CN118240087A - Anti-RNAse R antibody and application thereof - Google Patents

Abstract

The invention relates to the technical field of immunodetection, in particular to an Anti-RNAse R antibody and application thereof; the invention obtains a pair of Anti-RNase R antibodies, 15C3F4 and 11A3E4 through immune screening of RNase R, further develops a high-sensitivity kit for detecting RNase R residues by using the paired antibodies, and is suitable for quality control of detecting RNase R residues in the circRNA in the fields of circRNA therapeutic drugs and vaccine production.

Description

Anti-RNAse R antibody and application thereof

Technical Field

The invention relates to the technical field of immunodetection, in particular to an Anti-RNAse R antibody and application thereof.

Background

Ribonuclease R (RNase R, uniprot: P21499) is derived from E.coli and belongs to a magnesium ion-dependent 3 '. Fwdarw.5' riboexonuclease of RNase II family. RNase R consists of an exonuclease domain (RNB) with the ability to unwind and degrade RNA and a plurality of auxiliary domains that function to bind RNA. RNase R can bind, unwind and degrade RNA having a secondary structure, including linear RNA and double stranded RNA molecules with 3' -terminal overhangs of at least 7 nucleotides. However, RNase R is not readily digestible to circular RNA, lasso structures or double stranded RNA molecules with 3' -terminal overhangs of less than 7 nucleotides. In recent years, in vitro synthesized circular RNA (circRNA) has become an emerging RNA therapeutic drug and vaccine by virtue of the advantages of strong stability, low immunogenicity and the like. The circRNA is a closed RNA without a 5 'end or a 3' end, and after the in vitro transcription and cyclization reaction is finished, various RNA impurities exist in the product, mainly including uncyclized precursor (pre) RNA; the circRNA produces nicked (nicked) RNA, a single-stranded linear RNA of the same length as CircRNA; and the upstream and downstream intron sequences generated by the cyclized cleavage. RNase R can be applied to industrial production of digestion linear RNA and enrichment of circRNA.

In the purified circRNA, however, there may be RNase R residues, which may cause a safety risk if they follow the biological product into the human body, such as immunogenicity. Therefore, it is necessary to accurately analyze and detect the residue of RNase R so as to control it within a safe range.

Disclosure of Invention

The invention obtains a pair of Anti-RNase R antibodies, 15C3F4 and 11A3E4 through immune screening of RNase R, further develops a high-sensitivity kit for detecting RNase R residues by using the paired antibodies, and is suitable for quality control of detecting RNase R residues in the circRNA in the fields of circRNA therapeutic drugs and vaccine production.

In a first aspect of the invention, there is disclosed a pair of high affinity partner antibodies comprising 15C3F4 and 11A3E4;

according to the IMGT numbering system, the antibody 15C3F4 has a light chain complementarity determining region CDR1 as shown in SEQ ID NO. 1, a light chain complementarity determining region CDR2 as shown in SEQ ID NO. 2 and a light chain complementarity determining region CDR3 as shown in SEQ ID NO. 3;

CDR-1QSLLDSEGKTY(SEQ ID NO:1)

CDR-2LVS(SEQ ID NO:2)

CDR-3WQGTHFPWT(SEQ ID NO:3)；

And a heavy chain complementarity determining region CDR1 as shown in SEQ ID NO. 4, a heavy chain complementarity determining region CDR2 as shown in SEQ ID NO. 5 and a heavy chain complementarity determining region CDR3 as shown in SEQ ID NO. 6;

CDR-1GYSFTGYT(SEQ ID NO:4)

CDR-2INPYNGGT(SEQ ID NO:5)

CDR-3ATMIFDY(SEQ ID NO:6)。

The antibody 11A3E4 has a light chain complementarity determining region CDR1 as shown in SEQ ID NO. 7, a light chain complementarity determining region CDR2 as shown in SEQ ID NO. 8 and a light chain complementarity determining region CDR3 as shown in SEQ ID NO. 9;

CDR-1QDINRY(SEQ ID NO:7)

CDR-2RAN(SEQ ID NO:8)

CDR-3LQYDEFPFT(SEQ ID NO:9)

And a heavy chain complementarity determining region CDR1 as shown in SEQ ID NO. 10, a heavy chain complementarity determining region CDR2 as shown in SEQ ID NO. 11 and a heavy chain complementarity determining region CDR3 as shown in SEQ ID NO. 12.

