CN115710314B - Antibody combination for detecting tumor necrosis factor-α and its application - Google Patents
Antibody combination for detecting tumor necrosis factor-α and its applicationInfo
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Abstract
The invention provides an antibody combination for detecting tumor necrosis factor-alpha, which comprises a first antibody and/or a second antibody, wherein the antibodies have unique CDR fragments, and also provides specific application of the antibody combination. The invention has the advantages that the antibody combination has strong affinity to the tumor necrosis factor-alpha antigen, high detection sensitivity, simple and convenient use and rapid and stable detection, the content of serum of normal people is 9.42+/-2.91 ng/mL, the conventional low-affinity antibody is difficult to realize effective detection under the condition, and the high-affinity antibody combination and the tumor necrosis factor-alpha quantitative detection kit containing the antibody combination adopting the double-antibody sandwich method can effectively realize the quantitative detection of the ultralow-concentration tumor necrosis factor-alpha, and the sensitivity of the kit can reach 2.2pg/mL through verification.
Description
Technical Field
The invention relates to the technical field of in-vitro diagnosis, in particular to an antibody combination for detecting tumor necrosis factor-alpha and application thereof.
Technical Field
Tumor necrosis factor (tumor necrosis factor, TNF) is a small molecule protein secreted by macrophages, TNF- α is mainly secreted by mononuclear-macrophages, an important inflammatory factor that can up-regulate other inflammatory factors, such as the presence of TNF- α in systemic lupus erythematosus patients with significant increases in the levels of TNF- α in the patient's serum associated with disease activity, TNF- β is mainly secreted by activated T lymphocytes. TNF stimulates IL-1 production in vivo and in vitro, is thermolabile, and is inactivated at 70℃for 30 min.
The biological effects of TNF- α and TNF- β are very similar and mainly include the following:
1) Killing or inhibiting tumor cells, namely, killing some tumor cells in vivo and in vitro by TNF, or inhibiting proliferation, wherein the tumor cell lines have great difference on sensitivity of TNF-alpha, and TNF-alpha has stimulation effect on few tumor cells;
2) Improving phagocytic capacity of neutrophils by increasing production of peroxide anions, enhancing ADCC function, stimulating degranulation of cells and secreting myeloperoxidase;
3) Anti-infective by inhibiting plasmodium growth, inhibiting viral replication (such as adenovirus type II, cytidine type II), inhibiting viral protein synthesis, production of viral particles and infectivity, and killing virus infected cells;
4) Endogenous pyrogen, which causes fever and induces the synthesis of acute phase proteins of hepatocytes;
5) Promoting differentiation of myeloid leukemia cells into macrophages, namely promoting differentiation of myeloid leukemia cells ML-1, monocytic leukemia cells U937 and promyelocytic leukemia cells HL 60;
6) Promoting cell proliferation and differentiation, TNF promotes MHCI antigen expression of T cells, enhances IL-2 dependent thymus cell and T cell proliferation capacity, promotes lymphokine differentiation of IL-2, CSF, IFN-gamma and the like, and enhances proliferation and Ig secretion of mitogen or foreign antigen stimulated B cells.
However, the content of serum of normal people is 9.42+/-2.91 ng/mL, so that the existing biological products aiming at TNF-alpha are mostly low-affinity antibodies, and the detection accuracy and sensitivity of the biological products aiming at TNF-alpha are low.
Disclosure of Invention
Aiming at the limitation of the prior art, the invention provides an antibody combination for detecting tumor necrosis factor-alpha and application thereof, and overcomes the defects in the prior art.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
The invention provides an antibody combination for detecting tumor necrosis factor-alpha (TNF-alpha), which comprises a first antibody and/or a second antibody, wherein the amino acid sequences of CDR1, CDR2 and CDR3 regions of a heavy chain variable region of the first antibody are respectively shown as 26-35, 55-63 and 105-114 amino acid sequences of SEQ ID No.1, the amino acid sequences of CDR1, CDR2 and CDR3 regions of a light chain variable region are respectively shown as 24-32, 48-55 and 88-98 amino acid sequences of SEQ ID No.2, the amino acid sequences of CDR1, CDR2 and CDR3 regions of a heavy chain variable region of the second antibody are respectively shown as 26-34, 54-63 and 105-117 amino acid sequences of SEQ ID No.3, and the amino acid sequences of CDR1, CDR2 and CDR3 regions of the light chain variable region are respectively shown as 24-32, 48-56 and 89-97 amino acid sequences of SEQ ID No. 4.
The first antibody was 18C5 and the second antibody was 3D5.
CDR1 of the first antibody heavy chain variable region is GMNDSLTSRN;
CDR2 of the first antibody heavy chain variable region is KSAPEDTTF;
CDR3 of the first antibody heavy chain variable region is AKRTSMFPTD;
CDR1 of the first antibody light chain variable region is VPNKDFGAD;
CDR2 of the first antibody light chain variable region is APNFNFQQ;
CDR3 of the first antibody light chain variable region is MGTRLIPPSTV;
CDR1 of the second antibody heavy chain variable region is GNFTGFFRP;
CDR2 of the second antibody heavy chain variable region is TVVAFNFDTK;
CDR3 of the second antibody heavy chain variable region is VSSLYPFGKLYQT;
CDR1 of the second antibody light chain variable region is RTHVILTHA;
CDR2 of the second antibody light chain variable region is LSAIRKPCQ;
CDR3 of the light chain variable region of the second antibody is QRYNRAPYT.
In a preferred embodiment, the combination of antibodies for detecting tumor necrosis factor-alpha is characterized in that the amino acid sequence of the heavy chain variable region of the first antibody is shown as SEQ ID No.1 or the amino acid sequence with the homology of more than 90% with the sequence of SEQ ID No.1, the amino acid sequence of the light chain variable region is shown as SEQ ID No.2 or the amino acid sequence with the homology of more than 90% with the sequence of SEQ ID No.2, the amino acid sequence of the heavy chain variable region of the second antibody is shown as SEQ ID No.3 or the amino acid sequence with the homology of more than 90% with the sequence of SEQ ID No.3, and the amino acid sequence of the light chain variable region is shown as SEQ ID No.4 or the amino acid sequence with the homology of more than 90% with the sequence of SEQ ID No. 4.
