Chicken antibody transformed into cysteine and site-specific conjugation using same

Abstract

A framework fragment derived from a chicken antibody, in which an amino acid at a particular position is replaced by cysteine, a heavy chain variable region or a light chain variable region including the framework fragment, or an antibody including the heavy chain variable region or the light chain variable region does not induce or prevents formation of disulfide bonds between antibody molecules while maintaining activity and reactivity of the antibody. The antibody introduced with cysteine or an antigen-binding fragment thereof easily binds with a conjugation compound such as a chemotherapeutic drug, an enzyme, an aptamer, a toxin, an affinity ligand, or a detection label, thereby being applied to various fields for diagnosis or treatment of diseases.

Claims

1. An isolated cysteine-modified chicken antibody that binds to prostate specific antigen comprising at least one modified light chain framework or at least one heavy chain framework, wherein the at least one modified light chain framework comprises an amino acid sequence selected from the group consisting of SEQ ID NO:9 to SEQ ID NO: 56 and the at least one modified heavy chain framework comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 57 to SEQ ID NO: 88.

2. The modified chicken antibody of claim 1, wherein the modified antibody is prepared by a method comprising introducing a mutation a nucleic acid sequence encoding the modified antibody; expressing the modified antibody; and isolating and purifying the modified antibody.

3. An antibody complex prepared by conjugating the modified chicken antibody of claim 1 with one or more compounds selected from the group consisting of a drug, an enzyme, an aptamer, a toxin, an affinity ligand, and a detection label.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1a shows a sequential numbering scheme (top row) in comparison with the Kabat numbering scheme (bottom row) for base sequences of wild-type and germ-line of a chicken antibody used in the present experiment;

(2) FIG. 1b shows a cartoon depiction of Cys-modified antibody phage binding to immobilized PSA with binding of anti-phage HRP antibody for absorbance detection;

(3) FIGS. 1c and 1d show binding measurements with detection of absorbance at 405 nm of Cys-modified antibody phages (upper: modified light chain, lower: modified heavy chain);

(4) FIG. 1e shows a three-dimensional representation of a chicken antibody fragment derived by X-ray crystal coordinates, in which the structure positions of 5 types of the engineered Cys residues of the heavy and light chains are shown;

(5) FIG. 2 shows binding measurements with detection of absorbance at 405 nm of antibody phages including two cysteine residues having thiol reactivity;

(6) FIGS. 3a and 3b show binding measurements with detection of absorbance at 405 nm of Cys-modified antibody phages with replaced with cationic amino acids (upper: modified light chain, lower: modified heavy chain);

(7) FIGS. 3c and 3d show binding measurements with detection of absorbance at 405 nm of Cys-modified antibody phages with replacement of anionic amino acid (upper: modified light chain, lower: modified heavy chain);

(8) FIG. 4a shows coomassie blue staining of Cys-modified single chain Fv (scFv)-type antibodies expressed from cell culture for conjugation;

(9) FIG. 4b shows coomassie blue staining of cationic or anionic Cys-modified scFv-type antibodies expressed from cell culture for conjugation;

(10) FIG. 5a shows coomassie blue staining of Cys-modified full IgG-type antibodies expressed from cell culture for conjugation;

(11) FIG. 5b shows coomassie blue staining of cationic or anionic Cys-modified full IgG-type antibodies expressed from cell culture for conjugation;

(12) FIG. 6 shows a cartoon depiction of conjugation of maleimide activated PEG to Cys-modified antibody;

(13) FIG. 7a shows coomassie blue staining of maleimide PEG-conjugated scFv;

(14) FIG. 7b shows iodine staining of maleimide PEG-conjugated scFv;

(15) FIG. 7c shows immunoblotting of maleimide PEG-conjugated scFv;

(16) FIG. 8a shows coomassie blue staining of maleimide PEG-conjugated full IgG;

(17) FIG. 8b shows iodine staining of maleimide PEG-conjugated full IgG;

(18) FIG. 8c shows immunoblotting of maleimide PEG-conjugated full IgG;

(19) FIG. 9a shows coomassie blue staining of maleimide PEG-conjugated scFv with replacement of cationic or anionic residue;

(20) FIG. 9b shows iodine staining of maleimide PEG-conjugated scFv with replacement of cationic or anionic residue;

(21) FIG. 9c shows immunoblotting of maleimide PEG-conjugated scFv with replacement of cationic or anionic residue;

