CN112255419A - Midkine bioanalysis method and detection kit - Google Patents
Midkine bioanalysis method and detection kit Download PDFInfo
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Abstract
The invention discloses a Midkine bioanalysis method and a detection kit. The method comprises the following steps: coating the Midkine antibody on a micropore enzyme label plate to form a solid phase carrier; preparing a quality control product, a standard product and a sample to be detected by using a biological matrix, and respectively adding an acidification liquid to incubate and acidify at room temperature so as to fully dissociate a Midkine-heparin combined compound under an acidic condition; placing the acidified quality control product, standard product, sample to be detected and neutralizing/detecting antibody working solution in a reaction hole corresponding to the enzyme label plate for sample loading and incubation so as to neutralize the acidified solution in the quality control product, standard product and sample to be detected and return to a slightly alkaline environment, and simultaneously forming an antibody-antigen-antibody sandwich compound by the detecting antibody, rhMidkine in the quality control product, standard product and sample to be detected and the antibody on the surface of the immobilized carrier.
Description
Technical Field
The invention relates to the fields of bioanalysis, pharmacokinetic research, drug safety evaluation and clinical research, in particular to a Midkine bioanalysis method and a detection kit suitable for a biological matrix.
Background
Midkine (MK), also known as Neurite growth promoting factor 2 (Neurite growth promoting factor 2), is a secreted heparin-binding growth factor discovered in 1988. Midkine forms the Midkine family with another heparin-binding growth factor pleiotrophin (ptn). Its structure and composition properties are that Midkine is a highly basic, non-glycosylated polypeptide containing 5 intrachain disulfide bonds. Specifically, human Midkine consists of 121 amino acids, with a molecular weight of about 13.3 KD; murine Midkine consists of 118 amino acids. In addition, the C-terminal half of Midkine contains the most prominent heparin binding site as well as the antigenic and neurite-promoting sequences of the molecule. At the same time, both the C-and N-terminal ends are essential for the neurotrophic effect of the molecule.
In recent years, MK is over-expressed in various solid tumors and is highly expressed in precancerous lesions and tumor latency, so that MK can be used as a potential tumor marker. The research also shows that the expression level of the midkine in the serum of the senile dementia patient is obviously increased, which indicates that the midkine may have a certain correlation with the pathological process of the senile dementia. In addition, MK is involved in inflammation through two distinct behaviors, first it enhances neutrophil and macrophage migration ability either directly or by inducing chemokine expression; second, MK may partially inhibit the differentiation of tolerogenic dendritic cells essential to the body. MKs also have antimicrobial activity and are considered to be part of the innate immune system. As described above, MK is involved in many physiological and pathological processes, and thus MK itself and drugs targeting MK are considered to have therapeutic value for many diseases.
Many companies have been interested in human MK molecules and started to develop MK related drug products, for example, foreign companies develop recombinant MK proteins which are expected to be used for treating heart diseases caused by myocardial ischemia, and domestic companies also develop recombinant MK products for preparing drugs for treating osteoarthritis. The detection and analysis of drug molecules cannot be separated in preclinical and clinical stages based on the development of recombinant human midkine (rh MK) as a drug, and particularly the pharmacokinetic analysis needs to be carried out. Pharmacokinetic analysis is also required during the safety assessment phase of the reported new drug clinical trial. The biological analysis based on pharmacokinetics and toxicology is the most critical technical link in the detection process and also a technical problem which needs to be solved in advance in the research and development stage of related drugs.
Most of the current commercially available kits belong to kits for academic research, and when a standard curve is prepared, the standard solution is usually prepared by adopting a pure Buffer solution, and only the standard curve can be fitted. However, the guidelines of the food and drug administration for bioanalysis and the guidelines of the chinese pharmacopoeia for the verification of bioanalysis methods clearly indicate that the standard solution should be prepared using the same matrix as the sample to be tested (for example, a biological sample matrix such as serum is usually used in pharmaceutical analysis). Therefore, the kit for academic scientific research cannot meet the analysis of the biological matrix sample and also does not meet the requirement of the biological analysis guiding principle of the food and drug supervision and management bureau. Moreover, the above-mentioned kit for academic research does not consider the main feature that Midkine is a heparin-binding growth factor, and does not exclude the detection interference caused by the binding of heparin thereto in the detection method, and can only be used for the endogenous detection of the rh MK sample, and cannot really solve the problem of how to accurately quantify the recombinantly expressed rh MK as an exogenous drug, or even solve the problems of pharmacokinetic and pharmacokinetic analysis of the rh MK as a drug, and thus is not suitable for the industrial application of drug development.
