Anti-biotin antibody and application thereof

Abstract

The present disclosure provides an anti-biotin antibody, and provides an amino acid sequence encoding the CDRs of the antibody. Studies have shown that the antibody only reacts with a biotin conjugate or derivative, and does not react with free biotin. The present disclosure further provides applications of the antibody in, including but not limited to, ELISA, cell capture, sorting and enrichment, western blotting, flow cytometry, immunocytofluorescent staining, and immunohistochemistry. The anti-biotin antibody conjugated immunomagnetic beads can specifically and directly recognize a biotin labeled antigen, and do not bind to free biotin which is often presented in clinical samples and culture media. In addition, the anti-biotin antibody-conjugated magnetic beads or anti-biotin antibody-fluorescein provide an ideal solution for the isolation of specific cells, and can even enrich and separate target cells from samples rich in debris or other rare biological materials.

Claims

1. An anti-biotin antibody, wherein the antibody includes complementary determinant regions (CDR) of a heavy chain and a light chain, the heavy chain of the antibody includes three complementary determinant regions including CDR-H1, CDR-H2, and CDR-H3, and the light chain of the antibody includes other three complementary determinant regions including CDR-L1, CDR-L2 and CDR-L3, the CDR-H1, the CDR-H2, the CDR-H3, the CDR-L1, the CDR-L2 and the CDR-L3 of the antibody comprising the following polypeptide sequences: (1) the CDR-H1 comprising at least 6 amino acid sequences of SEQ ID NO: 1, the sequence being SSYWIC; (2) the CDR-H2 comprising at least 18 amino acid sequences of SEQ ID NO: 2, the sequence being CIDAGSSGSTYYARWVNG; (3) the CDR-H3 comprising at least 14 amino acid sequences of SEQ ID NO: 3, the sequence being EGDWGAPIYYGVDL; (4) the CDR-L1 comprising at least 13 amino acid sequences of SEQ ID NO: 4, the sequence being QSSQSVYNNNQLS; (5) the CDR-L2 comprising at least 7 amino acid sequences of SEQ ID NO: 5, the sequence being YASTLAS; (6) the CDR-L3 comprising at least 12 amino acid sequences of SEQ ID NO: 6, the sequence being LGGYYDYADTSA, wherein, the anti-biotin antibody specifically binds to non-free biotin.

2. The anti-biotin antibody of claim 1, wherein the antibody is a rabbit monoclonal antibody.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a picture of indirect ELISA results of an anti-biotin antibody.

(2) FIG. 2 is a picture of indirect competitive ELISA results of an anti-biotin antibody.

(3) FIG. 3 is a picture of indirect ELISA results of an anti-biotin antibody-conjugated magnetic bead complex.

(4) FIG. 4 is a picture of western blotting results of an anti-PD-L1 monoclonal antibody against NCI-H226 positive cell lysate and MCF-7 negative cell lysate, obtained by anti-biotin antibody conjugated immunomagnetic beads capture, sorting and enrichment.

(5) FIG. 5 is a picture of flow cytometry sorting results of immunized rabbit PBMCs labeled with an anti-biotin antibody.

(6) FIG. 6 is a picture of western blotting results of an anti-biotin antibody.

(7) FIG. 7 is a picture of results of flow cytometry application of an anti-biotin antibody.

(8) FIG. 8 is a picture of results of immunocytochemistry application of an anti-biotin antibody.

(9) FIG. 9 is a picture of results of immunohistochemistry application of an anti-biotin antibody.

DETAILED DESCRIPTION OF THE DISCLOSURE

(10) The present disclosure is further described by the examples, but the present disclosure is not limited to the examples, nor can the examples be construed as limitations of the present disclosure.

Example 1

(11) An anti-biotin antibody disclosed in the technical solution is a rabbit monoclonal antibody. The antibody uses a Kabat method to analyze CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 including the following polypeptide sequences:

(12) (1) CDR-H1 including at least 6 amino acid sequences of SEQ ID NO: 1, the sequence being SSYWIC;

(13) (2) CDR-H2 comprising at least 18 amino acid sequences of SEQ ID NO: 2, the sequence being CIDAGSSGSTYYARWVNG;

(14) (3) CDR-H3 comprising at least 14 amino acid sequences of SEQ ID NO: 3, the sequence being EGDWGAPIYYGVDL;

(15) (4) CDR-L1 comprising at least 13 amino acid sequences of SEQ ID NO: 4, the sequence being QSSQSVYNNNQLS;

(16) (5) CDR-L2 comprising at least 7 amino acid sequences of SEQ ID NO: 5, the sequence being YASTLAS; and

(17) (6) CDR-L3 comprising at least 12 amino acid sequences of SEQ ID NO: 6, the sequence being LGGYYDYADTSA.