(22) FIG. 10a shows coomassie blue staining of maleimide PEG-conjugated full IgG with replacement of cationic or anionic residue;

(23) FIG. 10b shows iodine staining of maleimide PEG-conjugated full IgG with replacement of cationic or anionic residue;

(24) FIG. 10c shows immunoblotting of maleimide PEG-conjugated full IgG with replacement of cationic or anionic residue;

(25) FIG. 11a shows a cartoon depiction of conjugation of GMBS-cotinine to Cys-modified antibody;

(26) FIG. 11b shows a cartoon depiction of GMBS-cotinine-conjugated, Cys-modified antibody binding to immobilized PSA with binding of anti-cotinine HRP antibody for absorbance detection;

(27) FIG. 12 shows detection of absorbance at 450 nm of binding of GMBS-cotinine to C.sub.kappa-fused, Cys-modified scFv (upper: detection with anti-cotinine IgG-HRP, lower: detection with anti-C.sub.kappa IgG-HRP);

(28) FIG. 13a shows injection time of cotinine-Cys into mice and blood collection time; and

(29) FIG. 13b shows detection of absorbance at 450 nm to examine the presence or absence of cotinine-Cys-modified antibody in the serum before and after injection of cotinine-Cys-modified antibody into mice.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(30) The present invention will be described in more detail with reference to specific Examples of the present invention. However, the present invention is not limited to the following Examples, and it will be apparent to those skilled in the art that various modifications and changes may be made thereto without departing from the scope and spirit of the present invention. Therefore, the appended claims should be interpreted broadly, as may be consistent with the spirit and scope of the present invention.

Example 1: Preparation of Antibody Having One Cys Modification

(31) 1-1. Preparation of Modified Antibody Phage

(32) An antibody used in Example was a chicken antibody against PSA (prostate specific antigen), and FIG. 1a shows base sequences of frameworks of anti-PSA antibody (wild-type) and chicken germ-line ([literature [Andris-Widhopf J. et al., J Immunol. Methods (2000) 242: 159-181]). Kabat numbering system is available at http://www.bioinf.org.uk/abs/.

(33) For preparation of Cys-modified antibody, PCR was first performed to insert NNK (N:A/C/G/T, K:G/T) into 78 light chain domain framework (V.sub.L domain framework) residues and 84 heavy chain domain framework (V.sub.H domain framework) residues in order to randomize the respective residues, in accordance with the literature [Chung J. et al., FASEB J. (2004) 18: 361-383]. Thereafter, in accordance with a method described in Barbas, C. F. (2001) Phage display: a laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, the obtained scFv (single chain Fv) DNA was inserted into a phagemid vector pcomb3X, which was transformed into E. coli ER2738. Then, 92 colonies were selected according to each residue to obtain a culture supernatant containing phage-displayed scFv (single chain Fv).

(34) 1-2. Selection of Modified Antibodies and Measurement of Their Affinity to Antigen

(35) The culture supernatant containing the phage antibodies was used to perform enzyme-linked immunosorbent assay (ELISA) as in a method illustrated in FIG. 1b. A microtiter plate was coated with an antigen PSA, and the culture supernatant containing phages was added thereto, followed by incubation for 1 hour. After washing three times, HRP (horse radish peroxidase)-conjugated anti-M13 antibody was added thereto. After incubation for 1 hour and washing three times, H.sub.2O.sub.2-added ABTS was added to examine color development. Then, absorbance at 405 nm was measured. As a control group, a wild-type phage was used. Even after 21 light chain residues and 27 heavy chain residues were replaced by cysteine, they showed affinity similar to that of the wild-type antibody (FIGS. 1c and 1d). In Tables 1a and 1b, sequential numbering and Kabat numbering of the Cys-modified antibodies, and a base sequence of the wild-type of each modified antibody are given.

