CN116500256A - Method for rapidly detecting content of serum retinol - Google Patents
Method for rapidly detecting content of serum retinol Download PDFInfo
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- CN116500256A CN116500256A CN202310135172.9A CN202310135172A CN116500256A CN 116500256 A CN116500256 A CN 116500256A CN 202310135172 A CN202310135172 A CN 202310135172A CN 116500256 A CN116500256 A CN 116500256A
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- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 title claims abstract description 82
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 title claims abstract description 41
- 210000002966 serum Anatomy 0.000 title claims abstract description 40
- 229960003471 retinol Drugs 0.000 title claims abstract description 37
- 235000020944 retinol Nutrition 0.000 title claims abstract description 37
- 239000011607 retinol Substances 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 25
- 108091000053 retinol binding Proteins 0.000 claims abstract description 45
- 102000029752 retinol binding Human genes 0.000 claims abstract description 45
- 238000003556 assay Methods 0.000 claims abstract description 8
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- 238000007405 data analysis Methods 0.000 claims abstract description 5
- 238000011088 calibration curve Methods 0.000 claims abstract description 4
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- 239000008280 blood Substances 0.000 claims description 17
- 238000002965 ELISA Methods 0.000 claims description 16
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- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 6
- 229940098773 bovine serum albumin Drugs 0.000 claims description 6
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- 238000006243 chemical reaction Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
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- 241001552669 Adonis annua Species 0.000 claims description 3
- 108010010803 Gelatin Proteins 0.000 claims description 3
- 101000665882 Homo sapiens Retinol-binding protein 4 Proteins 0.000 claims description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 3
- 108091005804 Peptidases Proteins 0.000 claims description 3
- 239000004365 Protease Substances 0.000 claims description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 3
- 239000000427 antigen Substances 0.000 claims description 3
- 102000036639 antigens Human genes 0.000 claims description 3
- 108091007433 antigens Proteins 0.000 claims description 3
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- 229910021641 deionized water Inorganic materials 0.000 claims description 3
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- 235000019322 gelatine Nutrition 0.000 claims description 3
- 235000011852 gelatine desserts Nutrition 0.000 claims description 3
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- 239000012089 stop solution Substances 0.000 claims description 3
- 238000012360 testing method Methods 0.000 claims description 3
- 239000003480 eluent Substances 0.000 claims description 2
- 101710137011 Retinol-binding protein 4 Proteins 0.000 claims 5
- 239000011717 all-trans-retinol Substances 0.000 abstract description 4
- 229940100609 all-trans-retinol Drugs 0.000 abstract description 4
- 235000019169 all-trans-retinol Nutrition 0.000 abstract description 4
- 238000011896 sensitive detection Methods 0.000 abstract description 2
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- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 238000003018 immunoassay Methods 0.000 description 7
- 239000012086 standard solution Substances 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 230000008569 process Effects 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 229960000342 retinol acetate Drugs 0.000 description 3
- 235000019173 retinyl acetate Nutrition 0.000 description 3
- 239000011770 retinyl acetate Substances 0.000 description 3
- QGNJRVVDBSJHIZ-QHLGVNSISA-N retinyl acetate Chemical compound CC(=O)OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C QGNJRVVDBSJHIZ-QHLGVNSISA-N 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000010268 HPLC based assay Methods 0.000 description 2
- 208000010011 Vitamin A Deficiency Diseases 0.000 description 2
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- 102000004169 proteins and genes Human genes 0.000 description 2
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- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- OTMSDBZUPAUEDD-UHFFFAOYSA-N Ethane Chemical compound CC OTMSDBZUPAUEDD-UHFFFAOYSA-N 0.000 description 1
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- 239000002245 particle Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
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- 210000002700 urine Anatomy 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/02—Devices for withdrawing samples
- G01N1/10—Devices for withdrawing samples in the liquid or fluent state
- G01N1/20—Devices for withdrawing samples in the liquid or fluent state for flowing or falling materials
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses a method for rapidly detecting the content of serum retinol, which is replaced by detecting the content of serum retinol binding protein, and comprises the following steps: sample collection, sample pretreatment, and quantification of RBP by using SCANLISA RBP assay; sample processing, namely, a step four: data analysis, calculation of results from best fit calibration curves for each plate using the macleaya software. The Retinol Binding Protein (RBP) in the serum of the invention is used as a sensitive detection index, has higher binding property with all-trans retinol, and has a binding property with retinol of about 1:1 molar correlation, and RBP has excellent correlation with serum retinol in different populations, can be used as a surrogate marker of retinol.