The first antibody heavy chain variable region amino acid sequence SEQ ID No.1 is:
EVQLVESGGGLVQPGGSLRLSCAVSGMNDSLTSRNSVNWIRQAPGKGLEWVGLIKSAPEDTTFTDYNSAIKSRFTISRDTSKSTVYLQMNSLRAEDTAVYYCARAKRTSMFPTDWGQGTLVTVSS;
The amino acid sequence of the first antibody light chain variable region SEQ ID No.2 is:
DIQMTQSPSSLSASVGDRVTITCVPNKDFGADWYQQKPGKAPKLLIYAPNFNFQQGVPSRFSGSGSATDYTLTISSLQPEDFATYYCMGTRLIPPSTVFGQGTKVEVKRTVA;
The second antibody heavy chain variable region amino acid sequence SEQ ID No.3 is:
EVQLVESGGGLVQPGRSLRLSCAASGNFTGFFRPAMHWVRQAPGKGLEWVSAITVVAFNFDTKIDYADSVEGRFTISRDNAKNSLYLDMNSLRAEDTAVYYCAKVSSLYPFGKLYQTWGQGTLVTVSS;
The second antibody light chain variable region amino acid sequence SEQ ID No.4 is:
DIQMTQSPSSLSASVGDRVTITCRTHVILTHAWYQQKPGKAPKLLIYLSAIRKPCQGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVA.
In a preferred embodiment, the combination of antibodies for detecting tumor necrosis factor-alpha is characterized in that the amino acid sequence of the heavy chain constant region of the first antibody is shown as SEQ ID No.5 or the amino acid sequence with the homology of more than 90% with the sequence of SEQ ID No.5, the amino acid sequence of the light chain constant region is shown as SEQ ID No.6 or the amino acid sequence with the homology of more than 90% with the sequence of SEQ ID No.6, the amino acid sequence of the heavy chain constant region of the second antibody is shown as SEQ ID No.7 or the amino acid sequence with the homology of more than 90% with the sequence of SEQ ID No.7, and the amino acid sequence of the light chain constant region is shown as SEQ ID No.8 or the amino acid sequence with the sequence of more than 90% with the sequence of SEQ ID No. 8.
The first antibody heavy chain constant region amino acid sequence SEQ ID No.5 is:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;
the amino acid sequence SEQ ID No.6 of the light chain constant region of the first antibody is:
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS;
The second antibody heavy chain constant region amino acid sequence SEQ ID No.7 is:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;
the amino acid sequence of the light chain constant region of the second antibody SEQ ID No.8 is:
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS.
The first antibody and the second antibody are independently coated antibodies or detection antibodies.
Further, the antibody combination for detecting tumor necrosis factor-alpha is selected from any one of the following combination forms, namely, a first antibody is used as a detection antibody, a second antibody is used as a detection antibody, a first antibody is used as a coating antibody and a second antibody is used as a detection antibody, a second antibody is used as a coating antibody and a first antibody is used as a detection antibody, and preferably, the first antibody is used as a coating antibody and the second antibody is used as a detection antibody.
That is, the first antibody and the second antibody can be used as the detection antibodies of tumor necrosis factor-alpha separately, and the conventional monoclonal antibody detection method can be used for detecting tumor necrosis factor-alpha by using the first antibody or the second antibody, and meanwhile, the first antibody can be used as a coated antibody and the second antibody can be used as detection antibodies, and the detection of tumor necrosis factor-alpha can be performed by using a double-antibody sandwich method, or the second antibody can be used as a coated antibody and the first antibody can be used as detection antibodies, and the detection of tumor necrosis factor-alpha can be performed by using a double-antibody sandwich method.
The second invention provides a polynucleotide for coding the heavy chain and the light chain of the antibody combination for detecting the tumor necrosis factor-alpha, wherein the polynucleotide sequence for coding the heavy chain variable region of the first antibody is shown as SEQ ID No.9 or a nucleotide sequence with the sequence homology of more than 90 percent with the SEQ ID No.9, the polynucleotide sequence for coding the light chain variable region of the first antibody is shown as SEQ ID No.10 or a nucleotide sequence with the sequence homology of more than 90 percent with the SEQ ID No.10, the polynucleotide sequence for coding the heavy chain variable region of the second antibody is shown as SEQ ID No.11 or a nucleotide sequence with the sequence homology of more than 90 percent with the sequence of SEQ ID No.11, and the polynucleotide sequence for coding the light chain variable region of the second antibody is shown as SEQ ID No.12 or a nucleotide sequence with the sequence homology of more than 90 percent with the sequence of SEQ ID No. 12.
The polynucleotide sequence SEQ ID No.9 encoding the heavy chain variable region of the first antibody is:
gaagtgcagctggtggaaagcggcggcggcctggtgcagccgggcggcagcctgcgcctgagctgcgcggtgagcggcatgaacgatagcctgaccagccgcaacagcgtgaactggattcgccaggcgccgggcaaaggcctggaatgggtgggcctgattaaaagcgcgccggaagataccacctttaccgattataacagcgcgattaaaagccgctttaccattagccgcgataccagcaaaagcaccgtgtatctgcagatgaacagcctgcgcgcggaagataccgcggtgtattattgcgcgcgcgcgaaacgcaccagcatgtttccgaccgattggggccagggcaccctggtgaccgtgagcagc;
The polynucleotide sequence SEQ ID No.10 encoding the light chain variable region of the first antibody is:
gatattcagatgacccagagcccgagcagcctgagcgcgagcgtgggcgatcgcgtgaccattacctgcgtgccgaacaaagattttggcgcggattggtatcagcagaaaccgggcaaagcgccgaaactgctgatttatgcgccgaactttaactttcagcagggcgtgccgagccgctttagcggcagcggcagcgcgaccgattataccctgaccattagcagcctgcagccggaagattttgcgacctattattgcatgggcacccgcctgattccgccgagcaccgtgtttggccagggcaccaaagtggaagtgaaacgcaccgtggcg;
The polynucleotide sequence SEQ ID No.11 encoding the heavy chain variable region of the second antibody is:
gaagtgcagctggtggaaagcggcggcggcctggtgcagccgggccgcagcctgcgcctgagctgcgcggcgagcggcaactttaccggcttttttcgcccggcgatgcattgggtgcgccaggcgccgggcaaaggcctggaatgggtgagcgcgattaccgtggtggcgtttaactttgataccaaaattgattatgcggatagcgtggaaggccgctttaccattagccgcgataacgcgaaaaacagcctgtatctggatatgaacagcctgcgcgcggaagataccgcggtgtattattgcgcgaaagtgagcagcctgtatccgtttggcaaactgtatcagacctggggccagggcaccctggtgaccgtgagcagc;
the polynucleotide sequence SEQ ID No.12 encoding the light chain variable region of the second antibody is:
gatattcagatgacccagagcccgagcagcctgagcgcgagcgtgggcgatcgcgtgaccattacctgccgcacccatgtgattctgacccatgcgtggtatcagcagaaaccgggcaaagcgccgaaactgctgatttatctgagcgcgattcgcaaaccgtgccagggcgtgccgagccgctttagcggcagcggcagcggcaccgattttaccctgaccattagcagcctgcagccggaagatgtggcgacctattattgccagcgctataaccgcgcgccgtatacctttggccagggcaccaaagtggaaattaaacgcaccgtggcg.