(36) TABLE-US-00005 TABLE 1a Light chain framework Sequence Kabat Wild type Light chain Cys mutant numbering numbering amino acid Framework 1 LF1-2 L3 L5 T LF1-3 L4 L6 Q LF1-4 L5 L7 P LF1-11 L12 L14 N LF1-15 L16 L18 T Framework 2 LF2-3 L31 L37 Q LF2-12 L40 L45 V Framework 3 LF3-1 L52 L57 N LF3-2 L53 L58 I LF3-5 L56 L61 R LF3-7 L58 L63 S LF3-8 L59 L64 G LF3-9 L60 L65 S LF3-10 L61 L66 T LF3-13 L64 L69 S LF3-14 L65 L70 T LF3-20 L71 L76 T LF3-27 L78 L83 A LF3-31 L82 L87 Y Framework 4 LF4-5 L98 L102 T LF4-9 L102 L106 V

(37) TABLE-US-00006 TABLE 1b Heavy chain framework Sequence Kabat Wild type Heavy chain Cys mutant numbering numbering amino acid Framework 1 HF1-1 H1 H1 A HF1-3 H3 H3 T HF1-5 H5 H5 D HF1-6 H6 H6 E HF1-7 H7 H7 S HF1-11 H11 H11 L HF1-12 H12 H12 Q HF1-13 H13 H13 T HF1-16 H16 H16 G HF1-18 H18 H18 L HF1-19 H19 H19 S HF1-24 H24 H24 A HF1-25 H25 H25 S Framework 2 HF2-7 H42 H42 G HF2-13 H48 H48 V Framework 3 HF3-4 H70 H69 I HF3-7 H73 H72 D HF3-9 H75 H74 G HF3-10 H76 H75 Q HF3-12 H78 H77 T HF3-21 H87 H83 R HF3-22 H88 H84 A HF3-23 H89 H85 E HF3-25 H91 H87 T HF3-27 H93 H89 T Framework 4 HF4-8 H123 H110 I HF4-11 H126 H113 S

(38) In order to examine solvent accessibility of the selected Cys-modified antibodies, solvent accessible surface area was examined by a three-dimensional structure of a chicken antibody fragment derived by X-ray crystal coordinates (literature [Shih H. H. et al., J Biol. Chem. (2012) 287:44425-44434; PDB ID: 4GLR]), as shown in Tables 2a and 2b. Of them, structural positions of 5 types of base (LF1-2, LF1-3, LF1-4, HF1-13, HF1-16) residues are shown in FIG. 1e.

(39) TABLE-US-00007 TABLE 2a Solvent accessibility of light chain Light chain Cys mutant Solvent accessibility area (A.sup.2) Framework 1 LF1-2 95.52 LF1-3 17 LF1-4 55.89 LF1-11 116.46 LF1-15 7.5 Framework 2 LF2-3 17.89 LF2-12 69.64 Framework 3 LF3-1 139.13 LF3-2 26.94 LF3-5 42.19 LF3-7 46.25 LF3-8 12.95 LF3-9 62.79 LF3-10 45.55 LF3-13 48.57 LF3-14 49.06 LF3-20 72.89 LF3-27 59.26 LF3-31 10.64 Framework 4 LF4-5 0 LF4-9 6.5

(40) TABLE-US-00008 TABLE 2b Solvent accessibility of heavy chain Heavy chain Cys mutant Solvent accessibility area (A.sup.2) Framework 1 HF1-1 127.31 HF1-3 73.16 HF1-5 71.13 HF1-6 6.42 HF1-7 62.7 HF1-11 147.06 HF1-12 29.81 HF1-13 91.96 HF1-16 21.38 HF1-18 12.61 HF1-19 42.57 HF1-24 0.88 HF1-25 45.66 Framework 2 HF2-7 80.67 HF2-13 1.53 Framework 3 HF3-4 4.97 HF3-7 67.97 HF3-9 72.49 HF3-10 121.23 HF3-12 28.12 HF3-21 110.03 HF3-22 70.68 HF3-23 118.19 HF3-25 28.23 HF3-27 5.82 Framework 4 HF4-8 89.14 HF4-11 101.82

Example 2: Preparation of Antibody Having Two Cys Modifications

(41) From the residues of light chain (L3, L4, L5) and heavy chain (H13, H16) according to sequential numbering, each one residue of the light chain and the heavy chain was replaced by cysteine in combination, and total 6 types (L3H13, L3H16, L4H13, L4H16, L5H13, L5H16) of Cys-modified antibody genes with two reactive cysteines were prepared by PCR. Next, they were expressed in the form of phage-displayed scFv as in Example 1, and then their affinity to PSA antigen was measured by enzyme-linked immunosorbent assay (FIG. 2).

