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Method for detecting concentration of FGF-21 in cynomolgus monkey serum

A technology of FGF-21 and detection method, applied in the field of detection of FGF-21 concentration in serum, achieving high precision and accuracy, low measurement interference, and wide detection range

Pending Publication Date: 2021-01-22
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, researchers often use cynomolgus monkeys to study the pharmacokinetics of FGF21, but there is no patent report for the rapid determination of FGF21 content in cynomolgus monkey serum

Method used

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  • Method for detecting concentration of FGF-21 in cynomolgus monkey serum
  • Method for detecting concentration of FGF-21 in cynomolgus monkey serum
  • Method for detecting concentration of FGF-21 in cynomolgus monkey serum

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1 minimum required dilution (MRD) test

[0041] Three different biological matrices were used to prepare FGF-21 standard curves, namely (1): Assay buffer (sample diluent provided by Kit); (2) 50% cynomolgus monkey blank serum (Monky Serum×2): first use food Prepare a series of high-concentration (2-fold concentration) serum samples from cynomolgus monkey blank serum, and then measure them after doubling dilution with Assay buffer; (3) 10% cynomolgus monkey blank serum (Monky Serum×10): first use cynomolgus monkey blank serum A series of high concentration (10-fold concentration) serum samples were prepared from the serum, and then diluted 10-fold with Assay buffer for determination. Prepare three standard curves of FGF-21 according to the above method, so that the final concentrations of the standard curves are 4000, 2000, 1000, 500, 250, 125, 62.5, 31.25 pg / mL. Within the measured concentration range (31.25-4000pg / mL), the drug concentration (C) and absorba...

Embodiment 2

[0047] Example 2 standard curve and quantitative range

[0048] First prepare a series of high-concentration (10-fold concentration) FGF-21 standard solutions with cynomolgus monkey blank serum, and then dilute them 10-fold with Assaybuffer to make the final concentrations: 4000, 2000, 1000, 500, 250, 125, 62.5 , 31.25pg / mL, measure the absorbance according to "under 2.8", and take the average value of the duplicate wells for each concentration. Within the measured concentration range (31.25-4000pg / mL), the drug concentration (C) and absorbance (Abs) are fitted into a Sigmoidal curve by a four-parameter Log-Logistic (4-PL) model, with a weight of 1 / Abs 2 . Table 3 is the average OD value of the standard curve of FGF-21 (n=9), and its equation is: Abs=5.800-5.786 / [1+(C / 3681.309) 1.129 ],R 2 = 0.9998. figure 2 is the mean standard curve graph for FGF-21 (n=9). The individual data of the 9 standard curves are shown in Table 4-Table 7. The accuracy (RE%) of the back-calcula...

Embodiment 3

[0062] Embodiment 3 specificity test

[0063] Select insulin-like growth factor 1 (IGF-1), epidermal growth factor (EGF), recombinant human interleukin-11 (rhIL-11), recombinant human granulocyte colony-stimulating factor (rhG-CSF), recombinant human interferon for injection Seven kinds of endogenous cytokines including γ(rhIFN-γ), recombinant human granulocyte macrophage stimulating factor for injection (rhGM-CSF), recombinant human tumor necrosis factor II receptor-antibody fusion protein (rhTNFR:Fc) Or related proteins to examine the specificity of this method. Add higher concentrations of the above-mentioned proteins to the blank serum of cynomolgus monkeys to make the final concentration 100ng / mL, which is equivalent to 25 times the highest concentration of the FGF-21 standard curve. The ELISA method established in this experiment was used for determination. The results are shown in Table 8. The test results showed that except for EGF, the OD values ​​of the other six p...

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Abstract

The invention discloses a method for detecting the concentration of FGF21 in cynomolgus monkey serum. The method comprises the following steps: (1) adding a to-be-detected serum sample on a 96 microwell plate pre-coated with rabbit anti-human FGF21 polyclonal antibody, and incubating; (2) washing the plate, adding a detection antibody, and incubating; (3) washing the plate, adding an enzyme-labeled antibody, and incubating; (4) after washing the plate, adding an HRP substrate tetramethyl benzidine, carrying out dark incubation, then adding a stop solution to stop the enzyme reaction, and determining the absorbance at 450 nm; and (5) calculating according to the absorbance value to obtain the FGF21 concentration in the serum. The detection method has high accuracy and precision, is slightlyinterfered, and can be used for better researching the action mechanism of the fibroblast growth factor 21.

Description

technical field [0001] The invention belongs to the field of biochemical detection, and in particular relates to a method for detecting FGF-21 concentration in serum. Background technique [0002] Fibroblast growth factor 21 (fibroblast growth factor 21, FGF21) is a new blood sugar regulating factor discovered in recent years. Since there is no specific glucose uptake cell, the development of its biological activity method has always been difficult. [0003] In the process of research on fibroblast growth factor 21, we often need to know the exact content of FGF21 in serum in order to further study its metabolic process and its influence on related diseases. At present, researchers often use cynomolgus monkeys to study the pharmacokinetics of FGF21, but there is no patent report on the rapid determination of FGF21 content in cynomolgus monkey serum. Contents of the invention [0004] The invention provides a method for detecting the concentration of FGF-21 in cynomolgus m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/58G01N33/543G01N21/31
CPCG01N21/31G01N33/54306G01N33/581G01N33/74G01N2333/50
Inventor 余丙洁惠琦杨选鑫
Owner WENZHOU MEDICAL UNIV
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