CN112394166A - Application of hexafluorophosphate in preparation of preparation for inhibiting ELISA reaction matrix effect - Google Patents
Application of hexafluorophosphate in preparation of preparation for inhibiting ELISA reaction matrix effect Download PDFInfo
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- CN112394166A CN112394166A CN202011242142.0A CN202011242142A CN112394166A CN 112394166 A CN112394166 A CN 112394166A CN 202011242142 A CN202011242142 A CN 202011242142A CN 112394166 A CN112394166 A CN 112394166A
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- -1 hexafluorophosphate Chemical compound 0.000 title claims abstract description 66
- 238000002965 ELISA Methods 0.000 title claims abstract description 28
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 28
- 239000011159 matrix material Substances 0.000 title claims abstract description 20
- 230000000694 effects Effects 0.000 title claims abstract description 17
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 11
- 239000000126 substance Substances 0.000 claims abstract description 4
- 230000009897 systematic effect Effects 0.000 claims abstract description 3
- 239000000872 buffer Substances 0.000 claims abstract 3
- 210000002966 serum Anatomy 0.000 claims description 17
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical group P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 12
- 239000002953 phosphate buffered saline Substances 0.000 claims description 12
- HZNVUJQVZSTENZ-UHFFFAOYSA-N 2,3-dichloro-5,6-dicyano-1,4-benzoquinone Chemical compound ClC1=C(Cl)C(=O)C(C#N)=C(C#N)C1=O HZNVUJQVZSTENZ-UHFFFAOYSA-N 0.000 claims description 7
- 238000009472 formulation Methods 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 238000003118 sandwich ELISA Methods 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 230000002860 competitive effect Effects 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 2
- 239000002904 solvent Substances 0.000 abstract 1
- 229910019398 NaPF6 Inorganic materials 0.000 description 17
- 210000002381 plasma Anatomy 0.000 description 16
- 108010074328 Interferon-gamma Proteins 0.000 description 14
- 102000008070 Interferon-gamma Human genes 0.000 description 13
- 241000699666 Mus <mouse, genus> Species 0.000 description 12
- 238000001514 detection method Methods 0.000 description 10
- 229960003130 interferon gamma Drugs 0.000 description 8
- 238000011084 recovery Methods 0.000 description 8
- 239000003085 diluting agent Substances 0.000 description 7
- 102000004889 Interleukin-6 Human genes 0.000 description 6
- 108090001005 Interleukin-6 Proteins 0.000 description 6
- 239000012895 dilution Substances 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 229940044627 gamma-interferon Drugs 0.000 description 5
- 238000008157 ELISA kit Methods 0.000 description 4
- 238000000034 method Methods 0.000 description 3
- 239000012898 sample dilution Substances 0.000 description 3
- 229910021135 KPF6 Inorganic materials 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 229940100601 interleukin-6 Drugs 0.000 description 2
- OOIBFPKQHULHSQ-UHFFFAOYSA-N (3-hydroxy-1-adamantyl) 2-methylprop-2-enoate Chemical compound C1C(C2)CC3CC2(O)CC1(OC(=O)C(=C)C)C3 OOIBFPKQHULHSQ-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 208000002151 Pleural effusion Diseases 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 150000003278 haem Chemical class 0.000 description 1
- 208000003906 hydrocephalus Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6866—Interferon
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/555—Interferons [IFN]
- G01N2333/57—IFN-gamma
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Abstract
The invention relates to an application of hexafluorophosphate in preparing a preparation for inhibiting ELISA reaction, wherein the matrix effect refers to a systematic error in detecting the same substance to be detected in the ELISA reaction in the same group of samples prepared in parallel, and the preparation comprises the hexafluorophosphate with the concentration of 0.01-2% (wt) and necessary buffer solvent.
Description
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to application of hexafluorophosphate in preparation of a preparation for inhibiting ELISA (enzyme-linked immunosorbent assay) reaction.
Background
Enzyme-linked immunosorbent assay (ELISA) is a common scientific research and clinical detection means and is used for quantitatively detecting the concentration of a target substance in a liquid sample.
Due to the diversity of detection samples, protein concentration, ionic strength, pH and the like of different detection samples (serum, plasma, pleural effusion, hydrocephalus, urine and the like) have certain differences, even if the same sample is used, some special differences can exist among different individuals, for example, HAMA (human anti-rat) reactions of more people are researched, the different differences can cause substances to be detected with the same concentration, and ELISA (enzyme-linked immuno sorbent assay) reactions are different in the same group of samples prepared in parallel, so that the detection result deviates from a true value.
Therefore, there is a need for more effective and accurate products and methods for inhibiting the non-specific deviating effects of ELISA reactions in both practical research and clinical work.