Example 3: Modified Antibody Having Replacement of Amino Acid Residues Adjacent to Cys

(42) To replace amino acids flanking both sides of 5 types of Cys-modified antibodies (LF1-2, LF1-3, LF1-4, HF1-13 and HF1-16) prepared in Example 1-1 for efficient conjugation by cationic amino acids (arginine, lysine) or anionic amino acids (aspartic acid, glutamic acid), DNA base sequences (arginine; CGT, lysine; AAG, aspartic acid; GAT, glutamic acid; GAA) corresponding to the respective residues were inserted by PCR. scFv DNAs thus obtained were inserted into a phagemid vector pcomb3X, respectively and then transformed into E. coli ER2738. Next, the antibodies were incubated in the form of phage-displayed scFv in SB culture medium. Base sequences of the Cys-modified antibodies with replacement of cationic or anionic residues are given in Tables 3a and 3b.

(43) For measurement of antigen affinity, a microtiter plate was coated with an antigen PSA, and then the culture supernatant containing phages was added. After incubation for 1 hour and washing three times, HRP (horse radish peroxidase)-conjugated anti-M13 antibody was added thereto. After incubation for 1 hour and washing three times, H.sub.2O.sub.2-added ABTS was added to examine color development. Then, absorbance at 405 nm was measured. The result of cationic Cys-modified antibodies is shown in FIGS. 3a and 3b and the result of anionic Cys-modified antibodies are shown in FIGS. 3c and 3d.

(44) TABLE-US-00009 Sequence SEQ Sequence SEQ Sequence SEQ Mutant Kabatnumbering ID Mutant Kabatnumbering ID Mutant Kabatnumbering ID name (L3-L4-L5-L6-L7) NO name (L4-L5-L6-L7-L8) NO name (L5-L6-L7-L8-L9) NO L2CR ALCRP 9 T3CR LTCRS 25 Q4CR TQCRS 41 L2CK ALCKP 10 T3CK LTCKS 26 Q4CK TQCKS 42 R2CQ ARCQP 11 R3CP LRCPS 27 R4CS TRCSS 43 R2CR ARCRP 12 R3CR LRCRS 28 R4CR TRCRS 44 R2CK ARCKP 13 R3CK LRCKS 29 R4CK TRCKS 45 K2CQ AKCQP 14 K3CP LKCPS 30 K4CS TKCSS 46 K2CR AKCRP 15 K3CR LKCRS 31 K4CR TKCRS 47 K2CK AKCKP 16 K3CK LKCKS 32 K4CK TKCKS 48 L2CD ALCDP 17 T3CD LTCDS 33 Q4CD TQCDS 49 L2CE ALCEP 18 T3CE LTCES 34 Q4CE TQCES 50 D2CQ ADCQP 19 D3CP LDCPS 35 D4CS TDCSS 51 D2CD ADCDP 20 D3CD LDCDS 36 D4CD TDCDS 52 D2CE ADCEP 21 D3CE LDCES 37 D4CE TDCES 53 E2CQ AECQP 22 E3CP LECPS 38 E4CS TECSS 54 E2CD AECDP 23 E3CD LECDS 39 E4CD TECDS 55 E2CE AECEP 24 E3CE LECES 40 E4CE TECES 56

(45) TABLE-US-00010 Sequence SEQ Sequence SEQ Mutant Kabatnumbering ID Mutant Kabatnumbering ID name (H11-H12-H13-H14-H15) NO name (H14-H15-H16-H17-H18) NO Q13CR LQCRG 57 G16CR PGCRL 73 Q13CK LQCKG 58 G16CK PGCKL 74 R13CP LRCPG 59 R16CA PRCAL 75 R13CR LRCRG 60 R16CR PRCRL 76 R13CK LRCKG 61 R16CK PRCKL 77 K13CP LKCPG 62 K16CA PKCAL 78 K13CR LKCRG 63 K16CR PKCRL 79 K13CK LKCKG 64 K16CK PKCKL 80 Q13CD LQCDG 65 G16CD PGCDL 81 Q13CE LQCEG 66 G16CE PGCEL 82 D13CP LDCPG 67 D16CA PDCAL 83 D13CD LDCDG 68 D16CD PDCDL 84 D13CE LDCEG 69 D16CE PDCEL 85 E13CP LECPG 70 E16CA PECAL 86 E13CD LECDG 71 E16CD PECDL 87 E13CE LECEG 72 E16CE PECEL 88

Example 4: Cys-Modified scFv Antibody

(46) 4-1. Expression and Purification of Cys-Modified scFv Antibody

(47) To prepare a fusion protein of Cys-modified scFv and human C.sub.kappa, Cys-modified scFv was inserted into a mammalian expression vector pCEP4 containing human C.sub.kappa by cloning, and its expression was induced in HEK293F cells to obtain a final culture broth. Purification of the scFv-C.sub.kappa fusion protein from the culture broth was performed using kappa select beads. After purification, the protein was loaded on a polyacrylamide gel (SDS-PAGE gel), followed by electrophoresis. Expression and purification thereof were confirmed by Coomassie blue staining FIG. 4a shows the result of examining expression and purification of wild-type, LF1-2, LF1-3, LF1-4, HF1-13, and HF1-16 scFv proteins.

