CN112394166B - 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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- CN112394166B CN112394166B CN202011242142.0A CN202011242142A CN112394166B CN 112394166 B CN112394166 B CN 112394166B CN 202011242142 A CN202011242142 A CN 202011242142A CN 112394166 B CN112394166 B CN 112394166B
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- -1 hexafluorophosphate Chemical compound 0.000 title claims abstract description 40
- 238000002965 ELISA Methods 0.000 title claims abstract description 25
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 23
- 230000000694 effects Effects 0.000 title claims abstract description 17
- 239000011159 matrix material Substances 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 9
- 239000000126 substance Substances 0.000 claims abstract description 4
- 230000009897 systematic effect Effects 0.000 claims abstract description 3
- 210000002966 serum Anatomy 0.000 claims description 15
- 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 5
- 230000002860 competitive effect Effects 0.000 claims description 2
- 239000008055 phosphate buffer solution Substances 0.000 claims 2
- 238000001514 detection method Methods 0.000 abstract description 11
- 239000000872 buffer Substances 0.000 abstract description 2
- 239000002904 solvent Substances 0.000 abstract 1
- 210000002381 plasma Anatomy 0.000 description 16
- 241000699666 Mus <mouse, genus> Species 0.000 description 12
- 229910019398 NaPF6 Inorganic materials 0.000 description 12
- 108010074328 Interferon-gamma Proteins 0.000 description 9
- 102000008070 Interferon-gamma Human genes 0.000 description 8
- 229940044627 gamma-interferon Drugs 0.000 description 8
- 238000011084 recovery Methods 0.000 description 8
- 102100037850 Interferon gamma Human genes 0.000 description 7
- 239000003085 diluting agent Substances 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 238000010790 dilution Methods 0.000 description 7
- 101000599940 Homo sapiens Interferon gamma Proteins 0.000 description 6
- 238000000034 method Methods 0.000 description 5
- 238000008157 ELISA kit Methods 0.000 description 4
- 102000004889 Interleukin-6 Human genes 0.000 description 4
- 108090001005 Interleukin-6 Proteins 0.000 description 4
- 239000012898 sample dilution Substances 0.000 description 4
- 238000009472 formulation Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229910021135 KPF6 Inorganic materials 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 229940100601 interleukin-6 Drugs 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000003118 sandwich ELISA Methods 0.000 description 2
- 238000012360 testing method Methods 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
- 206010048612 Hydrothorax Diseases 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000010171 animal model Methods 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
- 230000002490 cerebral effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent 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
- 238000002474 experimental method Methods 0.000 description 1
- 150000003278 haem Chemical class 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
- 239000008363 phosphate buffer Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Classifications
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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)
-
- 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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- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
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Abstract
The invention relates to application of hexafluorophosphate in preparation of preparation for inhibiting ELISA reaction, wherein matrix effect refers to systematic error in detection of the same substance to be detected in the same group of samples prepared in parallel in ELISA reaction, and the preparation comprises hexafluorophosphate with 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), which is a common scientific research and clinical detection means, is used for quantitatively detecting the concentration of a target in a liquid sample.
Because of the diversity of the detection samples, different detection samples (serum, plasma, hydrothorax, cerebral effusion, urine and the like) have certain differences in protein concentration, ionic strength, pH and the like, even though the same samples are different, special differences exist among different individuals, such as the research on more human anti-mouse HAMA reactions, which can cause substances to be detected with the same concentration, and ELISA reactions are different inside the same group of samples prepared in parallel, so that the detection results deviate from the true values.
Therefore, there is a need in both practical scientific and clinical work for more effective and accurate products and methods for inhibiting the non-specific bias effects of ELISA reactions.
Disclosure of Invention
The invention firstly relates to application of hexafluorophosphate in preparation of a preparation for inhibiting ELISA reaction matrix effect,
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 reasons for the reaction of the matrix include: components other than the target analyte in the sample interfere with the analysis process, affecting the accuracy of the analysis results. Common factors responsible for the matrix effects of serum, plasma species are: the sample contains the allophilic antibody, complement, heme, residual drug components, and the like.
The ELISA reaction comprises a double antibody sandwich ELISA reaction, an indirect ELISA reaction and a competitive ELISA reaction;
The hexafluorophosphate is sodium hexafluorophosphate, potassium hexafluorophosphate, calcium hexafluorophosphate, magnesium hexafluorophosphate and aluminum hexafluorophosphate;
most preferably, the hexafluorophosphate is sodium hexafluorophosphate.
The invention also relates to a formulation for inhibiting the effect of an ELISA reaction matrix, said formulation comprising hexafluorophosphate at a concentration of 0.01-2% (wt) and optionally a buffer; preferably, it comprises hexafluorophosphate at a concentration of 0.5-1% (wt); most preferably, it comprises hexafluorophosphate at a concentration of 0.5% (wt);
The preparation also comprises 5% 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 is sodium hexafluorophosphate.
The invention also relates to application of the hexafluorophosphate in preparing ELISA reaction kits, wherein the hexafluorophosphate is used as a reagent for inhibiting matrix reaction in the kits;
The hexafluorophosphate is sodium hexafluorophosphate, potassium hexafluorophosphate, calcium hexafluorophosphate, magnesium hexafluorophosphate and aluminum hexafluorophosphate;
most preferably, the hexafluorophosphate is sodium hexafluorophosphate.
Drawings
FIG. 1, recovery of gamma Interferon (IFNG) in plasma was measured, with only group A samples of mouse serum (FIG. 1A), group B samples of 0.1% NaPF6 (FIG. 1B), group C samples of 0.5% NaPF6 (FIG. 1C), and group D samples of 1% NaPF6 (FIG. 1D).
