CN117567635B - Antibodies against Cas9 enzymes and uses thereof - Google Patents

Antibodies against Cas9 enzymes and uses thereof Download PDF

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CN117567635B
CN117567635B CN202410059087.3A CN202410059087A CN117567635B CN 117567635 B CN117567635 B CN 117567635B CN 202410059087 A CN202410059087 A CN 202410059087A CN 117567635 B CN117567635 B CN 117567635B
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antibody
detection
cas9 enzyme
coated
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CN117567635A (en
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程智
尚延平
王玉满
程若东
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Kaikai Biotechnology Shanghai Co ltd
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Kaikai Biotechnology Shanghai Co ltd
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Abstract

The invention provides an antibody against a Cas9 enzyme, which is characterized by comprising a detection antibody and a coating antibody. The antibody for resisting the Cas9 enzyme has a good linear range and sensitivity for measuring the Cas9 concentration. The kit has better specificity, accuracy and stability, is not combined with other related proteins, has precision CV of less than 5 percent, and has basically unchanged performance through an acceleration experiment at 37 ℃, thus indicating that the kit can be stably stored for a long time.

Description

Antibodies against Cas9 enzymes and uses thereof
Technical Field
The invention relates to the field of biotechnology, in particular to an antibody for resisting Cas9 enzyme and application thereof.
Background
The Cas9 nuclease is cloned from wild streptococcus pyogenes Streptococcus pyogenes, can realize the gene modification of hematopoietic stem cells, T cells and the like, can also be applied to the field of molecular diagnosis, and can realize the high-sensitivity and high-specificity detection of pathogens.
Cas9 nucleases have attracted worldwide attention as a genomic engineering tool to induce site-directed double-strand breaks in DNA that can inactivate genes or introduce heterologous genes by non-homologous end joining and homologous recombination, respectively. In addition, cas9 nucleases can cleave almost any sequence complementary to their associated guide RNAs. Both gene deletion and gene replacement have been demonstrated for use in human cells using the CRISPR/Cas9 system. How to quickly and effectively realize quantitative detection of trace Cas9 nuclease is particularly important in practical application. However, the conventional kit has a problem of insufficient sensitivity.
Disclosure of Invention
The invention aims to overcome the defects, provide a pair of antibodies against Cas9 enzyme and an Elisa kit for measuring the concentration of Cas9 containing the antibodies, and realize the quantitative detection of high-sensitivity and high-specificity Cas9 protein.
The antibody for resisting the Cas9 enzyme is characterized by comprising a detection antibody;
The heavy chain CDR of the detection antibody is shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3;
The light chain CDR of the detection antibody is shown as SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO. 12.
Further, the antibody against Cas9 enzyme provided by the present invention is further characterized in that:
The heavy chain FR of the detection antibody is shown as SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO. 7;
The light chain FR of the detection antibody is shown as SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO. 16.
Further, the antibody against Cas9 enzyme provided by the present invention is further characterized in that:
The heavy chain amino acid sequence of the detection antibody is shown as SEQ ID NO. 8;
The light chain amino acid sequence of the detection antibody is shown as SEQ ID NO. 17.
Further, the antibody against Cas9 enzyme provided by the present invention is further characterized in that:
the heavy chain gene sequence of the detection antibody is shown as SEQ ID NO. 9;
the light chain gene sequence of the detection antibody is shown as SEQ ID NO. 18.
Further, the antibody for resisting the Cas9 enzyme is further characterized by further comprising a coated antibody;
the heavy chain CDR of the coated antibody is shown as SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO. 21;
the light chain CDR of the coated antibody is shown as SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO. 30.
Further, the antibody against Cas9 enzyme provided by the present invention is further characterized in that:
The heavy chain FR of the coated antibody is shown as SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24 and SEQ ID NO. 25;
the light chain FR of the coated antibody is shown as SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33 and SEQ ID NO. 34.
Further, the antibody against Cas9 enzyme provided by the present invention is further characterized in that:
the heavy chain amino acid sequence of the coated antibody is shown as SEQ ID NO. 26;
The light chain amino acid sequence of the coated antibody is shown as SEQ ID NO. 35.
Further, the antibody against Cas9 enzyme provided by the present invention is further characterized in that:
The heavy chain gene sequence of the coated antibody is shown as SEQ ID NO. 27;
the light chain gene sequence of the coated antibody is shown as SEQ ID NO. 36.
In addition, the invention also provides application of the antibody for resisting the Cas9 enzyme in quantitative detection of Cas9 protein, which is characterized in that: the content of the Cas9 nuclease in the sample is measured by adopting the detection antibody and the coated antibody and adopting a double-antibody sandwich ELISA method.
