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Monoclonal antibody for ketamine detection and immune detection board

A ketamine and detection plate technology, applied in the field of enzyme-linked immunoassay detection of ketamine, can solve the problems of incompetent reaction of complete antigen, low coupling efficiency of hapten and carrier protein, difficult preparation and the like

Active Publication Date: 2007-02-07
SHANGHAI CRIMINAL SCI TECH RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] However, studies have shown that the coupling efficiency of the hapten to the carrier protein is low, and it is difficult to prepare a complete antigen
In addition, the complete antigen prepared by it cannot compete with the ketamine standard, so it cannot be used for the preparation of colloidal gold rapid diagnostic reagents

Method used

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  • Monoclonal antibody for ketamine detection and immune detection board
  • Monoclonal antibody for ketamine detection and immune detection board
  • Monoclonal antibody for ketamine detection and immune detection board

Examples

Experimental program
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preparation example Construction

[0090] The preparation method of the complete antigen of the present invention is as follows:

[0091]

[0092] Firstly, ketamine is activated to obtain its derivative: p-amino-ketamine, and then p-amino-ketamine is connected to a suitable protein carrier (eg, KLH, BSA) to obtain a complete antigen. Wherein X is a protein carrier, preferably hemocyanin (KLH) or bovine serum albumin (BSA) in the present invention; The part that is covalently cross-linked with X carrier is the derivative p-amino-ketamine (p-NH 2 -Ketamine).

[0093] The nitration and reduction reactions described therein can be carried out by any method known to those skilled in the art under any suitable conditions. For example, the nitration reaction of the present invention can be carried out under the following conditions: the reaction temperature is -10-10°C, preferably -5-0°C; the reaction time is 1-24 hours, preferably 2-12 hours, more preferably 2-6 hours The reduction reaction conditions of the pre...

Embodiment 1

[0148] Embodiment 1 Activation of ketamine and preparation of hapten

[0149] Add 0.52g of ketamine raw material and 10ml of 98wt% concentrated sulfuric acid in the reaction flask, stir to dissolve it, place it in an ice-salt bath and cool to -5°C, add 0.25ml of 70wt% nitric acid and A mixed acid solution consisting of 0.75ml of 98wt% sulfuric acid. After the addition, react at 0°C for 2 hours, then pour it into 10g of crushed ice, wait for the crushed ice to melt, and then react at 0°C for half an hour, when a white solid precipitates, filter it with suction to obtain a white solid 0.62g (crude product, directly used in the next reaction without purification). Reaction yield: 53%. The reaction product was analyzed by MNR as the target object: p-nitro-N-ketamine.

[0150] Dissolve 0.5 g of the reaction product (i.e., p-nitroketamine) in 1.4 ml of water, add 0.55 ml of 38 wt % concentrated hydrochloric acid under stirring, gradually raise the temperature, and start adding 0.6...

Embodiment 2

[0152] Example 2 Preparation of Ketamine-KLH Complete Antigen

[0153] Weigh 100mg of p-amino-ketamine, dissolve it in 10ml of water under stirring, then slowly add 400mg of EDC (carbodiimide), shake while adding, adjust the pH value to 4.5 with 0.1M hydrochloric acid, at 25°C Continue to react for 10 minutes. 100 mg of hemocyanin (KLH) was dissolved in 5 ml of double-distilled water, added to the p-amino-ketamine solution, and the reaction was continued at 25° C. for 3 hours. The reaction product was dialyzed against 0.01M phosphate buffer at 4° C. to remove unreacted p-amino-ketamine and EDC, and the buffer was replaced 3 to 4 times. Obtain Ketamine-KLH Complete Antigen 120mg.

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Abstract

The invention discloses a hapten and complete antigen used for ketamine detection and antibody preparation. The invention also discloses an anti-ketamine monoclonal antibody prepared by the complete antigen and a colloidal gold-labeled ketamine monoclonal antibody immunoassay plate used for detecting ketamine in drugs or urine, etc., human samples. Compared with HPLC method, the invented detection plate is simple, portable and easy to carry, and can be used for spot detection without need of expensive equipment. The whole detection for ketamine by using the detection plate can be completed in 10 minutes with sensitivity up to 50 ng and with no cross reaction with 39 kinds of common pharmaceuticals, drugs, and ketamine metabolites in vivo.

Description

technical field [0001] The invention relates to the detection of contraband drugs, in particular to the enzyme-linked immunological detection of ketamine. Background technique [0002] Ketamine is a short-acting anesthetic administered intravenously. After entering the blood circulation, most of it enters the brain tissue, and then distributes in the tissues of the whole body. The drug concentration in the liver, lungs and fat is also relatively high. The drug is mainly biotransformed into norketamine in the liver, and then gradually metabolized into inactive compounds and excreted by the kidneys, only 2.5% of which is excreted in the urine in the original form. The drug can provide deep analgesia at a sub-anesthesia dose, and it does not have the side effects of inhibiting heart and respiratory function associated with most other conventional anesthetics. It has been clinically tested for many years. [0003] As early as June 2001, ketamine had been included in the nationa...

Claims

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Application Information

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IPC IPC(8): C07C225/22C07K14/765C07K14/435G01N33/53G01N33/577
Inventor 曾立波
Owner SHANGHAI CRIMINAL SCI TECH RES INST
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