Disclosure of Invention
The invention firstly relates to the application of hexafluorophosphate in preparing a preparation for inhibiting the effect of an ELISA reaction matrix,
the matrix effect refers to the systematic error when detecting the same substance to be detected in the same group of samples prepared in parallel in ELISA reaction,
the main causes of matrix reactions include: the components in the sample except the target analyte interfere the analysis process, and the accuracy of the analysis result is influenced. The factors which are commonly responsible for the matrix effect of serum and plasma products are: heterophilic antibodies, complement, heme, residual drug components, and the like contained in the sample.
The ELISA reaction comprises double-antibody sandwich ELISA reaction, indirect ELISA reaction and competitive ELISA reaction;
the hexafluorophosphate is sodium hexafluorophosphate, potassium hexafluorophosphate, calcium hexafluorophosphate, magnesium hexafluorophosphate and aluminum hexafluorophosphate;
most preferably, the hexafluorophosphate salt is sodium hexafluorophosphate.
The invention also relates to a formulation for inhibiting the effect of an ELISA reaction matrix, said formulation comprising hexafluorophosphate in a concentration of 0.01-2% (wt) and the necessary buffering agents; preferably, hexafluorophosphate salts are included at a concentration of 0.5 to 1% (wt); most preferably, hexafluorophosphate salts are included at a concentration of 0.5% (wt);
the preparation also comprises 5% of mouse serum;
preferably, the preparation is: PBS containing hexafluorophosphate at a concentration of 0.1-1% (wt) and 5% mouse serum;
most preferably, the formulation is: PBS containing hexafluorophosphate at a concentration of 0.5% (wt) and 5% mouse serum;
the hexafluorophosphate is sodium hexafluorophosphate, potassium hexafluorophosphate, calcium hexafluorophosphate, magnesium hexafluorophosphate and aluminum hexafluorophosphate;
most preferably, the hexafluorophosphate salt is sodium hexafluorophosphate.
The invention also relates to application of the hexafluorophosphate in preparation of an ELISA reaction kit, wherein the hexafluorophosphate is used as a reagent for inhibiting matrix reaction in the kit;
the hexafluorophosphate is sodium hexafluorophosphate, potassium hexafluorophosphate, calcium hexafluorophosphate, magnesium hexafluorophosphate and aluminum hexafluorophosphate;
most preferably, the hexafluorophosphate salt is sodium hexafluorophosphate.
Drawings
FIG. 1, the recovery of interferon-gamma (IFNG) in plasma was examined, and only the group A sample (FIG. 1A) to which mouse serum was added, the group B sample (FIG. 1B) to which 0.1% NaPF6 was added, the group C sample (FIG. 1C) to which 0.5% NaPF6 was added, and the group D sample (FIG. 1D) to which 1% NaPF6 was added.
FIG. 2 shows the recovery rate of group A samples to which only mouse serum was added (2A) and the recovery rate of group C samples to which 0.5% NaPF6C was added (2B) when IL-6 was detected in plasma.
FIG. 3 shows the recovery of interferon-. gamma. (IFNG) in plasma, to which 0.5% NaPF6(3A) and 0.5% KPF6(3B) were added.
Detailed Description
Experimental materials:
experimental animals (mice): purchased from the center of medical laboratory animals in Guangdong province,
hexafluorophosphates (sodium hexafluorophosphate, potassium hexafluorophosphate, etc.): purchased from Sigma-Aldrich and,
enzyme label plate: purchased from nunc maxisorp,
ELISA standard antibody: from R & D anti-human IFN-gamma
Example 1, NaPF6 can significantly reduce the matrix error of ELISA detection process
1. Sample information, detection antigen information, ELISA kit information,
venous peripheral blood samples, from volunteers of the company,
the ELISA kit for detecting the gamma-interferon used in the experiment is a product (product code: 5020001301) self-developed by the company, and the content of the gamma-interferon in blood plasma is detected by using a double-antibody sandwich ELISA reaction;
the interleukin 6(IL6) ELISA kit was also a product from this company (cat # 5020014501).