Drawings
FIG. 1 is a schematic diagram of the operation method provided in the present embodiment;
Fig. 2 is a schematic diagram of a standard curve provided in this embodiment.
Detailed Description
The invention is capable of many modifications and various embodiments and its several specific embodiments are illustrated in the drawings and described herein. It is not intended to limit the invention to the particular embodiments but is to be understood to include all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.
Example 1 obtaining of paired antibodies
Mice (source: kai cuo organisms) are immunized by using high-activity Cas9 protein, monoclonal antibodies are obtained through a hybridoma technology, after the monoclonal antibodies are obtained, two monoclonal antibodies are randomly taken out, one monoclonal antibody is taken as a capture antibody, the other monoclonal antibody is taken as a labeling antibody (biotin labeling), the capture antibody is coated on an antigen plate, antigen is firstly added, after incubation, unbound antigen is washed away, then the labeling antibody is added, after incubation, unbound labeling antibody is washed away, and finally a color development solution is added for color development. If color development is possible, it is indicated that the labeled antibody specifically binds to the antigen, and the capture antibody and the labeled antibody are a pair of paired antibodies. After multiple rounds of screening and pairing, the effective pairing antibody with high sensitivity is finally obtained as follows:
A. detection of antibodies: KI366-A/KI367, anti-Cas9 Anti-ibody 45D8mIgG2 b
Heavy chain gene sequence (SEQ ID NO. 9):
ATGTATCTTGGACTGAATTACGTTTTCATCGTGTTTCTGTTGAATGGTGTGCAGTCCGAGGTTAAACTTGAGGAGTCCGGAGGTGGTCTGGTCCAGCCCGGAGGATCTATGAACCTGTCTTGCGCTGCCAGCGGTTTCACGTTTTCCGACGCCTGGATGGATTGGGTGAGACAGTCCCCTGAGAAGGGCCTGGAATGGGTGGCGGAAATTCGGTCTAAGGCAAATAACCACGCTACCTATTACGCCGAAAGCGTGAAGGGGAGGTTCACAATTAGTCGCGATGACAGTAAGAGCAGTGTATACCTGCAGATGAACTCCCTGAGAGCCGAAGACACAGGTATCTACTATTGTACAAGATTCGATGTGTGGGGCGCCGGCACCACCGTCACCGTTAGTAGTAAAACAACCCCTCCCAGTGTCTACCCCCTCGCTCCCGGTTGTGGGGACACTACAGGTTCATCTGTCACCCTGGGCTGTCTGGTCAAGGGCTACTTCCCAGAGTCTGTGACCGTGACTTGGAACAGCGGCTCTCTGAGCTCTTCCGTGCACACCTTTCCTGCACTTTTGCAGTCCGGATTGTATACTATGTCTTCCAGTGTAACTGTGCCTAGCTCTACATGGCCTAGTCAGACCGTGACTTGCAGTGTAGCTCATCCGGCTTCCAGCACGACTGTGGACAAGAAACTGGAACCTAGTGGCCCTATCTCCACGATTAATCCCTGTCCTCCTTGCAAAGAGTGCCATAAATGCCCAGCTCCTAACTTGGAGGGGGGACCAAGTGTGTTTATCTTCCCCCCAAATATCAAGGACGTGCTCATGATCAGCCTCACCCCAAAAGTTACGTGCGTCGTGGTGGACGTTAGCGAAGACGACCCCGACGTGCAGATCTCCTGGTTCGTGAATAACGTAGAAGTGCATACAGCTCAGACCCAGACACACAGGGAAGATTACAACAGTACGATCAGGGTTGTGAGCACACTTCCCATACAGCACCAGGATTGGATGAGCGGTAAAGAGTTTAAGTGCAAGGTGAACAATAAAGATCTCCCCAGCCCAATTGAAAGAACAATCTCCAAGATCAAGGGGCTGGTGCGAGCGCCTCAGGTGTACATTCTGCCACCGCCTGCTGAGCAGCTGTCACGAAAGGACGTCTCTCTGACCTGCCTTGTCGTAGGTTTCAACCCTGGAGATATATCCGTGGAATGGACCAGTAACGGCCATACCGAGGAGAACTACAAGGATACCGCCCCGGTCCTCGACTCCGACGGGTCTTATTTTATTTACTCTAAGTTGAATATGAAAACATCCAAGTGGGAGAAAACTGACTCCTTTTCCTGCAATGTTAGACATGAGGGGCTGAAAAACTATTACTTGAAAAAAACAATTTCCAGGTCTCCCGGGAAG
