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Method for measuring leuprorelin acetate in minute quantities and application

A technology of leuprolide acetate and trace amounts, which can be used in measuring devices, biological tests, instruments, etc., and can solve problems such as expensive, unsuitable for clinical use, and environmental pollution

Inactive Publication Date: 2009-08-19
INST OF PHARMACOLOGY & TOXICOLOGY ACAD OF MILITARY MEDICAL SCI P L A
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these detection methods have relatively high sensitivity, they all have a common disadvantage, that is, the use of isotopes is easy to cause environmental pollution, and the operation is complicated and expensive, and must be equipped with advanced equipment, which is not suitable for clinical use.

Method used

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  • Method for measuring leuprorelin acetate in minute quantities and application
  • Method for measuring leuprorelin acetate in minute quantities and application
  • Method for measuring leuprorelin acetate in minute quantities and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] The preparation of embodiment 1 leuprolide acetate antiserum

[0014] 1 Materials and reagents

[0015] 1.1 Animals: White rabbit, 2.5 kg, male, provided by our hospital.

[0016] 1.2 0.1M pH 7.0 Phosphate Buffered Saline

[0017] Take 0.2M NaH 2 PO 4 39ml, 0.2M Na 2 HPO 4 61ml, add distilled water to 200ml.

[0018] 1.3 0.021M glutaraldehyde solution

[0019] Take 0.042ml of 50% glutaraldehyde, add 0.1M phosphate buffer solution pH 7.0 to 10ml.

[0020] 1.4 2% bovine serum albumin solution:

[0021] Dissolve 100mg bovine serum albumin (BSA) in 5ml 0.1M pH 7.0 phosphate buffer

[0022] 1.5 0.4% leuprolide acetate solution:

[0023] Dissolve 20mg of leuprolide acetate in 5ml of 0.1M pH 7.0 phosphate buffer.

[0024] 1.6 Freund's incomplete adjuvant:

[0025] 1 part of lanolin, 4 parts of liquid paraffin, autoclaved for 20 minutes.

[0026] 1.7 BCG 80mg.

[0027] 2 Artificial Antigen (LA-BSA) Synthesis

[0028] Take 5ml of 2% bovine serum albumin and 5ml of...

Embodiment 2

[0035] Embodiment 2 leuprolide acetate enzyme-linked immunosorbent assay

[0036] 1 Instruments and reagents

[0037] 1.1 Instrument

[0038] Micropipette (GILSON), microplate reader (BIO-RAD), -70°C refrigerator (FormaScientific, INC), 96-well microplate plate (Jiangsu Haimen Sanhe Yamei Glass Products Factory), 37°C incubator ( Tianjin Medical Device Factory), 4℃, -20℃ refrigerator (Qingdao Haier Co., Ltd.), LD5-2A medical centrifuge (Beijing Medical Centrifuge Factory), etc.

[0039] 1.2 Reagents

[0040] Antiserum against leuprolide acetate, leuprolide acetate (batch number: 011008, with a purity of 99.57%, provided by Room 7 of the Institute), goat anti-rabbit horseradish peroxidase (batch number: 56403, Beijing Zhongshan Biotechnology Co., Ltd. company), 3,3′,5,5′-tetramethylbenzidine (Sigma), skimmed milk powder (batch number: 8631550, Inner Mongolia Yili Industrial Group Co., Ltd.), and other reagents were domestically produced and of analytical grade.

[0041] 1.3...

Embodiment 3

[0082] The making of embodiment 3 standard curve

[0083] Get different concentrations of leuprolide acetate 0.07mL (final concentration is 0.04375-8750ng / mL), add 0.08mL of rabbit anti-leuprolide acetate antiserum prepared as in Example 1 diluted 1:3428 (final concentration The concentration is 1:15000), add 0.2mL of normal beagle dog serum, and make a blank control at the same time, the total reaction volume is 0.35mL, and measure it as described in "Enzyme Immunoassay Method", take the measured O.D.450nm value as the ordinate, The logarithmic value of the standard sample concentration was plotted on the abscissa, and the experimental data were fitted using a competitive inhibition equation (SigmaPlot 2000) at one site to obtain a standard curve (see figure 2 ). Its equation is:

[0084] y = min + max + min 1 + ...

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Abstract

The present invention relates to a novel assay method for trace amount of leuprolide acetate, which adopts enzyme-linked immunosorbent assay method, wherein anti-leuprolide acetate antiserum is used to complete the assay, and the effective detection range of leuprolide acetate content in serum samples 0.025-1000ng / ml. The present invention also relates to the use of the determination method for qualitative or quantitative detection of leuprolide acetate content in biological samples, development of new leuprolide acetate drugs and pharmacokinetic research thereof.

Description

technical field [0001] The present invention relates to a novel assay method for trace amount of leuprolide acetate, which adopts enzyme-linked immunosorbent assay method, wherein anti-leuprolide acetate antiserum is used to complete the assay, and the effective detection range of leuprolide acetate content in serum samples 0.025-1000ng / ml. The present invention also relates to the use of the determination method for qualitative or quantitative detection of leuprolide acetate content in biological samples, development of new leuprolide acetate drugs and pharmacokinetic research thereof. Background technique [0002] Leuprorelin acetate (hereinafter referred to as LA) is a potent gonadotropin-releasing agonist. In the early stage of use, it can stimulate the pituitary gland to secrete gonadotropins and induce the reproductive organs to produce steroids. Long-term use can desensitize and / or downregulate pituitary gonadotropin receptors, reducing gonadotropin release, thereby...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/53G01N33/535
Inventor 董泗建郑健全池木根张振清魏淑香王晓英梁远军刘克良
Owner INST OF PHARMACOLOGY & TOXICOLOGY ACAD OF MILITARY MEDICAL SCI P L A