Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1989-01-06
1994-05-31
Rosen, Sam
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 71, 435 72, 435 721, 435 793, 435 794, C12Q 100, G01N 33532, G01N 33535
Patent
active
053169115
DESCRIPTION:
BRIEF SUMMARY
FIELD OF INVENTION
The present invention relates to a method of determining the presence of endotoxin or endotoxin-like substances in a sample, as well as to a monoclonal antibody and test kit useful in the method.
BACKGROUND OF THE INVENTION
The horseshoe crabs Limulus polyphemus, Tachypleus tridentatus, Tachypleus gigas and Carcinoscorpius rotundicauda are phylogenetically primitive marine arthropods which have not evolved significantly over the past 300 million years (1).
The horseshoe crab has an open circulatory system containing blue haemolymph, and the only formed element present in the haemolymph is a cell called the amoebocyte (2).
The use of the Limulus amoebocyte lysate (LAL) as an in vitro test for endotoxin resulted directly from the important observation made by Bang (3). He observed that the horseshoe crab underwent a type of disseminated intravascular coagulation (DIC) when generalized infection occurred with marine gram-negative bacteria. This initial in vivo observation was later extended by the discovery that clotting of Limulus haemolymph could be produced in vitro by addition of either viable gram-negative bacteria or purified endotoxin from the cell wall of gram-negative bacteria (2). It was also discovered by the same researchers (1) that the amoebocyte was the source of all of the factors necessary for haemolymph coagulation.
The current application of the test as an in vitro assay for endotoxin is based on the fact that physical disruption of amoebocytes which have been isolated from the haemolymph by centrifugation yields a suspension (LAL) containing the coagulation components which may then only be activated by bacterial endotoxin.
Application of this principle has made the LAL test the most sensitive method available for the detection of gram-negative bacteria, endotoxin and lipopolysaccharide (LPS). LAL prepared by current purification methods can reliably detect 0.1 ng/ml of purified Escherichia coli standard endotoxin. LAL has been shown to detect either bound (cell-associated) or free endotoxin (4) incorporated in the cell walls of virtually all gram-negative bacteria. However, endotoxin prepared by different extraction procedures, from different species, may vary widely in reactivity (5,6). For these reasons, a reference standard endotoxin (RSE) has been prepared by the U.S. Food and Drug Administration (USFDA) as a means for standardization of LAL and rabbit pyrogenicity tests (United States Pharmacopoiea XX) (7).
Amoebocyte lysate of similar reactivity to endotoxin can be prepared from all 4 species of horseshoe crabs.
The clotting processes in Limulidae
The currently known clotting processes in Limulidae are summarized in Table 1. ##STR1##
Cellular coagulogen: Coagulogen consists of a polypeptide chain with internal disulphide bonds which are important for the stability of the polymerizable form of the molecule (8). In Limulus polyphemus, coagulogen was found to consist of 220 amino acids with half-cystine content of 18 residues (9). No free SH group was detected; glycine appears to be the only N-terminal residue, and serine its C-terminal residue (9). Coagulogen is always converted by a serine protease enzyme. After clotting, the gel protein display a helical structure in electron microscopy (10). In Tachypleus tridentatus, the coagulogen comprises 132-135 amino acids including high levels of basic amino acids, with N-terminal alanine and C-terminal phenylalanine. In Limulus, clot formation seems to involve the cleavage of the single Arg-Lys peptide bond on the coagulogen (11,9). The N-peptides interact among themselves in a non-covalent fashion to form the insoluble clot. In Tachypleus tridentatus, the enzymatic formation of gel involves limited proteolysis of the Arg-Gly and Arg-Thr peptide linkages located in the N-terminal portion of coagulogen, thus releasing peptide (12,13). The C-fragment of Limulus mainly contains glutamic and aspartic acids (14). Whereas Liu et al. (9) detected a C-peptide with 45 amino acids in this species, Nakamura et al. ( 13) and Shis
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Draft Guideline for Validation of the Limulus amebocyte lysate test as an End Product Endotoxin Test for Human and Animal Paren
Baek Leif
Koch Claus
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