amino acid sequence: (SEQ ID NO. 8)
MYLGLNYVFIVFLLNGVQS<EVKLEESGGGLVQPGGSMNLSCAASGFTFS>DAWMD<WVRQSPEKGLEWVA>EIRSKANNHATYYAESVKG<RFTISRDDSKSSVYLQMNSLRAEDTGIYYC>TRFDV<WGAGTTVTVSS>KTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK
Wherein the symbols "" "respectively represent heavy chain CDR-H1 (SEQ ID NO. 1), CDR-H2 (SEQ ID NO. 2), CDR-H3 (SEQ ID NO. 3)
The heavy chain FR-H1 (SEQ ID NO. 4), FR-H2 (SEQ ID NO. 5), FR-H3 (SEQ ID NO. 6), FR-H4 (SEQ ID NO. 7) are shown in "< >", respectively
Light chain gene sequence: (SEQ ID NO. 18)
ATGAGATTCCAGGTACAGGTGTTGGGGCTGCTGCTGCTGTGGATTTCTGGGGCCCAATGTGACGTGCAAATAACCCAGTCACCTTCTTATCTCGCTGCCTCACCAGGCGAAACAATTACAATCAATTGTCGCACCTCTAAAAATATATCAAAACACTTGGCCTGGTACCAGGAGAAAGCCGGCAAGACCAAGAAGCTGCTGATCACATCAGGATCTACTCTGCGCTCTGGCATCCCGAGTCGATTCTCCGGAAGCGGCTCTGGCACGGACTTCACACTCACCATCTCTAGCCTGGAACCAGAGGATTTTGCTATGTACTATTGTCAGCAGCACAACGAGTACCCCTATACCTTCGGCGGCGGAACCAAGCTCGAGATCAAACGGGCAGATGCAGCCCCCACCGTGTCCATTTTCCCCCCATCATCTGAGCAACTTACATCAGGAGGAGCCAGCGTCGTATGTTTCCTGAATAATTTTTATCCCAAAGACATTAATGTTAAGTGGAAGATTGATGGCTCCGAAAGACAGAACGGGGTTCTCAATTCATGGACCGATCAGGACTCCAAGGATTCTACCTATTCCATGTCATCTACTCTGACCCTCACTAAAGATGAGTATGAGCGACATAATTCTTACACATGCGAGGCAACCCACAAAACCTCTACAAGCCCCATTGTTAAGTCCTTTAATCGGAATGAGTGC
Amino acid sequence: (SEQ ID NO. 17)
MRFQVQVLGLLLLWISGAQC<DVQITQSPSYLAASPGETITINC>RTSKNISKHLA<WYQEKAGKTKKLLIT>SGSTLRS<GIPSRFSGSGSGTDFTLTISSLEPEDFAMYYC>QQHNEYPYT<FGGGTKLEIK>RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC
Wherein the symbols "" "respectively represent light chain CDR-H1 (SEQ ID NO. 10), CDR-H2 (SEQ ID NO. 11), CDR-H3 (SEQ ID NO. 12)
"< >" Respectively indicate light chain FR-H1 (SEQ ID NO. 13), FR-H2 (SEQ ID NO. 14), FR-H3 (SEQ ID NO. 15), FR-H4 (SEQ ID NO. 16)
B. Coating an antibody: KI345-A/KI346, anti-Cas9 anti-body <61A3>
Heavy chain gene sequence: (SEQ ID NO. 27)
ATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGGTCCTGTCCCAGGTGCAGCTGAAACAGTCAGGCGCAGAATTGATGAAGCCCGGTGCAAGTGTTAAGATTTCCTGCAAAGCTACCGGCTACACGTTTTCAAGCTACTGGATCGAGTGGGTCAAACAGAGGCCAGGTCATGGGCTCGAATGGATTGGGGAAATCAGCCCTGGGAGCGGGTACACTAATTATAACGAAAAGTTCAAAGGCAAGGCCACCTTCACCGCCGATACCTCCTCTAATACAGCTTATATGCAGCTTAGCAACCTCACTTCCGAGGACTCAGCTGTGTACTACTGTGCCCGCTGGACCTATTACGGTAATTACGGCCCATTCGACTATTGGGGTCAGGGCACCACCCTGACCGTGAGCTCCGCCAGCACCAAGGGCCCATCCGTGTTCCCTCTGGCTCCTAGCAGCAAGTCCACCTCCGGCGGCACCGCCGCTCTGGGATGTCTGGTGAAGGATTACTTCCCTGAGCCTGTGACAGTGTCCTGGAACAGCGGCGCCCTGACCTCCGGCGTGCACACATTCCCTGCCGTGCTGCAGAGCTCCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCTAGCTCCAGCCTGGGCACACAGACATACATCTGCAATGTGAATCACAAGCCCAGCAATACCAAGGTGGATAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACACACACATGTCCTCCCTGCCCAGCTCCTGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTTCCTCCTAAGCCTAAGGATACCCTGATGATCTCCAGAACACCTGAGGTGACATGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAGGTGAAGTTTAATTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCTAGGGAGGAGCAGTACAATTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCTATCGAGAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGAGAGCCTCAGGTGTACACCCTGCCTCCCTCCAGAGAGGAGATGACCAAGAACCAGGTGAGCCTGACATGCCTGGTGAAGGGCTTCTACCCATCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAATAATTACAAGACCACACCTCCCGTGCTGGATTCCGATGGCAGCTTCTTCCTGTACTCCAAGCTGACCGTGGACAAGAGCAGGTGGCAGCAGGGCAACGTGTTCTCCTGTAGCGTGATGCACGAGGCCCTGCACAACCACTACACACAGAAGTCCCTGTCCCTGAGCCCTGGCAAGGGCGGCGGCAGCGGCCTGAATGATATCTTCGAGGCCCAGAAGATCGAGTGGCACGAG
Amino acid sequence: (SEQ ID NO. 26)
MKHLWFFLLLVAAPRWVLS<QVQLKQSGAELMKPGASVKISCKATGYTFS>SYWIE<WVKQRPGHGLEWIG>EISPGSGYTNYNEKFKG<KATFTADTSSNTAYMQLSNLTSEDSAVYYCAR>WTYYGNYGPFDY<WGQGTTLTVSS>ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKgggsGLNDIFEAQKIEWHE
Wherein the symbols "" "respectively represent heavy chain CDR-H1 (SEQ ID NO. 19), CDR-H2 (SEQ ID NO. 20), CDR-H3 (SEQ ID NO. 21)
The heavy chain FR-H1 (SEQ ID NO. 22), FR-H2 (SEQ ID NO. 23), FR-H3 (SEQ ID NO. 24), FR-H4 (SEQ ID NO. 25) are shown in "< >", respectively
Light chain gene sequence: (SEQ ID NO. 36)
ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGACATTCAACTTACCCAGTCACCTTCTTCCCTGAGCGCTAGCCTTGGGGGTAAGGTCACAATCACCTGCAAAGCTTCCCAGGACATTAATAAATACATTGCCTGGTACCAATATAAGCCTGGCAAGGGTCCAAGACTGTTGATCCATTACACCAGCACCCTGCAGCCTGGAATACCCTCACGGTTTAGCGGCAGCGGGTCTGGCCGGGACTATAGCTTCAGCATTATCAACCTGGAGCCTGAAGACATCGCCACCTACTACTGCCTGCAGTACGACAATCTGCTTACATTCGGAGGGGGAACTAAACTGGAGATTAAAAGAACAGTGGCCGCTCCTAGCGTGTTCATCTTCCCTCCTAGCGACGAGCAGCTGAAGTCCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTATCCTAGGGAGGCCAAGGTGCAGTGGAAGGTGGATAACGCCCTGCAGAGCGGCAATAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGATTCCACCTACAGCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCTGTGACAAAGTCCTTCAACAGAGGCGAGTGT
Amino acid sequence: (SEQ ID NO. 35)
METPAQLLFLLLLWLPDTTG<DIQLTQSPSSLSASLGGKVTITC>KASQDINKYIA<WYQYKPGKGPRLLIH>YTSTLQP<GIPSRFSGSGSGRDYSFSIINLEPEDIATYYC>LQYDNLLT<FGGGTKLEI>KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Wherein the symbols "" "respectively represent heavy chain CDR-H1 (SEQ ID NO. 28), CDR-H2 (SEQ ID NO. 29), CDR-H3 (SEQ ID NO. 30)
The heavy chain FR-H1 (SEQ ID NO. 31), FR-H2 (SEQ ID NO. 32), FR-H3 (SEQ ID NO. 33), FR-H4 (SEQ ID NO. 34) are shown in "< >", respectively
Example 2 determination of Cas 9 nuclease content in samples Using double-antibody Sandwich ELISA
2.1 The product composition is shown in Table 1 below:
TABLE 1 product composition
Wherein, the coating lath: diluting Cas9 anti 61A3 to 5ug/mL with CBS (Na 2CO3:Na2HCO3: nacl=1.6:2.9:9) and stirring thoroughly; 100 uL/well is added into an ELISA plate, and incubated overnight (about 14 h) at 2-8 ℃; washing the ELISA plate by a plate washer for 4 times, beating up water, adding a sealing liquid into 200 uL/hole, and incubating for 2h at 37 ℃; throwing off the sealing liquid, beating to dry, and drying at a constant temperature of 37 ℃ for 4 hours; vacuum packaging, and storing at 2-8deg.C.