In a preferred embodiment, the polynucleotide encoding the heavy chain constant region of the first antibody has a nucleotide sequence shown in SEQ ID No.13 or having a nucleotide sequence having a homology of greater than 90% with the sequence shown in SEQ ID No.13, the polynucleotide encoding the light chain constant region of the first antibody has a nucleotide sequence shown in SEQ ID No.14 or having a nucleotide sequence having a homology of greater than 90% with the sequence shown in SEQ ID No.14, and the polynucleotide encoding the heavy chain constant region of the second antibody has a nucleotide sequence shown in SEQ ID No.15 or having a nucleotide sequence having a homology of greater than 90% with the sequence shown in SEQ ID No.15, and the polynucleotide encoding the light chain constant region of the second antibody has a nucleotide sequence shown in SEQ ID No.16 or having a nucleotide sequence having a homology of greater than 90% with the sequence shown in SEQ ID No. 16.
The polynucleotide sequence SEQ ID No.13 encoding the heavy chain constant region of the first antibody is:
gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa;
the polynucleotide sequence SEQ ID No.14 encoding the light chain constant region of the first antibody is:
ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaagcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtggaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc;
the polynucleotide sequence SEQ ID No.15 encoding the heavy chain constant region of the second antibody is:
gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa;
The polynucleotide sequence SEQ ID No.16 encoding the light chain constant region of the second antibody is:
ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaagcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtggaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc.
the third aspect of the present invention provides an expression system of the polynucleotide for detecting heavy and light chains of an antibody combination for tumor necrosis factor- α, wherein the expression system is a mammalian expression vector.
In a preferred embodiment, the expression system described above, the antigen expression system for tumor necrosis factor- α is the Expi 293 expression system.
In a preferred embodiment, the expression system described above, the antibody expression system is a CHO expression system.
A fourth aspect of the invention provides a host cell comprising the expression system described above.
The fifth invention provides the application of the antibody combination in preparing biological products for quantitatively detecting the tumor necrosis factor-alpha content in vitro.
In a preferred embodiment, the use as described above, the in vitro quantitative assay is for detecting the amount of tumor necrosis factor-alpha in serum, plasma, whole blood and/or peripheral blood.
In a preferred embodiment, the above-described use, the biologic is a kit.
In a preferred embodiment, the use as described above, the kit is a double antibody sandwich assay kit.
Compared with the prior art, the invention has the following advantages:
The antibody combination for detecting the tumor necrosis factor-alpha and the application thereof provided by the invention have the advantages that the affinity of the antibody combination for the tumor necrosis factor-alpha antigen is strong, the detection sensitivity is high, the use is simple and convenient, the detection is rapid and stable, the content of serum of normal people is 9.42+/-2.91 ng/mL, the effective detection is difficult to realize by the conventional low-affinity antibody against the condition, and the high-affinity antibody combination and the tumor necrosis factor-alpha quantitative detection kit containing the antibody combination and adopting the double-antibody sandwich method can effectively realize the quantitative detection of the tumor necrosis factor-alpha with ultralow concentration, and the sensitivity of the kit can reach 2.2pg/mL through verification.
The double antibody sandwich method adopted by the invention is used for measuring the TNF-alpha level in the sample, namely, the sandwich compound structure of the coated antibody-antigen-detection antibody is formed by the antibody coated on the ELISA plate, the detection antibody and the TNF-alpha of the detected sample, thereby being more beneficial to the detection of the TNF-alpha. In addition, the invention effectively adopts the avidin-biotin-enzyme complex to realize high sensitivity of detection.
The detection kit of the invention not only can rapidly and accurately measure the content of TNF-alpha in serum and provide reliable clinical reference value for early diagnosis and early treatment of inflammation, but also can greatly improve the detection sensitivity of the TNF-alpha due to the combination of two specific monoclonal antibodies.
Drawings
FIG. 1 is a standard curve of ELISA kit containing antibody combinations for detecting tumor necrosis factor-alpha according to an embodiment of the present invention.
Detailed Description
The present invention will be described in further detail below in order to make the objects, technical solutions and advantages of the present invention more apparent. It is to be understood that the description is only intended to illustrate the invention and is not intended to limit the scope of the invention.
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, and the terms used herein in this description of the invention are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention. Reagents and instruments used herein are commercially available, and reference to characterization means is made to the relevant description of the prior art and will not be repeated herein.
For a further understanding of the present invention, the present invention will be described in further detail with reference to the following preferred embodiments.