Description
Technical Field
The invention relates to a method for rapidly detecting the content of retinol in serum, which mainly detects the content of retinol in serum through enzyme immunoassay, and belongs to the technical field of molecular detection or medical science.
Background
Vitamin a deficiency is an important public health problem worldwide. Retinol, one of the active forms of vitamin a, has a serum content closely related to vitamin a levels. Retinol Binding Proteins (RBPs) are transport proteins of vitamins in the blood, synthesized by the liver, widely distributed in the blood, cerebrospinal fluid, urine and other body fluids. Studies have shown that RBP binds highly to all-trans retinol, and is present in serum at about 1:1 mole, and thus is considered to be a surrogate marker for retinol. In addition, RBP is more resistant to environmental conditions than retinol, blood spots can be collected by peripheral capillary blood sampling, can be detected using ELISA, and RBP has excellent correlation with serum retinol in different populations, so that the calculation of serum retinol content using RBP content detection has higher reliability.
High Performance Liquid Chromatography (HPLC) analysis is a traditional detection method for quantifying serum retinol, and is commonly used for the detection of serum vitamin levels. However, HPLC assays suffer from limitations including complex analytical procedures, high operational requirements, high costs, long post-treatment times, etc., resulting in low operability in rapid screening of large-scale populations, greatly limiting the epidemiological assessment rate of vitamin a deficiency. Therefore, there is a need to explore a rapid, economical and reliable detection method to address the limitations of HPLC analysis. The ELISA method combines specific antigen-antibody immunological reaction with enzyme catalysis reaction, and performs qualitative or quantitative analysis according to the color development depth of enzyme reaction substrate, and has high sensitivity, objective and accurate result judgment and strong practicability.
The measurement of Retinol Binding Protein (RBP) using the enzyme-linked immunosorbent assay has the following advantages over conventional HPLC assays: (1) fast, simple: HPLC analysis is widely recognized as a method for detecting retinol content with high reliability and accuracy. However, conventional HPLC detection procedures are complicated and require expensive equipment, have low versatility, and require a high degree of skill in the early stages. The enzyme immunoassay has simple requirements on the operation process, and has low operation difficulty of the whole experiment, and is particularly suitable for detecting the retinol content during large-scale epidemiological screening. (2) easy transportation and storage: blood samples required by HPLC analysis are required to be timely subjected to serum separation and placed in a strict light-shielding environment, and the preservation requirement is high. The filter paper card blood spot sample and the drying box used for enzyme immunoassay do not need special preservation environment and are easy to transport.
Therefore, based on the above detection problems, researchers in the field have a need to optimize the existing retinol binding protein detection procedure based on the enzyme immunoassay, so as to more rapidly and accurately detect the serum retinol content.
Disclosure of Invention
The invention provides a method for rapidly detecting the content of serum retinol, which is a method for detecting serum retinol binding protein based on enzyme immunoassay, so as to solve part of problems existing in the traditional detection process, reduce the interference effect of confounding factors in the detection process of the content of the serum retinol binding protein and improve the reaction sensitivity.