(18) The anti-biotin antibody specifically binds to non-free biotin.

(19) The following Examples 2-8 are applications of the antibody in, including but not limited to, ELISA, B cell cloning by magnetic bead method, western blotting, flow cytometry, immunocytofluorescent staining, and immunohistochemistry.

(20) The antibody can recognize the biotin motif of a protein-biotin conjugation complex coated on an ELISA plate by an indirect ELISA method; and the antibody does not recognize free biotins in an indirect competitive ELISA method.

Example 2: Direct Recognition of Biotin Labeled Antigens

(21) A biotin conjugate biotin-X was diluted to a suitable concentration, e.g., 1 μg/mL. An ELISA plate was coated with the biotin conjugate biotin-X, sealed with a sealing film, and incubated at 4° C. overnight.

(22) The next day, the ELISA plate was placed on a plate washer, washed with PBST 3 times, and blocked with skimmed milk or a BSA blocking buffer. The ELISA plate was incubated on a shaker at 30° C. for 1 about h.

(23) The anti-biotin antibody was pre-diluted to a suitable concentration, e.g., 1 μg/mL. Then serial dilution was made by 8 times. After block, the plate was washed 3 times. 50 μL of diluted anti-biotin antibody was added to each well. The plate was incubated at 30° C. on a shaker for about 1 h.

(24) After the plate was washed, 50 μL of HRP-conjugated goat anti-rabbit IgG secondary antibody was added to each well. The plate was incubated at 30° C. on a shaker for about 45 min.

(25) After the plate was washed, 50 μL of TMB was added to each well. The plate was kept at 30° C. in dark for coloration on a shaker for about 15 min.

(26) 50 μL of H.sub.2SO.sub.4 stop solution was added to stop the reaction, and the absorbance was read on a microplate reader after about 5-30 min.

(27) The concentration of the anti-biotin antibody was taken as the abscissa and the OD value was taken as the ordinate to make a four-parameter fitting curve (as shown in FIG. 1), and the EC50 (i.e., the half effective concentration) value of the antibody was determined as 2.52 ng/mL. Therefore, the ELISA sensitivity of the antibody against the biotin-X antigen is 2.52 ng/mL.

Example 3: Specificity to Biotin Labeled Antigens without Reaction with Free Biotin

(28) A biotin conjugate biotin-X was diluted to a suitable concentration, e.g., 1 μg/mL. An ELISA plate was coated with the biotin conjugate biotin-X, sealed with a sealing film, and incubated at 4° C. overnight.

(29) The next day, the ELISA plate was placed on a plate washer, washed with PBST 3 times, and blocked with skimmed milk or a BSA blocking buffer. The ELISA plate was incubated on a shaker at 30° C. for 1 h.

(30) The anti-biotin antibody was pre-diluted to an appropriate concentration for use. Free molecules of biotin were pre-adjusted to a suitable starting concentration. Then serial dilution was made by 10 times. 50 μL of the anti-biotin antibody and 50 μL of the diluted free molecules of biotin were mixed on another clean dilution plate. Then the plate was incubated at 30° C. and 100 rpm on a shaker for about 30 min.

(31) After the ELISA plate was washed, 50 μL of the mixture in step (3) was added to each well. The plate was incubated at 30° C. on a shaker for about 1 h.

(32) After the plate was washed, 50 μL of HRP-conjugated goat anti-rabbit IgG secondary antibody was added to each well. The plate was incubated at 30° C. on a shaker for about 45 min.

(33) After the plate was washed, 50 μL of TMB was added to each well. The plate was kept at 30° C. in dark for coloration on a shaker for about 15 min.

(34) 50 μL of H.sub.2SO.sub.4 stop solution was added to stop the reaction, and the absorbance was read on a microplate reader after about 5-30 min.

(35) According to FIG. 2, the absorbance basically does not change after the addition of the free biotin at different concentrations. It can be seen that the addition of free biotin does not affect the binding of the antibody to the biotin conjugate biotin-X, which shows that the antibody only binds to conjugated biotin, while does not bind to free biotin. the anti-biotin antibody shows very good specificity.

Example 4: Cell Capture, Sorting and Enrichment, and Monoclonal Antibody Preparation

(36) Cell capture and sorting include but are not limited to a magnetic bead method and a flow cytometry method.