Example 1
The antibody combination for detecting tumor necrosis factor-alpha (TNF-alpha) comprises a first antibody and/or a second antibody, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of a heavy chain variable region of the first antibody are respectively shown as amino acid sequences of 26-35, 55-63 and 105-114 of SEQ ID No.1, the amino acid sequences of CDR1, CDR2 and CDR3 of a light chain variable region are respectively shown as amino acid sequences of 24-32, 48-55 and 88-98 of SEQ ID No.2, and the amino acid sequences of CDR1, CDR2 and CDR3 of a heavy chain variable region of the second antibody are respectively shown as amino acid sequences of 26-34, 54-63 and 105-117 of SEQ ID No.3, and the amino acid sequences of CDR1, CDR2 and CDR3 of a light chain variable region are respectively shown as amino acid sequences of 24-32, 48-56 and 89-97 of SEQ ID No. 4.
The first antibody was 18C5 and the second antibody was 3D5.
CDR1 of the first antibody heavy chain variable region is GMNDSLTSRN;
CDR2 of the first antibody heavy chain variable region is KSAPEDTTF;
CDR3 of the first antibody heavy chain variable region is AKRTSMFPTD;
CDR1 of the first antibody light chain variable region is VPNKDFGAD;
CDR2 of the first antibody light chain variable region is APNFNFQQ;
CDR3 of the first antibody light chain variable region is MGTRLIPPSTV;
CDR1 of the second antibody heavy chain variable region is GNFTGFFRP;
CDR2 of the second antibody heavy chain variable region is TVVAFNFDTK;
CDR3 of the second antibody heavy chain variable region is VSSLYPFGKLYQT;
CDR1 of the second antibody light chain variable region is RTHVILTHA;
CDR2 of the second antibody light chain variable region is LSAIRKPCQ;
CDR3 of the light chain variable region of the second antibody is QRYNRAPYT.
In the antibody combination, the amino acid sequence of the heavy chain variable region of the first antibody is shown as SEQ ID No.1 or the amino acid sequence with the homology of more than 90 percent with the sequence of SEQ ID No.1, the amino acid sequence of the light chain variable region is shown as SEQ ID No.2 or the amino acid sequence with the homology of more than 90 percent with the sequence of SEQ ID No.2, the amino acid sequence of the heavy chain variable region of the second antibody is shown as SEQ ID No.3 or the amino acid sequence with the homology of more than 90 percent with the sequence of SEQ ID No.3, and the amino acid sequence of the light chain variable region is shown as SEQ ID No.4 or the amino acid sequence with the homology of more than 90 percent with the sequence of SEQ ID No. 4.
The first antibody heavy chain variable region amino acid sequence SEQ ID No.1 is:
EVQLVESGGGLVQPGGSLRLSCAVSGMNDSLTSRNSVNWIRQAPGKGLEWVGLIKSAPEDTTFTDYNSAIKSRFTISRDTSKSTVYLQMNSLRAEDTAVYYCARAKRTSMFPTDWGQGTLVTVSS;
The amino acid sequence of the first antibody light chain variable region SEQ ID No.2 is:
DIQMTQSPSSLSASVGDRVTITCVPNKDFGADWYQQKPGKAPKLLIYAPNFNFQQGVPSRFSGSGSATDYTLTISSLQPEDFATYYCMGTRLIPPSTVFGQGTKVEVKRTVA;
The second antibody heavy chain variable region amino acid sequence SEQ ID No.3 is:
EVQLVESGGGLVQPGRSLRLSCAASGNFTGFFRPAMHWVRQAPGKGLEWVSAITVVAFNFDTKIDYADSVEGRFTISRDNAKNSLYLDMNSLRAEDTAVYYCAKVSSLYPFGKLYQTWGQGTLVTVSS;
The second antibody light chain variable region amino acid sequence SEQ ID No.4 is:
DIQMTQSPSSLSASVGDRVTITCRTHVILTHAWYQQKPGKAPKLLIYLSAIRKPCQGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVA.
In the antibody combination, the amino acid sequence of the heavy chain constant region of the first antibody is shown as SEQ ID No.5 or the amino acid sequence with the homology of more than 90 percent with the sequence of SEQ ID No.5, the amino acid sequence of the light chain constant region is shown as SEQ ID No.6 or the amino acid sequence with the homology of more than 90 percent with the sequence of SEQ ID No.6, the amino acid sequence of the heavy chain constant region of the second antibody is shown as SEQ ID No.7 or the amino acid sequence with the homology of more than 90 percent with the sequence of SEQ ID No.7, and the amino acid sequence of the light chain constant region is shown as SEQ ID No.8 or the amino acid sequence with the homology of more than 90 percent with the sequence of SEQ ID No. 8.
The first antibody heavy chain constant region amino acid sequence SEQ ID No.5 is:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;
the amino acid sequence SEQ ID No.6 of the light chain constant region of the first antibody is:
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS;
The second antibody heavy chain constant region amino acid sequence SEQ ID No.7 is:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;
the amino acid sequence of the light chain constant region of the second antibody SEQ ID No.8 is:
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS.
the primary antibody and the secondary antibody are independently coated antibodies or detection antibodies.
The antibody combination is selected from any combination form of a first antibody serving as a detection antibody, a second antibody serving as a detection antibody, a first antibody serving as a coating antibody, a second antibody serving as a detection antibody, a second antibody serving as a coating antibody and a first antibody serving as a detection antibody;
preferably, the primary antibody 18C5 is used as a coating antibody and the secondary antibody 3D5 is used as a detection antibody.
That is, the first antibody and the second antibody can be used as the detection antibodies of tumor necrosis factor-alpha separately, and the conventional monoclonal antibody detection method can be used for detecting tumor necrosis factor-alpha by using the first antibody or the second antibody, and meanwhile, the first antibody can be used as a coated antibody and the second antibody can be used as detection antibodies, and the detection of tumor necrosis factor-alpha can be performed by using a double-antibody sandwich method, or the second antibody can be used as a coated antibody and the first antibody can be used as detection antibodies, and the detection of tumor necrosis factor-alpha can be performed by using a double-antibody sandwich method.
