Method of determining the presence of endotoxin in a sample
First Claim
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1. A monoclonal antibody which specifically binds to coagulogen of a limulus amoebocyte lysate, but does not bind to coagulogen of said limulus amoebocyte lysate which has been contacted with activated clotting enzyme of said limulus amoebocyte lysate.
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Abstract
There is disclosed a method for determining the presence of endotoxin or an endotoxin like substance in a sample, as well as a monoclonal antibody and test kit useful in the method.
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Citations
27 Claims
- 1. A monoclonal antibody which specifically binds to coagulogen of a limulus amoebocyte lysate, but does not bind to coagulogen of said limulus amoebocyte lysate which has been contacted with activated clotting enzyme of said limulus amoebocyte lysate.
- 2. A monoclonal antibody which specifically binds to a product of a coagulogen of a limulus amoebocyte lysate wherein said product is formed upon contact of aid coagulogen with activated clotting enzyme of said limulus amoebocyte lysate, but which does not bind to said coagulogen that has not been contacted with activated clotting enzyme of said limulus amoebocyte lysate.
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3. A monoclonal antibody produced by a hybridoma isolated by a method comprising:
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i) producing a pool of hybridomas secreting monoclonal antibodies by immunizing an animal with coagulogen of a limulus amoebocyte lysate or with an antigen comprising an immunologic determinant of aid coagulogen; ii) testing said pool of hybridomas for secretion of a monoclonal antibody that specifically binds to said coagulogen of a limulus amoebocyte lysate or with an antigen comprising an immunologic determinant of said coagulogen; iii) testing said pool of hybridomas for secretion of a monoclonal antibody that does not bind to said coagulogen of a limulus amoebocyte lysate wherein said limulus amoebocyte lysate has been contacted with a lipopolysaccharide of a gram-negative bacterium; and iv) isolating those hybridomas which secrete a monoclonal antibody that specifically binds to said coagulogen of a limulus amoebocyte lysate or with an antigen comprising an immunologic determinant of said coagulogen and that does not bind to said coagulogen of a limulus amoebocyte lysate wherein said limulus amoebocyte lysate has been contacted with a lipopolysaccharide of a gram-negative bacterium.
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4. A monoclonal antibody produced by a hybridoma isolated by a method comprising:
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i) producing a pool of hybridomas secreting monoclonal antibodies by immunizing an animal with coagulogen of a limulus amoebocyte lysate or with an antigen comprising an immunologic determinant of aid coagulogen; ii) testing said pool of hybridomas for secretion of a monoclonal antibody that specifically binds to said coagulogen of a limulus amoebocyte lysate or with an antigen comprising an immunologic determinant of said coagulogen; iii) testing said pool of hybridomas for secretion of a monoclonal antibody that does not bind to said coagulogen of a limulus amoebocyte lysate wherein said limulus amoebocyte lysate has been contacted with a lipopolysaccharide of a gram-negative bacterium; and iv) isolating those hybridomas which secrete a monoclonal antibody that specifically binds to said coagulogen of a limulus amoebocyte lysate wherein said limulus amoebocyte lysate has been contacted with a lipopolysaccharide of a gram-negative bacterium and that does not bind to said coagulogen of a limulus amoebocyte lysate or with an antigen comprising an immunologic determinant of said coagulogen.
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26. A method for producing a monoclonal antibody which specifically binds to coagulogen of a limulus amoebocyte lysate, but does not bind to coagulogen of said limulus amoebocyte lysate which has been contacted with activated clotting enzyme of said limulus amoebocyte lysate, comprising:
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i) producing a pool of hybridomas secreting monoclonal antibodies by immunizing an animal with coagulogen of a limulus amoebocyte lysate or with an antigen comprising an immunologic determinant of said coagulogen; ii) testing said pool of hybridomas for secretion of a monoclonal antibody that specifically binds to said coagulogen of a limulus amoebocyte lysate or with an antigen comprising an immunologic determinant of said coagulogen; iii) testing said pool of hybridomas for secretion of a monoclonal antibody that does not bind to said coagulogen of a limulus amoebocyte lysate wherein said limulus amoebocyte lysate has been contacted with a lipopolysaccharide of a gram-negative bacterium; and iv) isolating those hybridomas which secrete a monoclonal antibody that specifically binds to said coagulogen of a limulus amoebocyte lysate or with an antigen comprising an immunologic determinant of said coagulogen and that does not bind to said coagulogen of a limulus amoebocyte lysate wherein said limulus amoebocyte lysate has been contacted with a lipopolysaccharide of a gram-negative bacterium; v) culturing a hybridoma obtained from step (iv); and vi) isolating a monoclonal antibody from the hybridoma culture.
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27. A method for isolating a monoclonal antibody which specifically binds to a product of a coagulogen of a limulus amoebocyte lysate wherein said product is formed upon contact of said coagulogen with activated clotting enzyme of said limulus amoebocyte lysate, but which does not bind to said coagulogen that has not ben contacted with activated clotting enzyme of said limulus amoebocyte lysate, comprising:
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i) producing a pool of hybridomas secreting monoclonal antibodies by immunizing an animal with said product of a coagulogen of a limulus amoebocyte lysate or with an antigen comprising an immunologic determinant of said coagulogen product; ii) testing said pool of hybridomas for secretion of a monoclonal antibody that specifically binds to said product of a coagulogen of a limulus amoebocyte lysate or with an antigen comprising an immunologic determinant of said coagulogen product; iii) testing said pool of hybridomas for secretion of a monoclonal antibody that does not bind to said coagulogen of a limulus amoebocyte lysate wherein said limulus amoebocyte lysate has been contacted with a lipopolysaccharide of a gram-negative bacterium; and iv) isolating those hybridomas which secrete a monoclonal antibody that specifically binds to said product of a coagulogen of a limulus amoebocyte lysate wherein said limulus amoebocyte lysate has been contacted with a lipopolysaccharide of a gram-negative bacterium and that does not bind to said coagulogen of a limulus amoebocyte lysate or with an antigen comprising an immunologic determinant of said coagulogen; v) culturing a hybridoma obtained from step (iv); and vi) isolating a monoclonal antibody from the hybridoma culture.
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Specification