(37) The embodiments of cell capture, sorting and enrichment, and monoclonal antibody preparation by the magnetic bead method are as follows:

(38) The anti-biotin antibody was cross-linked to immunomagnetic beads to obtain anti-biotin antibody-immunomagnetic beads. After ELISA, as shown in FIG. 3, the antibody activity remains the same as before conjugation. After being filtered with a 0.45 μm filter membrane, the anti-biotin antibody-immunomagnetic beads were stored at 4° C. for later use.

(39) Biotin was cross-linked to a peptide or a protein in a proper way to obtain a biotinylated polypeptide/protein complex (biotin-Y). The biotinylated polypeptide/protein complex (biotin-Y) was filtered with a 0.22 μM filter membrane and stored at −20° C. for later use.

(40) Lymphocytes from animals immunized with antigen Y were taken, and the biotin-Y complex was added. The lymphocytes were incubated at 4-37° C. for 1-120 min, or a longer time accordingly, cells were washed with DPBS or a culture medium several times, and the supernatant was removed by centrifugation.

(41) The washed cells were resuspended in DPBS or the culture medium, and a certain number of immunomagnetic beads were added and mixed at 4-37° C. for about 1-120 min, or a longer time accordingly.

(42) After the incubation, the sample was applied to a magnetic field, and the magnetic bead-anti-biotin antibody-biotin-cell complex was enriched at the bottom. The supernatant was pipetted and discarded, then the magnetic field was removed. DPBS or the culture medium was added to wash the cells several times. Then a complete culture medium was added to resuspend the cells.

(43) After the cells were counted, the cells were adjusted to a suitable concentration and inoculated into a cell culture plate or culture dish, and the cell culture plate or culture dish was supplemented with sufficient culture medium.

(44) The cells were cultured for several days and then the supernatant was taken for detection.

(45) B cell clones meeting the criteria were collected to prepare monoclonal antibodies. The WB detection result of a PD-L1 monoclonal antibody prepared according to the method is shown in FIG. 4. A membrane was prepared with a positive cell lysate NCI-H226 and a negative cell lysate for the PD-L1, and the protein loading quantity of the two lysates both was 40 μg/lane. According to the result in the figure, a clear band at 40-60 KDa can be observed on the NCI-H266 membrane by probing of PD-L1 B cell clone supernatant, while no blotting bands on the MCF-7 membrane. The result shows that the monoclonal antibody prepared by enrichment and sorting with the anti-biotin antibody by the magnetic bead method has excellent specificity.

(46) Free biotin is often presented in the culture medium. The anti-biotin antibody-conjugated magnetic beads described in the experiment have the advantage of not binding to free biotin. The feature provides the most sensitive labeling and cell isolation. The anti-biotin antibody-conjugated magnetic beads provide an ideal solution for the isolation of specific cells, and can even separate cells from samples rich in debris or other rare biological materials.

(47) The embodiments of cell capture, sorting and enrichment, and monoclonal antibody preparation by the flow cytometry method are as follows:

(48) The anti-biotin antibody was cross-linked to a fluorescein to obtain an anti-biotin antibody-fluorescein. The anti-biotin antibody-fluorescein was filtered with a 0.22 μm filter membrane and stored at 4° C. for later use.

(49) Biotin was cross-linked to a peptide or a protein in a proper way to obtain a biotinylated polypeptide/protein complex (biotin-Y). The biotinylated polypeptide/protein complex (biotin-Y) was filtered with a 0.22 μM filter membrane and stored at −20° C. for later use.

(50) Lymphocytes from animals immunized with antigen Y were taken, and the biotin-Y complex was added. The lymphocytes were incubated at 4-37° C. for 1-120 min, or a longer time accordingly, cells were washed with DPBS or a culture medium several times, and the supernatant was removed by centrifugation.

(51) The washed cells were resuspended in DPBS or the culture medium, and a certain amount of anti-biotin antibody-fluorescein and/or other antibodies, such as anti-CD19 antibody, anti-CD138 antibody and anti-IgG antibody were added. The mixture was mixed at 4-37° C. for 1-120 min, or incubated for a longer time accordingly, cells were washed with DPBS or the culture medium several times, and the supernatant was removed by centrifugation. The cells were resuspended, with an appropriate amount of DPBS or culture medium, to be loaded for flow cytometry.

(52) Negative gating is set by the cells without the anti-biotin antibody fluorescein, then cells labeled with the anti-biotin antibody fluorescein are loaded to the sorter. Finally, cells are collected to the tube by combination of the signals of anti-CD19, anti-CD138 and anti-IgG antibodies.