Concentrated washing liquid :Na2HPO4•12H2O 71.6g/L,KH2PO4 4.8g/L,KCl 4g/L,NaCl 164g/L,Tween-20 10mL/L.
Assay buffer 10*PBS 100mL,BSA 10.0g,Tween-20 0.5mL,Proclin300 1mL.
2.2 Principle of operation: and (3) pre-coating the Cas9 monoclonal antibody in a 96-hole reaction plate by adopting the operation standard of a traditional double-antibody sandwich ELISA method, and performing coating treatment to ensure the activity of the Cas9 monoclonal antibody. After the standard substance or the sample to be detected is added, cas9 nuclease in the standard substance or the sample can be specifically bound into a reaction plate, then a detection antibody and an HRP label are added to form an antibody-antigen-detection antibody-HRP labeled complex, and the redundant detection antibody and the HRP label are removed through washing operation. After adding the chromogenic solution, HRP catalyzes its chromogenic reaction, and the chromogenic intensity is proportional to the concentration of Cas9 nuclease. After terminating the reaction with the termination solution, the absorbance at the wavelength of 450nm is read on the microplate reader to calculate the concentration of Cas9 nuclease in the sample.
2.3 Method of operation, as shown in figure 1,
1) The kit is taken out from the preservation environment at the temperature of 2-8 ℃ in the experiment, placed in the room temperature environment and balanced to the room temperature.
2) Preparing 1 Xwashing solution, namely diluting 20 Xconcentrated washing solution to 1 Xwith pure water for later use.
3) 1 Xanalysis buffer was prepared and 10 Xanalysis buffer was diluted to 1 Xwith pure water for use.
4) Preparation of 1 Xdetection antibody 300 Xdetection antibody was buffered with 1 Xassay buffer to 1 Xfor use.
5) Preparing standard substance, taking out standard substance, centrifuging at above 1000rpm for 30s, and concentrating the liquid at the bottom of the tube. 3.2 mu standard is added into 1000 mu 1 Xanalysis buffer solution to prepare cas9 standard with the concentration of 16ng/m, and then the 16ngm standard is diluted into 7 concentrations of 8ng/ml, 4ng/ml, 2ng/ml, 1ng/ml, 0.5ng/ml and 0.25ng/ml according to the 2-fold ratio, and 1 Xanalysis buffer solution is used as a zero standard, wherein each concentration standard is provided with multiple holes.
6) Pre-washing, namely taking out the 96-hole reaction plate according to the required test amount, washing the plate with a washing plate for 1 time, adding 300 mu of washing liquid into each hole, drying after washing, putting unused strips into a sealing bag as soon as possible, and storing at 4-8 ℃.
7) Sample adding, namely adding the standard substance and the sample into a 96-well reaction plate respectively, and carrying out constant-temperature oscillation reaction for 1h at the temperature of 37 ℃ and the constant-temperature oscillation reaction for 600pm at 100 mu l of each well
8) Adding 1 Xdetection antibody, washing the 96-well plate after the previous reaction for 4 times, drying 300 mu-well, adding detection antibody into each well after beating, and carrying out constant-temperature oscillation reaction for 1h at 37 ℃ and 600 rpm.
9) HRP label was added to the wells, the wells were washed 4 times with 300. Mu.l/well after the previous reaction, and after drying, HRP label was added to the wells, and the wells were subjected to a constant temperature shaking reaction at 37℃and 600rpm for 1 hour.
10 Washing the 96-well plate after the previous reaction for 4 times, 300 μl/well, drying, adding the color development liquid, 100 μl/well, developing at 37deg.C in dark for 10min, adding the stop liquid, and stopping the reaction. The OD value at 450nm wavelength was read immediately using a microplate reader.
11 Data processing.
11-1) Making a standard curve: the concentration of the standard substance is the cross-coordinate, and the OD value is the ordinate. If a complex hole is arranged, the average value of the complex holes is calculated. Various mapping and statistical software can be used to assist in mapping the standard curve and making the calculation of the unknown sample concentration. The general polynomial fitting effect is better, and other methods such as a linear and double-logarithmic method can obtain better fitting results, and the judgment is needed according to specific experimental data. An example of a calibration curve in one embodiment is shown in fig. 2.
11-2) Calculating the concentration of the sample, substituting the OD value of the sample into a fitting equation of a standard curve, and calculating the concentration of the sample, namely the actual concentration of the sample. The lowest limit of quantitation (LOQ) was 0.25ng/ml, below 0.25ng/ml reported as <0.25ng/ml. If the OD value of the sample is higher than the upper limit of the standard curve, the sample is re-measured after being properly diluted, and the final concentration is calculated by multiplying the final concentration by the corresponding dilution times.