CDR-1GFTFTDYY(SEQ ID NO:10)

CDR-2IRDKANGFTT(SEQ ID NO:11)

CDR-3ARDVGVY(SEQ ID NO:12)

The antibody 15C3F4 comprises a light chain variable region with a sequence shown as SEQ ID NO. 13 and a heavy chain variable region with a sequence shown as SEQ ID NO. 14.

DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSEGKTYLNWLLQRPGQSPQSLMYLVSE LDSGVPDRFTGSGSGTDFTLRISRLEAEDLGVYYCWQGTHFPWTFGGGTKLEIK(SEQ ID NO:13)

EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYN GGTSYNQKFRDKATLTVDKSSSTAYMELLSLTSEDSAVYYCATMIFDYWGQGTTLTVSS(SEQ ID NO:14)

The antibody 15C3F4 comprises a light chain amino acid sequence (light chain constant region mouse Igkappa) as shown in SEQ ID NO. 15; and the heavy chain amino acid sequence shown as SEQ ID NO. 16 (heavy chain constant region mouse IgG 1).

MSPAQFLFLLVLWIRETNGDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSEGKTYLNWLLQRPGQSPQSLMYLVSELDSGVPDRFTGSGSGTDFTLRISRLEAEDLGVYYCWQGTHFPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:15)

MGWSWIFLFLLSGTAGVHSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYNGGTSYNQKFRDKATLTVDKSSSTAYMELLSLTSEDSAVYYCATMIFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO:16)

The antibody 11A3E4 comprises a light chain variable region with a sequence shown as SEQ ID NO. 17 and a heavy chain variable region with a sequence shown as SEQ ID NO. 18.

DIKMTQSPSSMYASLGERVTFTCKASQDINRYLGWFQQKPGKSPKTLIYRANRLLDG VPSRFSGSGSGQDFSLTISTLDYEDVGFYYCLQYDEFPFTLGSGTKLEIK(SEQ ID NO:17)

EVKLVESGGGLVQPGGSLRLSCAVSGFTFTDYYMTWVRQPPGKALEWLAIIRDKAN GFTTDYSASVKGRFTISRDNSQSILYLQMNTLRAEDSATYYCARDVGVYWGQGTLVTVS AES(SEQ ID NO:18)

The antibody 11A3E4 comprises a light chain amino acid sequence (light chain constant region mouse Igkappa) as shown in SEQ ID NO. 19; and the heavy chain amino acid sequence shown as SEQ ID NO. 20 (heavy chain constant region mouse IgG 1).

MRTPAQFLGILLLWFPGFKCDIKMTQSPSSMYASLGERVTFTCKASQDINRYLGWFQQKPGKSPKTLIYRANRLLDGVPSRFSGSGSGQDFSLTISTLDYEDVGFYYCLQYDEFPFTLGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:19)

MKLWLNWILLVTLINGIQCEVKLVESGGGLVQPGGSLRLSCAVSGFTFTDYYMTWVRQPPGKALEWLAIIRDKANGFTTDYSASVKGRFTISRDNSQSILYLQMNTLRAEDSATYYCARDVGVYWGQGTLVTVSAESARTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO:20) The term "antibody" (Ab) shall include, but is not limited to, an immunoglobulin that specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains, or antigen binding portions thereof, interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain CL. VH and VL regions can be further subdivided into regions of hypervariability termed Complementarity Determining Regions (CDRs) interspersed with regions that are more conserved termed Framework Regions (FR). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

It should be understood that the amino acid names are identified by international single English letters, and the corresponding three English letters of the amino acid names are respectively ：Ala(A)、Arg(R)、Asn(N)、Asp(D)、Cys(C)、Gln(Q)、Glu(E)、Gly(G)、His(H)、I1e(I)、Leu(L)、Lys(K)、Met(M)、Phe(F)、Pro(P)、Ser(S)、Thr(T)、Trp(W)、Tyr(Y)、Val(V).