(48) 4-2. Expression and Purification of Cys-Modified scFv Antibody Having Replacement of Amino Acid Residues Adjacent to Cys

(49) From the Cys-modified antibodies having replacement of amino acid residues adjacent to Cys with cationic or anionic amino acids, clones maintaining antigen affinity were selected and their fusion proteins with human C.sub.kappa were expressed in HEK293F cells as in Example 4-1, and purified using kappa select beads. Thereafter, electrophoresis was performed on a polyacrylamide gel, and their expression and purification were confirmed by coomassie blue staining FIG. 4b shows the result of examining expression and purification of wild-type, HF1-13, R13CK, D13CE, E13CD, and E13CE scFv-C.sub.kappa fusion proteins.

Example 5: Cys-Modified Full IgG Antibody

(50) 5-1. Expression and Purification of Cys-Modified Full Antibody

(51) To prepare chicken-human chimeric full IgG protein, light chain and heavy chain DNAs of the variable region of Cys-modified antibody prepared in Example 1 were obtained by PCR and inserted into an IgG expression vector, followed by expression and culture in HEK293F cells. Thereafter, full IgG antibodies were purified using protein A beads. The purified proteins were electrophoresed, and their expression and purification were confirmed by coomassie blue staining. FIG. 5a shows the result of examining expression and purification of wild-type, LF1-2, LF1-3, LF1-4, HF1-13, and HF1-16 full IgG proteins.

(52) 5-2. Expression and Purification of Cys-Modified Full IgG Antibody Having Replacement of Amino Acid Residues Adjacent to Cys

(53) To express Cys-modified antibody having replacement of amino acid residues adjacent to Cys prepared in Example 3 in the form of chicken-human chimeric full IgG antibody as in Example 5-1, the antibody gene was inserted into an IgG expression vector, and expressed and cultured in HEK293F cells. Next, full IgG antibodies were purified from the culture broth using protein A beads. The purified proteins were loaded on a polyacrylamide gel, followed by electrophoresis. Expression and purification thereof were confirmed by coomassie blue staining. FIG. 5b shows the result of examining expression and purification of R13CK, E13CD, and E13CE full IgG proteins.

Example 6: PEG-Conjugated, Modified Antibody

(54) 6-1. Preparation of PEG-Cys-Modified Antibody by PEG Conjugation and Assay Thereof

(55) In accordance with a method described in the literature [Junutula et al., (2008) nature biotechnology 925-32], Cys-modified antibody prepared in Example 1 was reduced by addition of 10-fold TCEP, and then TCEP was removed. For partial oxidation, DHA was added in an amount corresponding to 2-fold of TCEP. Thereafter, maleimide PEG was added in an amount corresponding to 10-fold of the antibody, and incubated at 4 C. for 12 hours to induce conjugation of antibody with PEG. FIG. 6 shows a cartoon depiction of conjugation of maleimide PEG to sulfhydryl antibody.

(56) Antibody-PEG was loaded on a polyacrylamide gel, and then electrophoresed. Next, the gel was subjected to coomassie or iodine staining and immunoblotting to examine conjugation of PEG to antibody. For coomassie staining, the gel was stained with coomassie brilliant blue R250, and destained with a destaining solution containing methanol and acetic acid to identify the corresponding protein. FIG. 7a shows the result of wild-type, LF1-2, LF1-3, LF1-4, HF1-13, and HF1-16 scFv, and FIG. 8a shows the result of wild-type, LF1-2, LF1-3, LF1-4, HF1-13, and HF1-16 full IgG.

(57) For iodine staining, the polyacrylamide gel was washed with distilled water for 15 minutes, and then left in 5% BlCl.sub.2 for 15 minutes. Next, the gel was stained with 0.1 M iodine for 10 minutes, and destained with distilled water for 10 minutes to detect only PEG. FIG. 7b shows the result of wild-type, LF1-2, LF1-3, LF1-4, HF1-13, and HF1-16 scFv, and FIG. 8b shows the result of wild-type, LF1-2, LF1-3, LF1-4, HF1-13, and HF1-16 full IgG.

