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CEA TRFIA (time-resolved fluoroimmunoassay) kit based on IMB (immunomagnetic beads)

A time-resolved fluorescence and immunoassay technology, applied in the fields of nanobiology and bioanalytical chemistry, can solve the problem that tumor-associated antigens have not been reported in the literature, and achieve the effects of shortening the reaction time, widening the range of the standard curve, and improving the detection sensitivity

Active Publication Date: 2015-05-27
GUANGZHOU BIOKEY HEALTH TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, immunomagnetic beads have been widely used in the fields of chemiluminescent immunoassay and nucleic acid extraction, but there is no literature report on the detection of some tumor-associated antigens by combining immunomagnetic beads with time-resolved fluorescent immunoassay.

Method used

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  • CEA TRFIA (time-resolved fluoroimmunoassay) kit based on IMB (immunomagnetic beads)
  • CEA TRFIA (time-resolved fluoroimmunoassay) kit based on IMB (immunomagnetic beads)
  • CEA TRFIA (time-resolved fluoroimmunoassay) kit based on IMB (immunomagnetic beads)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 Preparation of the kit of the present invention

[0036] (1) Specific steps for the preparation of immunomagnetic beads:

[0037]The first step, pretreatment of magnetic beads: use a vortex mixer to fully mix and suspend magnetic beads (Merck company product, number: 39572001), take 1ml (10mg) of the suspension in a centrifuge tube, and place it on a magnetic separator 1min (or longer), carefully remove the supernatant. Add 1ml Binding Buffer, mix thoroughly with a vortex mixer to suspend the magnetic beads, and place on a magnetic separator for 1min (or longer) to carefully remove the supernatant, repeat this step twice, then add 25 μl EDC (10mg / ml) and 40 μl NHS (10mg / ml), use a sample rotary mixer and rotate at room temperature for 30min. Add 1ml Binding Buffer, mix well with a vortex mixer to suspend the magnetic beads, and place on a magnetic separator for 1min (or longer) to carefully remove the supernatant, repeat this step 2 times.

[0038] The ...

Embodiment 2

[0057] Embodiment 2 The usage method of the kit of the present invention

[0058] (1) Sample collection

[0059] Take 1-2ml of venous blood into the coagulation tube, place it at 4°C for more than 2 hours, and take 25ml of serum after the serum is precipitated. Serum samples can be stored at 2-8°C for 7 days. If long-term storage is required, please store at -20°C to avoid repeated freezing and thawing. Samples need to be transported in vacuum flasks or other devices containing dry ice.

[0060] (2) Preparation of reagents

[0061] 1) Washing solution: Mix 50 mL of concentrated washing solution and 1200 mL of deionized water as a working washing solution.

[0062] 2) Marker working solution: One hour before use, dissolve each bottle of marker with 1 mL of deionized water, and dilute it 1:50 times with analysis buffer as the europium-labeled antibody working solution.

[0063] 3) Immunomagnetic beads: shake and suspend before use.

[0064] (3) Operation steps

[0065] 1) ...

Embodiment 3

[0070] Embodiment 3 The methodological test of the kit of the present invention

[0071] The test kit prepared in Example 1 is tested according to the routine manufacturing and testing procedures in the art, and the results are as follows:

[0072] 1) Accuracy

[0073] After analyzing and measuring the calibrator and the corresponding national standard substance at the same time, the two dose-response curves are basically parallel ( figure 1 : is the dose-response curve of the national standard; figure 2 : is the dose-response curve of the calibrator; Cps is the fluorescence count per second), the slopes of the two curves are 0.461 and 0.458 ( t test P >0.05). With the CEA national standard as the reference substance, the ratio of the measured titer to the marked potency of the calibrator is between 0.9 and 1.1. The linear correlation coefficient of the dose-response curve ( r )=0.998.

[0074] 2) Analytical sensitivity and linear range

[0075] The zero reference sta...

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Abstract

The invention discloses a CEA TRFIA (time-resolved fluoroimmunoassay) kit based on IMB (immunomagnetic beads). The kit includes a calibrator, immunomagnetic beads connected with CEA antibodies, europium labeled CEA antibodies, an assay buffer solution, a cleaning solution and an enhanced solution. Through double antibody sandwich immunoreaction, IMB-CEA-europium labeled-CEA monoclonal antibody complexes are formed, IMB which adsorb CEA and supernate are separated through magnetic seperation and washed, the enhanced solution is added, and the value is measured through a time-resolved instrument. Besides the TRFIA technology, the invention further has the advantages as follows: through IMB enrichment and full diffusion of IMB in the solution, the combination superficial area is enlarged, the reaction time is greatly shortened, and the detection sensitivity is improved. IMB and antibodies are directionally connected through chemical groups, so that the consumption of paired antibodies is greatly reduced and the detection precision is improved. The technology realizes automation easily, and overcomes the problem that the traditional micropore plate type TRFIA technology can only carry out detection after samples are accumulated to a certain number, thereby realizing real-time sample detection.

Description

technical field [0001] The invention belongs to the fields of bioanalytical chemistry and nano-biotechnology, and in particular relates to a CEA time-resolved fluorescent immunoassay kit based on immunomagnetic beads. Background technique [0002] Carcinoembryonic antigen (CEA) was first discovered by Gold and Freedman in 1965 from fetal and colon cancer tissues. The coding gene of CEA is located on chromosome 19, which is a polysaccharide-protein complex with a molecular weight of 22,000, 45% of which is protein. In general, CEA is synthesized by fetal gastrointestinal epithelial tissue, pancreas and liver cells. Usually, the CEA content increases within the first 6 months of pregnancy, and the serum level is already very low after birth. Under normal circumstances, CEA is metabolized by the gastrointestinal tract, but in a tumor state, CEA enters the blood and lymphatic circulation, causing an abnormal increase in serum CEA. At present, it is believed that CEA is widely...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/531G01N21/64
Inventor 吴英松
Owner GUANGZHOU BIOKEY HEALTH TECH CO LTD
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