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Trimethoprim hapten as well as colloidal gold detection device and preparation method of trimethoprim hapten

A trimethoprim and detection device technology, which is applied in the field of trimethoprim hapten and its colloidal gold detection device and its preparation, can solve the problem of high technical requirements for operators, complex safety regulations of instruments and equipment, and inability to display results immediately, etc. problem, to achieve the effect of high detection sensitivity, easy production and high accuracy

Active Publication Date: 2016-06-08
SHENZHEN BIOEASY BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The instruments and equipment used in the above method have complex safety regulations, high cost, high technical requirements for operators, and cannot immediately display the results, so they are not suitable for rapid online detection and monitoring of suspected objects by commodity inspection, epidemic prevention, and livestock producers

Method used

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  • Trimethoprim hapten as well as colloidal gold detection device and preparation method of trimethoprim hapten
  • Trimethoprim hapten as well as colloidal gold detection device and preparation method of trimethoprim hapten
  • Trimethoprim hapten as well as colloidal gold detection device and preparation method of trimethoprim hapten

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The preparation of the trimethoprim hapten was prepared by the following steps:

[0043] In a 100mL three-necked flask, 2.5g of trimethoprim and 30mL of 48% by mass hydrobromic acid were sequentially added, reacted at 100°C for 3 hours, and then quenched by adding 6mL of 50% by mass sodium hydroxide solution. After the crystals were precipitated by cooling, the crystals were then dissolved in 20 mL of boiling water, and then concentrated ammonia water was added to adjust the pH to 7, followed by recrystallization and purification.

[0044] Take 0.5g of the crystal from the previous step and dissolve it in 10ml of DMF, add 0.5g of potassium carbonate, and 0.43mL of bromopropionic acid, and react at 40°C for 6 hours. Ethyl acetate extraction and column purification to obtain trimethoprim hapten.

[0045] The obtained trimethoprim hapten is detected, MASS[M+H] is 349.2, such as figure 1 Shown; its melting point is 214 degrees.

Embodiment 2

[0047] Using trimethoprim hapten to prepare the synthesis of trimethoprim immune antigen, the following steps are used to prepare:

[0048] Dissolve 0.1 mmol of trimethoprim hapten in 2 mL of DMF, and add 27.5 mg of DCC and 14.4 mg of NHS with stirring. Magnetic stirring was carried out overnight at 4°C. After centrifugation, the supernatant was liquid A, and 140 mg of human serum albumin (KLH) was weighed and dissolved in 10 mL of PBS with a concentration of 0.1 mol / L and a pH of 8.0. Add 1 mL of DMF, stir and dissolve to prepare liquid B. Under magnetic stirring, liquid A is gradually dropped into liquid B, and react at 4°C for 12 hours. After centrifugation, the supernatant was taken, dialyzed with normal saline at 4°C for 3 days, and the dialysate was changed 3 times a day. The obtained whole antigen was dispensed into 0.5 mL centrifuge tubes at a concentration of 1 mg / mL, and frozen in a -20°C refrigerator.

Embodiment 3

[0050] Using trimethoprim to immunize antigen to prepare trimethoprim monoclonal antibody, adopt the following steps to prepare:

[0051] Use trimethoprim to immunize the antigen and identify and immunize four 6-week-old Kunming mice. After boosting the immunization three times, blood is collected to measure the titer. When the serum titer no longer rises, the mice are immunized with twice the dose of the antigen without adjuvant , three days later, the mice were killed by neck dislocation, the spleen was taken under aseptic conditions to prepare splenocytes, and the vigorously growing mouse myeloma cells were mixed in a 50mL centrifuge tube at a ratio of 8:1, and 30mL of serum-free IPMI1640 was added to culture Base, centrifuge at 1100r / min for 5 minutes, discard the supernatant, shake the cell mass gently, and place it in a water bath at 37 degrees Celsius. Slowly add 1 mL of 50% PEG-4000 by volume to the cells, drop it within 1 minute, and at the same time gently stir the s...

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Abstract

The invention provides a trimethoprim hapten as well as a colloidal gold detection device and a preparation method of the trimethoprim hapten. The trimethoprim hapten has a chemical structural formula shown as the formula (1). With the adoption of the technical scheme provided by the invention, the trimethoprim hapten and the colloidal gold detection device are strong in specificity, high in detection sensitivity, high in accuracy and good in repeatability, and can be used for rapidly and simply detecting the residues of trimethoprim; any instrument and equipment are not needed and the trimethoprim hapten and the colloidal gold detection device are convenient to carry and low in detection cost; a test strip is simple to use and is not required to be operated by professionals; test paper is easy to produce and low in cost, and the detection requirements on the residual amount of the trimethoprim in food safety detection are met.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a trimethoprim hapten, a colloidal gold detection device and a preparation method thereof. Background technique [0002] Trimethoprim is a lipophilic weak base pyrimethamine antibacterial agent. Its antibacterial spectrum is similar to that of sulfonamides, but its antibacterial effect is stronger than that of sulfonamides. It is effective against Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Staphylococcus saprophyticus, and various Gram-positive and negative bacteria, but it is ineffective against Pseudomonas aeruginosa infection , the minimum inhibitory concentration is often lower than 10mg / L, single use is likely to cause bacterial resistance, so it is generally not used alone, mainly composed of compound preparations with sulfa drugs. Therefore, it is also called sulfonamide synergist, which is used to expand the antibacterial spectru...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/558G01N33/531G01N33/532G01N21/78
CPCG01N21/78G01N33/531G01N33/532G01N33/558G01N33/577
Inventor 严义勇朱海付辉李君秀李细清毕思远汪凤林
Owner SHENZHEN BIOEASY BIOTECHNOLOGY CO LTD
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