(53) After the cells were counted, the cells were adjusted to a suitable concentration and inoculated into a cell culture plate or culture dish, and the cell culture plate or culture dish was supplemented with sufficient culture medium.

(54) The cells were cultured for several days and then the supernatant was taken for detection.

(55) B cell clones meeting the criteria were collected for monoclonal antibody generation. As shown in FIG. 5, the anti-biotin antibody was used for fluorescein activated cell sorting. The left picture shows the control group (without addition of the anti-biotin antibody-FITC), and the right picture shows the experimental group (with addition of the anti-biotin antibody-FITC). After the test, the control group had about 0.18% background cells, and the experimental group had 1.97% specific lymphocytes that could be sorted out for monoclonal antibody preparation. So, monoclonal antibodies can be generated by FACs using the fluorescein labeled anti-biotin antibody, which makes the monoclonal antibody generation more effective.

(56) Free biotin is often presented in the culture medium. The fluorescein labeled anti-biotin antibody described in the experiment has the advantage of not binding to free biotin. The fluorescein labeled anti-biotin antibody provides an ideal solution for cell isolation by FACs.

Example 5: Western Blotting

(57) The biotin conjugate biotin-X was loaded to an SDS-PAGE at 2 ng/lane for electrophoresis.

(58) The prepared gel was then transferred onto a PVDF membrane.

(59) After the PVDF membrane was activated with methanol, the PVDF membrane was blocked with 5% skimmed milk powder/PBS at room temperature for about 1 h. 2 μL of 0.5 mg/mL free D-biotin was mixed with 1:2000 and 1:5000 prediluted anti-biotin antibodies (the original concentration of the antibody was 0.500 mg/mL) and incubated at 30° C. for about 40 min, then the membrane was rinsed 3 times with TBST, and then the membrane was washed with TBST for about 10 min.

(60) One lane of the PVDF membrane was probed with 75 μL of the complex of the antibody and free D-biotin, and another lane was probed with 75 μL of the anti-biotin antibody only, and incubated at room temperature for 1 h on a shaker, After the incubation, the membrane was washed twice with TBST for about 10 min each.

(61) The 1:4000 prediluted goat anti-rabbit IgG-HRP secondary antibody was added, and incubated at room temperature for 1 h on a shaker. The membrane was washed 3 times with TBST for about 10 min each.

(62) 1.5 mL of ECL was added to each membrane. The membranes were allowed for coloration for 5 min at room temperature, and then the signal was read in an imager by exposure for 60 s.

(63) The picture taken by the gel imager is shown in FIG. 6. Lanes 1, 2, 3 and 4 indicate that the antibody can react with conjugated biotin, and show a single band with the size of about 45 kD. Lane 2 shows that the antibody does not react with free biotin, but only reacts with biotin conjugated to protein X on the PVDF membrane, and the band size and position have no significant difference compared with lane 1. Lane 4 shows that the antibody does not react with free biotin, but only reacts with biotin conjugated to protein X on the PVDF membrane, and the band size and position have no significant difference compared with lane 3.

Example 6: Flow Cytometry

(64) Hela cells were washed with PBS, and centrifuged at 1500 rpm for 5 min at 4° C., and the cell density was adjusted to 3 M/mL.

(65) 100 μL of 4% paraformaldehyde solution was added to 1 M cells and mixed gently. The cells were fixed at room temperature for about 10 min.

(66) PBS was added to stop fixation, the cells were centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. Cell pellets were resuspended and washed with PBS, then the cells were centrifuged at 1500 rpm for 5 min again.

(67) 100 μL of 0.1% TritonX-100 (2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol) was added to 1 M cells, and mixed gently. Then cells were incubated at room temperature for about 15 min to permeabilize cell membrane.

(68) The step (3) was repeated to wash the cells.

(69) 3 clean 1.5 mL centrifuge tubes were prepared and 100 μL of 3 M/mL Hela cells were added to each tube, that is, each tube contains 0.3 M Hela cells. After centrifugation, the supernatant was discarded. 100 μL of PBS was added to one tube as a negative control, 100 μL of 1:200 biotin-conjugated anti-cytokeratin 8 (CK8) rabbit monoclonal antibody was added to another tube, 100 μL of 5 μg/mL rabbit isotype IgG was added to the third tube, and all cells were incubated at room temperature for about 1 h.

(70) The step (3) was repeated to wash the cells.