Plum gem (heparin-ELISA method for detecting serum midkine and primary application thereof to liver cancer diagnosis, second department of medical university, 2006, 26(7):631 and 633) designs heparin as a coating so as to capture MK molecules in blood and establish an ELISA method for detecting MK. The detection system of midkine in serum is carried out in buffer system, can be only used for detecting endogenous free MK, and can not recognize binding MK. The method utilizes the characteristic that heparin and Midkine can be combined, and uses heparin as a reagent for capturing Midkine, but does not exclude the interference of the combined Midkine formed by combining heparin and Midkine in an organism on Midkine detection. Since Midkine in the bound state is previously bound to heparin and thus cannot be captured by heparin coated on a 96-well plate, the measured Midkine content is greatly different from the actual value. The method can be used by reference in the early scientific research field and under the condition of meeting the requirement of relative quantification, but the method is far from meeting the requirement of Midkine detection as an exogenous drug in the medical technology field which needs absolute accurate quantification and accurate calculation of pharmacokinetic parameters and toxicity pharmacokinetic parameters. Therefore, the above method is still in need of improvement in practical industrial application.
Based on the above, development of a Midkine bioanalysis method and a detection kit suitable for serum or plasma matrix can eliminate heparin and Midkine combination and other detection interference in the serum or plasma matrix, realize accurate quantitative analysis of Midkine drug concentration, and meet serial requirements of drug approval on pharmacokinetic and toxicity pharmacokinetic analysis, and is imminent.
Disclosure of Invention
As mentioned previously, Midkine is an important heparin binding molecule. The concentration of heparin in human blood is about 9ug/mL, which greatly exceeds the concentration of rh MK into the circulation by exogenous administration. In view of the detection limit and sensitivity of pharmacokinetic analysis must reach the ng/mL level, the binding of heparin and drug rhMK in blood will seriously interfere with the detection of the concentration of drug rhMK, even result in great deviation of the result of pharmacokinetic and toxicity pharmacokinetic analysis, thus bringing potential risks to the research and development and application of drugs.
Aiming at the defects, the invention provides a Midkine bioanalysis method and a detection kit suitable for a biological matrix, which meet the related requirements of pharmacokinetics/toxokinetics bioanalysis of MK drug detection. The detection method and the detection kit provided by the invention can be used for dissociating the combined Midkine from heparin by acidifying and dissociating a serum or plasma matrix, so that the influence or deviation on an actual detection result caused by the combination of MK and endogenous heparin is avoided, a more real and accurate detection result is obtained, and absolute quantification is realized.
In a first aspect, the present invention provides a Midkine bioassay method for use with a biological substrate, comprising the steps of:
step A: coating the Midkine antibody on a micropore enzyme label plate to form a solid phase carrier;
and B: preparing a quality control product, a standard product and a sample to be detected by using a biological matrix, and respectively adding an acidification liquid to incubate and acidify at room temperature so as to fully dissociate a Midkine-heparin combined compound under an acidic condition;
and C: placing the acidified quality control product, the acidified standard product, the sample to be detected and a neutralizing/detecting antibody working solution in a reaction hole corresponding to the enzyme label plate for sample loading and incubation, wherein the neutralizing/detecting antibody working solution consists of an alkaline buffer solution and a detecting antibody working solution, the acidified solution in the acidified quality control product, the acidified standard product and the sample to be detected is used for neutralizing the alkaline environment, the analyte Midkine is subjected to renaturation storage in the alkaline environment, and meanwhile, the detecting antibody, the rhMidkine in the quality control product, the standard product, the sample to be detected and the antibody on the surface of the immobilized carrier form an antibody-antigen-antibody sandwich compound;
step D: after the reaction in the step C is finished, removing the residual solution in the reaction hole, adding a conjugate of horseradish peroxidase and streptavidin, sealing and incubating at room temperature;
step E: after the incubation is finished, removing the residual solution in the reaction hole, and adding a TMB solution for incubation in a dark place; then adding a stop solution, and reading an OD value at 450nm by using an enzyme-labeling instrument;
step F: and (3) taking the log of the concentration of the Midkine as the abscissa and the OD value as the ordinate, and performing regression on the relation between the instrument response value and the theoretical concentration of each concentration point of the standard curve so as to determine the standard curve.