Example 3 correlation quality detection
3.1. Precision detection
Sample dilution: 3 tubes of 5mlEP are taken for marking 1,2 and 3, 4ml of analysis buffer solution is sucked into a No. 1 EP tube by a pipette, 3ml of analysis buffer solution is respectively sucked into a No. 2 EP tube and a No. 3 EP tube, 8ul of standard substance is taken and added into a No. 1 EP tube, the mixture is repeatedly blown and beaten, a vortex instrument is used for uniformly mixing, 1ml of the mixture is taken into a No. 2 tube, the mixture is repeatedly blown and beaten, a vortex instrument is used for uniformly mixing 1ml of the mixture into a No. 3 tube, and the corresponding concentration of the No. 3 tube is 10ng/ml,2.5ng/ml and 0.625ng/ml at the moment.
Standard substance dilution: 9.6 μl of standard is added into 3000 μl of 1 Xanalysis buffer, repeatedly blown, and mixed with vortex for use, and prepared into a concentration of 16ng/ml for use.
Pre-washing: taking out the 96-well reaction plate, washing the plate for 2 times by using a plate washing machine, adding 300 mu l of washing liquid into each well, washing, and drying for later use.
Adding a standard substance: pouring the analysis buffer solution into a sample adding groove, respectively adding 100ul of 1 Xanalysis buffer solution into the 2 nd-9 th columns of A and B in a 96-well plate by using a row gun, adding 200ul of diluted standard substance with the concentration of 16ng/ml into the 1 st column of A and B in the 96-well plate by using a row gun, adjusting the gun to 100ul, sucking 100ul from the 1 st column to the 2 nd column of a corresponding hole, repeatedly blowing and mixing by using the gun, sucking 100ul from the 2 nd column to the 3 rd column of a corresponding hole again, sequentially carrying out 2 times ratio dilution until diluting to the 7 th column, repeatedly blowing and mixing by using the gun, sucking 100ul into a waste liquid groove, and then taking the concentrations of the 1 st-7 th columns as blank control, wherein the concentrations of the corresponding columns are 16ng/ml, 8ng/ml, 4ng/ml, 2ng/ml, 1ng/ml, 0.5ng/ml and the corresponding concentrations of the 8 th and 9 th columns are 0.
Adding a sample to be tested: 3 samples to be tested are respectively poured into a sample adding groove, are added to the 3 rd, 4 th and 5 th rows of a 96-well plate by a row gun, and each sample is provided with 12 compound wells and marked.
Incubation: the well plate with the sample added is placed in a constant temperature reactor at 37 ℃ for oscillating reaction for 1h.
Diluting the labeled antibody: diluting to 9ml according to the determined dilution ratio, and uniformly mixing by a vortex machine for later use
Adding a detection antibody: after 1h incubation, the liquid in the 96-well plate is rapidly poured out, the plate is put into a plate washer, the plate is washed for 4 times, 300 μl/well is added with detection antibody after beating, 100 μl of detection antibody is added into each well by a row gun, and the reaction is carried out for 1h under constant temperature oscillation at 37 ℃.
Adding HRP label: after 1h incubation, the liquid in the 96-well plate was rapidly poured off, placed on a plate washer, washed 4 times with 300. Mu.l/well, and after drying, HRP label was added with a row gun, 100. Mu.l/well was reacted for 1h with constant temperature shaking at 37 ℃.
Color reading: after 1h incubation, the liquid in the 96-well plate is rapidly poured out, the plate is placed on a plate washer, the plate is washed for 4 times, 300 μl/hole is added with a color development liquid by a discharge gun after the plate is patted dry, 100 μl/hole is developed for 10min at 37 ℃ in a dark place, and a stop solution of 50 μl/hole is added to stop the reaction. The OD value at the wavelength of 450nm was read immediately using a microplate reader.