2. The experimental procedures were as follows,
(1) preparing a sample diluent: group A is Phosphate Buffered Saline (PBS) containing 5% mouse serum, pH7.4;
(2) b, C, D, adding NaPF6 at concentrations of 0.1%, 0.5% and 1% on the basis of A;
(3) taking 7 samples (human plasma) to be detected, and setting 4 dilution times for each group of sample diluent for detection: stock solution (without sample diluent), 2 times diluted (1: 1 with sample diluent), 4 times diluted (1: 3 with sample diluent), 8 times diluted (1: 7 with sample diluent);
(3) all groups of samples to be tested were as follows:
group A: 7 samples to be tested (human plasma) + group A sample dilutions (PBS containing 5% mouse serum, pH7.4)
Group B: 7 samples to be tested (human plasma) + group B sample dilutions (PBS containing 5% mouse serum, pH7.4+ 0.1% NaPF6)
Group C: 7 samples to be tested (human plasma) + group C sample dilutions (PBS containing 5% mouse serum, pH7.4+ 0.5% NaPF6)
Group D: 7 samples to be tested (human plasma) + dilution of group D samples (PBS containing 5% mouse serum, pH7.4+ 1% NaPF6)
(4) ELISA detection was performed on each of the 4 groups of samples according to the standard kit protocol using an ELISA kit manufactured by this company for detecting gamma-Interferon (IFNG) or interleukin 6(IL6) in plasma.
Specifically, the matrix removal effect of the diluent is evaluated through the recovery rate of the detection result, and the calculation formula of the recovery rate is as follows:
the recovery rate is [ (measured value of sample after dilution × dilution multiple)/measured value of sample before dilution ] × 100%, and the closer the recovery rate is to 100%, the smaller the influence of matrix effect on the detection system is.
The results show that it is possible to display,
(1) when the gamma-Interferon (IFNG) in the plasma is detected, compared with the traditional A group sample (figure 1A) added with mouse serum only, the B group sample (figure 1B) added with 0.1 percent NaPF6, the C group sample (figure 1C) added with 0.5 percent NaPF6 and the D group sample (figure 1D) added with 1 percent NaPF6, the matrix effect is better removed;
(2) when detecting gamma-Interferon (IFNG) in plasma, a group B sample (figure 1B) added with 0.1% NaPF6, a group C sample (figure 1C) added with 0.5% NaPF6 and a group D sample (figure 1D) added with 1% NaPF6, for the removal effect of matrix effect, the comprehensive evaluation result shows that the purpose of optimally removing the matrix effect can be achieved by using 0.5% NaPF 6;
(3) when detecting interferon-gamma (IFNG) in plasma, replacing NaPF6 (fig. 3A) with KPF6 (fig. 3B) at the same concentration also resulted in more consistent matrix removal.
In another example, IL-6 in plasma was measured, and group B samples with 0.5% NaPF6 added (FIG. 2B) were also better at removing matrix effects than group A samples with only mouse serum added (FIG. 2A).
Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not intended to limit the protection scope of the present invention.
Claims (8)
1. The application of hexafluorophosphate in preparing a preparation for inhibiting ELISA reaction matrix effect refers to the systematic error when the same substance to be detected is detected in parallel prepared samples in the same group in ELISA reaction.
2. The use of claim 1, wherein the ELISA reaction comprises a double antibody sandwich ELISA reaction, an indirect ELISA reaction and a competitive ELISA reaction.
3. Use according to claim 1 or 2, wherein said hexafluorophosphate salt is sodium hexafluorophosphate, potassium hexafluorophosphate, calcium hexafluorophosphate, magnesium hexafluorophosphate, aluminium hexafluorophosphate; preferably, the hexafluorophosphate is sodium hexafluorophosphate.
4. An agent for inhibiting the effects of an ELISA reaction matrix, said agent comprising:
(1) hexafluorophosphate in a concentration of 0.01-2% (wt); preferably, hexafluorophosphate salts are included at a concentration of 0.5 to 1% (wt); most preferably, hexafluorophosphate salts are included at a concentration of 0.5% (wt);
(2) the necessary buffer, preferably, the buffer is Phosphate Buffered Saline (PBS).
5. The formulation of claim 4, further comprising: 5% mouse serum.
6. The formulation of claim 5, wherein the formulation is: PBS containing hexafluorophosphate at a concentration of 0.1-1% (wt) and 5% mouse serum; preferably, the preparation is: PBS containing hexafluorophosphate at a concentration of 0.5% (wt) and 5% mouse serum.
7. The formulation according to claim 4 or 5, wherein said hexafluorophosphate salt is sodium hexafluorophosphate, potassium hexafluorophosphate, calcium hexafluorophosphate, magnesium hexafluorophosphate, aluminum hexafluorophosphate; preferably, the hexafluorophosphate is sodium hexafluorophosphate.
8. The application of hexafluorophosphate in preparing an ELISA reaction kit, wherein the hexafluorophosphate is used as a reagent for inhibiting matrix reaction in the kit; the hexafluorophosphate is sodium hexafluorophosphate, potassium hexafluorophosphate, calcium hexafluorophosphate, magnesium hexafluorophosphate and aluminum hexafluorophosphate; preferably, the hexafluorophosphate is sodium hexafluorophosphate.
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