The invention also provides a polynucleotide for coding the heavy chain and the light chain of the antibody combination for detecting the tumor necrosis factor-alpha, the polynucleotide sequence for coding the heavy chain variable region of the first antibody is shown as SEQ ID No.9 or a nucleotide sequence with the sequence homology of more than 90 percent with the SEQ ID No.9, the polynucleotide sequence for coding the light chain variable region of the first antibody is shown as SEQ ID No.10 or a nucleotide sequence with the sequence homology of more than 90 percent with the SEQ ID No.10, the polynucleotide sequence for coding the heavy chain variable region of the second antibody is shown as SEQ ID No.11 or a nucleotide sequence with the sequence homology of more than 90 percent with the SEQ ID No.11, and the polynucleotide sequence for coding the light chain variable region of the second antibody is shown as SEQ ID No.12 or a nucleotide sequence with the sequence homology of more than 90 percent with the SEQ ID No. 12.
The polynucleotide sequence SEQ ID No.9 encoding the heavy chain variable region of the first antibody is:
gaagtgcagctggtggaaagcggcggcggcctggtgcagccgggcggcagcctgcgcctgagctgcgcggtgagcggcatgaacgatagcctgaccagccgcaacagcgtgaactggattcgccaggcgccgggcaaaggcctggaatgggtgggcctgattaaaagcgcgccggaagataccacctttaccgattataacagcgcgattaaaagccgctttaccattagccgcgataccagcaaaagcaccgtgtatctgcagatgaacagcctgcgcgcggaagataccgcggtgtattattgcgcgcgcgcgaaacgcaccagcatgtttccgaccgattggggccagggcaccctggtgaccgtgagcagc;
The polynucleotide sequence SEQ ID No.10 encoding the light chain variable region of the first antibody is:
gatattcagatgacccagagcccgagcagcctgagcgcgagcgtgggcgatcgcgtgaccattacctgcgtgccgaacaaagattttggcgcggattggtatcagcagaaaccgggcaaagcgccgaaactgctgatttatgcgccgaactttaactttcagcagggcgtgccgagccgctttagcggcagcggcagcgcgaccgattataccctgaccattagcagcctgcagccggaagattttgcgacctattattgcatgggcacccgcctgattccgccgagcaccgtgtttggccagggcaccaaagtggaagtgaaacgcaccgtggcg;
The polynucleotide sequence SEQ ID No.11 encoding the heavy chain variable region of the second antibody is:
gaagtgcagctggtggaaagcggcggcggcctggtgcagccgggccgcagcctgcgcctgagctgcgcggcgagcggcaactttaccggcttttttcgcccggcgatgcattgggtgcgccaggcgccgggcaaaggcctggaatgggtgagcgcgattaccgtggtggcgtttaactttgataccaaaattgattatgcggatagcgtggaaggccgctttaccattagccgcgataacgcgaaaaacagcctgtatctggatatgaacagcctgcgcgcggaagataccgcggtgtattattgcgcgaaagtgagcagcctgtatccgtttggcaaactgtatcagacctggggccagggcaccctggtgaccgtgagcagc;
the polynucleotide sequence SEQ ID No.12 encoding the light chain variable region of the second antibody is:
gatattcagatgacccagagcccgagcagcctgagcgcgagcgtgggcgatcgcgtgaccattacctgccgcacccatgtgattctgacccatgcgtggtatcagcagaaaccgggcaaagcgccgaaactgctgatttatctgagcgcgattcgcaaaccgtgccagggcgtgccgagccgctttagcggcagcggcagcggcaccgattttaccctgaccattagcagcctgcagccggaagatgtggcgacctattattgccagcgctataaccgcgcgccgtatacctttggccagggcaccaaagtggaaattaaacgcaccgtggcg.
The polynucleotide for detecting the heavy chain and the light chain of the tumor necrosis factor alpha antibody combination is characterized in that the polynucleotide sequence of the heavy chain constant region of the first antibody is shown as SEQ ID No.13 or the nucleotide sequence with the sequence homology of more than 90 percent with SEQ ID No.13, the polynucleotide sequence of the light chain constant region of the first antibody is shown as SEQ ID No.14 or the nucleotide sequence with the sequence homology of more than 90 percent with SEQ ID No.14, the polynucleotide sequence of the heavy chain constant region of the second antibody is shown as SEQ ID No.15 or the nucleotide sequence with the sequence homology of more than 90 percent with SEQ ID No.15, and the polynucleotide sequence of the light chain constant region of the second antibody is shown as SEQ ID No.16 or the nucleotide sequence with the sequence homology of more than 90 percent with SEQ ID No. 16.
The polynucleotide sequence SEQ ID No.13 encoding the heavy chain constant region of the first antibody is:
gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa;
the polynucleotide sequence SEQ ID No.14 encoding the light chain constant region of the first antibody is:
ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaagcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtggaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc;
the polynucleotide sequence SEQ ID No.15 encoding the heavy chain constant region of the second antibody is:
gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa;
The polynucleotide sequence SEQ ID No.16 encoding the light chain constant region of the second antibody is:
ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaagcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtggaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc.
An expression system for detecting polynucleotides of heavy and light chains of an antibody combination of tumor necrosis factor-alpha, the expression system being a mammalian expression vector.
The antigen expression system for tumor necrosis factor-alpha (TNF-alpha) is the Expi 293 expression system.
The antibody expression system is a CHO expression system.
Host cells comprising the expression system described above.
Use of an antibody combination for detecting tumor necrosis factor-alpha in the preparation of a biological product for in vitro quantitative detection of the content of tumor necrosis factor-alpha.
In vitro quantitative detection refers to the detection of the amount of tumor necrosis factor-alpha in serum, plasma, whole blood and/or peripheral blood.
The biological product is a kit.
The kit is a double-antibody sandwich method detection kit.
Example 2
The first antibody and the second antibody are both obtained by screening by a phage display method, purified proteins are marked by NHS-Biotin reagent, tumor necrosis factor-alpha (TNF-alpha) antigen is expressed by an Expi293 expression system, and the expression sequence is shown as SEQ ID No.17 or an amino acid sequence with the sequence homology of more than 90% with the SEQ ID No. 17.
SEQ ID No.17 sequence:
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL*.
the nucleotide sequence of the tumor necrosis factor-alpha (TNF-alpha) antigen is shown as SEQ ID No.18 or the nucleotide sequence with the homology of more than 90 percent with the sequence of SEQ ID No. 18.
