Homogeneous time-resolved fluorescence analysis method of alpha-fetalprotein
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A time-resolved fluorescence and alpha-fetoprotein technology, which is applied in the fields of bioanalytical chemistry and nano-biology, can solve the problems of short marker placement time, short validity period of reagents, and influence on further use, so as to improve stability and sensitivity, and overcome non-specificity. Adsorption, the effect of improving energy transfer efficiency
Inactive Publication Date: 2012-02-15
SOUTHERN MEDICAL UNIVERSITY
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Among them, the enzyme labeling method is a semi-quantitative reagent, which has great limitations in accuracy and sensitivity, and the activity of the enzyme is easily affected by factors such as labeling reaction, temperature, pH value, and ion concentration in the solution. Narrow, troublesome and other disadvantages; radioimmunoassay with radioactive operation and marker placement time is short, short reagent validity period and other disadvantages; the advantages of chemiluminescence are high sensitivity, wide linear range, short analysis time, but its disadvantages: chemiluminescence The generation of luminescence is usually completed instantaneously, the luminescence peak decays quickly, and the cost is high; temperature and pH value have a great influence on luminescence, etc. These factors affect the further use of this type of method
On the other hand, the existing HTRFA technology can be realized thanks to the use of rare earth metal chelates. The general energy donor is rare earth metal europium chelate, which requires the energy acceptor to emit near-infrared waves (such as 665nm), which requires expensive Near infrared sensitive detection device
Even if terbium chelate is used, due to the wide fluorescence emission spectrum of most organic dyes and obvious tailing, there is a large overlap with the time-resolved fluorescence spectrum of terbium chelate, which is only suitable for analysis in the long-wavelength or near-infrared band, which is expensive NIR-sensitive detector requirements remain
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Embodiment 1
[0033] Experimental Materials
[0034] The oil-soluble quantum dots were purchased from Invitrogen, the product number is Q21701MP, and the concentration is 1uM.
[0035] The energy donor LanthScreen Amino Labeling Kit was purchased from Invitrogen, and its item number is PV3582.
[0036] Chemical reagents such as 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) and trioctylphosphine oxide were purchased from Sigma-Aldrich, USA.
[0037] Alpha-fetoprotein monoclonal antibodies E010 and E014 and alpha-fetoprotein antigen standard (contains six samples, labeled A, B, C, D, E, F, and their concentrations are 0, 2, 10, 50, 200, 800U / ml, the buffer system of each sample is: 1.5% BSA, 0.15% NaN 3 50mM Tris-HC1, pH7.2 buffer) was purchased from Sun Yat-Sen University Da'an Gene Company.
[0038] Polystyrene-divinylbenzenecarboxylic acid modified latex was purchased from Thermo Fisher Scientific Company, and its item number is 8300-0520100390. The particle diameter...
Embodiment 2
[0070] 1. Preparation of specimens to be tested
[0071]The sample to be tested is divided into two, one of which uses 25ul and 55ul of alpha-fetoprotein standard C and D respectively, and mixes it evenly to make it theoretically should be (10×25+50×55) / 80=37.5U / ml , with a total volume of 80ul, marked G. Another sample to be tested is AFP antigen serum donated by Nanfang Hospital, the concentration is unknown, and it is marked as H.
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Abstract
The invention relates to fields of biochemical analysis, and concretely relates to a homogeneous time-resolved fluorescence analysis method of alpha-fetalprotein. The method comprises the following steps: 1, preparing a quantitative standard curve: incubating an HFRFA energy receptor, an HFRFA energy donor and an alpha-fetalprotein standard substance together, and carrying out time-resolved fluorescence detection and analysis to obtain the standard curve between the concentration of the alpha-fetalprotein and the fluorescence value; and 2, detecting a sample: incubating the HFRFA energy receptor, the HFRFA energy donor and the alpha-fetalprotein sample to be detected together, carrying out time-resolved fluorescence detection and analysis, comparing the fluorescence value obtained through detecting the sample with the standard curve obtained in step 1, and calculating to obtain the concentration of the alpha-fetalprotein sample to be detected. The method of the invention, which has the advantages of strong specificity and high detection sensitivity, has important meanings in the biochemical analysis of clinical molecular diagnosis and food detection.
Description
technical field [0001] The invention belongs to the fields of bioanalytical chemistry and nano-biotechnology, and in particular relates to an immunoassay method for biological substances. Background technique [0002] Human alpha-fetoprotein (AFP) is derived from fetal glycoprotein, with a molecular weight of about 70KDa. Adult alpha-fetoprotein can be produced by malignant tumors, especially liver cancer and embryonic cell tumors. 70-95% of primary liver cancer patients have AFP in serum The content is significantly increased, and alpha-fetoprotein is often slightly increased in patients with hepatitis and cirrhosis. [0003] Therefore, the clinical molecular diagnosis of AFP is very important, and the detection with the lowest sensitivity is particularly important for early detection of tumors and formulation of treatment plans. At present, there are many immunoassay methods for the determination of AFP, the most commonly used serum detection methods are: enzyme immunoass...
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