In a second aspect the invention discloses a nucleic acid composition comprising a nucleic acid molecule encoding the antibodies 15C3F4 and 11A3E4 of any one of claims 1-5.

The nucleotide sequence of the antibody light chain gene sequence of the 15C3F4 is shown as SEQ ID NO:21, as shown in:

ATGTCTCCTGCCCAATTCCTGTTTCTGCTTGTGCTTTGGATAAGAGAGACCAATGGAGACGTCGTGATGACTCAAACCCCGCTCACTCTGTCTGTTACGATTGGCCAGCCCGCCAGCATTTCATGTAAAAGCTCCCAGAGCCTGCTGGACTCTGAAGGTAAGACCTACCTTAACTGGCTGCTCCAGAGACCTGGACAGAGTCCCCAGAGCCTGATGTACTTGGTCAGCGAATTGGATTCCGGCGTCCCAGACAGGTTTACCGGATCTGGATCAGGCACCGACTTCACACTGAGAATAAGTCGATTGGAAGCTGAGGACCTTGGGGTGTACTATTGTTGGCAGGGAACTCATTTCCCTTGGACATTTGGAGGCGGGACTAAGCTCGAAATAAAACGCGCCGATGCCGCTCCTACAGTGAGCATCTTTCCTCCTTCCTCCGAGCAGCTGACAAGCGGCGGCGCCAGCGTGGTGTGTTTCCTGAACAACTTCTATCCTAAGGACATCAATGTGAAGTGGAAGATCGACGGCAGCGAGAGACAGAACGGCGTGCTGAACTCCTGGACCGACCAGGATTCCAAGGACTCCACCTACTCCATGTCCTCCACACTGACCCTGACCAAGGATGAGTACGAGAGGCACAACAGCTACACATGCGAGGCCACACACAAGACCTCCACCAGCCCTATCGTGAAGAGCTTCAATAGAAACGAGTGC(SEQ ID NO：21);

the nucleotide sequence of the heavy chain gene sequence of the antibody for 15C3F4 is shown in SEQ ID NO:22, as shown in:

ATGGGATGGTCATGGATCTTTCTCTTCCTCCTTAGCGGAACCGCCGGGGTTCATTCAGAGGTGCAGCTTCAGCAGTCCGGACCAGAATTGGTTAAGCCTGGTGCAAGTATGAAGATTAGTTGTAAGGCCTCCGGGTATAGTTTTACCGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGTAAAAACCTCGAGTGGATAGGGCTGATTAACCCATACAATGGCGGCACTTCTTACAATCAGAAGTTCCGCGATAAGGCCACTCTGACAGTTGATAAGAGCTCATCCACCGCCTACATGGAACTGTTGAGTCTTACATCCGAGGACTCCGCAGTCTACTATTGTGCCACCATGATTTTCGACTATTGGGGCCAGGGGACGACGCTCACTGTATCAAGCGCCAA GACCACCCCTCCTAGCGTGTACCCCCTGGCCCCTGGATCCGCCGCTCAGACAAACTCTATGGTGACCCTGGGCTGCCTGGTGAAGGGCTACTTCCCCGAGCCTGTGACAGTCACATGGAACTCCGGCAGCCTGTCTAGCGGCGTGCACACCTTCCCAGCCGTGCTGCAGAGCGACCTGTACACCCTGAGCAGCAGCGTTACCGTGCCTAGCAGCCCTAGACCCTCCGAGACAGTGACATGTAATGTGGCCCACCCAGCCTCCTCCACCAAGGTGGACAAGAAGATCGTGCCTAGGGACTGCGGCTGCAAGCCCTGTATCTGTACAGTGCCTGAGGTGTCCTCCGTGTTCATCTTCCCACCTAAGCCTAAGGATGTGCTGACCATCACACTGACACCTAAGGTGACATGTGTGGTGGTGGATATCTCCAAGGACGATCCTGAGGTGCAGTTTAGCTGGTTTGTGGACGACGTGGAGGTGCACACCGCCCAGACCCAGCCCAGAGAGGAGCAGTTCAATTCCACATTCAGGAGCGTGAGCGAGCTGCCTATCATGCACCAGGATTGGCTGAATGGCAAGGAGTTCAAGTGTAGAGTGAACAGCGCCGCCTTCCCAGCTCCTATCGAGAAGACCATCAGCAAGACAAAGGGCAGACCTAAGGCTCCTCAGGTGTACACCATCCCACCTCCTAAGGAGCAGATGGCCAAGGACAAGGTGAGCCTGACCTGTATGATCACCGATTTCTTCCCAGAGGATATCACCGTGGAGTGGCAGTGGAATGGCCAGCCCGCCGAGAATTACAAGAACACCCAGCCCATCATGAATACAAACGGCTCCTACTTTGTGTACTCCAAGCTGAACGTGCAGAAGTCCAACTGGGAGGCCGGCAATACATTCACCTGCTCCGTGCTGCACGAGGGCCTGCACAATCACCACACCGAGAAGAGCCTGTCCCACAGCCCAGGCAAG(SEQ ID NO：22).