Preferably, the Midkine antibody is a monoclonal Hunan Midkine Mab. The capture antibody is a monoclonal antibody, so that the specificity of the detection method can be ensured.
Preferably, the detection antibody is a biotin-labeled polyclonal antibody, Human Midkine Biotinylated affinity purification Pab. The detection antibody is a biotin-labeled polyclonal antibody, which is beneficial to improving detection signals so as to obtain high detection sensitivity.
The invention is designed based on a double-antibody sandwich method, uses a Hunan Midkine Mab as a capture antibody and a Human Midkine Biotinylated affinity purification Pab as a detection antibody, uses the capture antibody (monoclonal antibody) to ensure the specificity of the method, uses the detection antibody (polyclonal antibody) to improve the reactivity of the antigen antibody, further enhances signals by biotin labeling, and is relatively easy to improve the sensitivity. The double-antibody sandwich design in the mode is compared with the conventional method that heparin is used as a capture reagent, unmarked polyclonal antibody is used as a detection antibody, and the heparin and the polyclonal antibody form a double sandwich, so that the interference of the binding state Midkine formed by the combination of heparin and Midkine in an organism on the Midkine detection is effectively eliminated. Compared with the double sandwich of multi-antibody and multi-antibody pair Midkine, the double-antibody sandwich design of the invention can be used for blood plasma prepared by serum and/or non-heparin related anticoagulant, and widens the detection range of the Midkine bioanalysis method. More importantly, the invention combines the characteristic that the Midkine molecule is an overbased molecule rich in basic amino acid, promotes the dissociation of a complex formed by the Midkine and heparin through acidification, and then neutralizes by using an alkaline solution, so that on one hand, the acidity of a residual acidification solution after acidification is neutralized, and on the other hand, the addition of the alkaline neutralization solution is also beneficial to the renaturation of the Midkine molecule and the maintenance of the Midkine molecule in a slightly alkaline environment. Therefore, the detection method and the detection kit provided by the invention can be used for dissociating the combined Midkine from heparin by acidifying and dissociating the serum or plasma matrix, so that the influence or deviation on the actual detection result caused by the combination of MK and endogenous heparin can be avoided, a more real and accurate detection result can be obtained, and absolute quantification can be realized.
Preferably, the acidizing fluid is acetic acid with the pH value of 3-3.5. By way of example, the concentration of the acidification liquid is 100-500mM, preferably 300 mM.
Preferably, the volume ratio of the quality control product, the standard product or the sample to be detected and the acid solution is 1 (1-10).
Preferably, the ELISA plate is a 96-well ELISA plate or a 384-well ELISA plate.
Preferably, the pH of the alkaline buffer solution may be 8.5 to 9.0. Further preferably, the alkaline buffer solution is a Tris-HCl solution.
Preferably, Assay Buffer is plated in all reaction wells prior to step C loading incubation.
Preferably, the sample to be tested is serum and/or plasma prepared from a non-heparin related anticoagulant.
Preferably, the working solution of the detection antibody is a diluted solution obtained by diluting the detection antibody by Assay buffer according to the volume ratio of 1: 2000-1: 5000.
Preferably, in the step D, the streptavidin labeled by the horseradish peroxidase is diluted by an Assay Buffer according to the volume ratio of 1: 20000-1: 50000.
Preferably, the relationship between the instrument response value and the theoretical concentration at each concentration point of the standard curve is regressed by using a four-parameter equation or a five-parameter equation and Fixed weight factors.
Preferably, the incubation time in step B, step C and step D is independently set to 1 h ± 5 min.
Preferably, the step a of coating the enzyme label plate with the Midkine antibody to form the immobilized carrier comprises the following specific processes: diluting the Midkine antibody with a PBS (phosphate buffer solution) to a concentration of 1-2 mug/mL, fully and uniformly mixing, adding the mixture into an ELISA plate, and incubating for at least 15h at 2-8 ℃; after the coating is finished, removing residual liquid in the ELISA plate, and washing the ELISA plate; adding a sealing solution into the ELISA plate, and incubating for 2 h +/-15 min at room temperature to complete sealing.
