Antibody-immobilized carrier, method of producing antibody-immobilized carrier, and use of said antibody-immobilized carrier
The present invention provides an antibody-immobilized carrier that can be used in antibody screening, a method of producing the antibody-immobilized carrier, and use of the antibody-immobilized carrier. Efficient antibody screening can be carried out particularly by an antibody-immobilized carrier including two or more antibody immobilized regions onto each of which a heavy-chain low-molecular-weight antibody and a light-chain low-molecular-weight antibody are separately immobilized, the two or more antibody immobilized regions each being included in an independent manner, the heavy-chain low-molecular-weight antibody including a heavy-chain variable region, the light-chain low-molecular-weight antibody including a light-chain variable region, the heavy-chain low-molecular-weight antibody and the light-chain low-molecular-weight antibody each being derived from an antibody recognizing a different antigen.
1. A method of producing an antibody-immobilized carrier surface, comprising:
immobilizing one or more heavy-chain low-molecular-weight antibody fragments comprising a heavy-chain variable region (VH) fused to the N-terminus of a carrier binding peptide wherein the carrier binding peptide is selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19; and
immobilizing one or more light-chain low-molecular-weight antibody fragments comprising a light-chain variable region (VL) fused to the N-terminus of a carrier binding peptide wherein the carrier binding peptide is selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19 sequentially onto a carrier surface, wherein the material of the carrier surface is selected from the group consisting of a polystyrene plastic resin, a polycarbonate plastic resin, a polymethyl methacrylate plastic resin, a plastic resin obtained through hydrophilizing a polystyrene plastic resin, a plastic resin obtained through hydrophilizing a polycarbonate plastic resin, and a plastic resin obtained through hydrophilizing a polymethyl methacrylate plastic resin,
so that an antibody immobilized carrier surface is prepared.
2. The method as set forth in claim 1 , wherein the steps of immobilizing is repeated at least two times.
3. The method as set forth in claim 1 , wherein the step of immobilizing comprising:
(a) denaturing insoluble aggregates of said heavy-chain low-molecular-weight antibody fragments and said light-chain low-molecular-weight antibody fragments with a denaturing agent prior to said immobilizing; and
(b) refolding said denatured heavy-chain low-molecular-weight antibody fragments and said denatured light-chain low-molecular-weight antibody fragments, by removing the denaturing agent from said immobilized low-molecular-weight antibody fragments.
4. The method as set forth in claim 3 , wherein steps (a) and (b) are carried out separately for each of the heavy-chain low-molecular-weight antibody fragments and the light-chain low-molecular-weight antibody fragments.
5. The method as set forth in claim 4 , wherein
steps (a) and (b) are carried out for the light-chain low-molecular-weight antibody fragment prior to the heavy-chain low-molecular-weight antibody fragments.
6. The method as set forth in claim 3 , wherein
the denaturing agent is urea at a concentration range of 0.5 M to 4 M.
7. An antibody-immobilized carrier surface obtained by the method as set forth in claim 2 .
8. The method as set forth in claim 1 , wherein:
the heavy-chain low-molecular-weight antibody fragment and the light-chain low-molecular-weight antibody fragment are each being from an antibody recognizing a different antigen.