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A fluorescence-enhanced immunoassay method and immunoassay kit for detecting glycocholic acid
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A technology of immune analysis and fluorescence enhancement, applied in the field of immunology, can solve the problems of cumbersome analysis steps, expensive reagents, unfavorable development, etc., and achieve the effect of enhanced detection signal and sensitive and stable detection effect
Active Publication Date: 2018-07-24
广东南粤药业有限公司
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Problems solved by technology
The RIA method requires the use of supporting professional radioimmunotherapy facilities. The reagents are expensive, the analysis steps are cumbersome, and the waste is difficult to dispose of. Radioactive pollution will cause great harm to the health of operators, and it is rarely used in the world at present.
The CLTA method has high sensitivity, but the measurement speed is slow, the accuracy of the test results and the stability of the reagents are poor, and it requires expensive special chemiluminescence detection equipment, which is not conducive to the development of routine laboratories and has obvious limitations in clinical application.
In addition to the above methods, there are high-performance liquid chromatography (HPLC), gas-liquid chromatography (GLC), gas chromatography and mass spectrometry (GC-MS) to detect glycocholic acid, but these methods are not suitable for wide application. clinical test
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Embodiment 1
[0028] Embodiment 1: a kind of preparation method of glycocholic acid complete antigen, comprises the following steps:
[0029] CG (0.1 mmol) and NHS (0.15 mmol) were completely dissolved in DMF (1 mL), then DCC (0.15 mmol) was added, and the reaction was stirred at 4°C for 12 hours; after centrifugation (1000 g / min, 10 min), the supernatant was slowly dropped Add to PBS (8 mL, 10 mM, pH 7.4) dissolved with OVA (2 mmol), and stir for 12 hours at 4 °C; (10 mM, pH 7.4) for 3 days, and the medium was changed every 12 hours to obtain the complete glycocholic acid antigen. The flow of its synthetic route is as followsfigure 1
Embodiment 2
[0030] Embodiment 2: the preparation method of fluorescently labeled goat anti-rabbit, comprising the following steps:
[0031] Goat anti-rabbit was dissolved in PBS (0.01M, pH=7.4), and then FITC solution (CBS, 0.05M, pH=9.2) was added. The molar ratio of goat anti-rabbit and FITC was 1:10, and the mixture was stirred overnight at 4°C. The reaction solution was put into a dialysis bag, dialyzed in PBS (0.01M, pH=7.4) for three days, and the solution was changed every 12 hours to obtain FITC-labeled goat anti-rabbit.
Embodiment 3
[0032] Example 3: Determination of optimal concentrations of coated antigens and antibodies by checkerboardtitration, including the following steps:
[0033] (1) Dilute the complete antigen of glycocholic acid obtained in Example 1 by gradient dilution with coating buffer CBS (0.05M, pH 9.6), add 100 μL per well to a 96-well black plate, coat overnight at 4°C, and dry the liquid in the well , washed once with PBST, and patted dry with absorbent paper.
[0034] (2) 200 μL / well of 30 mg / mL nonfat dry milk (dissolved in PBS) was added to the well, and incubated at 37° C. for 1 h.
[0035] (3) Dry the liquid in the wells, wash three times with PBST, and pat dry with absorbent paper.
[0036] (4) Dilute the immune serum with PBS, doubling the dilution at a specific concentration, and add 100 μL / well to the well, and set 2 wells of negative control and blank control at the same time. After 2 hours incubation at 37°C, wash and pat dry.
[0037] (5) Dilute the FITC-labeled goat an...
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Abstract
The invention belongs to the technical field of immunology, and discloses a fluorescent enhanced immune analysis method and immune analysis kit for detecting glycocholic acid. The invention aims to develop the fluorescent enhanced immune analysis kit for detecting glycocholic acid, wherein the kit has the advantages of simple pretreatment, fastness and accuracy, low cost and stability, and can be used for on-site mass detection. Through coating of an antigen with glycocholic acid and preparation of a fluorescent labeled sheep anti rabbit, and under protective agents, such as glycerol, Tween and the like, a signal is enhanced and amplified, so as to achieve the detection of the content of glycocholic acid. The immune analysis method for detecting the glycocholic acid is a one-step competition fluorescent enhancement immune analysis method, and is a simple, fast and high-throughput detection method.
Description
technical field [0001] The invention belongs to the technical field of immunology, and particularly relates to a fluorescence-enhanced immunoassay method and an immunological kit for detecting glycocholic acid. Background technique [0002] Glycocholic acidhydrate (CG) is one of the conjugated cholic acids formed by the combination of cholic acid and glycine, with a relative molecular mass of 466.3. Cholesterol undergoes an extremely complex enzymatic reaction in hepatocytes and is converted into primary bile acids, including cholic acid (CA) and chenodeoxycholic acid (CDCA). The steroidnucleus of cholic acid has three hydroxyl groups (C3, C7, C12 ), the hydroxyl group at the end of the side chain is combined with glycine by a peptide bond to form glycocholic acid. Under normal circumstances, the content of glycocholic acid in peripheral blood is very small, and the concentration of glycocholic acid in normal people is stable at a low level regardless of fasting or after ...
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