A method for rapidly detecting the content of serum retinol is replaced by detecting the content of serum retinol binding protein, which comprises the following steps:
step one: sample collection, alcohol disinfection is carried out on a third finger or a fourth finger of a participant, a sterile telescopic bayonet is used for puncturing, the flowing blood drops freely fall into a preprinted circle on a filter paper card to be used as a sample, and the sample filter paper card is placed in an opaque drying box containing a drying agent so as to accelerate drying; monitoring the humidity in the box by using a humidity indicator card, and adding an additional desiccant bag into the box if necessary to keep the filter paper card dry; after drying overnight in the drying box, the filter paper card was individually packaged in a low permeability plastic self-sealing bag containing a desiccant and a humidity indicator card and stored in a battery operated portable refrigerator at 4 ° to 8 ℃; the dried blood spots are sent to a laboratory for storage in an environment of-20 ℃ within 7 to 10 days after collection until analysis;
step two: sample pretreatment, quantifying RBP using a scanisa RBP assay; all reagents except deionized water were provided as part of the assay kit; this assay uses purified human RBP to adsorb into micro-test strip wells to compete with native RBP found in serum; for analysis, a 1/4 inch punch was taken from the center of the two dried blood spot circles, placed in a microcentrifuge tube, and 300 μl of sample diluent was added, 150 μl of punch per 1/4 inch; the sample is vortexed for 20 seconds, centrifuged for 2 minutes at a low Wen Zhongsu and eluted at 4 ℃ to 8 ℃ for 18 to 20 hours, and if precipitation occurs during storage, the sample should be centrifuged again;
step three: sample processing, taking out the sample and the reagent from the refrigerator, and reaching room temperature before analysis; firstly, carrying out serial dilution of 640 mug/L standard with diluent to obtain 7 point standard of 5, 10, 20, 40, 80, 160 and 320 mug/L, respectively taking 0.1ml multiplied by 2, and taking 0.1ml multiplied by 2 diluent as blank control; then the standard and the control are vortexed for 20 seconds, centrifuged for 2 minutes at a low speed Wen Zhongsu, and then added into a serum retinol binding protein coating plate respectively; immediately adding a monoclonal anti-RBP antibody coupled with horseradish peroxidase; incubation at room temperature for 15 min, washing with Phosphate Buffer (PBS), and adding a blocking solution containing gelatin or Bovine Serum Albumin (BSA) to block the portion of the elisa plate to which the antibody was not bound; adding 100 mu L of dry blood sample eluent, and incubating for 1-2 hours at 37 ℃; at the moment, the antibody on the ELISA plate is specifically identified and combined with the sample to be detected; washing the unbound antigen with phosphate buffer, repeatedly washing for 4 times, and drying; adding a retinol-binding protease marker, then incubating for 10 minutes, adding a stop solution hydrochloride solution to stop the reaction, and finally immediately reading the OD values of a standard substance and a sample on an ELISA (enzyme-linked immunosorbent assay) at 450nm, and calibrating zero by using a blank control tube;
step four: data analysis, namely calculating a result according to a best fit calibration curve of each plate by using the Mazelon software, wherein the result is expressed in micrograms per milliliter RBP; and drawing a standard curve by using the OD value of the standard substance and the corresponding concentration thereof and using the OD value as an abscissa and the concentration as an ordinate, substituting the OD value of the sample as an X value, and obtaining the concentration of the sample with the Y value.
Preferably, in the second step, the measured precision between batches and the measured precision in batches are 8.9% and 6.7% respectively; the limit of quantitation was 7.7 μg/mL RBP with a linearity of 99.7%.
Compared with the conventional HPLC detection of the retinol content, the enzyme-linked immunosorbent assay method for detecting the Retinol Binding Protein (RBP) has the advantages that:
(1) Retinol Binding Protein (RBP) in serum is a sensitive assay indicator with high binding to all-trans retinol, and is present in serum at about 1:1 molar correlation, and RBP has excellent correlation with serum retinol in different populations, can be used as a surrogate marker of retinol.
(2) RBP can collect blood spots through peripheral capillary blood sampling method, and enzyme immunoassay for detection has the advantages of simpler operation process requirement, lower overall experiment operation difficulty, rapidness and simplicity.
(3) RBP is more resistant to environmental conditions than retinol, and filter paper card blood spot samples and dry cartridges used in enzyme immunoassays do not require special storage environments and are easier to transport.
Drawings
FIG. 1 is a flow chart of a method for detecting retinol binding protein content in serum provided by the present invention;
FIG. 2 is a flow chart of a method for detecting retinol content in serum provided by the present invention;
FIG. 3 shows the chemical structure of retinol as provided in the present invention.
