CN110988361B - Human serum albumin removing kit - Google Patents
Human serum albumin removing kit Download PDFInfo
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- CN110988361B CN110988361B CN201911290897.5A CN201911290897A CN110988361B CN 110988361 B CN110988361 B CN 110988361B CN 201911290897 A CN201911290897 A CN 201911290897A CN 110988361 B CN110988361 B CN 110988361B
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Abstract
The invention discloses a human serum albumin removal kit which consists of an immunoaffinity material, a buffer solution, a washing solution, a disposable consumable and a human serum albumin standard substance, wherein the immunoaffinity material comprises a carrier matrix and a nano antibody. The human serum albumin removal kit provided by the invention has the advantages that due to the specific nano antibody and the carrier matrix with a special structure, the human serum albumin can be specifically identified and adsorbed, the kit can be used for removing albumin in human plasma or serum, and the detection capability of the biological mass spectrum on the low-abundance protein index of peripheral blood is improved, so that the application of the biological mass spectrum in the peripheral blood proteomics detection is promoted. In addition, the nano antibody has the characteristics of high affinity, stable properties, acid and alkali resistance, high salt resistance, easiness in preparation and the like, so that the kit has a wide application range.
Description
Technical Field
The invention belongs to the technical field of immunology and biological mass spectrometry, relates to a nano antibody and an immunoaffinity material based on the nano antibody, and particularly relates to a human serum albumin removal kit.
Background
With the introduction of precise medical concepts and the development of precise medicine, proteomics based on biological mass spectrometry has become more and more important in clinical diagnosis and precise treatment. A high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is used as a novel detection platform and is widely applied to the field of biomedical research. With the continuous innovation of the technology, LC-MS/MS gradually turns to clinical laboratory detection, and provides a more reliable detection result for the diagnosis and treatment monitoring of clinical endocrine related diseases. Compared with the commercialized immunological method widely used in the market at present, the method has the advantages of co-detection of various target analytes, strong anti-interference capability (heterophilic antibodies, autoantibodies and cross reaction), high specificity and sensitivity and the like. Several years ago, the application of LC-MS/MS in clinical diagnosis is limited to the detection of some trace elements, vitamins, hormones, lipids and other small molecular substances and the detection of pathogenic microorganisms. The biological mass spectrometry technology is rarely applied to the detection of a large number of clinical laboratory indexes of proteins. With the continuous development of mass spectrometry technology in recent years, the detection sensitivity, detection flux and data processing capability are continuously improved. In addition, the discovery of a large number of biological targets makes it possible to apply proteomic detection to large-scale routine clinical detection. Through the method of biological mass spectrometry and proteomics, tens of even hundreds of protein indexes can be detected by only 1 drop of blood (about tens of microliters), so that the detection cost is greatly reduced, the detection efficiency is improved, meanwhile, the targets of comprehensive diagnosis and individualized treatment can be realized, and the method is a great progress of precise medicine.
However, an important factor that currently restricts the application of the biological mass spectrometry in the detection of peripheral blood proteomics is the preparation of samples. Peripheral blood is used as a biological sample which is most commonly used in clinical detection, has the characteristics of convenience, rapidness, small wound, rich targets and the like, and has a high reference value for clinical diagnosis. The total protein content in human serum is about 60-80g/L, wherein the albumin content is about 40-55g/L, which accounts for about 50-70% of the total protein content, the globulin content is about 20-30g/L, which accounts for about 30% of the total protein content, and the remaining protein content is < 5%. A large amount of albumin occupies the analysis capacity of a mass spectrometer, so that a plurality of protein components with lower content cannot be detected, and the application of a biomass spectrum in the detection of peripheral blood protein biological indexes is limited. Therefore, the rapid and effective removal of albumin in human plasma or serum greatly improves the detection capability of the biological mass spectrum on the low-abundance protein index of peripheral blood, and powerfully promotes the application of the biological mass spectrum in the peripheral blood proteomics detection.
Disclosure of Invention
The invention aims to provide a human serum albumin removal kit, which is a human serum albumin removal kit based on an immunoaffinity material of a nano antibody.
The kit for removing the human serum albumin comprises an immunoaffinity material, a buffer solution, a washing solution, a disposable consumable and a human serum albumin standard substance.
The immunoaffinity material comprises an anti-human serum albumin nano antibody and a carrier matrix. The amino acid sequence of the antihuman serum albumin nano antibody is shown in SEQ ID NO.1, and the nucleotide sequence of the coding nucleic acid molecule thereof is shown in SEQ ID NO. 2. The nano antibody can be combined with human serum albumin with high affinity, and is not combined with other proteins in human plasma or serum. The nanobody can be produced by introducing an expression vector containing the encoding nucleic acid molecule into escherichia coli or yeast cells for expression.
