Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Monoclonal antibody or fragment thereof
Reexamination Certificate
1998-06-09
2002-04-23
Nolan, Patrick (Department: 1644)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Monoclonal antibody or fragment thereof
C435S007100, C436S518000, C436S536000, C436S541000, C530S388250
Reexamination Certificate
active
06375949
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a monoclonal antibody recognizing amyloid A in human serum (hereinafter abbreviated as SAA) and cause the agglutination reaction, a reagent for immunoassay comprising the monoclonal antibody, and a method for immunoassay using the monoclonal antibody.
BACKGROUND ART
In such fields as clinical examinations, immunoassay is often utilized in order to measure certain substances simply and conveniently or with high sensitivity and specificity. Antibodies are required for immunoassay. Monoclonal antibodies are indispensable tools for immunoassay because of their numerous advantages: that they can be continuously supplied with uniform characteristics; that an amount of antigens used can be small since the antibody-producing cells can be established as cell lines; and among others, that antibodies of high specificity can be easily obtained. Monoclonal antibodies are important tools not only for assay but also for purification of substances and for studies on their physiological activities and in vivo behavior.
SAA is a serum protein with a molecular weight of about 12,000, which is considered as a precursor protein of amyloid protein A (hereinafter abbreviated as AA protein) that is deposited in tissues in a certain type of amyloidosis (J. Clin. Invest. 53: 1054-1061, 1974). Recently, it was reported that the serum SAA value rises in inflammatory disorders, and therefore it has been recognized as a sensitive marker for inflammations (Rinshokensa 32: 2, p 168, 1988).
There are some reports on the monoclonal antibodies recognizing SAA (J. Immunol. Methods 144, 149-155, 1991; Clin. Chem. 40/7, 1284-1290, 1994). In these reports, ELISA is constituted with the monoclonal antibodies recognizing SAA.
Generally speaking, those monoclonal antibodies that are sufficiently usable in ELISA often are not practically useful in the agglutination method. This is because the antibody to be used must have the following characteristics in either case of utilizing carrier particles like latex or of not requiring a carrier as in the case of immunological turbidimetric analysis.
First of all, the agglutination method requires higher affinity. Compared to ELISA, it requires generally a higher level of affinity in order to achieve the agglutination reaction and to maintain a physically stable aggregate. Especially with monoclonal antibodies, one would need to prepare those with high affinities because the reaction needs to be constituted solely with the antibody molecules that react specifically with certain epitopes. However, conventional monoclonal antibodies cannot satisfy this requirement. If one attempts to constitute a reaction system with a monoclonal antibody with insufficient affinity, it is impossible to conduct measurement by the agglutination method because the antibody does not form an aggregate that can be analyzed with practical sensitivity. If the quantity of the antibody is increased to compensate for the low affinity, sufficient sensitivity cannot be obtained since the number of antibody molecules that can bind to one antigen molecule does not change. The measure to increase the quantity of the antibody is also unsatisfactory when using carriers such as latex by binding it to the antibody because the amount of the antibody bound is limited.
Furthermore, the agglutination reaction requires multiple existence of the epitope recognized by the monoclonal antibody on a single antigen and positional relationship among the epitope at multiple sites must be suitable for agglutination. Therefore, the positional relationship, which is not an issue in ELISA, may become an obstacle in the agglutination method. Although the same condition is required in the sandwich ELISA method, it is disadvantageous to rely solely on the contiguous epitopes even if their physical sites are different because, as described earlier, the agglutination method requires a physically stronger binding. This is because steric hindrance tends to occur, which makes it difficult to obtain a large, stable aggregate. Thus, also from the standpoint of epitope selection, conventional monoclonal antibodies are not suitable for the agglutination method.
An object of the present invention is to provide a novel monoclonal antibody that enables measurement of SAA based on the agglutination reaction. Another object of the invention is to provide a novel reagent for immunoassay comprising the monoclonal antibody, and a novel method for immunoassay using the monoclonal antibody.
DISCLOSURE OF THE INVENTION
Using highly purified SAA and enhancing the antigenicity of SAA by combining SAA with various adjuvant components to make it into a unique form of immunogen, the present inventors have obtained several kinds of hybridomas producing the monoclonal antibodies recognizing human SAA with the following reactivity characteristics: (1) that they recognize the epitope of human serum amyloid A, and (2) that they agglutinate by reacting with human serum amyloid A in the absence/presence of other monoclonal antibodies, to isolate several monoclonal antibodies from the hybridomas. The present inventors also conducted agglutination experiments of SAA using the monoclonal antibodies, and have established a novel method for immunoassay utilizing the monoclonal antibodies, thereby completing the invention.
The monoclonal antibodies of the present invention enable immunoassay by the agglutination reaction. The use of currently available monoclonal antibodies against SAA has been limited to such measurement methods as ELISA because they have insufficient affinities for the agglutination reaction. Since the monoclonal antibodies of the present invention have new characteristics that they agglutinate by reacting with SAA, it is possible to provide a convenient method for measuring SAA based on the agglutination reaction.
The monoclonal antibodies of the present invention can be obtained by immunizing mice, rats, etc. with the purified SAA, and immortalizing the antibody-producing cells by some means. As to the techniques for immortalization, known techniques include cell fusion with tumor cells such as myelomas and transformation with Epstein-Barr virus.
When preparing hybridomas by cell fusion, the myeloma cells from the same animal species as the immunized animal may be used, or they can originate from some other animals, thereby creating hetero-hybridoma cells. SAA is a protein also found in serum of non-human animals, and it increases by immunization as a stimulus. Consequently it is advantageous to use an animal species that does not show such a phenomenon, e.g., rats, as the animal to be immunized because the antibody titer is likely to rise.
SAA to be utilized as the immunogen can be obtained by purification using a known method. Specifically, SAA can be recovered as a pure protein by obtaining a high density lipoprotein (hereinafter abbreviated as HDL) fraction from crude serum by ultracentrifugation, subjecting it to delipidation, and purifying by means of, e.g., ion exchange chromatography. It is desirable to use such highly purified protein in order to obtain the cells that produce monoclonal antibodies specific to SAA in a high yield. Although there is a report that the unpurified HDL fraction obtained through ultracentrifugation was used as an immunogen, the antibody-producing cells obtained using such immunogens often produce antibodies recognizing various apolipoprotein antigens, which may be disadvantageous in terms of, e.g., cloning.
When using purified SAA as an immunogen, a variety of techniques can be applied to enhance its antigenicity. Freund's complete adjuvant (hereinafter abbreviated as FCA) is one of the necessary components to enhance immunogenicity. It is also effective to use the SAA adsorbed to lipid liposomes to serve as an immunogen. It is pointed out that SAA exhibits poor antigenicity in the immunized animals since SAA is a protein inherently found in many mammals' blood. One of the reasons why there is no report on the monoclonal antibody usable in the agglutination
Hirano Norihito
Yamada Michiko
Eiken Kagaku Kabushiki Kaisha
Nolan Patrick
Pepper Hamilton LLP
Villacorte Gilberto M.
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