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Chemiluminescence immunoassay based on labeling amplification of intelligent nano-luminescent particles and application of chemiluminescence immunoassay

A technology of chemiluminescent immunity and luminescent particles, which is applied in the direction of analyzing materials through chemical reactions, chemiluminescence/bioluminescence, and analyzing materials. It can solve the problems of limited number of coupling functional groups, limited labeling amplification effect, and low labeling efficiency. To achieve high sensitivity, improve the labeling efficiency of luminescent molecules and antibodies, and wide detection range

Active Publication Date: 2018-01-09
GETEIN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since the luminescent molecules and proteins are simultaneously labeled on the microspheres, the luminescent molecules and antibodies will compete for the coupling functional groups on the microspheres, resulting in low labeling efficiency, and the luminescent substances will still block the antibody sites, resulting in loss of activity
In addition, the number of coupling functional groups on the surface of the particle is limited, resulting in limited labeling amplification effect

Method used

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  • Chemiluminescence immunoassay based on labeling amplification of intelligent nano-luminescent particles and application of chemiluminescence immunoassay
  • Chemiluminescence immunoassay based on labeling amplification of intelligent nano-luminescent particles and application of chemiluminescence immunoassay
  • Chemiluminescence immunoassay based on labeling amplification of intelligent nano-luminescent particles and application of chemiluminescence immunoassay

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] 1. Preparation of temperature-sensitive nano-luminescent particles:

[0036] Preparation of A.P(NIPAm-co-MAA) nanoparticles

[0037] Copolymerized nanoparticles (P(NIPAm-co-MAA)) of isopropylacrylamide and methacrylic acid were prepared by emulsion polymerization: 50 mg of sodium dodecyl sulfate (SDS) and 100 mg of peroxide were added to a three-necked flask. Potassium sulfate, add 100mL deionized water to dissolve. Take 2mL of monomer isopropylacrylamide, 0.1mL of methacrylic acid and 0.05mL of cross-linking agent methylenebisacrylamide (MBA), mix them uniformly in advance, add them into a three-necked flask, emulsify evenly and stir at 70°C The reaction was heated for 8 hours. After the product was collected, it was centrifuged (10,000 rpm, 30 min) to remove the liquid, and then resuspended with deionized water. This was repeated 3 times to obtain P(NIPAm-co-MAA) nanoparticles.

[0038] B. Preparation of P(NIPAm-co-MAA) Nanoparticles / Acridinium Ester Luminescent Co...

Embodiment 2

[0055] In order to confirm the significant advantages of the labeling amplification system based on temperature-sensitive nano-luminescent particles of the present invention, we designed the following parallel comparative experiments. The concentration gradient of the CTNI standard substance at the same concentration was detected with the traditional double-antibody sandwich system, the labeling amplification system only through polystyrene nanospheres, and the labeling amplification system of the present invention, and the luminescence value was recorded to fit the standard curve. The concentrations of CTNI detection antibodies used in the above three systems are equal.

[0056] A. Traditional CLIA double-antibody sandwich method:

[0057] Dilute with PBS buffer to prepare CTNI standard with concentration gradient 0, 0.003, 0.008, 0.023, 0.069, 0.206, 0.617, 1.852, 5.556, 16.667, 50.000ng / mL. In the cuvette, add 10 μL of streptavidin-coated magnetic microspheres, 50 μL of a ...

Embodiment 3

[0070] Embodiment 3 Correlation comparative experiment between the concentration of CTNI in the serum sample measured by the system test of the present invention and the measured value of Siemens

[0071] In order to verify the accuracy of the system kit of the present invention, after testing the CTNI content with Siemens CTNI chemiluminescent detection reagent for multiple serum samples, 60 samples with a suitable gradient were selected. Use the system of the present invention to measure the CTNI content respectively, and draw a graph to compare the correlation. Depend on image 3 It can be seen that the two systems show a good correlation, R 2 Values ​​as high as 0.9896. It has been proved that the system of the present invention and the kit based on the system have excellent accuracy in addition to ultra-high sensitivity and wider detection range.

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Abstract

The invention discloses chemiluminescence immunoassay based on labeling amplification of intelligent nano-luminescent particles and application of the chemiluminescence immunoassay. The chemiluminescence immunoassay is realized by the following steps: covalently coupling an antibody of a to-be-tested antigen to carboxyl groups on the surfaces of the intelligent nano-luminescent particles to form intelligent nano-luminescent particles with a labeling amplification effect; directly or indirectly coating a solid-phase carrier with the other antibody of the to-be-tested antigen, adding the intelligent nano-luminescent particles and a sample containing the to-be-tested antigen, and forming a solid-phase carrier-antibody-to-be-tested antigen-antibody-intelligent nano-luminescent particle immunecomplex by interaction of antibody-antigen-antibody; and separating out the immune complex, then adding an excimer, detecting the luminous intensity by using a chemiluminescence detector, and furthercalculating to obtain the concentration of the to-be-tested antigen. According to the chemiluminescence immunoassay disclosed by the invention, the labeling efficiency of luminescence molecules and the antibody can be improved, and the luminescent intensity is enhanced. Therefore, the chemiluminescence immunoassay has the advantages of high sensitivity, wider detection range and the like.

Description

technical field [0001] The invention belongs to the technical field of chemiluminescence immunoassay, and relates to a chemiluminescence immunoassay based on intelligent nano-luminescent particle label amplification and its application. Background technique [0002] Chemiluminescence immunoassay (CLIA) is a combination of highly sensitive chemiluminescent assay technology and highly specific immune response, used for various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins and The detection and analysis technology of drugs is a newest immunoassay technology developed after radioimmunoassay, enzyme immunoassay, fluorescent immunoassay and time-resolved fluorescent immunoassay. Compared with the traditional enzyme immunoassay, CLIA has the characteristics of higher sensitivity, shorter detection time, simpler labeling method and lower cost of raw materials. Nevertheless, with the gradual popularization of analytical testing technology and the further im...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/577G01N33/532G01N21/76
Inventor 苏恩本赵欢林琦峰陈赢胡昕
Owner GETEIN BIOTECH
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