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Preparation method and using method of nodularin (NODLN) polyclonal antibody immunoaffinity column (IAC)

A technology of nodularia and immunopro, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., and can solve the problems of high price, low detection limit, interference detection results, etc.

Inactive Publication Date: 2011-06-29
XUCHANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Our previous studies have shown that HPLC and LC-MS methods can be used to detect NODLN in water. Because of its low content, solid phase extraction (SPE) is generally used for enrichment before sample injection. However, SPE is not specific, and there are many impurities in water. After collection, many impurities will be introduced, which will interfere with the detection results, especially with HPLC method, sometimes the measured value is far from the actual value
Although the LC-MS method has a low detection limit and relatively accurate measurement results, it is expensive and generally not available in laboratories.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Preparation of NODLN polyclonal antibody

[0036] 1. Synthesis of complete antigen

[0037] (1) Synthesis of NODLN-BSA immunogen

[0038] The synthesis of the complete antigen NODLN-BSA adopts the water-soluble carbodiimide method, using 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC) as a "bridging agent" to make the carboxyl group of NODLN (-COOH) reacts with EDC to generate an intermediate product, and then reacts with the amino group on the BSA protein molecule to make a complete antigen NODLN-BSA conjugate as an immunogen. The synthesis method is as follows:

[0039]Dissolve 0.4mg NODLN in 0.5mL methanol, divide it into two equal portions of 0.25mL, pass nitrogen gas, and evaporate to dryness at 35°C. A portion of the residue containing 0.2 mg NODLN was dissolved in 1 mL PBS (pH7.4) buffer, 5 mg EDC was added, shaken until completely dissolved, the pH value was adjusted to 5 with 0.1 M hydrochloric acid solution, and stirred slowly at room temper...

Embodiment 2

[0049] Example 2: Activation of Sepharose 4B

[0050] Sepharose 4B was activated by the cyanogen bromide method. The cyanogen bromide method has good activation effect, high coupling rate, and little effect on antibodies. The activation process is as follows:

[0051] (1) Take 10mL Sepharose 4B and put it in a Buchner funnel to drain it, wash it twice with 30mL of water and drain it, add a small amount of 0.1mol / L NaHCO at pH8.3 3 After washing, immediately transfer to a 100mL beaker and stir slowly under ice bath.

[0052] (2) Weigh 1g of cyanogen bromide in a fume hood, add 10mL of water to dissolve, then pour into agarose in batches, stir while adding, and measure the pH value at the same time, and keep the pH at 10.5 by adding 2mol / L NaOH dropwise about. After the cyanogen bromide has completely reacted and the pH remains basically unchanged, the stirring can be stopped.

[0053] (3) Add the activated Sepharose 4B into small ice cubes, quickly pour it into the Buchner ...

Embodiment 3

[0054] Example 3: Preparation of NODLN polyantibody IAC

[0055] The NODLN polyclonal antibody was coupled with Sepharose 4B to obtain an affinity adsorbent, which was filled into a column to prepare the NODLN polyclonal antibody IAC. The operation steps were as follows:

[0056] (1) Coupling

[0057] Put the above prepared and purified NODLN polyclonal antibody in 0.1M NaHCO containing 0.5M NaCl at pH 8.3 3 The buffer was dialyzed against the coupling buffer for 12 h. Put the above-mentioned activated Sepharose 4B gel in a sand core funnel and quickly wash it with the coupling buffer, then quickly pour it into the NODLN polyclonal antibody solution for coupling, and monitor the coupling process by UV scanning. Wash away the unconjugated anti-NODLN polyclonal antibody with more than 5 times the volume of coupling buffer to obtain the agarose-antibody coupling complex. Collect all the eluate, and calculate the amount of uncoupled NODLN polyclonal antibody by measuring its pr...

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Abstract

The invention relates to a preparation method and a using method of a nodularin (NODLN) polyclonal antibody immunoaffinity column (IAC), and belongs to the technical field of immunoaffinity chromatography and algae toxin detection. In the prepared IAC, an NODLN polyclonal antibody is fixed in the column to adsorb NODLN in sample solution, enrichment and purification effects are achieved after elution, and qualitative and quantitative analysis is performed through high performance liquid chromatography (HPLC); in the pretreatment process, IAC enrichment is superior to the conventional solid-phase extraction (SPE) method; an HPLC graph shows that the NODLN polyclonal antibody IAC can separate the peak of the NODLN from the peak of other impurities so as not to influence a measurement result, and excessive impurities exist in SPE purification, so that the peak of the NODLN is even difficult to distinguish; and the NODLN polyclonal antibody can be specifically combined with the NODLN, so that the IAC has high specificity, and most interfering substances can be removed through one-time purification, while the SPE does not have specificity, and the interfering substances cannot be removed, so that the final measurement result is influenced.

Description

technical field [0001] A method for preparing and using a nodular toxin polyantibody immunoaffinity column belongs to the technical field of immunoaffinity chromatography and nodular toxin detection. Background technique [0002] In recent years, due to the development of production and living activities, human beings have discharged a large amount of waste and wastewater into the water body, resulting in eutrophication of the water body, a large number of algae, and the number of toxic algae has also increased sharply. my country's drinking water sources such as Taihu Lake, Chaohu Lake, Dianchi Lake and other lakes, some reservoirs and rivers are all attacked by cyanobacteria blooms. According to the "Yangtze River Protection and Development Report" released in April 2007, after the Three Gorges Reservoir was impounded to 135 meters in 2003, 12 primary tributaries of the Yangtze River had algal blooms in varying degrees in the backwater areas, and in recent years there have...

Claims

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Application Information

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IPC IPC(8): G01N33/531G01N30/00G01N30/08
Inventor 肖付刚吕春霞王德国刘海英高雪丽张晓伟郭卫芸李凌乐
Owner XUCHANG UNIV
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