In a second aspect, the present invention provides an ELISA detection kit for Midkine, the detection kit comprising:
a: coating a Midkine antibody on a solid phase carrier formed by an enzyme label plate;
b: and (3) calibrating product diluent: animal or human serum, or animal or human non-heparin anticoagulated plasma;
C：Assay Buffer；
d: acidifying the solution with acetic acid with pH of 3-3.5;
e: neutralizing/detecting antibody working solution, preferably mixed solution of alkaline buffer solution with pH of 8.5-9.0 and detecting antibody working solution in a volume ratio of 1: 2; wherein the working solution for detecting the antibody is a diluent which is diluted by using an Assay buffer according to the volume ratio of 1: 2000-1: 5000;
f: horseradish peroxidase and streptavidin conjugate or diluent thereof;
g: TMB color developing agent;
h: stop solution, 2M sulfuric acid solution;
i: stock standard, rhMidkine (2 ug/mL).
Has the advantages that:
1. the method is different from a Midkine bioanalysis method and a kit which are set by using a pure Buffer system, constructs a Midkine detection method which can be directly used for biological matrixes of mice, monkeys, people and the like, is suitable for endogenous Midkine detection, can also be used for detection of recombinant Midkine, and more importantly can be directly used for detection of clinical and preclinical pharmacokinetics or toxicity pharmacokinetic samples, and has industrial practical value.
2. The biological detection method and the kit provided by the invention can be used for pretreating a real biological matrix sample in a chemical reagent acidification and neutralization manner, so that the scientific problem of heparin interference is solved, the method is economical and economical, and the influence of heparin and Midkine combination can be eliminated, so that the total Midkine amount can be more scientifically and accurately measured, and the method and the kit are firstly provided and realized.
3. The biological detection method and the kit can give consideration to the detection of serum and non-heparin anticoagulated plasma samples of animals and people of different species, and can be put into use only by correspondingly replacing a matrix for preparing a standard curve and QC in the actual detection process, thereby greatly saving time and economic cost.
4. The detection sensitivity of the bioanalysis method and the kit can reach below 1pg level, and excellent sensitivity can be realized in both endogenous Midkine detection and exogenous Midkine detection.
Drawings
FIG. 1 is a standard curve of the medium phase factor Midkine bioassay method of the present invention, in which Std1 is a standard curve formulated in pure Buffer, Std2 is a standard curve formulated in 10% serum, and Std3 is a standard curve formulated in 100% serum.
Detailed Description
The following is an exemplary illustration of the method of the present invention for detecting rhMidkine in serum using enzyme-linked immunosorbent assay (ELISA).
The establishment of the analysis method is based on the technology of determining the drug content in the matrix by a double-antibody sandwich method. For example, Human Midkine Mab is coated on a 96-microwell microplate to form a solid-phase antibody, and the antibody bound on the surface of the solid-phase carrier still maintains its immunological activity. The rhMidkine in the standard substance, the QC sample and the serum sample which are prepared in the matrix reacts with the antibody on the surface of the solid phase carrier through incubation to form an antigen-antibody complex. The antigen-antibody complex formed on the solid phase carrier is separated from other substances in the reaction solution by washing, and then a Human Midkine Biotinylated affinity Purified Pab is added to continue the incubation reaction to form a double-antibody sandwich complex. After washing to remove unbound antibody, Streptavidin-HRP is added, and the detection antibody reacts with the complex on the solid phase carrier and forms a new complex through incubation. In this case, the amount of the enzyme on the solid-phase carrier is in a certain ratio to the amount of the analyte in the specimen. An enzymatic substrate (TMB) is added, which is catalyzed by the enzyme to a colored product, and the color development is stopped by the addition of a stop solution. The color depth of the product is positively correlated with the amount of the detected substance in the sample, so that the quantitative analysis can be carried out according to the color depth, and the rhMidkine or endogenous midkine concentration values in the quality control sample and the tested serum sample can be calculated according to a standard curve of the regression fitting of the standard substance.
