Method for preparing high-sensitivity immunity quantitative latex testing reagent

An immunoquantitative and sensitive technology, applied in biological tests, measuring devices, material testing products, etc., can solve the problems of insufficient sensitivity, inability to form immune binding networks, and inability of protein molecules to improve sensitivity and increase secondary immune responses. speed, and the effect of improving the sensitivity of the reaction

Inactive Publication Date: 2010-01-13
JIANGSU FLON BIOTECH
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AI-Extracted Technical Summary

Problems solved by technology

However, its disadvantage is that it is not as sensitive as enzyme labeling and fluorescent labeling methods. It cannot detect protein molecules with very low content, and cannot form an effective immune binding network for sm...
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Method used

(4) the microsphere liquid of above-mentioned stretched arm is added EDC by 15%r (0.005*192*15%) amount, after activating 20 minutes, add the ethylene glycol of 50% volume, and pH is raised to 7.0-7.2, after equilibrating, quickly pour in the antibody solution, react at room temperature for 2 hours, while controlling the pH to not less than 7.0, and equilibrate at 4°C overnight; the next day, adjust the pH to 7.0, and then equilibrate at 4°C overnight After 48 hours, add bovine serum albumin (dissolved in 0.025M, NaCL, pH 6.8) in the ratio of 2.0mg/1% ...
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Abstract

The invention relates to a method for preparing a high-sensitivity immunity quantitative latex testing reagent. The reagent comprises the following components: microsphere particles of which the concentration is between 0.2 and 0.4 percent (w/w), 0.12 M of glycine buffer solution of which the pH is 7.2, 30 percent of ethylene glycol (v/v), 2mM EDTA.2Na disodium EDTA, and 0.1 percent of NaN3. The preparation method comprises the following steps: preparing vector immunity quantitative latex micro-particles; preparing an antibody and an antigen; adding EDC at normal temperature; activating carboxyl under a condition that the pH is less than 6.5; and after certain period of time, raising the pH value and slowing down the reaction. Compared with the prior art, the method for preparing the high-sensitivity immunity quantitative latex testing reagent improves the uniformity of the diameters of the vector micro-particles and the uniformity of the antigen or the antibody distributed on the surfaces of the particles, and simultaneously improves the degree of freedom of the coated antigen or the antibody under a homogeneous phase condition, reduces the spatial combination steric hindrance, improves the secondary immunological reaction rate and improves the reaction sensitivity, and the detection sensitivity is improved by 100 times compared with that reported by the literatures in the past.

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  • Method for preparing high-sensitivity immunity quantitative latex testing reagent

Examples

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Example Embodiment

[0015] (1) Preparation of Carrier Immunoquantitative Latex Microparticles Pure water is heated to boiling to remove most of the oxygen in the water. After cooling, take 750ml and add 1.0g sodium dodecylsulfonate SDS to keep the temperature at 65°C for 10 minutes. After the SDS is in a suspended solution state, add 30ml of styrene monomer with a content greater than 98%, then raise the temperature to 75°C, and add 5.2ml of acrylic acid (pH adjusted to 5.5-6.5) after equilibrating for 15 minutes, and then add potassium persulfate after equilibrating. Start timing and gradually raise the temperature to 84°C. The reaction time is 30 minutes. Add neutralized acrylic acid in two times according to 4 times the amount of carboxyl groups on the surface of the microparticle size until the surface of the microspheres is fully carboxylated. Aging at 84°C for two hours , natural cooling. Calculate the concentration of the reaction based on the effective amount added to the reaction and the total volume of the reaction, and convert the OD value into the average diameter based on the standard concentration at a wavelength of 550nm by turbidimetry. The diameter of the microspheres can also be directly measured with an electron microscope, and synthesized according to this operation The microsphere diameter uniformity is greater than 98%.
[0016] (2) Preparation of antibodies and antigens: purchased from the market, purified by 33% (W/V) ammonium sulfate saturated precipitation, this method is milder for antibodies, and the antibodies are not easily denatured.
[0017] (3) At room temperature, add water-soluble carbodiimide EDC (0.005*192*40%) mg amount to the microparticle sphere liquid according to the 1% concentration containing 0.005 mM effective carboxyl group, and activate it under the condition of pH<6.5 After 20 minutes, adjust the pH to 7.0-7.2, add the amount of n-aminocaproic acid (0.005*132*60%) mg, and adjust the pH to 7.5 at room temperature for 2 hours, overnight at 4 ° C, the next day to 0.025M, NaCl solution with pH 7.0 was dialyzed twice and set aside. In this reaction, the activation pH condition of the carboxyl group must be controlled between 4.5-6.5. When the pH>6.5, EDC will fail to activate the carboxyl group. But when the carboxyl group is activated, the reaction speed is obviously slowed down when the pH>6.5, but the reaction is not terminated. This operation will greatly improve the distribution uniformity of the antibody antigen, and can increase the detection sensitivity by an order of magnitude.
[0018] (4) Add 15%r (0.005*192*15%) of the above-mentioned microsphere solution into EDC, activate it for 20 minutes, add 50% volume of ethylene glycol, and adjust the pH to 7.0-7.2 After equilibrating, quickly pour the antibody liquid into it, and act for 2 hours at room temperature, while controlling the pH to not less than 7.0, and equilibrate at 4°C overnight; the next day, adjust the pH to 7.0, and then equilibrate at 4°C overnight; 48 hours Finally, add bovine serum albumin (dissolved in 0.025M, NaCL, pH 6.8) in the ratio of 2.0mg/1%ml to block overnight, and finally add glycine, ethylene glycol, NaN 3 , so that the final concentration reached 0.12M, 30%, 0.1%, pH 7.4; stored at 4°C for 3 days before use. In order to increase the degree of freedom of the coated antibody, a certain amount of arms is appropriately added on the surface of the microsphere particles to increase the degree of freedom of the antibody in space and reduce steric hindrance. The approach of adding arms via the surface of the microspheres can increase the sensitivity of the reaction by another order of magnitude.
[0019] (5) According to this, high-sensitivity immunoquantitative latex assay reagents were prepared.
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