the nucleotide sequence of the antibody light chain gene sequence of the coding 11A3E4 is shown as SEQ ID NO:23, as shown in:

ATGCGCACGCCTGCTCAATTTTTGGGAATACTGCTCCTCTGGTTTCCCGGCTTCAAGTGTGATATAAAGATGACACAGTCCCCCTCAAGCATGTACGCATCCCTCGGAGAGAGAGTTACCTTCACCTGTAAAGCCTCCCAGGACATCAATCGGTACCTTGGGTGGTTTCAGCAGAAGCCAGGGAAGAGCCCAAAAACGTTGATATACAGAGCGAACAGGCTTCTGGACGGCGTGCCTTCACGATTTTCAGGAAGCGGTAGCGGACAGGACTTCTCCTTGACAATCTCAACCCTCGACTATGAGGACGTGGGGTTCTATTACTGCCTGCAGTACGACGAGTTTCCCTTCACCCTGGGTTCCGGGACTAAGCTCGAAATAAAACGCGCCGATGCCGCTCCTACAGTGAGCATCTTTCCTCCTTCCTCCGAGCAGCTGACAAGCGGCGGCGCCAGCGTGGTGTGTTTCCTGAACAACTTCTATCCTAAGGACATCAATGTGAAGTGGAAGATCGACGGCAGCGAGAGACAGAACGGCGTGCTGAACTCCTGGACCGACCAGGATTCCAAGGACTCCACCTACTCCATGTCCTCCACACTGACCCTGACCAAGGATGAGTACGAGAGGCACAACAGCTACACATGCGAGGCCACACACAAGACCTCCACCAGCCCTATCGTGAAGAGCTTCAATAGAAACGAGTGC(SEQ ID NO：23);

the nucleotide sequence of the heavy chain gene sequence of the antibody for encoding 11A3E4 is shown in SEQ ID NO:24, as shown in:

ATGAAACTCTGGCTCAATTGGATACTTCTTGTGACGCTTATTAACGGGATCCAGTGCGAAGTCAAACTCGTTGAGTCCGGTGGAGGACTGGTCCAGCCAGGCGGTAGTCTCCGCTTGAGTTGTGCCGTCTCCGGCTTTACCTTTACAGATTACTACATGACCTGGGTCCGGCAACCACCCGGTAAAGCTCTTGAGTGGCTTGCTATAATCCGGGACAAAGCTAACGGCTTTACTACTGACTACAGTGCCTCTGTCAAGGGCCGGTTCACCATAAGCAGAGACAACTCCCAGAGCATCCTCTACCTGCAGATGAATACCCTGCGAGCCGAGGACTCTGCAACTTACTACTGTGCCCGAGACGTTGGGGTGTACTGGGGCCAGGGTACCCTGGTGACAGTTTCTGCCGAGAGCGCCAGGACCACCCCTCCTAGCGTGTACCCCCTGGCCCCTGGATCCGCCGCTCAGACAAACTCTATGGTGACCCTGGGCTGCCTGGTGAAGGGCTACTTCCCCGAGCCTGTGACAGTCACATGGAACTCCGGCAGCCTGTCTAGCGGCGTGCACACCTTCCCAGCCGTGCTGCAGAGCGACCTGTACACCCTGAGCAGCAGCGTTACCGTGCCTAGCAGCCCTAGACCCTCCGAGACAGTGACATGTAATGTGGCCCACCCAGCCTCCTCCACCAAGGTGGACAAGAAGATCGTGCCTAGGGACTGCGGCTGCAAGCCCTGTATCTGTACAGTGCCTGAGGTGTCCTCCGTGTTCATCTTCCCACCTAAGCCTAAGGATGTGCTGACCATCACACTGACACCTAAGGTGACATGTGTGGTGGTGGATATCTCCAAGGACGATCCTGAGGTGCAGTTTAGCTGGTTTGTGGACGACGTGGAGGTGCACACCGCCCAGACCCAGCCCAGAGAGGAGCAGTTCAATTCCACATTCAGGAGCGTGAGCGAGCTGCCTATCATGCACCAGGATTGGCTGAATGGCAAGGAGTTCAAGTGTAGAGTGAACAGCGCCGCCTTCCCAGCTCCTATCGAGAAGACCATCAGCAAGACAAAGGGCAGACCTAAGGCTCCTCAGGTGTACACCATCCCACCTCCTAAGGAGCAGATGGCCAAGGACAAGGTGAGCCTGACCTGTATGATCACCGATTTCTTCCCAGAGGATATCACCGTGGAGTGGCAGTGGAATGGCCAGCCCGCCGAGAATTACAAGAACACCCAGCCCATCATGAATACAAACGGCTCCTACTTTGTGTACTCCAAGCTGAACGTGCAGAAGTCCAACTGGGAGGCCGGCAATACATTCACCTGCTCCGTGCTGCACGAGGGCCTGCACAATCACCACACCGAGAAGAGCCTGTCCCACAGCCCAGGCAAG(SEQ ID NO：24).

Nucleic acid sequences encoding a desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from vectors known to include the gene, or by direct isolation from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest may be produced synthetically.

In a third aspect, the invention discloses the use of a companion antibody or nucleic acid composition as described above in the preparation of a product for detection of RNase R.

Wherein the product comprises a kit for detecting RNase R by using the paired antibodies

The invention has the following beneficial technical effects:

According to the invention, a pair of Anti-RNase R high-affinity paired antibodies is obtained through mouse immune screening, clone number 15C3F4 is used as an Elisa kit capture antibody, clone number 11A3E4 is used as an Elisa kit detection antibody; the sensitivity of detection of RNase R by the Anti-RNase R pairing antibody is 39pg/ml; RNase R residue detection Elisa kit detection linear range 2.5ng/ml-39pg/ml; the kit can be applied to quantitative and residual detection of RNase R. The method is suitable for quantitative and residual detection of wild RNase R produced by different companies in the market.

Drawings

FIG. 1 is a graph showing the titers of immune antisera after boosting with RNase R in example 1;

FIG. 2 is a graph showing titers of polyclonal hybridoma cells in example 1;

FIG. 3 is a sandwich Elisa plot of antibodies 15C3F4 and 11A3E4 of example 2: RNase R products from three companies;

FIG. 4 is a standard curve of Elisakit detection RNase R in example 3.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications. The starting materials and equipment used in the examples are well known to those skilled in the art and are commercially available or readily available or available.

EXAMPLE 1RNase R immunization and antibody screening

(1) Immunization of mice:

50ug of RNase R (Kactus, RNR-EE 001) was mixed with an equal volume of CFA adjuvant, emulsified and 6-8 week female Balb/C mice were selected for subcutaneous multipoint immunization at 50 ug/dose. The same dose is mixed with IFA adjuvant to emulsify subcutaneous multipoint injection for enhancing immunity after two weeks, and serum titer detection is carried out after three times of immunization every two weeks, wherein the titer is greater than 1: myeloma cell SP2/0 fusion was performed 1000000 later, and 50ug RNase R was used to boost the abdominal cavity once three days before fusion; the immune antisera titers shown in figure 1 >2187000.