Materials and instrumentation
1) An enzyme-labeling instrument;
2) a 96-hole enzyme label plate;
3) a pipettor;
4) a water purifier;
5) a balance;
6) a pH meter;
7) a vortex instrument;
8) a refrigerator at the temperature of less than or equal to minus 10 ℃;
9) a refrigerator at 2-8 ℃;
10) a refrigerator at the temperature of less than or equal to minus 60 ℃.
Biological and chemical agents
Biological reagent
1) rhMidkine (R & D or homemade, not more than-60 ℃);
2）Hunan Midkine Mab，（R&D，Cat#2284A，≤-60℃）；
3) human Midkine Biotinylated affinity Purified Pab, also known as "Human Midkine biotin affinity Purified antibody Pab" (R & D, Cat # BAF258, ≦ 60 ℃);
4) streptavidin HRP Conjugate, also known as "horseradish peroxidase conjugated Streptavidin" (MERCK, Cat # 18-152 or other source, 2-8 ℃);
5) serum;
6) BSA, also known as "Calf serum Albumin" (Sigma, Cat # A7030, 2-8 ℃);
7) skimmed milk powder (room temperature).
Chemical reagent
1）PBS；
2）Tween-20；
3）TMB；
4) Concentrated sulfuric acid.
Preparation of the solution
1) PBS (10 ×) solution: a bag of PBS powder was dissolved in 1000 mL of deionized water and mixed well for use.
2) PBS (1 ×) solution: 100 mL of 10 XPBS solution was added to 900 mL of deionized water and mixed until needed.
3) Wash (0.05% PBST): 100 mL of PBS (10X) solution was added to 900 mL of deionized water, followed by 0.5 mL of Tween-20, and mixed well for use.
4) Assay Buffer: 1.000 g BSA and 1.0 mL Tween-20 were added to 1000 mL PBS solution and mixed well for use.
5) Blocking solution (5% non-fat milk in PBS): 5.0000g of skim milk powder was added to 100 mL of PBS and mixed well for use.
6) 300mM acetic acid solution (acidified solution): measuring 17.16 mL of glacial acetic acid, diluting to 1000 mL with deionized water, and mixing uniformly for later use.
7) 1M Tris-HCl buffer (pH 8.5, neutralized): weighing 60.56 g of Tris, dissolving in 400 mL of deionized water, adjusting the pH value to 8.50 +/-0.1 by using concentrated hydrochloric acid, metering the volume to 500 mL, and mixing uniformly for later use.
8) Stop solution (2M H)2SO4): dissolving 108.7 mL of sulfuric acid into 800 mL of deionized water, diluting to 1000 mL, and uniformly mixing for later use.
The following is an example illustrating this implementation.
Preparing a solid-phase carrier
(1) Coating: diluting the Hunan Midkine Mab to 1-2 mug/mL by PBS (1 x), uniformly mixing, adding the mixture into a 96-micropore enzyme label plate, placing the plate in a refrigerator at 2-8 ℃ for overnight incubation (at least 15 h), wherein the sample is 100 mug/well;
(2) and (3) sealing: after coating is finished, throwing away residual liquid in the ELISA plate, washing the plate for three times (300 muL/hole) by using a Wash Buffer, then adding a sealing liquid for 200 muL/hole, placing the plate in a digital display type ELISA plate oscillator at 500 +/-50 rpm, and incubating for 2 h +/-15 min at room temperature;
(3) washing the plate: and (4) throwing off the confining liquid, washing the plate for three times (300 mu L/hole) by using a Wash Buffer, patting the plate dry, using the plate or sealing the plate with a sealing plate film, and placing the plate in a refrigerator at the temperature of 2-8 ℃ for use within one week.
Preparation of standard substance and quality control substance
1) Preparing a standard substance: rhMidkine was formulated to 200, 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078ng/mL using dilutions (pooled or single sera from the same species).
2) Preparation of quality control products (i.e. internal standard samples): rhMidkine was formulated to 7.5, 0.4, 1 ng/mL using dilutions (pooled from the same species or single serum).
Quality control and acidification dissociation of sample
Adding a quality control product, a standard product and a sample to be detected into a 96-hole V-bottom dilution plate or an eppendorf tube according to a microplate sample arrangement table, carrying out acidification in a 300mM acetic acid acidification solution (the sample and the 300mM acetic acid solution are acidified according to a ratio of 1: 2), placing the sample in a digital display type microplate oscillator at 500 +/-50 rpm, and incubating at room temperature for 1 h +/-5 min to fully dissociate an antigen-antibody complex. The actual volumes of the acid solution and the sample can be adjusted in the same proportion according to the requirement.
