mouse IgG HRP marker (diluted 5000 times) into each hole; 37 ℃ for 0.5h; washing for 3 times, adding 0.1ml of color development solution into each hole, and washing at 37 deg.C for 10min; adding stop solution 0.05ml per hole, and detecting OD450nm value; the test was positive when the P/N value (sample OD value/blank OD value) > 2.1. The detection result shows that the monoclonal antibodies 2, 3 and 4 have good potency (the potency is more than or equal to 10) 6 ) Monoclonal antibodies 1 and 5 were relatively low (titers of 10 each) 4 ) (ii) a Therefore, monoclonal antibodies 2, 3 and 4 were selected as monoclonal antibodies for subsequent kit development, but the antigenic polypeptides recognized by monoclonal antibodies 2 and 3 were relatively close, so the focus was on monoclonal antibody 2 and monoclonal antibody 4 as target antibodies for kit development.
The variable region sequences of monoclonal antibody 2 and monoclonal antibody 4 were analyzed (the specific analysis method is conventional, see CN 111393525B), wherein the sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 2 are shown in SEQ ID No.2 and SEQ ID No. 3; wherein the sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 4 are shown in SEQ ID NO.4 and SEQ ID NO. 5.
Example 3: preparation and detection of IL-12B protein detection kit
3.1 preparing standard products: the IL-12B protein (available from GmbH, PCK172, wuhan Pronay Ltd.) was used as the stock solution, which was diluted to 7 gradients of 3000pg/ml, 500pg/ml, 100pg/ml, 25pg/ml, 5pg/ml, 2.5pg/ml, 0pg/ml, respectively, using a diluent (2% BSA in TBST buffer).
3.2 magnetic particle coupling monoclonal antibody 2: 1) 1mg of the carboxyl magnetic particles (purchased from thermo) were washed 3 times with 0.1M MES buffer and resuspended in 0.5ml MES (pH 6.0) buffer. 2) Add 10. Mu.g of monoclonal antibody 2, mix well and incubate at room temperature for 20min. 3) Add 10. Mu.l EDC (10 mg/ml) and mix well and incubate for 2h at room temperature. 4) After centrifugation, the supernatant was discarded, and 500. Mu.l of a washing buffer (TBST) was added thereto, followed by 3 times of washing. 5) Blocking was performed with TBST buffer containing 1% BSA and blocking was performed at room temperature for 2h. 6) After washing three times with TBST, it was resuspended in 0.5ml of 1% BSA-containing TBST buffer and stored at 4 ℃ until use.
3.3 acridinium ester-labeled monoclonal antibody 4: 1) Mu.g of monoclonal antibody 4 was mixed with 190. Mu.l of acridinium ester solution (6.5 mM) under exclusion of light, reacted at room temperature for 1 hour, and 0.1ml of lysine (10 g/L) was added to continue the reaction for 10 minutes. 2) The labeled solution was injected into a Sephadex G-25 desalting column. Washing was performed with PBS. 3) Protein concentration was measured at 280 nm. When the chromatograph showed a distinct protein peak, the effluent was collected.
4) The effluent (about 0.5 ml) was stored at-20 ℃ until use.
3.4 kit detection
Preparation of a detection kit: diluting the magnetic particles of the 3.2 coupled monoclonal antibody 2 to 0.25mg/ml, and marking as a reagent R1; diluting 3.3-labeled acridinium ester monoclonal antibody 4 500-fold (monoclonal antibody 4 concentration about 40 ng/ml), and labeling as reagent R2; the dilutions were 0.1% BSA TBS pH7.4.
A detection step: 1) 10 μ l sample +50 μ l reagent R1+50 μ l reagent R2+90 μ l wash buffer; 2) Incubation with mixing at 37 ℃ for 15min (1000 rpm at 37 ℃); 3) washing buffer for 3 times, 500 mul each time; 4) 200 μ l of substrate (purchased from Yapei) was added and the RLU value was immediately detected by chemiluminescence. Wherein the wash buffer is TBST buffer solution, and the sample can be serum, whole blood, urine, interstitial fluid and the like.
3.5 kit Linear Range: standard prepared according to the test procedure of 3.4 for 3.1 using the prepared kitAnd (6) detecting. The results show (FIG. 3) that the kit has good linearity in the concentration range of 2.5pg/ml to 3000pg/ml, R 2 ＞0.995。
3.6 kit sensitivity: and (4) carrying out 20 times of repeated tests on the zero standard solution, and taking the average value of the zero calibration solution measurement plus 3 times of standard deviation to obtain the detection limit of the kit. Through detection and calculation, the detection limit of the kit is 0.5pg/ml.