(71) 100 μL of the 1:100 prediluted FITC-labeled anti-biotin antibody was added to each tube. The step (3) was repeated to wash the cells after incubation at room temperature for 40 min, and 200 μL of PBS was added to each tube to detect the fluorescein intensity of the cells in each tube on a flow cytometer.

(72) The results are shown in FIG. 7. The unfilled black histogram on the left is the isotype control group indicating that the Hela cells detected by isotype IgG-biotin conjugate and the FITC-labeled anti-biotin antibody. The histogram filled with black on the right is the experimental group indicating that the Hela cells detected by the anti-cytokeratin 8-biotin conjugate and the FITC-labeled anti-biotin antibody. From the figure, it can be seen that the peak of the experimental group shifts more to the right than the isotype control group, indicating that the anti-biotin antibody can be used in flow cytometry experiments.

Example 7: Immunofluorescent Staining

(73) Hela cells grown in a 12-well plate at confluency of 50%-60% were washed twice with PBS.

(74) 1 mL of 4% paraformaldehyde solution was added to each well, and allowed to stand at room temperature for about 30 min for fixation.

(75) The fixative solution was poured out. 1 mL of 1% BSA/PBST was added to each well. The cells were washed on a shaker for about 5 min, and then the washing solution was poured out. The step was repeated twice.

(76) 1 mL of 0.1% TritonX-100 (2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol) was added to each well. Then cells were incubated at room temperature for about 10 min to permeabilize cell membrane. The step (3) was repeated to wash the cells.

(77) 1 mL of 10% goat serum was added to each well and incubated at room temperature for about 30 min to block non-specific antigen sites on cells. The step (3) was repeated to wash the cells.

(78) 1 mL of 1% BSA/PBST was added to one well as a negative control, 1 mL of 1:200 biotin-conjugated anti-cytokeratin 8 rabbit monoclonal antibody was added to another well, 1 mL of 5 μg/mL rabbit isotype IgG antibody was added to the third well, and the cells were incubated at room temperature for about 1 h.

(79) The step (3) was repeated to wash the cells. 1 mL of the 1:100 prediluted FITC-labeled anti-biotin antibody was added to each well. The cells were incubated at room temperature for about 50 min and then step (3) was repeated to wash the cells.

(80) A drop of antifade mounting solution with DAPI was added on a blank glass slide. A cell slide was carefully clamped out of a well with tweezers. Then the cell slide (the side with the cells) was covered to the mounting solution, and let dry at room temperature in dark.

(81) The slide was observed under a fluorescence microscope, and photos were taken.

(82) The results are shown in FIG. 8. There are 4 small pictures in the figure. The picture in the upper left corner shows the signal of anti-cytokeratin 8 monoclonal antibody at 1:200 dilution. The picture was taken with a green fluorescein channel to locate the cytokeratin 8 protein in the cytoplasm. The picture in the upper right corner shows the signal of anti-cytokeratin 8 monoclonal antibody at 1:200 dilution, and the picture was taken with a blue fluorescein channel to locate the cell nucleus. The picture in the lower left corner shows the merged signal of the green and blue channel. The picture in the lower right corner shows the merged signal of negative control. It can be seen that only the anti-CK8 antibody showed green cytoplasma staining signal. The anti-biotin antibody can be used in immunofluorescent staining experiments.

Example 8: Immunohistochemistry

(83) Human endometrium tissue was selected for CK-8 staining in IHC experiment.

(84) The endometrium tissue slides were fixed in an oven at 62° C. for about 1 h.

(85) Tris-EDTA (pH 9.0) was used for antigen retrieval.

(86) 100 μL of the 1:50 prediluted biotin-conjugated anti-cytokeratin 8 rabbit monoclonal antibody was added to the slides. The slides were incubated at room temperature for about 30 min, and then washed.

(87) The 1:100 prediluted HRP labeled anti-biotin antibody was added to the slides. The slides were incubated at room temperature for about 30 min, and then washed.

(88) 100 μL of DAB was added. Color was developed at room temperature for about 2 min, and the slides were rinsed with water.

(89) The slides were put into a hematoxylin staining solution for 2 min to stain the nucleus and then washed with water. Then the slides were put in PBST to return to blue for 60 s.

(90) After dehydration and transparency, the slides were mounted and let dry in a fume hood. The slides were photographed under a microscope.

(91) The results are shown in FIG. 9. In the figure, cytokeratin 8 has specific cytoplasmic staining on the glandular epithelium of the endometrium. The staining result is located correctly, and no non-specific staining and background staining are presented. The antibody can be used in immunohistochemistry experiments.