Preparation of neutralizing/detecting antibody working solution
The 300mM acetic acid solution and the neutralizing/detecting antibody working solution (1M Tris-HCl buffer solution) are neutralized according to the actual volume ratio of acid to alkali of 2: 1. And (3) incubating the acidified sample with a neutralizing/detecting antibody working solution on the plate according to the volume ratio of 1: 2. According to the calculation, the final neutralizing/detecting antibody working solution comprises the following components: 1/3 volume 1M Tris-HCl Buffer +2/3 volume Assay Buffer +1:2000 Human Midkine Biotinylated affinity Purified Pab) for use.
The purpose of the above-mentioned acidification dissociation is to allow sufficient dissociation of the Midkine-heparin binding complex, whereas the neutralization is to remove the acid after acidification is complete. The pH of the reaction solution after neutralization is preferably 7.2 to 7.6. It is understood that the Tris-HCl solution may be replaced with other alkaline buffer solutions.
Neutralization and capture
And taking out the sealed 96-well ELISA plate, adding the prepared neutralizing/detecting antibody working solution into 75 muL/well, then adding the acidified quality control product, the sample to be detected and the standard product into 75 muL/well, sealing with a sealing plate membrane, placing in a digital display type ELISA plate oscillator at 500 +/-50 rpm, and incubating for 1 h +/-5 min at room temperature.
Adding an enzyme detection complex
After neutralization and antibody capture are finished, throwing off solution in the plate, washing the plate for three times (300 mu L/hole) by using a Wash Buffer, beating the plate to be dry, adding Streptavidin-HRP diluted by the Assay Buffer according to the proportion of 1:20000, 100 mu L/hole, sealing the plate by using a sealing plate membrane, placing the plate in a digital display type ELISA plate oscillator at 500 +/-50 rpm, and oscillating and incubating the plate for 1 h +/-5 min at room temperature. The purpose of this process is to promote a better reaction.
Color development
After the incubation is finished, liquid in the plate is thrown away, the plate is washed three times (300 mu L/hole) by a Wash Buffer, the plate is patted dry, then TMB which is balanced to the room temperature in advance is added, the plate is hatched at 100 mu L/hole, and the plate is incubated for 30 +/-10 min in a dark place.
Termination and read plate
Adding stop solution into the sample at 100 mu L/hole, placing the sample on an enzyme labeling instrument, and reading the sample at the wavelength of 450 nm.
Sample pretreatment
The sample to be tested is unfrozen at room temperature without external force influence and is balanced to the room temperature, the sample can be directly loaded without dilution, and if the sample needs to be diluted, the dilution multiple cannot exceed the maximum dilution multiple verified by the method.
Microplate reader parameter setting
Data analysis
The data of each analysis batch is processed by SoftMax Pro software (Molecular Devices) or other software versions, and the relationship between the instrument response value and the theoretical concentration of each concentration point of the standard curve is regressed by a 4-parameter equation and Fixed weight factors so as to determine the standard curve. The measured value of the quality control sample and/or the sample to be measured can be calculated by a standard curve, and if the QC and/or the sample to be measured is diluted, the measured value can be multiplied by the corresponding dilution times to obtain the final measured concentration.
Data analysis
Standard curve kinetic range: 0.156 ng/mL-10.000 ng/mL.
Quantitative range: 0.156 ng/mL-10.000 ng/mL.
Kit constitution
Based on the scheme, an ELISA detection kit of Midkine is synchronously constructed, and comprises the following components:
a, an ELISA plate is coated with a 96-hole ELISA plate of which the volume is 1-2 mug/mL and which can be directly loaded for use;
calibrator diluent, animal serum or human serum (which can be prepared according to specific experimental requirements);
assay Buffer, the component of which is PBS (pH 7.4), and contains 1/1000 BSA by volume and 1/1000 Tween20 by volume;
d: acidifying solution, acetic acid (pH 3-3.5) (optionally 300 mM);
e: neutralizing/detecting antibody working solution, 1M Tris-HCl buffer solution (pH 8.5-9.0), containing directly used detecting antibody;
f, directly using diluted SA-HRP;
g: a color-developing agent, TMB;
h: stop solution (2M H2SO4）；
I: stock standard, rhMidkine (2 ug/ml).
