Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Utility Patent
1997-10-21
2001-01-02
Swartz, Rodney P. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S013000, C435S018000, C435S019000, C435S023000, C435S024000, C435S038000, C435S175000, C435S183000, C435S184000, C436S175000, C436S825000
Utility Patent
active
06168924
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a limulus reaction for activating an amebocyte lysate, which is a hematocyte component of a horseshoe crab (Limulus) and is used for detecting endotoxin or (1→3)-&bgr;-D-glucan, and, more particularly, to an endotoxin-specific limulus reaction. More specifically, the present invention relates to a technical field related to (1) a non-endotoxin-like substance that reacts with and activates factor C, which is the first factor of the limulus reaction starting with the endotoxin; (2) means for eliminating the influence of the non-endotoxin-like substance in the limulus reaction; (3) means for measuring the amount of the endotoxin through use of the means for eliminating the influence; and (4) means for measuring the amount of the non-endotoxin-like substance by means of the limulus reaction.
The influence of the non-endotoxin-like substance particularly poses a problem for the measurement of the amount of endotoxin by means of the limulus reaction. Therefore, the present invention further relates to (5) means for measuring the true amount of endotoxin contained in a biological product.
2. Description of the Related Art
Endotoxin is a lipopolysaccharide present in the outer membrane of cell wall of a Gram-negative bacteria and is known to be a highly-pyrogenic substance. Even a trace amount of endotoxin is known to induce various types of morbidity attributable to bacterial infection, as well as fever; e.g., the release of inflammatory cytokines, such as TNF or interleukin-1 (IL-1), in association with the activation of macrophages or endotoxin shock. For these reasons, it is important to detect endotoxin contained in parenteral drugs, and an endotoxin test method is described in Pharmacopoeias in Japan and the U.S., as well as in the Minimum Requirements for Biological Products. Endotoxin is considered to be primarily responsible for the shock induced by Gram-negative infectious diseases. The amount of endotoxin contained in plasma is measured for the purposes of diagnosing Gram-negative infectious diseases, determining treatment effects or prognosis, or early diagnosis of endotoxin shock.
A limulus test method, or an endotoxin test method, is a method of detecting endotoxin by means of activation of an amebocyte lysate of a horseshoe crab performed by Gram-negative bacterial endotoxin (this reaction is called a limulus reaction, reagents using the limulus reaction are called limulus reagents, and assay using the limulus reaction is called limulus assay). At present, this limulus test is roughly classified into a gelation method, turbidimetry which uses as an indicator variations in the degree of turbidity of a gel, and colorimetry which uses as an indicator a color developed as a result of the hydrolysis of a synthetic chromogenic substrate.
In 1978, the U.S. Food and Drug Administration approved the limulus test as an alternative to a rabbit pyrogen test, because of a high degree of correlation between these tests. As a result, the limulus test has been widely applied to a test for the safety of medical devices or drugs which include biological products such as vaccines or blood products. Even in Japan, the limulus test is included, as an alternative to a rabbit pyrogen test, in Japanese pharmacopoeia and the Minimum Requirements for Biological Products.
As shown in
FIG. 1
, the amebocyte lysate of a horseshoe crab (hereinafter simply referred to as a lysate) contained in the limulus reagent has two coagulation cascades, i.e., A coagulation cascade in which factor C participates by reacting with an endotoxin to become active; and A coagulation cascade in which factor G participates by reacting with (1→3)-&bgr;-D-glucan (hereinafter may be referred to as &bgr;-D-glucan) to become active. A method of specifically measuring the amount of endotoxin by use of only the former cascade and a method of specifically measuring the amount of &bgr;-D-glucan by use of only the latter cascade have already been known (Obayashi T. et. al., Clin. Chem. Acta. 149, pp. 55-65 (1985); endotoxin-specific measurement methods disclosed in WO90/02951, U.S. Pat. No. 5,155,032, U.S. Pat. No. 5,179,006, WO92/03736, WO92/06381, Japanese Patent Laid-Open (kokai) No. 6-258326, Japanese Patent Publication (kokoku) No. 2-18080; &bgr;-D-glucan-specific measurement methods disclosed in WO91/19981, WO92/16651). Further, limulus reagents used for specifically measuring the amount of endotoxin and the &bgr;-D-glucan, respectively, are already commercially available.
Since there are many cases where the &bgr;-D-glucan is mixed in a sample to be subjected to a limulus test, the Japanese Minimum Requirements for Biological Products specify the use of endotoxin-specific limulus reagents.
The endotoxin-specific limulus reagent is also known to react with several substances other than endotoxin, depending on the content of a sample for limulus assay (which signifies a sample to be subjected to measurement which utilizes a limulus reaction). More specifically, like serine proteases such as trypsin, thrombin, factor Xa (the limulus reaction utilizes the reaction of serine-protease contained in the lysate; the action of the above-mentioned serine proteases is similar to that of coagulation enzymes included in the lysate, thereby testing positive) or chymotrypsin (which activates factor C under the absence of endotoxin, thereby testing positive), there are known substances other than endotoxin which specifically react with the endotoxin-specific limulus reagent (the substance will be hereinafter referred to as endotoxin-specific limulus reaction false-positive substances).
There is a known method of eliminating the limulus reaction false-positive substances without deteriorating the activity of endotoxin, which method is to be employed in the measurement of the amount of endotoxin contained in a sample for limulus assay which includes these substances, i.e., in a blood sample (whole blood, plasma, and serum).
For example, there is disclosed means which eliminates the effect of false-positive substances contained in a sample for limulus assay by diluting the sample with a surfactant-containing aqueous solution on the basis of the assumption that the sample is subjected to heat treatment (see Japanese Patent Laid-Open (kokai) No. 6-118086).
Other known methods include means which eliminates the effect of limulus reaction false-positive substances contained in a sample for limulus assay by treating the sample with acid such as perchloric acid (see Japanese Patent Publication (kokoku) No. 63-55671) and means which eliminates the effect of a limulus-reaction inhibition factor contained in the sample by treating the sample with hexadimethrines, such as polybrene, and alkali metal hydroxides (Japanese Patent Laid-Open (kokai) No. 6-70796).
Primarily in the field of biological products, a phenomenon has recently been observed in which drugs that test positive during the endotoxin-specific limulus reaction in spite of the fact that they do not exhibit pyrogenic properties when subjected to a rabbit pyrogen test specified by the Japanese pharmacopoeia. Thus, the use of an endotoxin test method (i.e., endotoxin-specific limulus reaction) described in the Minimum Requirements for Biological Products was found to involve the risk of inadequate evaluation of the safety of drugs.
By the analysis of this phenomenon, the present inventors discovered that the substances which falsely test positive during the endotoxin-specific limulus reaction are different from the conventionally-known limulus reaction-activating substances and are difficult to eliminate by means of any one of the foregoing means for eliminating limulus reaction false-positive substance.
This finding has made it considerably important to provide a method for eliminating the influence on the limulus reaction of the limulus reaction false-positive substances mixed in the biological products and for enabling accurate measurement of only endotoxin.
SUMMARY OF THE INVENTION
Accordingl
Tamura Hiroshi
Tanaka Shigenori
Watanabe Maki
Knobbe Marten Olson & Bear LLP
Seikagaku Kogyo Kabushiki Kaisha
Swartz Rodney P.
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