WO2015090168A1 - 生物载体及其在检测中的应用 - Google Patents

生物载体及其在检测中的应用 Download PDF

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WO2015090168A1
WO2015090168A1 PCT/CN2014/093838 CN2014093838W WO2015090168A1 WO 2015090168 A1 WO2015090168 A1 WO 2015090168A1 CN 2014093838 W CN2014093838 W CN 2014093838W WO 2015090168 A1 WO2015090168 A1 WO 2015090168A1
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antibody
detection
antigen
immunoturbidimetric
biological
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PCT/CN2014/093838
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French (fr)
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顾晋元
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顾晋元
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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
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin

Definitions

  • the invention relates to the application of a biological detection carrier, in particular to the application of a biological detection carrier obtained by using display technology in immunoturbidimetric detection.
  • the detection of antigen or antibody belongs to immunological detection technology.
  • the detection methods can be divided into: enzyme-linked immunoassay, isotope immunoassay, colloidal gold immunoassay, fluorescent immunoassay and chemiluminescence immunoassay, etc.
  • Different detection methods can be divided into: immunoenzyme labeling, immunological lateral chromatographic test paper and immunofiltration.
  • immunoassay technology has become a widely used and routinely used detection technology, in which enzyme-linked immunosorbent assay (ELISA) is currently used.
  • ELISA enzyme-linked immunosorbent assay
  • Chinese Patent Application No. 200910194749.3 discloses a method for semi-quantitative detection of rheumatoid factor, which is sensitized to the carrier latex particles by the physical adsorption principle of gamma globulin, and the serum to be tested with different dilutions is detected during the detection. Agglutination reaction with sensitized latex particles to achieve semi-quantitative detection of rheumatoid factor.
  • Chinese invention patent application 201210545717.5 discloses a human cystatin C chemiluminescence quantitative detection method, which combines magnetic particle separation technology and enzymatic chemiluminescence technology, adopts double antibody sandwich one-step reaction principle, and can be used for serum, plasma and urine. Determination of the content of human cystatin C in the liquid sample.
  • a magnetic separation reagent is prepared by covalently attaching a FITC antibody to the surface of a carboxyl group-containing magnetic particle.
  • an antigen reacts with an antibody in a special dilution system and the ratio is appropriate (generally specifying an excess of antibody)
  • the soluble immune complex formed is under the action of a coagulant (eg, polyethylene glycol) in the dilution system.
  • a coagulant eg, polyethylene glycol
  • the composite particles formed in the precipitation reaction have special optical properties and can be detected by instruments, which improves the detection speed, sensitivity and ease of operation.
  • This method of dynamic determination of antigen and antibody binding is an immunoturbidimetric detection technique (eg, CN1556408A), which is further divided into: immunoturbidimetry, immunoturbidimetry, and immunoturbidimetry.
  • Particle-enhanced turbidimetric immunoassay is also a relatively stable and accurate method for detecting humoral protein homogenization in recent years.
  • Chinese Patent Application No. 200510096787.7 discloses an aminated polymer microsphere for quantitative detection of immunoturbidimetry.
  • the seed particles made of polystyrene are chemically reacted with the aminated monomer to bind the surface of the seed particles to the amino group, and crosslink with the protein under the action of the crosslinking agent (glutaraldehyde) to form a stable crosslink. product.
  • the crosslinking agent glutaraldehyde
  • Chinese invention patent application 200810084390.X discloses a latex-enhanced immunoturbidimetric kit for detecting cystatin C in serum or plasma, using latex-coated anti-cystatin C antibody, latex through immune response
  • the layers are aggregated to form an insoluble immune particle complex, and the particle size of the latex changes to cause a significant change in turbidity, and the change is detected by light transmission intensity or light scattering intensity.
  • Chinese invention patent ZL201010140329.X discloses a kit for detecting fibrin (original) degradation product (FDP) content by latex immunoturbidimetry, acidified anti-human FDP antibody and polyvinyl benzyl chloride latex The anti-human FDP antibody latex reagent formed by chemical cross-linking of the particles and blocking.
  • Chinese invention patent application 201210200339.7 discloses a competition method latex particle enhanced immunoturbidimetric BNP detection kit and a preparation method thereof, and the FGRKMDR-X amino acid sequence is coated on the latex particles, thereby solving the difficulty of detecting BNP by the sandwich method. High and long detection time and other issues.
  • Another object of the present invention is to provide a biological carrier for use as a carrier particle for an immunoturbidimetric detection product.