Detailed Description
The invention is further illustrated below with reference to examples and comparative examples.
Example 1
As shown in FIG. 1, a method for rapidly detecting the content of serum retinol is replaced by detecting the content of serum retinol binding protein, which comprises the following steps:
step one: sample collection, alcohol disinfection is carried out on a third finger or a fourth finger of a participant, a sterile telescopic bayonet is used for puncturing, the flowing blood drops freely fall into a preprinted circle on a filter paper card to be used as a sample, and the sample filter paper card is placed in an opaque drying box containing a drying agent so as to accelerate drying; monitoring the humidity in the box by using a humidity indicator card, and adding an additional desiccant bag into the box if necessary to keep the filter paper card dry; after drying overnight in the drying box, the filter paper card was individually packaged in a low permeability plastic self-sealing bag containing a desiccant and a humidity indicator card and stored in a battery operated portable refrigerator at 4 ° to 8 ℃; the dried blood spots are sent to a laboratory for storage in an environment of-20 ℃ within 7 to 10 days after collection until analysis;
step two: sample pretreatment, quantifying RBP using a scanisa RBP assay; all reagents except deionized water were provided as part of the assay kit; this assay uses purified human RBP to adsorb into micro-test strip wells to compete with native RBP found in serum; for analysis, a 1/4 inch punch was taken from the center of the two dried blood spot circles, placed in a microcentrifuge tube, and 300 μl of sample diluent was added, 150 μl of punch per 1/4 inch; the sample is vortexed for 20 seconds, centrifuged for 2 minutes at a low Wen Zhongsu and eluted at 4 ℃ to 8 ℃ for 18 to 20 hours, and if precipitation occurs during storage, the sample should be centrifuged again;
step three: sample processing, taking out the sample and the reagent from the refrigerator, and reaching room temperature before analysis; firstly, carrying out serial dilution of 640 mug/L standard with diluent to obtain 7 point standard of 5, 10, 20, 40, 80, 160 and 320 mug/L, respectively taking 0.1ml multiplied by 2, and taking 0.1ml multiplied by 2 diluent as blank control; then the standard and the control are vortexed for 20 seconds, centrifuged for 2 minutes at a low speed Wen Zhongsu, and then added into a serum retinol binding protein coating plate respectively; immediately adding a monoclonal anti-RBP antibody coupled with horseradish peroxidase; because the ELISA plate is made of polystyrene, the benzene ring contained in the ELISA plate and the amino acid residue of the antibody have attraction similar to pi-pi stacking effect, and the antibody can be adsorbed on the surface of the ELISA plate by combining static electricity and hydrophobic effect; incubation at room temperature for 15 min, washing with Phosphate Buffer (PBS), and adding a blocking solution containing gelatin or Bovine Serum Albumin (BSA) to block the portion of the elisa plate to which the antibody was not bound; the purpose is to prevent other proteins from being adsorbed on a 96-well plate due to static electricity or hydrophobic effect, so as to cause false positive signals and interfere the follow-up experiment; 100. Mu.L of dry blood sample eluate (or serum diluted 1:25 with sample diluent) was added and incubated at 37℃for 1-2 hours; at the moment, the antibody on the ELISA plate is specifically identified and combined with the sample to be detected; washing the unbound antigen with phosphate buffer, repeatedly washing for 4 times, and drying; adding a retinol-binding protease marker, then incubating for 10 minutes, adding a stop solution hydrochloride solution to stop the reaction, and finally immediately reading the OD values of a standard substance and a sample on an ELISA (enzyme-linked immunosorbent assay) at 450nm, and calibrating zero by using a blank control tube;
step four: data analysis, namely calculating a result according to a best fit calibration curve of each plate by using the Mazelon software, wherein the result is expressed in micrograms per milliliter RBP; and drawing a standard curve by using the OD value of the standard substance and the corresponding concentration thereof and using the OD value as an abscissa and the concentration as an ordinate, substituting the OD value of the sample as an X value, and obtaining the concentration of the sample with the Y value.
