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Kit and method for detecting antibody by spatial proximity chemiluminescence two-step method

A chemiluminescence and spatially adjacent technology, which is applied in chemiluminescence/bioluminescence, analysis by making materials react chemically, measurement devices, etc., can solve problems such as inability to complete two-step detection and methodological inability to detect

Pending Publication Date: 2021-08-31
无锡壹闪生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The advantages of spatially adjacent chemiluminescence technology are obvious. The reaction process does not require coating and washing, and is simple and fast. However, there are also shortcomings in the clinical application process. For example, many clinical detection items need to be completed by two-step methods, namely Through the washing step, non-specific interference, such as autoantibody detection, etc., cannot be completed by the existing spatial proximity chemiluminescence technology, which makes the technology have limitations in clinical application, and many projects are due to methodological limitations. sex undetectable

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0055] I. Preparation of calibrator

[0056] Calibration dilution formulation: potassium dihydrogen phosphate weighed 14.1g, NaH 2 PO4 · 2H 2 O 3.0g, dissolved in deionised water was added, Proclin-300 0.5 ~ 1mL, after mixing, adding ultrapure water volume to 1000mL, calibrated dilution, 2 ~ 8 ℃ stored for future use;

[0057] A calibration: The purified antibody was diluted to the corresponding concentration, and the spare was stored 2 to 8 ° C.

[0058] Second, the preparation of HRP-labeled avidin / streptavidin, avidin

[0059] Add 60mmol / L Naio in 1mg HRP 4 0.1 ml was taken at 4 ° C for 30 minutes, then adding ether alglegol of 0.16 mol / L of concentration of concentration of concentration. After 30 minutes, 1 mg a or SA was added to 4 ° C for 24 hours; dialyzate overnight, addition and equal volume saturated ammonium sulfate The solution, 4000 r / min was centrifuged for 15 minutes, and the precipitate was dissolved in PBS having a pH of 7.4, and the absorbance was measur...

Embodiment 1

[0083] Embodiments of the present invention is a space adjacent to the two-step chemiluminescent Zika virus antibody detection kit, comprising:

[0084] Luminescent labels, enzyme labels, biotin-labeled Zika virus antigen coated magnetic beads, adjuvants, and Calibrator trigger;

[0085] Luminescent labels feed components comprises 9,10-dihydro-acridinium-labeled monoclonal antibodies anti-human immunoglobulin and 0.05M Tris buffer;

[0086] Feed components include enzyme labels HRP labeled avidin / streptavidin and avidin 0.05M phosphate buffer;

[0087] Trigger selection 0.05M pH 8.0Tris-HCl buffer;

[0088] Emitting adjuvants include adjuvants and citrate buffer, pH 6.0 emitting adjuvant is p-hydroxyphenyl acrylate;

[0089] Calibrator Calibrators and calibrator dilutions 0.1M include different concentrations of antibody;

[0090] Preparation calibrator

[0091] Calibration dilution formulation: potassium dihydrogen phosphate weighed 14.1g, NaH2PO 4 · 2h 2 O 3.0g, dissolved in ...

Embodiment 2

[0109] This example is a method of detecting a village card virus antibody using a kit in Example 1, including steps:

[0110] S1: Add 25 μL of the calibration and sample to the reaction tube, and then add a biotin-labeled virus antigen to 25 μl of the magnetic bead, mix well, incubate at 37 ° C for 15 min;

[0111] S2: Wash, 0.1 mol / L of PB buffer by pH 7.2; 3 times;

[0112] S3: 25 μl of the enzyme labeled label is added to the calibration and sample, 25 μl of the luminescent label, mix well, incubation at 37 ° C for 10 min;

[0113] S4: 5 μL of the adjuvant is added to the calibration and sample, and the oscillation is mixed, stands for 1 to 2 min, and then adds 75 μl of the trigger, immediately detects, read the signal;

[0114] S5 :: Detect the light-emitting strength of the calibration and sample, compared with the Cutoff value, calculates the COI, judging the result;

[0115] When COI ≧ 1.0, the detection result is determined as positive;

[0116] When COI <1.0, the detec...

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Abstract

The invention discloses a kit and a method for detecting an antibody by a spatial proximity chemiluminescence two-step method. The kit comprises a luminous marker, an enzyme marker, a biotin-labeled antigen solid-phase coating substance, an auxiliary agent, a triggering agent and a calibrator; the luminous marker comprises a 9,10-dihydroacridine labeled anti-human immune globulin monoclonal antibody; and the enzyme marker comprises the raw material component of HRP-labeled avidin / streptavidin. According to the invention, a streptavidin / biotin system is introduced into a detection system, and a plurality of clinical projects requiring a two-step method to complete detection can be completed by applying a spatial proximity chemiluminescence technology, so that the clinical application range and value of the spatial proximity chemiluminescence technology are improved; meanwhile, a biotin-avidin / streptavidin system is introduced, so that the chemiluminescence efficiency can be greatly improved, and the detection sensitivity is improved; and the system can be used for rapid quantitative analysis of an automatic immunochemiluminescence system, POCT, a micro-fluidic chip and the like.

Description

Technical field [0001] The present invention relates to the technical field of chemiluminescence kits, and more particularly to a kit and method for detecting an antibody in a space adjacent chemiluminescent die. Background technique [0002] Space neighboring chemiluminescence technology is a homogeneous chemiluminescence technology, inventors for the United States H · Ahawan - Tafi, "Non-separated assay method" one-step test. Patent Application No. 20101616899.1 discloses a non-separated assay method, which is the protracted macarcamole peroxidase (HRP) on an antibody, labeled 9,10-dihydroacid acridine (ACRIDAN) on another antibody. When the antigen in the sample is formed to form an antigen antibody sandwich complex in the sample, the horseradish peroxidase and 9,10-dihydrozepine (Acridan) are close to each other in space, add excitation solution. After the light is illuminated, the working curve is drawn by a known concentration of calibration and its light-emitting value, th...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N21/76
CPCG01N33/56983G01N21/76
Inventor 奚伟红奚梦娇朱丹丹
Owner 无锡壹闪生物科技有限公司
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