  • It is still another object of the present invention to provide an immunoturbidimetric detection composition comprising a biological carrier having a polymer on its surface.
  • a further object of the present invention is to provide an immunoturbidimetric detection method for adding a detection reagent containing a biological carrier to a sample to be tested to form a complex (eg, an immune complex formed by an antigen and an antibody), and can be performed by Quantitative or qualitative detection by turbidimetry.
  • a polymer is a compound in which a large number of atoms or atomic groups are mainly covalently bonded, and in particular, a polypeptide, a protein, a nucleic acid, an antibody or a Nanobody in which an amino acid or a ribonucleoside is used as a monomer.
  • the amino acid or ribonucleoside can be obtained directly from nature or indirectly by hydrolysis, enzymatic hydrolysis or chemical means, and its configuration is not intended to limit the invention.
  • these polymers should also include other necessary molecular modifications or linkages, such as, but not limited to, glycosylation, disulfide bonds, and sulfur modifications, as well as non-covalent bonds such as: But not limited to hydrogen bonding and hydrophobic interactions.
  • polymers are to be understood as molecules which are endogenous or exogenous, ie molecules which are obtained as a whole from the replication, transcription or expression of the biological information of the biological vector, such as nucleic acids, polypeptides, proteins and antibodies and A fragment or the like may also be a result of replication, transcription or expression of genetic information that is not entirely derived from a biological vector, a so-called exogenous property.
  • exogenous polymers can be obtained, expressed or expressed on the surface of biological carriers in vitro or in vivo by biological or chemical methods, such as: isolation, chemical synthesis of polypeptides, PCR amplification of nucleic acids, phage display, bacterial display and fungi. Show and so on.
  • an antigen is a substance that causes a specific immune response in the body. After the antigen enters the body, it can stimulate the body to produce antibodies and cause cellular immunity.
  • an antigen refers to a substance that binds to an antibody. The reactivity of an antigen depends on the antigenic determinant, also known as the epitope. An antigen molecule can carry different determinants.
  • epitopes can be spliced by genetic engineering or chemical ligation.
  • the antigens referred to in the present invention also include, but are not limited to, the commonly considered haptens (i.e., only reactive, non-immunogenic, hapten).
  • haptens i.e., only reactive, non-immunogenic, hapten.
  • molecules that are neither reactive nor immunogenic eg, heavy metals
  • an antibody is an immunoglobulin (Ig) that specifically binds to an antigen.
  • Ig is divided into five categories, namely IgG, IgA, IgM, IgD and IgE.
  • the Ig associated with immunoassays is primarily IgG and IgM.
  • the antibody further includes a molecule having an action of recognizing an antigen, such as, but not limited to, an antibody Fab fragment derived from Ig, a single-chain antibody, a single-domain antibody, and a Nanobody, and the like.
  • a specific embodiment of the Nanobody of the present invention is a single domain antibody consisting of a heavy chain variable region, which is obtained by cloning a variable region of a heavy chain antibody (eg, derived from a camel) Sex antigen-binding fragments, also known as single-domain antibodies.
  • the antibody fragment thereof can be obtained by means of polypeptide synthesis, or can be obtained by expressing the cloned sequence by a biological vector.
  • polypeptides, proteins, antibodies, antibody Fab fragments, single-chain antibodies, single-domain antibodies or Nanobodies can be carried out by isolation from natural organisms and chemical synthesis of polypeptides (Eur. J. Immunol. 1994, 24, 3188-3193; J. Org. Chem. 1972, 37, 3404-3409; peptide synthesis [P], Beijing: Science Press, 1985), prokaryotic microorganisms (eg E. coli) genetically engineered bacteria after expression, purification, eukaryotic microorganisms (eg: Genetically engineered bacteria such as S.
  • cerevisiae, Pichia pastoris, and Kluyveromyces lactis are purified or cultured from animal cells (eg, Chinese hamster CHO, hamster BHK, murine myeloma cell mouse fibroblasts, monkey CV1 cells, and human lymphocytes). Cells, etc. are expressed and purified.
  • animal cells eg, Chinese hamster CHO, hamster BHK, murine myeloma cell mouse fibroblasts, monkey CV1 cells, and human lymphocytes. Cells, etc. are expressed and purified.
  • nucleic acids can be obtained by in vitro amplification techniques, which have been described in the book "Molecular Cloning Handbook” and are also operational skills that a skilled artisan must possess and master.
  • Biological vectors such as, but not limited to, bacteriophage, viruses, bacteria, fungi, and cells, etc., which usually include Transfer material and coating.