Example 2
As shown in fig. 2 and 3, this example is a conventional method for detecting serum retinol content, which comprises the following steps:
and (2) preparing a standard storage solution A: retinol standard solution (107.1.+ -. 5.4 mg/L) was used immediately after unsealing. Before use, the liquid is corrected by an ultraviolet spectrophotometer.
And (3) preparing an internal standard storage solution B: taking 10mg of retinol acetate standard substance, placing in a 10m L volumetric flask, dissolving with absolute ethanol, and fixing the volume to 10m L to obtain retinol acetate mother liquor (1000 mg/L), and storing at-80deg.C in a dark environment.
And (3) preparing a standard working solution C: and (3) diluting a proper amount of standard stock solution A with absolute ethyl alcohol to obtain a standard working solution containing 2.0mg/L retinol, and preserving at-80 ℃.
And (3) preparing an internal standard working solution D: and (3) diluting an appropriate amount of internal standard stock solution B with absolute ethyl alcohol to obtain an internal standard working solution containing 7.5mg/L of retinol acetate, and preserving at-80 ℃ in a light-proof environment.
Preparing a standard solution E in the step (5): and (3) taking 90 mu L of standard working solution C and 10 mu L of mixed internal standard working solution D, respectively placing into a centrifuge tube, centrifuging for 1min at a centrifugation speed of 2000rpm, and taking supernatant to obtain the standard solution.
Step (6) preparation of a sample F to be tested: (1) 100. Mu.L of blood was placed in a 1.5-m L covered plastic centrifuge tube, 10. Mu.L of the mixed internal standard working solution was removed by a pipette, mixed for about 5 seconds on a mixer, and then 100. Mu.L of pure water was added as a diluent, followed by centrifugation at 2000rpm for 5 minutes. (2) 200. Mu.L of absolute ethanol was added as a reagent for precipitating proteins, and the mixture was centrifuged at 2000rpm for 1min. (3) Adding 500 mu L of normal ethane as an extractant, centrifuging at 2000rpm for 7min, centrifuging at 10000rpm for 10min, transferring a certain amount of supernatant after centrifugation to a clean 1.5m L centrifuge tube, transferring to a nitrogen blow-drying device, and blow-drying the supernatant. (5) Adding 100 mu L of methanol serving as a complex solution into a 1.5m L centrifuge tube, centrifuging at 2000rpm for 1min, centrifuging at 10000rpm for 5min, and removing the supernatant to obtain a sample to be tested.
Step (7): using high performance liquid chromatograph, shimadzu LC-20A. Under the same detection condition, detecting a certain amount of sample F to be detected, standard solution E and internal standard working solution D respectively to obtain and record corresponding chromatograms and chromatographic peak response values.
And (8) data analysis: calculation formula by internal standard method
f= (As/ms)/(Ar/mr) (formula-1)
Mi=f×Ai/(As/ms) (formula-2)
Wherein f in the formula-1 is a correction factor, as and Ar are peak areas or peak heights of the internal standard working solution D and the standard solution E, respectively, and ms and mr are amounts of the internal standard working solution D and the standard solution E added, respectively. In the formula-2, mi is the content of a sample to be detected, ai and As are the peak areas or peak heights of the sample to be detected F and the internal standard working solution D respectively, and ms is the amount of the internal standard working solution D added.
The whole treatment process of the standard solution and the sample should be carried out in a light-proof room.
The conditions of the used instrument are detected: (1) chromatographic column: a Porosiwell 120SB-C18 column from Agilent corporation, the length of the column was 50mm, the inner diameter was 3.0mm, and the packing particle size was 2.7. Mu.m. (2) Mobile phase: methanol and pure water. (3) Elution mode: gradient elution is used. (4) Fluorescence detector wavelength: the excitation wavelength of retinol is 325nm, the emission wavelength is 470nm, and the maximum absorption wavelength is selected.
In the two examples, retinol Binding Protein (RBP) in serum is used as a sensitive detection index, has high binding property with all-trans retinol, and has a binding property with retinol of about 1:1 molar correlation, and RBP has excellent correlation with serum retinol in different populations, can be used as a surrogate marker of retinol.
