Method for measuring immunologic colloidal gold particle fluorescence quenching

A technology of fluorescence quenching and measurement method, applied in the field of detection, can solve the problems of low sensitivity, high price and high cost, and achieve the effects of high detection sensitivity, wide quantitative range and simple method

Inactive Publication Date: 2006-05-17
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these two methods require that the solid-phase carrier membrane must have good protein binding ability, suitable pore size, and good tensile strength, and the requirements for the membrane are relatively high. Most of the currently used membranes are imported from abroad, and the cost is high. On the other hand, judging the results simply by the color depth of colloidal gold can only be qualitative or semi-quantitative, and the sensitivity is low. For some samples with extremely low antigen or antibody content, it will cause false negatives and the detection rate is low.
[0003] After searching the literature of the prior art, it was found that in order to improve the defects of the above method, the Chinese patent name: "Immuno-Colloidal Gold and Immunomagnetic Particles Double-Labeled Rapid Diagnostic Kit", Publication No.: CN 1335503A, this patent combines magnetic beads and colloidal Gold is simultaneously labeled on the antibody or antigen of the target object, and combines with the antigen or antibody coated on the membrane through an immune reaction to form a detection zone. Quantitative detection of the detection band is carried out by an instrument. This method provides an idea for quantitative detection, but requires an expensive ultra-trace magnetic quantitative detector, and the detection sensitivity for extremely small magnetic beads is low.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Embodiment 1, for nanoparticles, taking magnetic particles as an example, establish an immune colloidal gold fluorescence quenching measurement method, take the following steps:

[0013] 1. Preparation of immunomagnetic particles: first prepare nano-ferric oxide (Fe 3 o 4 ) particles, and then use organic molecules to treat nano-Fe 3 o 4 Particles have functional groups (carboxyl, amino, aldehyde, mercapto, etc.) on their surface. Here, magnetic particles with a carboxyl group on the surface are selected with a particle size of about 100 nanometers, and are linked with a monoclonal antibody against carcinoembryonic antigen (CEA) under the action of a linker carbodiimide to prepare solid-phase immunomagnetic particles.

[0014] 2. Preparation of colloidal gold-labeled antibody: first use chloroauric acid (HAuCl 4 ) prepare colloidal gold particles by trisodium citrate reduction method, can obtain the different gold nanoparticles of particle size by adjusting raw mate...

Embodiment 2

[0016] Embodiment 2, for micron particles, taking the thalline of Staphylococcus aureus as an example, an immunocolloidal gold fluorescence quenching measurement method is established, and the following steps are taken:

[0017] 1. Preparation of colloidal gold-labeled antigen: first use chloroauric acid (HAuCl 4 ) to prepare colloidal gold particles by trisodium citrate reduction method, use 0.1 molar concentration of Na 2 CO 3 Adjust the pH to 8, then add alpha-fetoprotein (AFP), react at room temperature for 1 hour, and centrifuge at 9500 rpm to obtain colloidal gold-labeled alpha-fetoprotein (AFP).

[0018] 2. To establish the measurement method: add 100 μL alpha-fetoprotein (AFP) standard (concentration gradient is 0, 15, 30, 100, 200, 400 ng / mL) or the sample to be tested (serum), 100 μL rabbit anti-human AFP polyclonal antibody (titer 1:15000), and 50 μL colloidal gold-labeled AFP, reacted at 37 ° C for 2 hours, then added 0.1 mL of a solution containing Staphylococcu...

Embodiment 3

[0019] Embodiment 3, for micropores, take the plastic test tube made of polystyrene as an example, establish the immunocolloidal gold fluorescence quenching measurement method, take the following steps:

[0020] 1. Coating of test tube antibody: Add 0.5 mL of anti-carcinoembryonic antigen (CEA) monoclonal antibody (4 μg / mL) formulated with carbonate buffer (pH9.5) into a polystyrene test tube, 4 °C After reacting overnight, discard the liquid, wash with phosphate buffer 3 times, add phosphate buffer containing 1% bovine serum albumin to block for 1 hour, and obtain a reaction test tube coated with antibodies.

[0021] 2. To establish a measurement method: add 100 μL of carcinoembryonic antigen (CEA) standard (concentration gradient of 0, 10, 20, 40, 80, 120 ng / mL) or test sample (serum) to the antibody-coated test tube, 100 μL of colloidal gold-labeled polyclonal antibody (prepared by the method in Example 1), reacted at room temperature for 3 hours, took 150 μL of supernatant...

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Abstract

The present invention relates to a measurement method of immune colloidal gold particle fluorescence quenching in the field of detection technology. The antibody or antigen of target compound, namely tested compound is connected on the microgranule or microwell to form solid-phase carrier, the tested sample is added, and excess colloidal gold lag also is added, so that the target compound in the sample and the antibody or antigen fixed on the microgranule or microwell plate are undergone the immunological reaction to form solid-phase carrier compound of antibody antigen, namely conjugated compound and unreacted colloidal gold lag, namely free compound, then the conjugated compound and free colloidal gold tag are separated, in the free compound a fluorescence substance is added to make fluorescence signal be quenched. The signal quenching extent is related to free gold lag quantity in the system, and the quantity of free compound is related to quantity of target compound, so that it can implement quantitative detection of target compound.

Description

technical field [0001] The invention relates to a method in the technical field of detection, in particular to a method for measuring fluorescence quenching of immune colloidal gold particles. Background technique [0002] The application of colloidal gold particles as markers in immunoassays was invented by Faulk et al. in the 1970s, mainly because of its optical properties, that is, colloidal gold particles themselves have color, and the results of immunoassays can be observed with the naked eye. At present, the most widely used analysis methods are dot immunogold diafiltration and colloidal gold immunochromatography. This technology has been widely used in clinical medical testing, and there are a large number of reports. The phase carrier, the antigen or antibody coated with the target object (ie, the analyte), is added to the sample to be tested, and the target object in the sample and the antigen or antibody coated on the membrane are combined by the diafiltration of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/542G01N33/52G01N33/531G01N33/543
Inventor 高峰崔大祥贺蓉敖丽梅潘碧峰
Owner SHANGHAI JIAO TONG UNIV
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