  • a coating is understood to be a generic term for a genetic material that can be encapsulated therein and that carries a polymer expressed by the genetic material or a class of substances that bind the polymer to its surface.
  • Suitable coatings such as, but not limited to, lipid membranes, liposomes, cell membranes, cell walls, organelle membranes, viral envelopes, and phage coats.
  • exogenous molecules are introduced into a biological vector by a biological expression system and displayed on the surface of the biological carrier.
  • a biological expression system is understood to be a class of substances capable of carrying genetic information, such as: phage display-related plasmids (eg, including but not limited to pCantab, pHen, and pComb, etc.), viral display-related plasmids (eg, including but not limited to pBACsurf- 1, pLenti6, pAAv and pLV.Des2d.P, etc.), bacteria display related plasmids (such as: including but not limited to pMB172, pINA1317, pETAg43, pTNIM and pBLIM, etc.) and fungal display related plasmids (eg: including but not limited to pCTcon2 pYD1 and pKFS, etc.).
  • phage display-related plasmids eg, including but not limited to pCantab, pHen, and
  • the present invention also includes the use of physical or chemical methods to label exogenous molecules to the surface of a biological carrier by adsorption, hydrophobic interaction or coupling.
  • the biological carrier provided by the present invention further comprises a polymer, that is, an aggregate formed by a plurality of biological carriers gathered together in an automatic manner (e.g., physical adsorption).
  • a polymer that is, an aggregate formed by a plurality of biological carriers gathered together in an automatic manner (e.g., physical adsorption).
  • an automatic manner e.g., physical adsorption
  • the biocarriers provided by the present invention further comprise a linker, i.e., one or several biological carriers are linked together by human manipulation using biological or chemical ligation techniques (BioConjugate Chemistry).
  • a linker i.e., one or several biological carriers are linked together by human manipulation using biological or chemical ligation techniques (BioConjugate Chemistry).
  • BioConjugate Chemistry biological or chemical ligation techniques
  • the present invention provides an immunoturbidimetric detection composition
  • an immunoturbidimetric detection composition comprising a biological carrier having a surface having a polymer, the other components being substantially the same as those disclosed.
  • the immunoturbidimetric detection composition is used for quantitative detection of a target molecule contained in a sample to be tested.
  • the invention provides an immunoturbidimetric detection method, which is characterized in that a biological carrier is added as a detection reagent to a sample to be tested to form an immune complex, or supplemented by a sensitizer, a surfactant and a buffer, and the like. Quantitative and qualitative detection of samples to be tested by turbidimetry.
  • sample to be tested or “sample” referred to in the present invention is derived from blood, secretions, tissue fluid, in vitro culture fluid or tissue of a subject or a patient.
  • patient refers to humans, wild animals and livestock (Livestock).
  • Wild animals are animals that have not been artificially domesticated in their natural state.
  • Livestock are animals that are artificially raised to provide a source of food, such as monkeys, dogs, rats, hamsters, pigs, rabbits, cows, buffalo, bulls, sheep, goats, geese, and chickens.
  • the "patient” given the diagnosis prefers mammals, especially humans.
  • the invention provides a biological carrier which is a phage having an antibody on its outer shell.
  • the invention provides a biological carrier which is a phage having an antigen on its outer shell.
  • Another biological carrier provided by the present invention is a bacterium having an antibody or antigen on the cell wall.
  • Another biological carrier provided by the present invention is a fungus having an antibody or antigen on its cell membrane or cell wall.
  • the present invention is directed to different kinds of specific molecules (antigens or antibodies), and may be added by adding a currently used immunoturbidimetric-related sensitizer (eg, PEG), a surfactant (such as, but not limited to, a nonionic surfactant).
  • a currently used immunoturbidimetric-related sensitizer eg, PEG
  • a surfactant such as, but not limited to, a nonionic surfactant.
  • buffers eg phosphate buffer, Tris buffer, imidazole buffer, triethanolamine ⁇ hydrochloric acid buffer, MES buffer, Bis-Tris
  • Buffer eg phosphate buffer, Tris buffer, imidazole buffer, triethanolamine ⁇ hydrochloric acid buffer, MES buffer, Bis-Tris Buffer, ADA buffer, PIPES buffer, Bis-Tris-propane buffer, ACES buffer, MOPS buffer, BES buffer, TES buffer, HEPES buffer, HEPPS buffer, Tricine buffer, Bicine buffer Adjust the detection time, detection range or detection sensitivity with TAPS buffer, etc. or other reagents.
