Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1995-02-10
2001-03-06
Ceperley, Mary E. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S962000, C436S500000, C436S811000, C436S825000
Reexamination Certificate
active
06197533
ABSTRACT:
This invention relates to a method for the chemical modification of antibodies and/or antigens in order to minimise sample interference in immunoassays, to an immunoassay method in which chemically modified antibodies and/or antigens are employed, to the use of the chemically modified antibodies and/or antigens in an immunoassay and to a kit for assaying biological specimens which comprises the chemically modified antibodies and/or antigens.
Sample interference in immunoassays involves deleterious interactions between specific components of immunoassays and interfering species in samples and is a well documented phenomenon. References to sample interference include (1) Levinson S., “Heterophilic antibodies and their role in immunoassay interference”; J. Clin. Immunoassay, 1992, 15, 108-115 and (2) Maxey K. M., Krishna R. M and Birkmeir J., “Interference in Enzyme Immunoassays”; J. Clin. Immunoassay, 1992 15, 116-120. Much of this interference can be attributed to the presence of species within samples that cross-link specific immunoassay components. Routinely this is caused by antibodies or other sample components that are directed against either (a) structural elements of the immunoglobulins of the immunoassay or (b) structural elements of native, recombinant or synthetic antigens.
Immunoglobulins are constructed from two heavy chains each consisting of three constant regions (CH-1, CH-2 and CH-3) and a variable region (VH). These are associated with two light chains each consisting of a constant region (CL) and a variable region (VL). There are also structural variants of immunoglobulin heavy and light chains termed isotypes—see for example Vlug A. and Van Remortal P., “The structure and function of human IgG subclasses”; European Clinical Lab., 1989, 8, 26-33. An interfering sample may show reactivity against one isotype but not another. Other interfering samples may show reactivity across isotypes. A significant proportion, although not all, of the interfering anti-antibody components are directed against the Fc region (CH2-CH3) of the assay specific immunoglobulins. Interference can also occur when labelled and unlabelled assay specific species, where one or more of these is the antigen, are cross-linked by an interfering species. Blocking of interference has been achieved in the past by addition of quantities of immunoglobulin containing sera from different species or purified immunoglobulins or aggregated immunoglobulins. This has been described in U.S. Pat. No. 4,914,040 to Boehringer Mannheim issued 1990 and entitled “Reagent and method for determination of a polyvalent substance using an immunoaggregate”. Reduction of the interference has also been achieved by removal of the Fc fragment from one or more of the assay specific immunoglobulins and also by blocking assay specific antibody with anti FC antibody as described in European Patent Publication No. 566205A.
It has been reported that rheumatoid factor and other interfering species are predominantly reactive with the CH-2 and CH-3 regions on immunoglobulin heavy chains—see for example Williams R. C., Malone C. and Solomon A., “Conformational dependency of human IgG heavy chain-associated Gm allotypes”; Mol. Immunol., 1993, 30, (4), 341-351. The existance of species reactive with the CH1 region of immunoglobulin heavy chains is also known—see for example Aguado M. T., Balderas R. S., Rubin R. L., Duchosal R. K., Kofler R., Birshtein B. K., Secher D. S., Dixon F. J. and Theofilopoulos A. N., “Specificity and molecular characteristics of monoclonal IgM rheumatoid factors from arthritic and non-arthritic mice”; J. Immunol., 1987, 139, 1080-1087. The presence of these factors in sample sera can cause incorrect antigen concentration determinations in immunoassays. Removal of the Fc from assay specific immunoglobulins will therefore not prevent factors reactive with the CH-1 region from binding.
According to the present invention we provide a method for the chemical modification of antibodies and/or antigens and/or fragments of antibodies and/or antigens characterised in that at least one amino acid selected from the group consisting of argenine, histidine, lysine, threonine and tyrosine located at an active site within the antibody, antigen and/or fragment thereof is modified by chemical treatment in such a manner that recognition of the antibody, antigen and/or fragment thereof by an interfering antibody is substantially prevented.
Further according to the present invention we provide a method for the immunoassay of a biological specimen which comprises a step in which there is used an antibody and/or antigen and/or a fragment of an antibody and/or antigen characterised in that the antibody and/or antigen and/or fragment thereof has at least one amino acid selected from the group consisting of argenine, histidine, lysine, threonine and tyrosine located at an active site within the antibody, antigen and/or fragment thereof which has been modified by chemical treatment in such a manner that recognition of the antibody, antigen and/or fragment thereof by an interfering antibody is substantially prevented.
Further according to the present invention we provide the use in a method for the immunoassay of a biological specimen of a modified antibody and/or antigen and/or fragment of an antibody and/or antigen characterised in that the antibody and/or antigen and/or fragment thereof has at least one amino acid selected from the group consisting of argenine, histidine, lysine, threonine and tyrosine located at an active site within the antibody, antigen and/or fragment thereof which has been modified by chemical treatment in such a manner that recognition of the antibody, antigen and/or fragment thereof by an interfering antibody is substantially prevented.
Further according to the present invention we provide a kit useful in an immunoassay which comprises an antibody and/or antigen and/or fragment of an antibody and/or antigen characterised in that the antibody and/or antigen and/or fragment thereof has at least one amino acid selected from the group consisting of argenine, histidine, lysine, threonine and tyrosine located at an active site within the antibody, antigen and/or fragment thereof which has been modified by chemical treatment in such a manner that recognition of the antibody, antigen and/or fragment thereof of an interfering antibody is substantially prevented.
The chemical modification of specific amino acid residues in the immunoglobulin or antigen enables blocking or reduction of the interactions mentioned above and other non-desirable interactions in the case of antigens to be achieved. Suitably the antibody, antigen and/or fragment (hereinafter referred to as the reagent particle) is chemically treated to modify the charge and/or structural properties of one or more of the group of amino acids concerned ie argenine, histidine, lysine, threonine and tyrosine so that recognition by the reagent particle of an interfering antibody is no longer possible. Suitably the active sites on the reagent particle which are targeted for modification of amino acids are those areas to which an interfering amino acid could bind to a significant extent. Preferred target regions of the reagent particle include the “constant” regions of antibodies ie the CH 1, CH 2 and CH 3 regions and between the CH 2 and CH 3 regions. Preferably, during the modification method, argenine, histidine, lysine, threonine and tyrosine moeties located on a reagent particle elsewhere than at active sites are protected in a manner suitable to prevent any significant modification of them from occurring.
Any suitable chemical modification of the amino acid moeties concerned on the reagent particle may be used in the modification method of the invention. The preferred modification technique in any instance will depend upon the amino acid to be modified.
Chemical compounds which can be used to modify reagent particles include compounds having the structure:
R—O—CO—X
where R can be any of a wide range of groups including alkyl, aryl and pyridyl; and
X is a lea
Dawkes Adrian Charles
Diment John Arthur
Yearwood Graham DeLisle
Ceperley Mary E.
Ortho-Clinical Diagnostics, Inc.
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