3.7 kit specificity: the prepared kit is used for detecting IL-12A, IL-3, IL-14, IFN-gamma, TNF-beta, GM-CSF (all purchased in the market) and the like, and the detection results show that the RLU values for detecting IL-12A, IL-3 and the like are all about zero value (less than 10000), and the detection results are all less than 0.5pg/ml, which indicates that the kit has good specificity.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> Guangzhou Shuntai biomedical science and technology Co., ltd
<120> kit for detecting protein expression level, preparation method and application thereof
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 306
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr
1 5 10 15
Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu
20 25 30
Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly
35 40 45
Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala Gly
50 55 60
Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu
65 70 75 80
Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys
85 90 95
Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys
100 105 110
Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr
115 120 125
Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln
130 135 140
Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly
145 150 155 160
Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala
165 170 175
Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala
180 185 190
Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg
195 200 205
Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu
210 215 220
Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp
225 230 235 240
Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln
245 250 255
Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr
260 265 270
Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala
275 280 285
Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro
290 295 300
Cys Ser
305
<210> 2
<211> 360
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gaatggcaat tgcaagagtc tcggtctggg gcagaactgg taagggcagg tatgagcgtg 60
aagatgtctt gcaaggcttc tcgctacacc ttcactagct atggaattaa ttgggtgaag 120
cagcggccag gcgctctgtt ggagtttatc gggtatatca atccacacct tggttataca 180
ctgtataatg agaaattcaa aggcaagatg accctgactg tcgacaaatc ctcttccacc 240
gcttatatgc agctgaattc actgacctcc gaagacagcg ctgtgtatga ctgcgcaaga 300
gagggggccg gatcatacta ttacgactat tggtgcgatg gaaccacact gacggtttca 360
<210> 3
<211> 300
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gacattgtca tgacacagag catgcagaag ttcatgtcta ccacagtctc aggctatcgc 60
gttcttataa cctgcaaggc aagcacccag aacgtgggta ccgcctgtgc ctggtaccag 120
gagagacaga aaccgggaca gtctcctgaa aagctgctga tctacagcgc atctaaccga 180
tactccgaag gggtgaagga taggttcacc ggctccaact ctttcggcac agacttcacc 240
gtcactatct ctgagatgca ggagtggttg gctgattatt tctgccagca atgccactct 300
<210> 4
<211> 357
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
caggtacagc ttaagtctga ctttagagga cccgggctcg ttgctccaag tcagagtttg 60
tccattacat gtacagtctc aggctttagc ctgtctagat acagcgtgca ctgggtgagg 120
cagcccccag gcaagggtct tgaatggctg gggatgatct ggggtggcgg gaataccgac 180
tacaacagcg ctcttaagtc ccggctgtcc atctctaaag ataacagcaa gtctcaggtg 240
ttcctgaaaa tgaacagtct ccagaccgat gacactgcca tgtactactg tgccagggac 300
ggctactacg attacgctat ggattactgg ggccagggaa catcagtcac tgtttca 357
<210> 5
<211> 342
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gacattctta tgacacagat ctcaccactt agcaagtctt ggtcactcgg catggatcag 60
gcctccatca gctgcagatc tagtcaatct atagtgcaca gcaacggtaa tactcgccag 120
gagtggtacc tgcaggtccc aggtcagtcc ccgaagcact tgatgtataa agtaagcaac 180
cgattctccg gtgtgccttt ccggttcagc ggccagggta gcggaactga tttcaccctg 240
aagatctcta gggtggaggc cgagcggctg ggcatctact attgctttca ggggagccac 300
tgcccgtata cctttggtgg cggcaccaag ctggagctga ag 342
Claims (6)
1. A kit for detecting protein expression level, the protein is human IL-12B protein, characterized in that, the kit is composed of effective dose of human IL-12B protein and two strains of effective dose of human IL-12B protein monoclonal antibodies, the two strains of human IL-12B protein monoclonal antibodies are monoclonal antibody 2 and monoclonal antibody 4, the heavy chain variable region and light chain variable region sequences of the monoclonal antibody 2 are shown as SEQ ID NO.2 and SEQ ID NO.3, and the heavy chain variable region and light chain variable region sequences of the monoclonal antibody 4 are shown as SEQ ID NO.4 and SEQ ID NO. 5.
2. The kit of claim 1, wherein the effective amount of the human IL-12B protein is a standard having a gradient of 3000pg/ml, 500pg/ml, 100pg/ml, 25pg/ml, 5pg/ml, 2.5pg/ml, 0pg/ml.
3. The kit of claim 1, wherein the epitope sequence of monoclonal antibody 2 is LLLHKKEDGIWSTDILKDQKEPKNK.
4. The kit of claim 1, wherein the epitope sequence of monoclonal antibody 4 is hklkyenytssffirdhikpdpkn.
5. The kit of claim 1, wherein said monoclonal antibody 2 is conjugated to magnetic microparticles, and the concentration of said magnetic microparticles conjugated with monoclonal antibody 2 is 0.25mg/ml.
6. The kit according to claim 1, wherein said acridinium ester is labeled by said monoclonal antibody 4, and the concentration of said acridinium ester-labeled monoclonal antibody 4 is 40ng/ml.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102741285A (en) * | 2009-10-26 | 2012-10-17 | 安姆根有限公司 | Human IL-23 antigen binding proteins |
| WO2017201350A1 (en) * | 2016-05-18 | 2017-11-23 | Modernatx, Inc. | Polynucleotides encoding interleukin-12 (il12) and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102741285A (en) * | 2009-10-26 | 2012-10-17 | 安姆根有限公司 | Human IL-23 antigen binding proteins |
| WO2017201350A1 (en) * | 2016-05-18 | 2017-11-23 | Modernatx, Inc. | Polynucleotides encoding interleukin-12 (il12) and uses thereof |
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