The carrier matrix is a porous material, and the porous material is selected from one of agarose gel microspheres, fiber microspheres, magnetic beads, silica gel microspheres, activated carbon and resin microspheres. The carrier matrix can specifically adsorb human serum albumin.
The buffer solution is phosphate buffer solution, has a pH value of 7.4 and does not contain calcium ions and magnesium ions.
The washing solution is phosphate buffer solution containing 0.1% Tween 20, has pH of 7.4, and is free of calcium ion and magnesium ion.
The disposable consumables are low-adsorption 1.5mL centrifuge tubes.
The human serum albumin standard is human serum albumin with the concentration of 100mg/mL, and is dissolved in a phosphate buffer solution with the pH value of 7.4.
The invention has the beneficial effects that: the human serum albumin removal kit provided by the invention has the advantages that due to the specific nano antibody and the carrier matrix with a special structure, the human serum albumin can be specifically identified and adsorbed, the kit can be used for removing albumin in human plasma or serum, and the detection capability of the biological mass spectrum on the low-abundance protein index of peripheral blood is improved, so that the application of the biological mass spectrum in the peripheral blood proteomics detection is promoted. In addition, the nano antibody has the characteristics of high affinity, stable properties, acid and alkali resistance, high salt resistance, easiness in preparation and the like, so that the kit has a wide application range.
Drawings
FIG. 1 shows the electrophoresis chart of the purification of nano-antibody.
FIG. 2 is a graph showing the adsorption capacity of immunoaffinity materials.
Figure 3 effect of human serum albumin removal kit on removal of albumin from human plasma.
Detailed Description
For a further understanding of the invention, preferred embodiments of the invention are described below in conjunction with the examples and the figures, but it should be understood that these descriptions are merely provided to further illustrate the features and advantages of the invention, and not to limit the claims of the invention.
Example 1 preparation of anti-human serum albumin Nanobodies
(1) Synthesizing nucleic acid molecules for coding the anti-human serum albumin nano antibody according to the sequence shown in SEQ ID NO. 2. At the same time, an Nde I cleavage site (CATATG) is added to the 5 'end of the nucleic acid molecule, and a Not I cleavage site (GCGGCCGC) is added to the 3' end.
(2) pET30a (+) prokaryotic expression plasmid is used as the expression vector of the nano antibody. The expression vector and the nucleic acid molecule fragment of the nano antibody are subjected to double enzyme digestion by using Not I and Nde I enzymes, wherein the enzyme digestion conditions are as follows: mu.g of DNA, 5. mu.L of 10 Xbuffer, 1. mu.L of each of Not I and Nde I enzymes, 50. mu.L of water, 20 minutes in a 37 ℃ water bath, 10 minutes of inactivation at 80 ℃ and recovery of the fragments after running the gel.
(3) Vector and fragment ligation: mu.g vector, 40. mu.g fragment, 1. mu.L T4 ligase, 2. mu.L 10 Xbuffer, water to 20. mu.L, room temperature for 1 hour.
(4) mu.L of the ligation product was taken, 50. mu.L of thawed BL21(DE3) E.coli competent cells were added, placed on ice for 30 minutes, heat shocked at 42 ℃ for 30 seconds, restored on ice for 5 minutes, and added 2mL of SOC medium and incubated at 37 ℃ for 1 hour. Centrifuging to collect cells, discarding most of the supernatant, leaving about 100 μ L of supernatant, blowing, mixing, spreading on ampicillin positive LB agar culture plate, and culturing at 37 deg.C overnight.
(5) 6-8 positive clones were selected, cultured overnight at 37 ℃ in 2mL of LB medium positive for ampicillin, 1mL of the extracted plasmid was extracted, and the size of the fragment was identified by agarose gel electrophoresis after double digestion with Not I and Nde I enzymes. The plasmid with correct connection is the carrier containing the nucleic acid molecule for encoding the nano antibody, and the BL21(DE3) escherichia coli cell containing the carrier is the host cell.
(6) Selecting a host cell which is subjected to double enzyme digestion to identify the correctness, and culturing the host cell in 2mL of LB culture medium with ampicillin positive at 37 ℃ overnight. Diluting the bacterial liquid into LB culture medium with ampicillin positive according to the ratio of 1:100, culturing at 37 ℃ until OD value is about 0.6, adding IPTG to 1mmol/L, culturing at 37 ℃ for 3-4 hours
(7) The cells were collected by centrifugation, washed once with PBS, and collected by centrifugation. The mixed bacteria are blown and beaten by PBS10mL containing 1% Triton X-100, and the bacteria are ultrasonically crushed on ice for 5s at an interval of 5s and with the power of 20 percent for 10 minutes in total.