And (3) data processing: exporting the data to a computer for data processing: a) Firstly, drawing a standard curve from a standard product compound Kong Qiu average value, b) respectively checking 12 compound holes of 3 samples, removing a highest point and a lowest point (also a jump hole point, wherein the numerical value is obvious and different from other most numerical values are regarded as jump holes), respectively bringing an OD value into a standard curve formula, and calculating the corresponding concentration; c) The average AV and standard deviation SD of 3 samples are calculated separately, and the standard deviation coefficient CV, i.e. standard deviation/average 100% is further calculated, where the formula of the average AV and standard deviation SD is:
wherein C 1···C10 is the concentration corresponding to 10 samples respectively
Where n=10, c is the concentration average of 10 samples
3.2. Recovery rate detection
Dilution of the samples: taking 515 ml centrifuge tubes, and numbering 0,1,2,3 and 4; 6ml of 1 x assay buffer was added to tube 0 using a pipette, and 4ml of 1 x assay buffer was added to each of tubes 1-4; adding 19.2ul standard substances into a No. 0 tube, repeatedly blowing, uniformly mixing by using a vortex meter, repeatedly blowing, uniformly mixing by using the vortex meter, adding 4ml into a No. 1 tube, uniformly mixing by using the vortex meter, adding 4ml into a No. 2 tube, and the like, carrying out equal-ratio dilution, wherein the final No. 4 tube is provided with 8ml of solution, and the concentrations corresponding to the No. 0,1,2,3 and 4 tubes are 16ng/ml,8ng/ml,4ng/ml and 1ng/ml. Wherein the pipe No. 4 is used as a basic concentration, and 1-3 is used as an additive concentration; taking 35 mlEP pipes, and numbering a, b and c; taking a tube No. 4 as a basic concentration, taking 1ml from the tube No. 4 by using a pipettor, adding the same to a tube A, b tube and c tube, taking 1ml from the tube No. 1, adding the same to a tube A, taking 1ml from the tube No. 2, adding the same to a tube b, taking 1ml from the tube No. 3, adding the same to a tube c, repeatedly blowing, uniformly mixing by using a vortex instrument, and reserving.
Pre-washing: taking out the 96-well reaction plate, washing the plate for 2 times by using a plate washing machine, adding 300 mu l of washing liquid into each well, washing, and drying for later use.
Adding a standard substance: pouring the analysis buffer solution into a sample adding groove, respectively adding 100ul of 1 Xanalysis buffer solution into the 2 nd-9 th columns of A and B in a 96-well plate by using a row gun, adding 200ul of diluted standard substance with the concentration of 16ng/ml, namely a0 th tube, into the 1 st column of the 96-well plate by using a row gun, adjusting the gun to 100ul, sucking 100ul from the 1 st column to the 2 nd column of a corresponding hole, repeatedly blowing and mixing by using the gun, sucking 100ul from the 2 nd column to the 3 rd column of a corresponding hole again, sequentially carrying out 2 times ratio dilution until the solution is diluted to the 7 th column, repeatedly blowing and mixing by using the gun, sucking 100ul into a waste liquid groove, and then the concentrations corresponding to the 1 st-7 th columns are 16ng/ml, 8ng/ml, 4ng/ml, 2ng/ml, 1ng/ml, 0.5ng/ml and 0.25ng/ml, and the concentrations corresponding to the 8 th column and 9 th column are 0, thereby being used as blank control.
Adding a sample to be tested: samples numbered a, b, c, and 4 were poured into the loading wells, 100ul was added to the 3 rd and 4 th rows of the 96 well plate with a row gun, 4 wells were multiplexed for each sample, and labeled.
Incubation: the well plate with the sample added is placed in a constant temperature reactor at 37 ℃ for oscillating reaction for 1h.
Diluting the labeled antibody: diluting to 9ml according to the determined dilution ratio, and uniformly mixing by a vortex instrument for later use
Adding a detection antibody: after 1h incubation, the liquid in the 96-well plate is rapidly poured out, the plate is put into a plate washer, the plate is washed for 4 times, 300 μl/well is added with detection antibody after beating, 100 μl of detection antibody is added into each well by a row gun, and the reaction is carried out for 1h under constant temperature oscillation at 37 ℃.
Adding HRP label: after 1h incubation, the liquid in the 96-well plate was rapidly poured off, placed on a plate washer, washed 4 times with 300. Mu.l/well, and after drying, HRP label was added with a row gun, 100. Mu.l/well was reacted for 1h with constant temperature shaking at 37 ℃.
Color reading: after 1h incubation, the liquid in the 96-well plate is rapidly poured out, the plate is placed on a plate washer, the plate is washed for 4 times, 300 μl/hole is added with a color development liquid by a discharge gun after the plate is patted dry, 100 μl/hole is developed for 10min at 37 ℃ in a dark place, and a stop solution of 50 μl/hole is added to stop the reaction. The OD value at the wavelength of 450nm was read immediately using a microplate reader.
The data processing is shown in Table 2: exporting the data to a computer for data processing: a) Checking 4 complex holes of 4 samples respectively, if a jump hole point exists, removing the 4 complex holes (the numerical value is obvious and the difference from other most numerical values is regarded as the jump hole), respectively bringing the OD value into a formula of the standard curve, calculating the corresponding concentration of the OD value, and calculating the average value; c) According to the theoretical concentration, taking a No. 4 tube as a basic concentration, taking 1,2 and 3 tubes as additive concentrations, and respectively calculating the theoretically added mass of a, b, c and 3 samples; d) According to the measured concentration, taking a tube No. 4 as a basic concentration, taking a tube No. 1,2 and 3 as additive concentrations, and respectively calculating the mass of actually added a, b, c and 3 samples; e) Calculating recovery rate R: actual added mass/theoretical added mass 100%.
