Gram negative bacteria screening method with horseshoe crab amebocyte lysate (LAL)
First Claim
1. A gram negative bacteria screening method for determining the presence of at least 104 gram negative bacteria cells per milliliter of a urine sample, comprising the steps of:
- (a) adding an undiluted urine sample to be tested to a test tube containing horseshoe crab amebocyte lysate and a first buffer capable of resisting a pH change in the pH range of from about 6.3 to about 7.5, to form a test tube mixture wherein the concentration of lysate is at least 3.5 milligrams per milliliter of the test tube mixture formed;
(b) incubating the test tube mixture for a time sufficient to activate the lysate;
(c) contacting a test device with the activated test tube mixture, said test device comprising a carrier matrix incorporated with a synthetic peptide substrate containing a chromogenic or fluorogenic leaving group capable of being cleaved by the activated lysate and a second buffer capable of resisting pH change in the pH range of from about 8.0 to about 8.9;
(d) removing the contacted test device; and
(e) determining the concentration of cleaved leaving group.
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Accused Products
Abstract
A screening method for the determination of 104 gram negative bacteria per milliliter of an undiluted urine sample and a unitary screening test device for the determination of at least 105 gram negative bacteria per milliliter of an undiluted urine sample. The method and test device make use of Limulus amebocyte lysate (LAL) and a synthetic substrate containing a chromogenic or fluorogenic leaving group capable of being cleaved by activated lysate. This screening method and unitary screening device provide a quick, convenient, inexpensive indication of the possible presence of a urinary tract infection caused by gram negative bacteria.
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Citations
16 Claims
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1. A gram negative bacteria screening method for determining the presence of at least 104 gram negative bacteria cells per milliliter of a urine sample, comprising the steps of:
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(a) adding an undiluted urine sample to be tested to a test tube containing horseshoe crab amebocyte lysate and a first buffer capable of resisting a pH change in the pH range of from about 6.3 to about 7.5, to form a test tube mixture wherein the concentration of lysate is at least 3.5 milligrams per milliliter of the test tube mixture formed; (b) incubating the test tube mixture for a time sufficient to activate the lysate; (c) contacting a test device with the activated test tube mixture, said test device comprising a carrier matrix incorporated with a synthetic peptide substrate containing a chromogenic or fluorogenic leaving group capable of being cleaved by the activated lysate and a second buffer capable of resisting pH change in the pH range of from about 8.0 to about 8.9; (d) removing the contacted test device; and (e) determining the concentration of cleaved leaving group. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A unitary gram negative bacteruria screening device for determining the presence of at least 105 gram negative bacteria per milliliter of a urine sample, comprising:
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(a) a carrier matrix; and (b) a test composition incorporated with the carrier matrix, which composition includes horseshoe crab amebocyte lysate, a divalent cation, a synthetic peptide substrate with a chromogenic or fluorogenic leaving group capable of being cleaved by the lysate, a buffer component capable of resisting a pH change in a pH range of from about 7.5 to about 8.5 and a stabilizing component capable of stabilizing the lysate. - View Dependent Claims (11, 12, 13, 14, 15)
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16. A method for preparing a unitary gram negative bacteruria unitary test device for determining the presence of at least 105 gram negative bacteria per milliliter of a urine sample, comprising the steps of:
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(a) incorporating a carrier matrix with a stabilizing component capable of stabilizing horseshoe crab amebocyte lysate; (b) drying; and (c) incorporating the dried matrix with a test composition including horseshoe crab amebocyte lysate, a divalent cation, a synthetic peptide substrate with a chromogenic or fluorogenic leaving group capable of being cleaved by the lysate and a buffer component capable of resisting a pH change in a pH range of from about 7.5 to about 8.5; and (d) drying.
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Specification