(8) Centrifuging the crushed bacteria solution at 10000g and 4 ℃ for 20 minutes, taking supernatant, and using HisPurTMAnd purifying the recombinant nano antibody with the His tag protein by using a Ni-NTASuperflow Agarose nickel ion affinity chromatography column. And dialyzing the eluted nano antibody by PBS, determining the protein concentration, and storing at-20 ℃ for later use.
(9) SDS-PAG gel electrophoresis identifies the purified antibody, and the result is shown in figure 1, and the purity of the nano antibody is higher.
Example 2 preparation of immunoaffinity Material
(1) Preparing 2mg of purified anti-human serum albumin nano antibody into 1mg/mL by using a crosslinking buffer solution;
(2) soaking 0.3mg of cyanogen bromide activated agarose in 0.1M hydrochloric acid for 20 minutes to prepare cyanogen bromide activated agarose gel microspheres;
(3) washing 3 times of cyanogen bromide activated agarose gel microspheres by using a crosslinking buffer solution, mixing a nano antibody solution with the agarose gel microspheres, and oscillating and incubating for 2 hours at room temperature;
(4) alternately washing the cyanogen bromide activated sepharose microspheres for 3 times by using a cross-linking buffer solution and a low-pH acetic acid solution;
(5) the cyanogen bromide-activated Sepharose microspheres were resuspended in 1mL PBS and stored at 4 ℃ until use.
Example 3 measurement of adsorption Capacity of immunoaffinity Material
(1) Diluting 25 μ g human serum albumin into 50 μ LPBS buffer solution, and preparing 5 parts in total;
(2) mixing 12.5. mu.L, 25. mu.L, 37.5. mu.L, 50. mu.L and 62.5. mu.L of cyanogen bromide activated agarose gel microspheres with a human serum albumin diluent, and performing shaking incubation for 20 minutes at room temperature;
(3) centrifuging at room temperature for 2 min at 100g, and taking the supernatant;
(4) taking 4 μ L of supernatant and 1 μ L of agarose gel microspheres, running SDS-PAGE gel electrophoresis, the result is shown in figure 2, wherein the loading amount of the gel microspheres is 0.5uL, and the loading amount of the supernatant is 4 uL;
(5) the amount of adsorbed human serum albumin was calculated to be 2ug for 1 uL of cyanogen bromide activated Sepharose beads.
Example 4 removal of Albumin from serum/plasma with human serum Albumin removal kit
(1) Taking 5 mu L of human plasma, quickly centrifuging for 2 minutes at 10,000g, taking supernatant, and diluting to 100 mu L with PBS;
(2) mixing 10 mu L of cyanogen bromide activated agarose gel microspheres with plasma diluent, and oscillating and incubating for 20 minutes at room temperature;
(3) centrifuging at room temperature for 2 min at 100g, and taking the supernatant;
(4) the collected cyanogen bromide activated sepharose microspheres were washed 3 times with 100. mu.L PBS containing 0.1% Tween 20;
(5) the plasma albumin removal effect was identified by SDS-PAGE, and the result is shown in FIG. 3, in which the plasma albumin removal efficiency was 95% or more.
Sequence listing
<110> Shandong Minkang Biotech Co., Ltd
Hangzhou heavy chain Technology Co.,Ltd.
