The assignee for this patent application is
Reporters obtained the following quote from the background information supplied by the inventors: "Cochlin-tomoprotein (hereinafter referred to as CTP), which is a 16 kDa isoform of Cochlin present in perilymph, is a promising diagnostic marker for perilymph fistula because it is not found in other body fluids which can be present in the middle ear such as cerebrospinal fluid, blood and saliva (Patent Literature 1, Nonpatent Literatures 1-5). Perilymph fistula is a disease in which inner ear dysfunction is manifested when labyrinthine window separating the inner ear from the middle ear is ruptured, and then perilymph with which the inner ear is filled leaks into the middle ear. Because the symptom may be ameliorated by closing labyrinthine window in the early stage of the onset, early diagnosis is very important.
"So far, the research group of the present inventors has produced four rabbit polyclonal antibodies using a synthetic peptide as an immunogen in order to measure CTP in a biological sample (Patent Literature 1). However, all of the antibodies only react with denatured CTP. An antibody reacting with native CTP has not been obtained. In Nonpatent Literatures 1-5, therefore, the above anti-LCCL3 antibody which recognizes denatured CTP (which does not react with native CTP) is used to measure CTP in a biological sample by Western blotting."
In addition to obtaining background information on this patent application, NewsRx editors also obtained the inventors' summary information for this patent application: "However, measurements under denatured conditions, in which CTP in a biological sample cannot be measured in its native state, may result in false negative and false positive, and may be susceptible to contaminating proteins. The measurements also require complicated procedures and are time-consuming before obtaining results. Therefore, a method of measuring CTP in a faster, simpler and more accurate fashion is demanded.
"In order to measure CTP in a biological sample more simply and accurately, the research group of the present inventors has tried to raise a monoclonal antibody using recombinant CTP expressed in E. coli as an immunogen in addition to the 4 polyclonal antibodies described in Patent Literature 1. Nonetheless, a high antibody titer was difficult to be achieved, and only few clones were obtained, because CTP is present in a wide variety of animal species such as mouse, swine and bovine, and its homology is high between animal species. The clones obtained were reactive only in denatured conditions. An antibody reacting with native CTP was not obtained.
"Because only a trace amount of CTP is present in a living organism, it is not possible to prepare CTP from a biological sample in an amount enough to use it as an immunogen for raising an antibody. Therefore, it is necessary to raise an antibody by using a synthetic peptide, a recombinant protein and the like as an immunogen. However, as mentioned above, neither the antibody against a synthetic peptide nor the antibody against recombinant CTP expressed in E. coli reacted with native CTP. This suggests that CTP forms a highly complex tertiary structure in a biological sample. Usually, a strain having good reactivity with an antigen used for immunization is selected at a screening process in antibody production. In the case of CTP, the three dimensional structure of the recombinant protein used for immunization differs from that of CTP present in a biological sample. Therefore, even if a done recognizing native CTP has been raised, it may not be selected at the screening process due to poor reactivity with the immunogen. As a result, an antibody which recognizes native CTP is much more difficult to be obtained.
"Accordingly, in order to solve the problem described above, the present invention provides an antibody which recognizes native CTP. The present invention further provides a method of measuring CTP in a biological sample using the antibody. Moreover, the present invention provides a method of screening for an antibody which recognizes native CTP.
"The present inventors found that native CTP can be measured by using an antibody which recognizes an epitope in a peptide sequence of CTP-C (amino acid numbers 118-132 of SEQ ID NO: 1). Further, the present inventors found that an antibody which recognizes native CTP can be obtained by selecting an antibody reacting with native CTP captured by anti CTP-C antibody solid-phased on a support. Then the present inventors completed the present Invention
"The present invention includes:
"(1) An antibody which recognizes an antigenic determinant included in the polypeptide represented by amino acid numbers 118-132 of SEQ ID NO: 1, and reacts with native Cochlin-tomoprotein (CTP).
"(2) The anti CTP-antibody according to (1), which is obtainable by using a peptide consisting of amino acid numbers 118-132 of SEQ ID NO: 1 as an immunogen.
"(3) A method of screening an antibody reacting with native CTP, comprising providing native CTP captured by a first antibody reacting with native CTP and selecting a second antibody which recognizes the native CTP.
"(4) The method of screening an antibody reacting with native CTP according to (3), wherein the first antibody is the antibody according to (1).
"(5) An method of immunologically measuring a protein comprising the polypeptide represented by amino acid numbers 118-132 of SEQ ID NO: 1, comprising the step of using at least one antibody according to (1) or (2).
"(6) The method according to (5), wherein the protein comprising the polypeptide represented by amino acid numbers 118-132 of SEQ ID NO: 1 is CTP.
"(7) A method of examining perilymph fistula, comprising the step of immunologically measuring CTP using at least one antibody according to (1) or (2).
"(8) A kit for measuring CTP, comprising at least one antibody according to (1) or (2).
"(9) The kit for measuring CTP according to (8), which is used for diagnosing perilymph fistula.
"Effects of Invention
"The antibody according to the present invention enabled measurement of native CTP. In addition, an antibody which recognizes native CTP can be obtained. This allows perilymph fistula to be diagnosed rapidly and accurately by a simple procedure.
BRIEF DESCRIPTION OF DRAWINGS
"FIG. 1 shows the positions of antigenic polypeptides for raising the antibody of the present invention in the amino acid sequence represented by SEQ ID NO: 1.
"FIG. 2 shows the results from the measurements of serially diluted human perilymph by the sandwich ELISA method using the anti-CTP-C antibody of the present invention and the 3C10 antibody."
For more information, see this patent application: Ikezono, Tetsuo; Shikaze,
Keywords for this news article include: Antibodies, Peptides, Immunology, Amino Acids, Blood Proteins, Immunoglobulins,
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