A chemiluminescent immunoassay based on intelligent nanoluminescent particle labeling amplification and its application

A technology of chemiluminescence immunity and luminescence particles, which is applied in the direction of chemical reaction of materials for analysis, chemiluminescence/bioluminescence, analysis of materials, etc., which can solve the problem of limited number of coupling functional groups, limited labeling amplification effect, and low labeling efficiency. and other problems, to achieve the effects of high sensitivity, improved labeling efficiency of luminescent molecules and antibodies, and a wide detection range

Active Publication Date: 2019-11-08
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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  • A chemiluminescent immunoassay based on intelligent nanoluminescent particle labeling amplification and its application
  • A chemiluminescent immunoassay based on intelligent nanoluminescent particle labeling amplification and its application
  • A chemiluminescent immunoassay based on intelligent nanoluminescent particle labeling amplification and its application

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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] Preparation of isopropylacrylamide and methacrylic acid copolymer nanoparticles (P(NIPAm-co-MAA)) by emulsion polymerization: 50mg of sodium dodecyl sulfate (SDS) and 100mg of permeate were added to a three-necked flask. Potassium sulfate, add 100mL deionized water to dissolve. Take 2 mL of monomer isopropyl acrylamide, 0.1 mL of methacrylic acid and 0.05 mL of cross-linking agent methylene bis acrylamide (MBA), mix it well in advance, add it to a three-necked flask, emulsify it and stir at 70°C The reaction was heated for 8 hours. After the product was collected, the liquid was removed by centrifugation (rotating speed 10000 rpm, centrifugation for 30 min), and then resuspended in deionized water. This was repeated 3 times to obtain P(NIPAm-co-MAA) nanoparticles.

[0038] Preparation of B.P (NIPAm-co-MAA) Nanoparticle / Acridinium Ester Lumine...

Embodiment 2

[0055] In order to verify the significant advantages of the temperature-sensitive nano-luminescent particle labeling amplification system of the present invention, we designed the following parallel comparative experiments. The concentration gradient of the CTNI standard substance of the same concentration is detected separately by the traditional double antibody sandwich system, the labeling amplification system using only polystyrene nanospheres, and the labeling amplification system of the present invention, and the luminescence value is 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] It was diluted with PBS buffer to prepare CTNI standards with concentration gradients of 0, 0.003, 0.008, 0.023, 0.069, 0.206, 0.617, 1.852, 5.556, 16.667, and 50.000 ng / mL. In the reaction cup, add 10 μL of streptavidin-coated magnetic microspheres,...

Embodiment 3

[0070] Example 3 The system of the present invention tests the correlation between the CTNI concentration in the serum sample and the Siemens measurement value.

[0071] In order to verify the accuracy of the system kit of the present invention, a plurality of serum samples were tested for CTNI content with Siemens CTNI chemiluminescence detection reagent, and 60 of them were selected with a suitable gradient. The system of the present invention was used to determine the content of CTNI, and the correlation was compared by plotting. by image 3 It can be seen that the two systems show good correlation, R 2 The value is as high as 0.9896. It is confirmed 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 method based on intelligent nano-luminescence particle label amplification and its application. Background technique [0002] Chemiluminescence immunoassay (CLIA) is a combination of highly sensitive chemiluminescence assay technology and highly specific immune response for various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins and The detection and analysis technology for drugs, etc. is the latest 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 raw material cost. Nevertheless, with the gradual popularization of analytical testing techniques and the further...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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