SEQ ID No.18 sequence:
gtgcgcagcagcagccgcaccccgagcgataaaccggtggcgcatgtggtggcgaacccgcaggcggaaggccagctgcagtggctgaaccgccgcgcgaacgcgctgctggcgaacggcgtggaactgcgcgataaccagctggtggtgccgagcgaaggcctgtatctgatttatagccaggtgctgtttaaaggccagggctgcccgagcacccatgtgctgctgacccataccattagccgcattgcggtgagctatcagaccaaagtgaacctgctgagcgcgattaaaagcccgtgccagcgcgaaaccccggaaggcgcggaagcgaaaccgtggtatgaaccgatttatctgggcggcgtgtttcagctggaaaaaggcgatcgcctgagcgcggaaattaaccgcccggattatctggattttgcggaaagcggccaggtgtattttggcattattgcgctgtaa.
example 3
The specific compositions and specific concentrations of the respective compositions of the kits containing 2 antibodies are shown in table 1.
TABLE 1
The reagents are as follows:
(1) ELISA assay coating buffer (pH 9.6.05M carbonate buffer):
NaHCO 3 1.59.59 g,
NaHCO 3 2.93.93 g,
Distilled water is added to 1000ml;
(2) ELISA assay washing buffer (pH 7.4 PBS) 0.15M
KH 2PO4 0.2.2 g of the total weight of the composition,
Na 2HPO4·12H2 O2.9 g,
8.0 G of NaCl, and the concentration of the sodium chloride in the solution is higher than that of the sodium chloride,
0.2 G of KCl, which is prepared from the raw materials of the formula,
Tween-20 0.05%0.5ml,
Distilled water is added to 1000ml;
(3) ELISA sample dilution:
bovine Serum Albumin (BSA) 0.1 g,
Adding a washing buffer solution to 100ml;
(4) ELISA assay stop solution (2M H 2SO4):
178.3ml of distilled water and 21.7ml of concentrated sulfuric acid (98%) are added dropwise;
(5) ELISA substrate buffer (pH 5.0 disodium phosphate citrate):
0.2M Na 2HPO4 (28.4 g/L) 25.7ml,
0.1M citric acid (19.2 g/L) 24.3ml,
Adding 50ml of distilled water;
(6) ELISA experiment TMB (tetramethylbenzidine) use solution:
TMB (10 mg/5ml absolute ethanol) 0.5ml,
10Ml of substrate buffer (pH 5.5),
0.75%H2O2 32μl;
(7) ELISA test blocking solution:
Bovine Serum Albumin (BSA) 5g,
Wash buffer was added to 100ml.
Example 4
1. The specific procedures for using the kit containing 2 antibodies for tumor necrosis factor-alpha detection are shown below.
The steps are as follows:
1) Antibody pre-coating, namely diluting an 18C5 antibody with a coating buffer solution to a concentration of 1 mug/mL, adding 100 mu L of the antibody into an ELISA plate per well, and standing at 4 ℃ overnight;
2) Sealing, namely adding 100 mu L of sealing liquid into each hole, sealing for 0.5-1 hour at 37 ℃, washing the plate 3 times by using washing buffer solution, and beating to dry each time for 30 seconds;
3) The dilution and sample adding of the standard substance, namely, a standard substance hole and a blank control hole are arranged on an enzyme-labeled coating plate, hTNF-alpha antigen is subjected to gradient dilution by using sample diluent, each concentration is 200 mu L, the concentrations are respectively 500pg/mL, 125pg/mL, 62.5pg/mL, 31.25pg/mL, 15.6pg/mL, 7.8pg/mL and 3.9pg/mL, each concentration is two complex holes, the sample adding amount of each hole is 100 mu L, and each blank hole is added with 100 mu L of sample diluent;
4) Diluting and sample adding of the sample to be tested, namely adding 240 mu L of sample diluent and 60 mu L of sample to be tested (the final dilution of the sample is 5 times) into the sample, adding 100 mu L of sample into the bottom of an enzyme-labeled plate hole in each hole;
5) Incubation, namely, placing the membrane close plate at 37 ℃ for incubation for 30 minutes, carefully removing the membrane close plate, discarding liquid, spin-drying, washing the plate 3 times with washing buffer for 30 seconds each time, and beating to dry;
6) The biotin-labeled antibody is incubated, namely, fresh diluted biotin-labeled antibody is added into each reaction well, each well is 100 mu L,37 ℃ and 0.5-1h, the plate is washed 3 times by a washing buffer solution for 30 seconds each time, and the plate is patted dry;
7) Color development, namely adding 50 mu l of TMB color developing agent into each hole, gently shaking and uniformly mixing, and developing for 15 minutes at 37 ℃ in a dark place;
8) Stop reaction (blue changes vertically to yellow) by adding 50 μl of stop solution to each well;
9) The absorbance (OD value) of each well was measured sequentially at a wavelength of 450nm with the blank air-conditioner at zero, and the measurement was performed within 15 minutes after the addition of the stop solution.
2. Sample requirements:
Serum is collected by a sterile tube, the blood is naturally coagulated at room temperature for 10-20 minutes, and centrifuged at 2-8 ℃ for about 20 minutes (2000-3000 rpm), and the supernatant is carefully collected and centrifuged again if precipitation occurs during storage.
The determination criteria of the results of the kit containing the antibody combination for in vitro quantitative determination of the content of tumor necrosis factor-alpha (TNF-alpha) in human serum, plasma, whole blood, peripheral blood are shown in Table 2 below.
TABLE 2
| TNF-α(ng/ml) | Clinical application advice |
| <0.006 | Reference value for 95% locus in apparent healthy population |
| 0.006-0.120 | Normal, or in the presence of minor inflammation, minor infection |
| 0.120-0.200 | Suggesting a general bacterial infection or systemic inflammatory response |
| >0.200 | The indication may be sepsis |
The standard curve of the kit of the invention is shown in FIG. 1.
Example 5
A sample was tested using a commercially available TNF- α assay kit as comparative product 1, separately from the kit provided in example 4 of the present invention.