The present invention will be described in detail by way of examples. It is also to be understood that the following examples are illustrative of the present invention and are not to be construed as limiting the scope of the invention, and that certain insubstantial modifications and adaptations of the invention by those skilled in the art may be made in light of the above teachings. The specific process parameters and the like of the following examples are also only one example of suitable ranges, i.e., those skilled in the art can select the appropriate ranges through the description herein, and are not limited to the specific values exemplified below. More than 100% of the data appears in some tables of the examples, which is the presentation of the original experimental data, resulting from the automatic fitting of the software to the standard curve. For example, the data sheet of example 1 is provided to show and illustrate the detection signals of standard curve formulations in Assay Buffer, 10% serum, and 100% serum. There were differences in the standard curve fit in the three environments. Concentration points on the standard curve in 10% serum and 100% serum are points where the signal values are flat or slightly different, and even points with low concentration (OD) higher than the concentration above them affect the accuracy of the standard curve fitting, and appear higher or lower. Accordingly, the quality control sample measured by the standard curve may have a higher or lower phenomenon. In the whole specification, the method and the process of optimizing the kit condition are further illustrated by a series of examples so as to comprehensively embody the implementation value and the implementation effect of the invention. The requirement of the industry for precision (or called accuracy and recovery rate) is 75-125%. The experimental data of some examples are beyond the range, which further indicates that it is difficult to achieve the final excellent effect if the subsequent modification optimization is not performed but the direct implementation is performed according to the experimental conditions.
Example 1
Standard curves were prepared using Assay Buffer, 10% serum (i.e., 1: 9 by volume of serum to Assay Buffer), and serum, respectively, and the differences in detection signals were compared. The details are given in the following table:
it can be seen that the signal was much higher with the standard formulated in Buffer than in serum, and the signal decreased the greater with increasing serum concentration. This again confirms that the current kit will have low measurement results for serum samples prepared by formulating standards directly into buffer. Therefore, the final standard curve and the quality control of the detection method and the kit and the sample to be detected are consistent, and the standard curve can not be prepared by Buffer directly to measure the blood sample as the conventional detection method.
FIG. 1 is a standard curve of the bioassay method for Midkine as a mid-stage factor of the present invention, wherein Std1 is a standard curve prepared in pure Buffer, Std2 is a standard curve prepared in 10% serum, and Std3 is a standard prepared in 100% serumCurve line. The fitting results are shown below. The fitting equation is a five-parameter equation, specifically y = (A-D)/(1 + (x/C)B）G) + D. The details are given in the following table:
example 2: a control run without acidification was set up to determine the effect of acidification on heparin removal. The details are given in the following table:
the standard yeast is prepared by respectively using the Assay Buffer and 10% serum, because the signal in the Assay Buffer is very high and is used as a positive control in the experiment, the standard yeast prepared by 10% serum is respectively acidified by using acetic acid and hydrochloric acid, and according to the data of the experimental result, the signal of the Assay can be obviously promoted when the acidification treatment is carried out by using 300mM acetic acid, the highest signal is equal to the Buffer, which shows that the interference of heparin can be eliminated, and the acidification effect of the hydrochloric acid is not ideal, so the method disclosed by the invention uses the acetic acid as the acidification liquid.
Example 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, and thus the standard curve and QC were prepared for recovery rate test. The details are given in the following table:
(NA does not participate in the fitting for this experiment)
The following two QCs were prepared from different individual sera. The details are given in the following table:
from the experimental data, the signals are normal after acidification treatment, the QC recovery rate prepared by 1 serum is well within 70-125%, and the other one fluctuates greatly, which indicates that the method also needs to solve the problems of fluctuation of different matrixes and matrix interference.
Example 4: before sample loading, namely placing the acidified quality control product, the standard product and the sample to be detected in the corresponding reaction holes of the ELISA plate for incubation, spreading 75uL of Assay Buffer on all the reaction holes. All quality control and standards were formulated in pure serum. The details are given in the following table:
when 75. mu.l of Assay Buffer was applied to the reaction well before loading, all QC recoveries returned to acceptable ranges, indicating that this step helped to improve the interference with other components in the matrix.