(2) Screening of hybridoma cell lines

After 3 days of mouse booster immunization, spleen cells of the mice are fused with myeloma cells SP2/0 in the logarithmic growth phase through PEG1500 to prepare hybridoma cells. After 10 days, cell lines positive for the binding of RNase R antigen and having an OD value of ELISA of more than 3 were screened by detection with an RNase R1. Mu.g/ml plate and indirect ELISA. The positive polyclonal hybridoma supernatants were further diluted 100-fold, and polyclonal hybridoma cells (1 #,11A3,3#,4#,5#,15c3#,7#,8 #) having the high supernatant titers of fig. 2 were selected for subcloning. The polyclonal hybridoma cells are subjected to expansion culture and frozen storage after being subjected to limited dilution to a monoclonal state. The specificity of binding of hybridoma cell line antibodies to RNase R was again verified by indirect ELISA from the supernatant of the expanded hybridoma monoclonal cells to obtain 8 monoclonal antibodies, which included 11A3E4 monoclonal strain obtained by screening from 11A3 polyclonal strain and 15C3F4 monoclonal strain obtained by screening from 15C3 polyclonal strain, and each antibody was purified for antibody pairing test.

Example 2 anti-RNase R monoclonal antibody pair screening and recombinant antibody expression purification

The purified 8 anti-RNase R monoclonal antibodies are used as non-fixed-point biotin labels, and the mol ratio of biotin to antibody is 20:1, biotin-labeled antibody is used as a paired detection antibody. 8 unlabeled monoclonal antibodies were coated onto polyvinyl chloride plates at 2. Mu.g/ml, respectively, as capture antibodies, RNase R was added to Elisa plates coated with 8 different antibodies in 1. Mu.g/ml and 3-fold gradient dilution amounts, respectively, and after one hour of binding, 7 biotin-labeled detection antibodies (2. Mu.g/ml) were added to each Elisa plate, respectively, in addition to the coated antibodies, and SA-HRP developed. The 8 antibodies were paired two by sandwich ELISA, and the pair of antibodies 15C3F4 and 11A3E4 with the highest signal capable of pairing was selected as shown in FIG. 3. Determining 15C3F4 antibody subtype using an antibody subtype identification kit (Wuhan Sanying organism), heavy chain IgG1 and light chain kappa (kappa); 11A3E4 antibody subtype, heavy chain IgG1, light chain kappa (kappa).

The 15C3F4 and 11A3E4 hybridoma cell lines were cultured to a cell density of 1E+07cell/ml and were sent for sequencing of hybridoma monoclonal antibodies (Sony Biotech Co., ltd.). Gene synthesis encodes signal peptide, and antibody heavy chain and light chain gene sequences of variable region and constant region are respectively constructed into mammalian cell expression vectors, and the expression vectors can be selected from any mammalian cell expression commercialized vectors such as pTT5, pCDNA3.1 and the like. Recombinant plasmid transfects an Expi293 mammalian cell, the antibodies 15C3F4 and 11A3E4 are secreted and expressed, and the cell expression supernatant is subjected to protein A affinity purification to obtain the RNase R specific recombinant antibodies 15C3F4 and 11A3E4. The method is used for antibody characterization and Elisa kit development.

Example 3RNAse R residue detection kit

The preparation of the RNase R residue detection kit and the standard curve experiment are briefly described as follows:

1) 15C3F4 capture antibody wrapper

15C3F4 antibody was diluted to 5. Mu.g/ml using 1XPBS, 100. Mu.l per well was added to 96-well polyvinyl chloride plates and after incubation at 37℃for 2 hours the plates were washed once with PBST. 200 μl of 3% BSA in PBST was added to each well, incubated at 37℃for 1 hour, and blocked.