(58) For immunoblotting, the polyacrylamide gel was separated according to the size of the protein, and then full IgG was transferred onto an NC membrane, and scFv-C.sub.kappa fusion protein and full IgG light chain region were detected using HRP-conjugated anti-C.sub.kappa antibody and full IgG heavy chain region was detected using HRP-conjugated anti-human Fc antibody. FIG. 7c shows the result of wild-type, LF1-2, LF1-3, LF1-4, HF1-13, and HF1-16 scFv, and FIG. 8c shows the result of wild-type, LF1-2, LF1-3, LF1-4, HF1-13, and HF1-16 full IgG.

(59) 6-2. Preparation of PEG-Cys-Modified Antibody Having Replacement of Amino Acid Residues Adjacent to Cys by PEG Conjugation and Assay Thereof

(60) The antibody having replacement of cationic or anionic residue was reduced using TCEP as in Example 6-1, and partially oxidized by addition of DHA. Next, PEG was added to the antibody to induce conjugation of PEG to the antibody. To examine antibody-PEG conjugation, coomassie blue staining, iodine staining and immunoblotting were performed.

(61) FIG. 9a shows the result of coomassie blue staining to examine conjugation of PEG to wild-type, HF1-13, R13CK, D13CE, E13CD, or E13CE scFv, and FIG. 10a shows the result of coomassie blue staining to examine conjugation of PEG to wild-type, HF1-13, R13CK, or E13CE full IgG.

(62) FIG. 9b shows the result of iodine staining to examine conjugation of PEG to wild-type, HF1-13, R13CK, D13CE, E13CD, or E13CE scFv, and FIG. 10b shows the result of iodine staining of conjugation of PEG to HF1-13, R13CK, or E13CE full IgG.

(63) FIG. 9c shows the result of immunoblotting to examine conjugation of PEG to wild-type, HF1-13, R13CK, D13CE, E13CD, or E13CE scFv, and FIG. 10c shows the result of immunoblotting of conjugation of PEG to HF1-13, R13CK, or E13CE full IgG.

Example 7: Preparation of Antibody-Cotinine by Cotinine Conjugation and Assay Thereof

(64) Cys-modified antibody prepared in Example 1 was reduced by addition of 10-fold TCEP, and then TCEP was removed. For partial oxidation, DHA was added in an amount corresponding to 2-fold of TCEP. Thereafter, GMBS-cotinine was added in an amount corresponding to 10-fold of the antibody, and incubated at 4 C. for 12 hours to induce conjugation of antibody with cotinine. FIG. 11a shows a cartoon depiction of conjugation of GMBS-cotinine to sulfhydryl antibody.

(65) Antibody-cotinine was assayed by enzyme-linked immunosorbent assay (ELISA) (FIG. 11b). A plate was coated with PSA antigen, and then antibody-cotinine was added thereto, followed by incubation for 1 hour. After washing three times, HRP-conjugated anti-cotinine IgG or anti-C.sub.kappa IgG antibody was added. After incubation for 1 hour and washing three times, TMB (3,3,5,5-Tetramethylbenzidine) was added to examine color development, and then 2M H.sub.2SO.sub.4 was used to terminate the reaction. Absorbance at 450 nm was measured. In the result of FIG. 12, cotinine conjugation was confirmed by anti-cotinine IgG, and anti-C.sub.kappa IgG was used to detect only antibody, irrespective of cotinine conjugation.

Example 8: Injection of Antibody-Cotinine into Mouse and Assay of Cotinine-Antibody

(66) 6-week-old Balb/c mice were randomly divided into three groups (HF1-13, E13CD, E13CE), and then retroorbital blood collection was performed. After 24 hours, each 100 g of 3 types of cotinine-scFv antibody C.sub.kappa fusion proteins prepared in Example 7 was intravenously injected into mice. 1 hour after cotinine-antibody injection, retroorbital blood collection was performed, and sera were separated using a centrifuge. The separated sera were diluted 50-fold in order to examine the presence or absence of cotinine-antibody in the sera, and enzyme-linked immunosorbent assay was performed as in Example 9. All procedures using mice were performed after inhalation anesthesia in order to minimize pain.