Monoclonal antibody against soluble transferrin receptor as well as preparation method and application thereof

Abstract

The invention discloses an anti-soluble transferrin receptor monoclonal antibody as well as a preparation method and application thereof, and belongs to the technical field of antibody detection. According to the antibody, an L101-F760 amino acid sequence of human TfR protein is used as an immunogen, specific screening of B cells, reverse RNA (Ribonucleic Acid) into cDNA (Complementary Deoxyribose Nucleic Acid) through RT-PCR (Reverse Transcription-Polymerase Chain Reaction), then homologous recombination is performed to construct heavy and light chain vectors, and finally multiple rounds of screening are performed to obtain the purified sTfR monoclonal antibody, so that the sTfR protein can be specifically recognized, and the antibody has the advantages of high specificity and high sensitivity and can be widely applied to the field of immunotherapy. The natural sTfR protein in human serum can be recognized. The kit is applied to the detection and screening fields of immunohistochemistry, immunofluorescence chemistry, western blot, co-immunoprecipitation, indirect ELISA, flow cytometry and the like, especially has low background and no interference of non-specific binding impure protein during immunohistochemistry detection, can obtain more accurate detection and evaluation results, avoids interference of false positive and false negative, and has high detection accuracy. The method has great application value in the aspects of basic scientific research and clinical pathology detection.

Claims

1. An anti-soluble transferrin receptor monoclonal antibody or an antigen-binding fragment thereof, characterized in that: include: (i) a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO. 2 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO. 3; or (ii) a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO. 4 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO. 5; or (iii) the heavy chain variable region having an amino acid sequence as shown in SEQ ID NO.6, and the light chain variable region having an amino acid sequence as shown in SEQ ID NO.

7.

2. The anti-soluble transferrin receptor monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that The antibody comprises a heavy chain constant region whose amino acid sequence is shown in SEQ ID NO.8 and a light chain constant region whose amino acid sequence is shown in SEQ ID NO.

9.

3. The anti-soluble transferrin receptor monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a single-chain Fv protein or a disulfide-stabilized Fv protein.

4. A nucleic acid, characterized in that The nucleic acid encodes the anti-soluble transferrin receptor or antigen-binding fragment thereof according to any one of claims 1 to 3, or the heavy chain variable region and / or light chain variable region thereof.

5. The nucleic acid according to claim 4, characterized in that The nucleotide sequence of the nucleic acid encoding the heavy chain variable region with the amino acid sequence shown in SEQ ID NO.2 is shown in SEQ ID NO.10; The nucleotide sequence of the nucleic acid encoding the light chain variable region with the amino acid sequence shown in SEQ ID NO.3 is shown in SEQ ID NO.11; The nucleotide sequence of the nucleic acid encoding the heavy chain variable region with the amino acid sequence shown in SEQ ID NO.4 is shown in SEQ ID NO.12; The nucleotide sequence of the nucleic acid encoding the light chain variable region with the amino acid sequence shown in SEQ ID NO.5 is shown in SEQ ID NO.13; The nucleotide sequence of the nucleic acid encoding the heavy chain variable region with the amino acid sequence shown in SEQ ID NO.6 is shown in SEQ ID NO.14; The nucleotide sequence of the nucleic acid encoding the light chain variable region with the amino acid sequence shown in SEQ ID NO.7 is shown in SEQ ID NO.

15.

6. A recombinant expression vector, characterized in that: The recombinant expression vector contains the nucleic acid according to claim 4 or 5.

7. A recombinant expression cell, characterized in that The recombinant expression cell comprises the recombinant expression vector according to claim 6, or the nucleic acid encoding the anti-soluble transferrin receptor monoclonal antibody or the antigen-binding fragment thereof according to claim 4 or 5.

8. Use of the nucleic acid encoding the anti-soluble transferrin receptor monoclonal antibody or the antigen-binding fragment thereof according to claim 4 or 5, or the recombinant expression vector according to claim 6, or the recombinant expression cell according to claim 7 in the preparation of the anti-soluble transferrin receptor monoclonal antibody or the antigen-binding fragment thereof.

9. A method for preparing an anti-soluble transferrin receptor monoclonal antibody or an antigen-binding fragment thereof, comprising: The recombinant expression vector of claim 6 is used to transfect cells to obtain recombinant expression cells, and the recombinant expression cells are cultured; or the recombinant expression cells of claim 7 are cultured; the supernatant is collected and purified to obtain an anti-soluble transferrin receptor monoclonal antibody or an antigen-binding fragment thereof.

10. Use of the anti-soluble transferrin receptor monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the nucleic acid encoding the anti-soluble transferrin receptor monoclonal antibody or antigen-binding fragment thereof according to claim 4 or 5, or the recombinant expression vector according to claim 6, or the recombinant expression cell according to claim 7 in detecting soluble transferrin receptor or preparing a product for detecting soluble transferrin receptor.

11. The use according to claim 10, characterized in that The detection includes any one or more of ELISA, immunoblotting, cell immunofluorescence and immunohistochemistry.

12. A kit for detecting soluble transferrin receptor, characterized in that: The kit comprises the anti-soluble transferrin receptor monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

13. The kit for detecting soluble transferrin receptor according to claim 12, characterized in that: The kit comprises: an anti-soluble transferrin receptor monoclonal antibody or an antigen-binding fragment thereof having a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.6 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.7; and An anti-soluble transferrin receptor monoclonal antibody or an antigen-binding fragment thereof having a heavy chain variable region as shown in SEQ ID NO.2 and a light chain variable region as shown in SEQ ID NO.3; or an anti-soluble transferrin receptor monoclonal antibody or an antigen-binding fragment thereof having a heavy chain variable region as shown in SEQ ID NO.4 and a light chain variable region as shown in SEQ ID NO.5.

Images