  • the test results may vary slightly depending on the instrument (eg, cuvettes with different light paths or path lengths) and the detection environment (eg temperature and pH).
  • the present invention alters the manner in which antibodies are currently obtained using hybridomas by displaying specific antigens or antibodies on a biological vector, which not only simplifies the process of preparation, but also the antibodies exhibited by a single biological vector (eg, phage) obtained. Or the antigen is monoclonal.
  • the biological carrier of the present invention is suitable for immunoturbidimetric detection of a sample applied in the visible light range, which not only greatly speeds up the detection speed (compared with ELISA), but also exhibits a good linear relationship over a wide concentration range. It is conducive to improving the accuracy of trace substance detection.
  • the biological carrier of the invention has the advantages of convenient preparation, strong stability, high specificity and easy preparation on a large scale.
  • Figure 1 is a graph showing the relationship between time and concentration of the biological carrier for detecting IgY of the present invention
  • Figure 2 is a graph showing the relationship between time and concentration of the biological carrier of the present invention for detecting unrelated protein BSA;
  • 3 is a result of immunoturbidimetric detection of a biological carrier of the present invention at a wavelength of 320 nm;
  • Figure 5 is a result of detecting the antibody in serum of the biological carrier of the present invention at a concentration of 3.525 ⁇ 10 5 ;
  • Figure 6 is a graph showing the results of detecting the antibody in serum of the biological carrier of the present invention at a concentration of 3.525 ⁇ 10 7 ;
  • Figure 7 is a graph showing changes in turbidity obtained by using a 5% PEG 4000 adjuvant in combination with a biological carrier of the present invention.
  • Figure 8 is a graph showing changes in turbidity obtained by using a biological carrier of the present invention in combination with PEG auxiliary agents having different molecular weights;
  • Figure 9 is a graph showing changes in turbidity obtained by using a biocarrier in combination with different concentrations of PEG6000 adjuvant
  • Figure 10 is a graph showing changes in turbidity obtained by chemical methods of the biocarrier of the present invention using different concentrations of PEG 6000 adjuvant;
  • Figure 11 is a graph showing changes in turbidity obtained by detecting a gradient antigen by the biological carrier of the present invention obtained by a chemical method.
  • the present invention relates to molecular biology experiments, and if not specifically noted, reference may be made to the book "Molecular Cloning” (J. Sambrook, EF Frici, T. Maniartis, Science Press, 1994). . This book and its subsequent published editions are the most commonly used instructive reference books by those skilled in the art in conducting experimental operations related to molecular biology. In addition, according to different experimental purposes, those skilled in the art can carry out corresponding experiments or commissioned specialized companies under the guidance of the operation manual attached to various commercial kits (Kit), such as: gene sequencing, plasmid sequencing and determination. Molecular weight, etc.
  • the alpaca nanobody primers were designed, and the alpaca nanobody gene was cloned by nested PCR to construct phagemids. After infecting the bacteria, the packaging was assisted by the helper phage. As a phage displaying Nanobody, a Nanobody library with a titer of 1011 was successfully obtained.
  • the main contents are as follows:
  • the cDNA reverse-transcribed from the mRNA of alpaca lymphocytes as a template was used as a template for the first round of PCR, and a total of 750 ng of PCR product (obtained with primer 1 as an amplification primer) was obtained.
  • a total of 750 ng of PCR product obtained with primer 1 as an amplification primer
  • a total of 1.4 ⁇ g of PCR product obtained with primer 2 as an amplification primer
  • was obtained Using the second round of PCR product as a template for the third round of PCR a total of 40 ⁇ g of PCR product (obtained with primer 3 as an amplification primer) was obtained.
  • the phage nanoparticle library was constructed by ligating the phagemid pCANTAB5E (the displayed protein was fused with the P3 protein) and the PCR product after ligation.
  • 3BSA blocked the potential protein binding site, and 4 ml of 5% BSA was placed in an immunotube, shaken slowly for 2 hours at room temperature, washed 3 times with PBS, and rinsed clean.
  • phage Nanobody reservoir solution 41 ml of phage Nanobody reservoir solution, a total of 10 14 phage were added to the immunotube, and the mixture was shaken slowly for 30 minutes at room temperature, then allowed to stand at room temperature for 90 minutes, and the immunotube was washed 10 times with PBST, and the immunotube was washed 10 times with PBS and dried.