FIG. 2, recovery of group A samples (2A) with only mouse serum and recovery of group C samples with 0.5% NaPF6C (2B) when IL-6 in plasma was detected.
FIG. 3 shows the recovery rate of gamma-Interferon (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 medical laboratory animal center in Guangdong province,
Hexafluorophosphate (sodium hexafluorophosphate, potassium hexafluorophosphate, etc.): purchased from Sigma-Aldrich,
ELISA plate: purchased from the company nunc maxisorp,
ELISA Standard antibody: purchased from R & D anti-human IFN-gamma
Example 1, naPF6 significantly reduced matrix error in ELISA detection procedures
1. Sample information, detection antigen information, ELISA kit information,
Venous peripheral blood samples from volunteers from this company,
ELISA kit for detecting gamma-interferon used in the experiment is a self-grinding product (product number: 5020001301) of the company, and the content of gamma-interferon in blood plasma is detected by using a double-antibody sandwich ELISA reaction;
interleukin 6 (IL 6) ELISA kit was also a self-grinding product of this company (cat# 5020014501).
2. In the experimental process flow, the preparation method comprises the following steps,
(1) Preparing a sample diluent: group A was Phosphate Buffer (PBS) containing 5% mouse serum, pH7.4;
(2) In B, C, D groups, on the basis of the A group, naPF6 with the concentration of 0.1%, 0.5% and 1% is additionally added;
(3) 7 samples to be tested (human plasma) are taken, each group is respectively tested by using sample diluents of each group, and 4 dilution multiples are set for testing: stock solution (no sample diluent added), 2-fold dilution (sample diluent added 1:1), 4-fold dilution (sample diluent added 1:3), 8-fold dilution (sample diluent added 1:7);
(3) All sample groups tested were as follows:
group A: 7 samples to be tested (human plasma) +group A sample dilutions (PBS containing 5% mouse serum, pH 7.4)
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) +group D sample dilutions (PBS containing 5% mouse serum, pH7.4+1% NaPF6)
(4) ELISA tests were performed on the 4 groups of samples according to the standard procedure of the kit using ELISA kits for detecting gamma-Interferon (IFNG) or interleukin 6 (IL 6) in plasma produced by the company.
Specifically, the matrix removal effect of the diluent is evaluated by detecting the recovery rate of the result, and the calculation formula of the recovery rate is as follows:
recovery = [ (sample detection value after dilution x dilution multiple)/sample detection value before dilution ] ×100%, the closer the recovery is to 100%, the less the detection system is affected by matrix effect.
The results show that the data obtained from the above-mentioned method,
(1) In the detection of gamma Interferon (IFNG) in plasma, the matrix effect was better removed than the prior group a samples with only mouse serum (fig. 1A), group B samples with 0.1% naff 6 (fig. 1B), group C samples with 0.5% naff 6 (fig. 1C), and group D samples with 1% naff 6 (fig. 1D);
(2) When gamma-Interferon (IFNG) in blood plasma is detected, a group B sample (figure 1B) added with 0.1 percent NaPF6, a group C sample (figure 1C) added with 0.5 percent NaPF6 and a group D sample (figure 1D) added with 1 percent NaPF6 are added, and as for the removal effect of the matrix effect, the comprehensive evaluation result shows that the purpose of optimally removing the matrix effect can be achieved by using 0.5 percent NaPF 6;
(3) When gamma-Interferon (IFNG) was detected in plasma, the same concentration of KPF6 (fig. 3B) was replaced with NaPF6 (fig. 3A), and a more consistent matrix-removal effect could be achieved.
In another example, IL-6 in plasma was detected, and group B samples (FIG. 2B) with 0.5% NaPF6 added were also better for matrix effect removal than the prior group A samples with only mouse serum (FIG. 2A).
Finally, it should be noted that the above embodiments are merely used to help those skilled in the art understand the essence of the present invention, and are not intended to limit the scope of the present invention.
Claims (2)
1. The application of hexafluorophosphate in preparing preparation for inhibiting ELISA reaction matrix effect, which is the systematic error of detecting the same substance to be detected in ELISA reaction inside the same group of plasma samples prepared in parallel;
the preparation is phosphate buffer solution only containing hexafluorophosphate with concentration of 0.5-1% (wt) and 5% mouse serum, and the pH value of the phosphate buffer solution is 7.4;
the hexafluorophosphate is sodium hexafluorophosphate or potassium hexafluorophosphate.
2. The use according to claim 1, wherein the ELISA reaction is a competitive ELISA reaction.
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Citations (1)
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| CN110462402A (en) * | 2017-03-27 | 2019-11-15 | 日本火腿株式会社 | The substance that antigen-antibody reaction caused by preventing because of body fluid hinders |
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| NL187998C (en) * | 1953-06-01 | Pusch Guenter | CAMOUFLAGE AGENT. | |
| US3203803A (en) * | 1964-01-20 | 1965-08-31 | Tecnifax Corp | Light-sensitive diazo hexafluoro-phosphate compositions |
| US6027890A (en) * | 1996-01-23 | 2000-02-22 | Rapigene, Inc. | Methods and compositions for enhancing sensitivity in the analysis of biological-based assays |
| JP2000211916A (en) * | 1999-01-20 | 2000-08-02 | Morita Kagaku Kogyo Kk | Treatment of organic solution containing lithium hexafluorophosphate or lithium tetrafluoroborate |
| JP2004219233A (en) * | 2003-01-15 | 2004-08-05 | Nisshinbo Ind Inc | Medium for fluorescence or chemiluminescence detection and method for fluorescence or chemiluminescence detection |
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