Example 5: and (3) preparing QC by using a mode based on acidification and pre-spreading Assay buffer and using serum from different sources to confirm whether the recovery rate is normal or not. The details are given in the following table:
to sum up: the improved measures and the process of the Assay are comprehensively utilized to solve the problems that the Assay cannot be carried out due to the reduction of heparin-interfered signals in a real matrix and other interference recovery rates in the matrix; sensitivity essentially ensures that even lower concentration levels can be detected at 0.781 pg/mL.
Claims (10)
1. A Midkine bioanalysis method is characterized by comprising the following steps of:
step A: coating the Midkine antibody on a micropore enzyme label plate to form a solid phase carrier;
and B: preparing a quality control product, a standard product and a sample to be detected by using a biological matrix, and respectively adding an acidification liquid to incubate and acidify at room temperature so as to fully dissociate a Midkine-heparin combined compound under an acidic condition;
and C: placing the acidified quality control product, the acidified standard product, the sample to be detected and a neutralizing/detecting antibody working solution in a reaction hole corresponding to the enzyme label plate for sample loading and incubation, wherein the neutralizing/detecting antibody working solution consists of an alkaline buffer solution and a detecting antibody working solution, the acidified solution in the acidified quality control product, the acidified standard product and the sample to be detected is used for neutralizing the alkaline environment, the analyte Midkine is subjected to renaturation storage in the alkaline environment, and meanwhile, the detecting antibody, the rhMidkine in the quality control product, the standard product, the sample to be detected and the antibody on the surface of the immobilized carrier form an antibody-antigen-antibody sandwich compound;
step D: after the reaction in the step C is finished, removing the residual solution in the reaction hole, adding a conjugate of horseradish peroxidase and streptavidin, sealing and incubating at room temperature;
step E: after the incubation is finished, removing the residual solution in the reaction hole, and adding a TMB solution for incubation in a dark place; then adding a stop solution, and reading an OD value at 450nm by using an enzyme-labeling instrument;
step F: and (3) taking the log of the concentration of the Midkine as the abscissa and the OD value as the ordinate, and performing regression on the relation between the instrument response value and the theoretical concentration of each concentration point of the standard curve so as to determine the standard curve.
2. The bioassay method as set forth in claim 1, wherein the Midkine antibody is a monoclonal Hunan Midkine Mab.
3. The bioassay method as set forth in claim 1, wherein the detecting antibody is a biotin-labeled polyclonal antibody Human Midkine Biotinylated affinity purification Pab.
4. The bioassay method as set forth in claim 1, wherein said acidifying solution is acetic acid having a pH of 3-3.5.
5. The bioassay method as set forth in claim 1, wherein the pH of said basic buffer solution is 8.5 to 9.0.
6. The bioassay method according to claim 1, wherein the Assay Buffer is plated in all the reaction wells before the sample-loading incubation in step C.
7. The bioanalytical method of claim 1 wherein the relationship between the instrument response and theoretical concentration at each concentration point of the standard curve is regressed using a four-or five-parameter equation and Fixed weight factors.
8. The bioassay method according to claim 1, wherein the sample to be tested is serum and/or plasma prepared from a non-heparin-related anticoagulant.
9. The bioassay method as set forth in claim 1, wherein the incubation time in step B, step C and step D is set to 1 h ± 5 min independently.
10. An ELISA detection kit for Midkine, comprising:
a: coating a Midkine antibody on a solid phase carrier formed by an enzyme label plate;
b: and (3) calibrating product diluent: animal or human serum, or animal or human non-heparin anticoagulated plasma;
C：Assay Buffer；
d: acidifying the solution with acetic acid with pH of 3-3.5;
e: the neutralizing/detecting antibody working solution is a mixed solution consisting of an alkaline buffer solution with the pH value of 8.5-9.0 and a detecting antibody working solution in a volume ratio of 1: 1-10; wherein the working solution for detecting the antibody is a diluent which is diluted by using an Assay buffer according to the volume ratio of 1: 2000-1: 5000;
f: horseradish peroxidase and streptavidin conjugate or diluent thereof;
g: TMB color developing agent;
h: stop solution, 2M sulfuric acid solution;
i: stock standard, rhMidkine (2 ug/mL).
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