Claims (3)
1. A method for rapidly detecting the content of serum retinol is characterized in that: it is replaced by detecting the content of serum retinol binding protein.
2. The method for detecting the content of the retinol binding protein according to claim 1, which comprises the following steps:
step one: sample collection, alcohol disinfection is carried out on a third finger or a fourth finger of a participant, a sterile telescopic bayonet is used for puncturing, the flowing blood drops freely fall into a preprinted circle on a filter paper card to be used as a sample, and the sample filter paper card is placed in an opaque drying box containing a drying agent so as to accelerate drying; monitoring the humidity in the box by using a humidity indicator card, and adding an additional desiccant bag into the box if necessary to keep the filter paper card dry; after drying overnight in the drying box, the filter paper card was individually packaged in a low permeability plastic self-sealing bag containing a desiccant and a humidity indicator card and stored in a battery operated portable refrigerator at 4 ° to 8 ℃; the dried blood spots are sent to a laboratory for storage in an environment of-20 ℃ within 7 to 10 days after collection until analysis;
step two: sample pretreatment, quantifying RBP using a scanisa RBP assay; all reagents except deionized water were provided as part of the assay kit; this assay uses purified human RBP to adsorb into micro-test strip wells to compete with native RBP found in serum; for analysis, a 1/4 inch punch was taken from the center of the two dried blood spot circles, placed in a microcentrifuge tube, and 300 μl of sample diluent was added, 150 μl of punch per 1/4 inch; the sample is vortexed for 20 seconds, centrifuged for 2 minutes at a low Wen Zhongsu and eluted at 4 ℃ to 8 ℃ for 18 to 20 hours, and if precipitation occurs during storage, the sample should be centrifuged again;
step three: sample processing, taking out the sample and the reagent from the refrigerator, and reaching room temperature before analysis; firstly, carrying out serial dilution of 640 mug/L standard with diluent to obtain 7 point standard of 5, 10, 20, 40, 80, 160 and 320 mug/L, respectively taking 0.1ml multiplied by 2, and taking 0.1ml multiplied by 2 diluent as blank control; then the standard and the control are vortexed for 20 seconds, centrifuged for 2 minutes at a low speed Wen Zhongsu, and then added into a serum retinol binding protein coating plate respectively; immediately adding a monoclonal anti-RBP antibody coupled with horseradish peroxidase; incubation at room temperature for 15 min, washing with Phosphate Buffer (PBS), and adding a blocking solution containing gelatin or Bovine Serum Albumin (BSA) to block the portion of the elisa plate to which the antibody was not bound; adding 100 mu L of dry blood sample eluent, and incubating for 1-2 hours at 37 ℃; at the moment, the antibody on the ELISA plate is specifically identified and combined with the sample to be detected; washing the unbound antigen with phosphate buffer, repeatedly washing for 4 times, and drying; adding a retinol-binding protease marker, then incubating for 10 minutes, adding a stop solution hydrochloride solution to stop the reaction, and finally immediately reading the OD values of a standard substance and a sample on an ELISA (enzyme-linked immunosorbent assay) at 450nm, and calibrating zero by using a blank control tube;
step four: data analysis, namely calculating a result according to a best fit calibration curve of each plate by using the Mazelon software, wherein the result is expressed in micrograms per milliliter RBP; and drawing a standard curve by using the OD value of the standard substance and the corresponding concentration thereof and using the OD value as an abscissa and the concentration as an ordinate, substituting the OD value of the sample as an X value, and obtaining the concentration of the sample with the Y value.
3. The method for detecting the content of the retinol binding protein according to claim 2, wherein the method comprises the following steps: in the second step, the measured precision between batches and the measured precision in batches are 8.9% and 6.7% respectively; the limit of quantitation was 7.7 μg/mL RBP with a linearity of 99.7%.
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| US5532166A (en) * | 1994-04-18 | 1996-07-02 | Ma; Yinfa | Quantitative retinol assay for serum and dried blood spots |
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