The commercial TNF-alpha detection kit is:
Comparative product 1:
The product name is Human TNF-alpha ELISA Kit (Human tumor necrosis factor-alpha ELISA Kit);
Product number PT518;
the specification of the product is 96T;
purchased from Shanghai Biyun biotechnology Co.
In the detection process, the basic characteristics of the kit are compared, and the basic characteristics are shown in the following table 3.
TABLE 3 Table 3
| Example 4 | Comparative product 1 | |
| Antigens | Human TNF-alpha | Human TNF-alpha |
| Coated antibodies | 18C5 | Human TNF-alpha antibodies |
| Detection antibodies | 3D5 | Human TNF-alpha biotinylated antibodies |
| Method of | ELISA | ELISA |
| Sensitivity of | 2.2pg/mL | 4.3pg/mL |
| Detection range | 10pg/mL-1000pg/mL | 50pg/mL-200pg/mL |
As can be seen from Table 3, the basic characteristics of the respective kits are different, the samples aimed at in example 4 and comparative product 1 are tumor necrosis factor-alpha (TNF-alpha) in human serum, but the coated antibody and the detection antibody of example 4 are completely different from those of comparative product 1, and on the premise that the detection sensitivity of the kit provided in example 4 is greatly higher than that of comparative product 1, the detection range is also wider. The kit of example 4 has in fact reached the highest sensitivity that can be achieved with the same class of products.
Further, the detection method of TNF-alpha was carried out by collecting 10 samples of serum from patients suffering from inflammation or infection and normal persons, and carrying out TNF-alpha detection by using the kit of example 4 of the present patent and a commercially available TNF-alpha detection kit (comparative product 1), respectively, as shown in Table 4, wherein the H group is healthy body serum and the P group is inflammation or infection body serum.
TABLE 4 Table 4
By combining the criteria in Table 2, it was determined that if the serum TNF-. Alpha.concentration was >6pg/mL, which indicates that inflammation or other infection may be present, a comparison of the test data in Table 4 indicated above shows that the commercial kit (comparative product 1) had a leak/false detection due to low sensitivity in the tests for H4, H7, P2, P9, and P10, and the leak/false detection rate was 25% which was a higher level than that in the test for H10, whereas the kit of the present invention, which was a false detection due to high leak/false detection rate, indicated that the kit of the present invention contained an antibody combination, had a sensitivity far higher than that of the prior art.
Example 6
The invention provides two antibodies for detecting tumor necrosis factor-alpha in a human serum sample, namely a first antibody 18C5 and a second antibody 3D5, which are combined and detected by adopting a double-antibody sandwich method, but in practice, the two antibodies obtained by screening by a phage display method also have the function of detecting tumor necrosis factor-alpha in the human serum sample when used independently and used in a replacement way, and in order to verify the effect of the antibodies when used independently and used in a replacement way, the applicant adopts the similar method of the examples 1-5 to detect (the monoclonal antibody is detected by adopting a conventional method and the double antibody is detected by adopting a sandwich method), and compares the results, in particular as follows.
The basic feature comparison results of the primary antibody 18C5 alone, the secondary antibody 3D5 alone, the antibody combination 18C5 (coating) +3d5 (assay), and the antibody combination 3D5 (coating) +18c5 (assay) constituting the kit are shown in table 5.
TABLE 5
| 18C5 alone | 3D5 alone | 3D5+18C5 | 18C5+3D5 | |
| Antigens | Human TNF-alpha | Human TNF-alpha | Human TNF-alpha | Human TNF-alpha |
| Coated antibodies | — | — | 3D5 (second antibody) | 18C5 (first antibody) |
| Detection antibodies | 18C5 (first antibody) | 3D5 (second antibody) | 18C5 (first antibody) | 3D5 (second antibody) |
| Method of | ELISA | ELISA | ELISA | ELISA |
| Sensitivity of | 3.2pg/mL | 3.0pg/mL | 2.5pg/mL | 2.2pg/mL |
| Detection range | 50pg/mL-500pg/mL | 50pg/mL-500pg/mL | 20pg/mL-800pg/mL | 10pg/mL-1000pg/mL |
As can be seen from table 5, the primary antibody 18C5 alone and the secondary antibody 3D5 alone can be used to effectively detect tumor necrosis factor- α in human serum samples, and the detection sensitivity and detection range of the primary antibody and secondary antibody used alone are also superior to those of the existing commercial products (comparative product 1 in table 3), while the effect of the primary antibody and secondary antibody exchanged antibody combination 3D5 (coating) +18c5 (detection) is superior to that of the primary antibody and secondary antibody used alone, and further, the detection sensitivity and detection range of the antibody combination 18c5+3d5 described in the present invention are the best of all choices, and are superior to those of the existing products, and are also superior to those of the single use and the exchanged use.
To test the effect of actual use, the applicant also performed TNF- α testing on random human blood samples, similar to the testing method used to draw the conclusions in table 4, with specific results shown in table 6.
TABLE 6
Also, by combining the criteria in Table 2, it was confirmed that if the serum TNF-. Alpha.concentration was >6pg/mL, it was possible to demonstrate that there was inflammation or other infection, and in the comparison of the detection data in Table 6, it was found that the antibody combination 18C5+3D5 kit had a false detection of 5% in the detection of H10, the antibody combination 3D5+18C5 kit had a false detection of H4, H8, and P5, that is, a false detection of 15%, the second antibody 3D5 alone had a false detection of 15%, the first antibody 18C5 alone had a false detection of 20%, and the comparison result showed that the detection sensitivity was slightly lower than that of the first and second antibodies alone.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.
Claims (16)
1. The antibody composition for detecting tumor necrosis factor-alpha is characterized by comprising a first antibody and a second antibody, wherein the amino acid sequences of CDR1, CDR2 and CDR3 regions of a heavy chain variable region of the first antibody are respectively shown as amino acid sequences at positions 26-35, 55-63 and 105-114 of SEQ ID No.1, the amino acid sequences of CDR1, CDR2 and CDR3 regions of a light chain variable region are respectively shown as amino acid sequences at positions 24-32, 48-55 and 88-98 of SEQ ID No.2, and the amino acid sequences of CDR1, CDR2 and CDR3 regions of a heavy chain variable region of the second antibody are respectively shown as amino acid sequences at positions 26-34, 54-63 and 105-117 of SEQ ID No.3, and the amino acid sequences of CDR1, CDR2 and CDR3 regions of the light chain variable region are respectively shown as amino acid sequences at positions 24-32, 48-56 and 89-97 of SEQ ID No. 4.
