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A kind of midkine midkine biological analysis method and detection kit

A technology for detection kits and midkine, applied in biological testing, biological material analysis, analytical materials, etc., can solve the problems of midkine content differences, heparin capture, midkine detection can not meet the requirements, etc., to broaden the detection range and improve reactivity , the effect of eliminating interference

Active Publication Date: 2021-03-12
SHANGHAI MEDICILON INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The bound Midkine cannot be captured by the heparin coated on the 96-well plate because it is pre-bound with heparin, so the measured Midkine content is quite different from the actual value
This method can be used as a reference in the early scientific research field and when it meets the requirements of relative quantification, but it can be used as an external source in the field of pharmaceutical technology that requires absolute accurate quantification and accurate calculation of pharmacokinetic parameters and toxicokinetic parameters. Midkine detection of sexual drugs is far from meeting the requirements

Method used

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  • A kind of midkine midkine biological analysis method and detection kit
  • A kind of midkine midkine biological analysis method and detection kit
  • A kind of midkine midkine biological analysis method and detection kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Use Assay Buffer, 10% serum (that is, the volume ratio of serum to Assay Buffer is 1:9), and serum to prepare a standard curve, and compare the differences in detection signals. See the table below for details:

[0127]

[0128] It can be seen that the signal of the standard prepared in Buffer is much higher than that in serum, and the signal decreases with the increase of serum concentration. This once again confirms that the measurement results of the serum samples obtained by directly preparing the standard in the buffer of the current kit will be low. Therefore, the final standard curve of the detection method and the kit of the present invention are consistent with the quality control and the sample to be tested, and the blood sample cannot be directly prepared with buffer as in the conventional detection method.

[0129] figure 1 It is the standard curve of the midkine Midkine biological analysis method of the present invention, wherein Std1 is the standard c...

Embodiment 2

[0131] Example 2: Setting up a control experiment without acidification to determine the effect of acidification on the removal of heparin. See the table below for details:

[0132]

[0133] We use Assay Buffer and 10% serum to prepare the standard song, because the signal in the Assay Buffer is very high, it is used as a positive control in this experiment, and the standard song prepared with 10% serum is acidified with acetic acid and hydrochloric acid respectively, according to the experiment According to the result data, it can be found that the signal of Assay can be obviously promoted when using 300mM acetic acid for acidification treatment, and the highest signal is equal to that of Buffer, indicating that this can eliminate the interference of heparin, but the acidification effect of hydrochloric acid is not very ideal, so the method protected in the invention Acetic acid is used as the acidifying solution.

Embodiment 3

[0134] Embodiment 3: Based on the above-mentioned groping test conditions, each concentration point of the standard curve was prepared with serum, and each concentration point of QC was also prepared with serum, so that the standard curve and QC were prepared for the recovery test. See the table below for details:

[0135]

[0136] (NA does not participate in the fitting in this experiment)

[0137] The following two groups of QC are prepared from different individual sera. See the table below for details:

[0138]

[0139] From this group of experimental data, it can be seen that the signal is normal after acidification treatment, and the QC recovery rate of one serum preparation is very good within 70-125%, but the other fluctuates greatly, indicating that the method also needs to deal with different matrices The problem of fluctuations, the problem of matrix interference.

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Abstract

The invention discloses a midkine Midkine biological analysis method and a detection kit. The method comprises: coating the Midkine antibody on a microwell enzyme plate to form a solid-phase carrier; using a biological matrix to prepare a quality control, a standard and a sample to be tested, adding an acidifying solution and incubating for acidification at room temperature, so that the Midkine- The heparin-bound complex is fully dissociated under acidic conditions; the acidified quality control, standard, and test samples, as well as the neutralizing / detection antibody working solution, are placed in the corresponding reaction wells of the microtiter plate and incubated. To neutralize the acidified solution in the quality control, standard and test samples and return to the alkaline environment, at the same time, the detection antibody and rhMidkine in the quality control, standard and test samples and the surface of the solid-phase carrier Antibodies form antibody-antigen-antibody sandwich complexes.

Description

technical field [0001] The invention relates to the fields of biological analysis, pharmacokinetic research, toxicokinetic research, drug safety evaluation and clinical research, in particular to a Midkine biological analysis method and detection kit suitable for biological substrates. Background technique [0002] Midkine (MK), also known as neurite growth promoting factor 2 (Neurite growth promoting factor 2), is a secretory heparin-binding growth factor discovered in 1988. Midkine and another heparin-binding growth factor, pleiotrophin (PTN), constitute the Midkine family. Its structure and composition properties are that Midkine is a highly alkaline, non-glycosylated polypeptide containing 5 intra-chain disulfide bonds. Specifically, human midkine consists of 121 amino acids, with a molecular weight of about 13.3 KD; mouse midkine consists of 118 amino acids. Additionally, the C-terminal half of Midkine contains the predominant heparin-binding site as well as the molec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/543G01N33/535G01N33/532
CPCG01N33/532G01N33/535G01N33/54306G01N33/577G01N33/74G01N2333/475
Inventor 章登吉任朋亮黄宗强章春燕梁冉冉潘桂梅
Owner SHANGHAI MEDICILON INC