<120> human serum albumin removal kit
<160>2
<170>SIPOSequenceListing 1.0
<210>1
<211>137
<212>PRT
<213> Nanobody amino acid sequence (Natural antibody)
<400>1
Glu Asn Leu Tyr Phe Gln Gly Ala Gln Val Gln Leu Val Glu Ser Gly
1 5 10 15
Gly Gly Leu Val Gln Ala Gly Asp Ser Leu Arg Leu Ser Cys Ala Ala
20 25 30
Ser Gly Arg Thr Phe Glu Thr His Ala Met Gly Trp Phe Arg Gln Ala
35 40 45
Pro Gly Lys Glu Arg Glu Phe Val Ala Thr Ile Thr Pro Ser Gly Arg
50 55 60
Ser Thr Ser Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ser
65 70 75 80
Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro
85 90 95
Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Phe Ala Tyr Gly Val Gly Leu
100 105 110
Tyr Lys Leu Ala Arg Gln Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val
115 120 125
Thr Val Ser Glu Pro Lys Thr Pro Lys
130 135
<210>2
<211>411
<212>DNA
<213> Nanobody nucleotide sequence (Natural antibody)
<400>2
gagaacctgt attttcaggg tgcacaggtt cagctggttg aaagcggtgg tggtctggtt 60
caggcaggcg atagcctgcg tctgagctgt gcagcaagcg gtcgtacctt tgaaacccat 120
gcaatgggct ggtttcgtca ggcaccgggt aaagaacgtg aatttgttgc aaccattaca 180
ccgagcggtc gcagcaccag ctatggtgat agcgttaaag gtcgttttac cattagcagc 240
gataatgcca aaaataccgt ttacctgcag atgaatagcc tgaaaccgga tgataccgca 300
atctattatt gtgcatttgc ctatggtgtg ggcctgtata aactggcacg tcagtatgat 360
tattggggtc agggcaccca ggttaccgtt agcgaaccga aaacaccgaa a 411
Claims (3)
1. A kit for removing human serum albumin is composed of an immunoaffinity material, a buffer solution, a washing solution, a disposable consumable and a human serum albumin standard substance, and is characterized in that the immunoaffinity material comprises an anti-human serum albumin nano antibody and a carrier matrix, the carrier matrix is a porous material, the buffer solution is a phosphate buffer solution, the washing solution is a phosphate buffer solution containing 0.1% Tween 20, the disposable consumable is a low-adsorption 1.5mL centrifuge tube, the amino acid sequence of the anti-human serum albumin nano antibody is shown as SEQ ID No.1, the nucleotide sequence of an encoding nucleic acid molecule is shown as SEQ ID No.2, the pH value of the phosphate buffer solution is 7.4, calcium ions and magnesium ions are not contained, the pH value of the washing solution is 7.4, the calcium ions and the magnesium ions are not contained, the human serum albumin standard substance is human serum albumin with the concentration of 100mg/mL, dissolved in phosphate buffer at pH 7.4.
2. The human serum albumin removal kit according to claim 1, wherein the porous material is selected from one of agarose gel microspheres, fiber microspheres, magnetic beads, silica gel microspheres, activated carbon, and resin microspheres.
3. A human serum albumin removal kit according to claim 1, wherein the immunoaffinity material is capable of specifically adsorbing human serum albumin.
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| WO2007112940A2 (en) * | 2006-03-31 | 2007-10-11 | Ablynx N.V. | Albumin-derived amino acid sequence, use thereof for increasing the half-life of therapeutic proteins and of other therapeutic compounds and entities, and constructs comprising the same |
| CN103254309A (en) * | 2005-05-18 | 2013-08-21 | 埃博灵克斯股份有限公司 | Improved nanobodies TM against tumor necrosis factor-alpha |
| CN103619878A (en) * | 2011-06-23 | 2014-03-05 | 埃博灵克斯股份有限公司 | Serum albumin binding proteins |
| CN105498704A (en) * | 2015-12-02 | 2016-04-20 | 南昌大学 | Nano antibody based immunoaffinity material for specific adsorption of aflatoxin |
| CN105524170A (en) * | 2016-02-03 | 2016-04-27 | 南昌大学 | Nanobody capable of specifically binding with PSMA (prostate specific membrane antigen) |
| CN108473564A (en) * | 2015-11-18 | 2018-08-31 | 埃博灵克斯股份有限公司 | Improved seralbumin bonding agent |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20110262348A1 (en) * | 2010-03-29 | 2011-10-27 | Vrije Universiteit Brussel | Selective targeting of intratumoral cells |
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| WO2007112940A2 (en) * | 2006-03-31 | 2007-10-11 | Ablynx N.V. | Albumin-derived amino acid sequence, use thereof for increasing the half-life of therapeutic proteins and of other therapeutic compounds and entities, and constructs comprising the same |
| CN103619878A (en) * | 2011-06-23 | 2014-03-05 | 埃博灵克斯股份有限公司 | Serum albumin binding proteins |
| CN108473564A (en) * | 2015-11-18 | 2018-08-31 | 埃博灵克斯股份有限公司 | Improved seralbumin bonding agent |
| CN105498704A (en) * | 2015-12-02 | 2016-04-20 | 南昌大学 | Nano antibody based immunoaffinity material for specific adsorption of aflatoxin |
| CN105524170A (en) * | 2016-02-03 | 2016-04-27 | 南昌大学 | Nanobody capable of specifically binding with PSMA (prostate specific membrane antigen) |
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