2. The antibody composition for detecting tumor necrosis factor-alpha according to claim 1, wherein the amino acid sequence of the heavy chain variable region of said first antibody is shown as SEQ ID No.1 or is the amino acid sequence having more than 90% homology with SEQ ID No.1, the amino acid sequence of the light chain variable region is shown as SEQ ID No.2 or is the amino acid sequence having more than 90% homology with SEQ ID No.2, the amino acid sequence of the heavy chain variable region of said second antibody is shown as SEQ ID No.3 or is the amino acid sequence having more than 90% homology with SEQ ID No.3, and the amino acid sequence of the light chain variable region is shown as SEQ ID No.4 or is the amino acid sequence having more than 90% homology with SEQ ID No. 4.
3. The antibody composition for detecting tumor necrosis factor-alpha according to claim 2, wherein the amino acid sequence of the heavy chain constant region of said first antibody is shown in SEQ ID No.5 or is an amino acid sequence having homology of more than 90% with SEQ ID No.5, the amino acid sequence of the light chain constant region is shown in SEQ ID No.6 or is an amino acid sequence having homology of more than 90% with SEQ ID No.6, the amino acid sequence of the heavy chain constant region of said second antibody is shown in SEQ ID No.7 or is an amino acid sequence having homology of more than 90% with SEQ ID No.7, and the amino acid sequence of the light chain constant region is shown in SEQ ID No.8 or is an amino acid sequence having homology of more than 90% with SEQ ID No. 8.
4. The antibody composition for detecting tumor necrosis factor- α according to any one of claims 1-3, wherein said first antibody and said second antibody are independently coated antibodies or detection antibodies.
5. The antibody composition for detecting tumor necrosis factor- α according to claim 4, wherein the antibody composition is selected from any one of a combination of a first antibody as a coated antibody and a second antibody as a detecting antibody or a second antibody as a coated antibody and a first antibody as a detecting antibody.
6. The antibody composition for detecting tumor necrosis factor- α as defined in claim 5, wherein said antibody composition is selected as a first antibody as a coating antibody and a second antibody as a detecting antibody.
7. A polynucleotide encoding the heavy chain and light chain of the antibody composition for detecting tumor necrosis factor-alpha according to any one of claims 1-6, wherein the polynucleotide encoding the heavy chain variable region of the first antibody has a nucleotide sequence shown in SEQ ID No.9 or having a nucleotide sequence having a homology of more than 90% with the sequence shown in SEQ ID No.9, the polynucleotide encoding the light chain variable region of the first antibody has a nucleotide sequence shown in SEQ ID No.10 or having a nucleotide sequence having a homology of more than 90% with the sequence shown in SEQ ID No.10, the polynucleotide encoding the heavy chain variable region of the second antibody has a nucleotide sequence shown in SEQ ID No.11 or having a nucleotide sequence having a homology of more than 90% with the sequence shown in SEQ ID No.11, and the polynucleotide encoding the light chain variable region of the second antibody has a nucleotide sequence shown in SEQ ID No.12 or having a nucleotide sequence having a homology of more than 90% with the sequence shown in SEQ ID No. 12.
8. The polynucleotide for detecting heavy and light chains of an antibody composition for tumor necrosis factor- α according to claim 7, wherein the polynucleotide sequence encoding the heavy chain constant region of said first antibody is represented by SEQ ID No.13 or is a nucleotide sequence having a sequence homology of more than 90% with SEQ ID No.13, the polynucleotide sequence encoding the light chain constant region of said first antibody is represented by SEQ ID No.14 or is a nucleotide sequence having a sequence homology of more than 90% with SEQ ID No.14, the polynucleotide sequence encoding the heavy chain constant region of said second antibody is represented by SEQ ID No.15 or is a nucleotide sequence having a sequence homology of more than 90% with SEQ ID No.15, and the polynucleotide sequence encoding the light chain constant region of said second antibody is represented by SEQ ID No.16 or is a nucleotide sequence having a sequence homology of more than 90% with SEQ ID No. 16.
9. An expression system for detecting polynucleotides of heavy and light chains of an antibody composition for tumor necrosis factor-alpha according to any one of claims 7-8, wherein said expression system is a mammalian expression vector.
10. The expression system of claim 9, wherein the tumor necrosis factor- α antigen expression system is an Expi 293 expression system.
11. The expression system of claim 9, wherein the antibody expression system is a CHO expression system.
12. A host cell comprising the expression system of any one of claims 9-11.
13. Use of an antibody composition according to any one of claims 1-6 for the preparation of a biological product for in vitro quantitative detection of tumor necrosis factor-alpha content.
14. The use according to claim 13, wherein the in vitro quantitative detection is the detection of the amount of tumor necrosis factor- α in serum, plasma or peripheral blood.
15. The use of claim 14, wherein the biologic is a kit.
16. The use according to claim 15, wherein the kit is a double antibody sandwich assay kit.
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| CN102539778A (en) * | 2010-12-24 | 2012-07-04 | 北京义翘神州生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting recombinant human tumor necrosis factor-alpha |
| CN102675460A (en) * | 2011-02-28 | 2012-09-19 | 珠海市丽珠单抗生物技术有限公司 | Humanized antibodies against tumor necrosis factor alpha |
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| CN113552368A (en) * | 2021-07-21 | 2021-10-26 | 苏州立禾生物医学工程有限公司 | Tumor necrosis factor alpha detection kit and detection method thereof |
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| CN102539778A (en) * | 2010-12-24 | 2012-07-04 | 北京义翘神州生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting recombinant human tumor necrosis factor-alpha |
| CN102675460A (en) * | 2011-02-28 | 2012-09-19 | 珠海市丽珠单抗生物技术有限公司 | Humanized antibodies against tumor necrosis factor alpha |
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