Homogeneous enzyme immunoassay for oral fluid

A technology of oral fluid and enzyme immunity, applied in the field of immune analysis

Inactive Publication Date: 2007-10-03
LIN ZHI INT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Homogeneous enzyme immunoassay for oral fluid
  • Homogeneous enzyme immunoassay for oral fluid
  • Homogeneous enzyme immunoassay for oral fluid

Examples

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Effect test

Embodiment 1

[0231] Example 1: Calculation of Enzyme Activity of Enzyme-Analyte Conjugates

[0232] In order to accurately determine the concentration of the analyte in a sample suspected of containing the analyte, the signal (expressed as ΔA / min) generated by G6PDH between the negative calibrator and the high calibrator should preferably be about 100 mA / min (rate mode ), the negative calibrator is a calibrator with 0 ng / ml analyte, and the high calibrator is, for example, a calibrator with 50 ng / ml analyte. Therefore, in the conventional homogeneous enzyme immunoassay of the present invention, G6PDH produced about 100 mA / min. The following equation shows the relationship between signal intensity, enzyme activity, and reaction volume.

[0233]

[0234] where ΔA is the signal generated by G6PDH (expressed in absorption or milliabsorbance units); V t is the total reaction volume in milliliters (ml) and includes the volume of assay sample, R1 (volume of antibody or receptor, substrate, c...

Embodiment 2

Example 2: G6PDH-PCP conjugates

[0248] G6PDH with an initial specific activity of 860 units / mg was purchased from USB Biochemical. Conjugation of 19 mg of G6PDH to the PCP hapten resulted in 45% inactivation. After purification, 13 ml of enzyme-PCP conjugate (1.4 mg / ml) was isolated. Purified enzyme-PCP conjugates were further inhibited by up to 60% after binding to PCP-reactive antibodies. 1-2,000-fold dilutions of the enzyme reagent containing 0.731 μg / ml enzyme-PCP conjugate were prepared. In the desired immunoassay, 20 μl-45 μl sample, 75 μl enzyme-PCP conjugate, and 150 μl antibody solution were used. The following calculations illustrate the importance of % inactivation, % inhibition and sample volume.

1. Enzyme concentration in the reagent: 0.000731mg / ml

2. Enzyme in each assay (75 μl / analysis): 0.0000548 mg / analysis

3. Standardized to 1.0ml (from an assay volume of 0.245ml): 0.000224mg / ml

4. Enzyme activity after 45% inactivation: 0.1058 units / ml

5. Enzym...

Embodiment 3

[0269]G6PDH enzyme (4 mg, starting specific activity 860 units / ml) was conjugated to the opiate hapten. 8.5 ml of G6PDH-opiate conjugate were purified. This conjugation resulted in a 45% inactivation of G6PDH. Opioid antibody binding to the G6PDH-opiate conjugate resulted in 52% inhibition. The conjugate was formulated into the enzyme reagent at a dilution factor of 1:450. Using the same calculation method as in Example 1, the following results were obtained:

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Abstract

The present invention discloses homogeneous enzyme immunoassay systems, methods and kits useful for the qualitatively and quantitatively determination of analytes in oral fluid samples. The system involves a competitive enzyme immunoassay employing a conjugate comprising glucose-6-phosphate dehydrogenase (G6PDH) and an analyte. The methods and kits are particularly useful in the detection of recent drug use and for fast determination of analytes using auto-analyzers.

Description

field of invention [0001] The present invention relates to the field of immunoassays. The present invention provides compositions and methods for determining the amount of an analyte in an oral fluid sample suspected of containing said analyte. In particular, the immunoassay composition of the invention comprises a glucose-6-phosphate dehydrogenase (G6PDH)-analyte conjugate, an antibody reactive with said analyte, an oral fluid sample suspected of containing said analyte, an enzyme Acting substrate, and coenzyme of G6PDH. The invention also relates to kits for the determination of analytes in oral fluid specimens using a homogeneous immunoassay. Background of the invention [0002] Currently, there is a continuing need to develop new, simpler, faster and more sensitive techniques for determining the presence of analytes in samples suspected of containing them. In particular, the determination of trace analytes, especially chemical substances, is critical for many healthca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/535G01N33/536G01N33/542G01N33/94C07K16/44
CPCG01N33/94G01N33/581G01N2333/904
Inventor 林正义林陈梅怡贾荟
Owner LIN ZHI INT
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