TABLE 2 data processing
3.3 Specificity experiments
The detection under the condition of the solvent contained in the kit (except the standard substance) and the condition of the serum of the mice, rabbits and sheep and the cell supernatant of the escherichia coli shows that the detection is not influenced by the interfering substances.
Example 4 performance comparison
Commercial NB-E1372PR with a linear range of 1.56ng/ml to 100ng/ml, a sensitivity of 1.5ng/ml, an effectiveness of 6 months and a preservation temperature of 2-8 ℃.
Cas9-1000 is commercially available, the linear range is 1.56ng/ml-100ng/ml, the sensitivity reaches 1.5ng/ml, the effectiveness is 3 weeks, and the preservation temperature is 2-8 ℃.
The product has a linear range of 0.25ng/ml-16ng/ml, a sensitivity of 0.125ng/ml, an effectiveness of 12 months, and a preservation temperature of 2-8deg.C (see Table 3-1 and Table 3-2).
TABLE 3-1, batch production release data
TABLE 3-2 data after 1 year of long term monitoring
The Cas9 concentration measurement has good linear range and sensitivity through comparison. The kit has better specificity, accuracy and stability, is not combined with other related proteins, has precision CV of less than 5 percent, and has basically unchanged performance through an acceleration experiment at 37 ℃, thus indicating that the kit can be stably stored for a long time.
Although the embodiments have been described above mainly, this is merely illustrative, and not restrictive of the invention, and it will be apparent to those skilled in the art that various modifications and applications not illustrated above can be made without departing from the essential characteristics of the present embodiments. For example, each component specifically shown in the embodiments can be implemented by being modified. Moreover, various points related to such modifications and applications should be construed as including the scope of the present invention as defined in the appended claims.

Claims (10)

1. An antibody against Cas9 enzyme, characterized by comprising a detection antibody;
The heavy chain CDR1, CDR2 and CDR3 of the detection antibody are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3;
The light chain CDR1, CDR2 and CDR3 of the detection antibody are respectively shown as SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO. 12.
2. The antibody against Cas9 enzyme of claim 1, wherein:
the heavy chains FR1, FR2, FR3 and FR4 of the detection antibody are respectively shown as SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO. 7;
The light chains FR1, FR2, FR3 and FR4 of the detection antibody are respectively shown as SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO. 16.
3. The antibody against Cas9 enzyme of claim 1, wherein:
The heavy chain amino acid sequence of the detection antibody is shown as SEQ ID NO. 8;
The light chain amino acid sequence of the detection antibody is shown as SEQ ID NO. 17.
4. The antibody against Cas9 enzyme of claim 1, wherein:
the heavy chain gene sequence of the detection antibody is shown as SEQ ID NO. 9;
the light chain gene sequence of the detection antibody is shown as SEQ ID NO. 18.
5. The antibody against Cas9 enzyme of claim 1, further comprising a coating antibody;
The heavy chain CDR1, CDR2 and CDR3 of the coated antibody are respectively shown as SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO. 21;
the light chain CDR1, CDR2 and CDR3 of the coated antibody are respectively shown as SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO. 30.
6. The antibody against Cas9 enzyme of claim 5, wherein:
The heavy chains FR1, FR2, FR3 and FR4 of the coated antibody are respectively represented by SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24 and SEQ ID NO. 25;
the light chains FR1, FR2, FR3 and FR4 of the coated antibody are respectively shown as SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33 and SEQ ID NO. 34.
7. The antibody against Cas9 enzyme of claim 5, wherein:
the heavy chain amino acid sequence of the coated antibody is shown as SEQ ID NO. 26;
The light chain amino acid sequence of the coated antibody is shown as SEQ ID NO. 35.
8. The antibody against Cas9 enzyme of claim 5, wherein:
The heavy chain gene sequence of the coated antibody is shown as SEQ ID NO. 27;
The light chain gene sequence of the coated antibody is shown as SEQ ID NO. 36.
9. The application of an antibody against Cas9 enzyme in the quantitative detection of Cas9 protein is characterized in that:
the antibody against Cas9 enzyme is the combination of the detection antibody of claim 5 and a coated antibody.
10. The use according to claim 9, wherein:
and determining the content of the Cas9 nuclease in the sample by adopting a double-antibody sandwich ELISA method.
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