2) RNase R standard preparation and capture

RNase R enzyme at a concentration of 1. Mu.g/ml was diluted to 2.5ng/ml with 1ml of a PBST solution containing 1% BSA to prepare a standard at a concentration of 2.5ng/ml, and then diluted to 1.25ng/ml, 0.625ng/ml, 0.313ng/ml, 0.156ng/ml, 0.078ng/ml, 0.039ng/ml at a 2-fold gradient to obtain 7 concentrations of the standard, and the 1% BSA PBST solution was used as a zero point, and 100. Mu.l was added to each well of the Elisa plate, and after incubation at 37℃for 1 hour, the plate was washed 3 times, and RNase R capturing was completed.

3) Preparation and binding of biotin-labeled 11A3E4 detection antibody

Biotin-labeled 11A3E4 antibody (antibody biotin-labeled as described in example 2) was diluted to 0.5. Mu.g/ml using 1% BSA in PBST, 100. Mu.l per well was added to the Elisa plate in 2), and the plate was washed 3 times after incubation at 37℃for 1 hour.

4) SA-HRP binding color development

SA-HRP was diluted to 0.5. Mu.g/ml using 1% BSA in PBST, 100ul was added to each well of the Elisa plate in 3), and the plate was washed three times after incubation at 37℃for 1 hour. 100 μl of TMB developing solution was added to each well of the Elisa plate, developed at 37℃in the dark for 10-15min, and 50 μl of stop solution was added to each well to terminate the reaction. The OD value at 450nm wavelength was read immediately using a microplate reader. The data of table 1 were obtained.

The standard curve of the RNase R residue detection kit is fitted by four parameters, the linear range of the standard curve is 2.5ng/ml-39pg/ml, the sensitivity of the standard curve is 39pg/ml as shown in FIG. 4, and the kit can quantitatively detect RNase R and residues.

The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.

Claims (7)

1. An Anti-RNAse R antibody comprising antibodies 15C3F4 and 11A3E4; the antibody 15C3F4 has a light chain complementarity determining region CDR1 as shown in SEQ ID NO. 1, a light chain complementarity determining region CDR2 as shown in SEQ ID NO. 2 and a light chain complementarity determining region CDR3 as shown in SEQ ID NO. 3, and a heavy chain complementarity determining region CDR1 as shown in SEQ ID NO. 4, a heavy chain complementarity determining region CDR2 as shown in SEQ ID NO. 5 and a heavy chain complementarity determining region CDR3 as shown in SEQ ID NO. 6;

The antibody 11A3E4 has a light chain complementarity determining region CDR1 as shown in SEQ ID NO. 7, a light chain complementarity determining region CDR2 as shown in SEQ ID NO. 8 and a light chain complementarity determining region CDR3 as shown in SEQ ID NO. 9, and a heavy chain complementarity determining region CDR1 as shown in SEQ ID NO. 10, a heavy chain complementarity determining region CDR2 as shown in SEQ ID NO. 11 and a heavy chain complementarity determining region CDR3 as shown in SEQ ID NO. 12.

2. The Anti-RNAse R antibody of claim 1 wherein the antibody 15C3F4 comprises a light chain variable region of sequence set forth in SEQ ID No. 13 and a heavy chain variable region of sequence set forth in SEQ ID No. 14.

3. The Anti-RNAse R antibody of claim 2 wherein the antibody 15C3F4 comprises the light chain amino acid sequence shown in SEQ ID No. 15; and the heavy chain amino acid sequence shown as SEQ ID NO. 16.

4. The Anti-RNAse R antibody of claim 1 wherein the antibody 11A3E4 comprises a light chain variable region of sequence set forth in SEQ ID No. 17 and a heavy chain variable region of sequence set forth in SEQ ID No. 18.

5. The Anti-RNAse R antibody of claim 4 wherein the antibody 11A3E4 comprises the light chain amino acid sequence shown in SEQ ID No. 19; and the heavy chain amino acid sequence shown as SEQ ID NO. 20.

6. A nucleic acid composition comprising a nucleic acid molecule encoding the antibodies 15C3F4 and 11A3E4 of any one of claims 1-5.

7. Use of an Anti-RNAse R antibody of any of claims 1-5 or a nucleic acid composition of claim 6 in the preparation of a product for the detection of RNAse R.