  • the phage in the supernatant of 7PEG4000 precipitation medium was centrifuged at 14,000 rpm, and 2 ml of 1 ⁇ TE was used to dissolve the phage, and the titer was about 10 11 .
  • Anti-phage antibody (rabbit source), dilution 1:5000, 100 ⁇ l/well, 37 ° C, 1 hour.
  • Nanoparticles capable of specifically recognizing chicken antibody protein IgY were gradually enriched as one round of screening was performed.
  • Antigen antibody preparation :
  • Antigen Take a certain amount of antigen and dilute it to a certain concentration with 1 ⁇ PBS, then dilute it to the desired concentration (starting at 10 ng/ml, 3 times doubling);
  • Antibody diluted to the required concentration (usually 10 9 / ⁇ l - 10 10 / ⁇ l) with 1 ⁇ PBS;
  • Antigen-antibody mixing Prepare the antigen and antibody manually and mix thoroughly, set at 37 ° C, and test every hour;
  • the experimental procedure was the same as in Example 3, adding a group of unrelated protein bovine serum albumin BSA as a control, and found that the turbidity of the unrelated protein did not change significantly with time (see Fig. 2). It can be seen that the biological carrier provided by the invention has high detection specificity and is not interfered by other substances.
  • the biocarrier of the invention can be adapted for detection in the visible range.
  • Chicken bursal disease virus VP2 protein was used to immunize 2 rabbits, while rabbit No. 1 was injected with complete adjuvant, and rabbit No. 2 was not injected, once a week, three times. At the fourth week, a small amount of blood was taken and antibodies were detected. Titer, the results are shown in Table 2.
  • Blood was drawn from the heart, serum was prepared, sodium azide was added, and the refrigerator was stored at 4 ° C until use.
  • the chicken bursal virus protein VP2 gene fragment is ligated to the phagemid vector pCANTAB5E-8 (carrying the phage g8 gene); the ligation product is transformed into the TG1 chemical transformation competent bacteria;
  • the overnight bacteria were transferred to a sterile 50 ml centrifuge tube, centrifuged at 4800 rpm for 20 minutes at 4 ° C, and the supernatant was transferred to a new tube.
  • 2.5 M NaCl solution containing 40% PEG 4000 was added in a volume ratio of 1/5, strongly After shaking, ice bath for 30 minutes. After centrifugation at 4,800 rpm for 20 minutes at 4 ° C, the supernatant was discarded, the pellet was resuspended in 1 ml of sterile PBS buffer, and then centrifuged at 12,000 rpm for 5 minutes at room temperature. The supernatant was taken to obtain a fragment of the VP2 protein of chicken bursal virus. Phage.
  • Antigen antibody preparation :
  • Antigen two concentrations of phage display antigen were used as detection, and the phage particle concentration was 3.525 ⁇ 10 5 / ⁇ l and 3.525 ⁇ 10 7 / ⁇ l;
  • Antibody diluted rabbit antiserum 1 with PBS, dilution factors of 10, 100, 1,000, 10,000, 100,000 and 1,000,000;
  • Antigen-antibody mixing Prepare the antigen and antibody manually and mix thoroughly, set at 37 ° C, and test every hour;
  • Reading the microplate reader select the plate holes on the microplate, and then add the samples to be detected separately, and read at different wavelengths of 340 nm;
  • Antigen antibody preparation :
  • the phage display antigen is used as a detection antigen, and the phage particle concentration is 3.525 ⁇ 10 9 / ⁇ l;
  • Antibody 1 diluted rabbit antiserum 1 ⁇ PBS, the dilution factor was 10 times; 2 with the unrelated protein BSA as a control group, to observe the specificity of detection and whether 5% PEG has an effect on specific detection;
  • Antigen-antibody mixing Prepare the antigen and antibody manually and mix thoroughly, set or not add 5% PEG4000 experimental group, set at 37 °C, and detect as follows: 0.5h, 1h, 1.5h, 2h, 3h, 4h, 5h and 6h;
  • Example 10 The effect of contrast turbidity formation on PEG of different molecular weights was observed.
  • Antigen antibody preparation :
  • the phage display antigen is used as a detection antigen, and the phage particle concentration is 3.525 ⁇ 10 9 / ⁇ l;
  • Antibody diluted rabbit antiserum 1 with PBS, the dilution factor was 10 times;
  • Antigen-antibody mixing Prepare the antigen and antibody manually and mix thoroughly. Set PEG 4000, PEG6000, PEG10000 and PEG20000 with different molecular weights of PEG, set at 37 °C, and detect as follows: 5min, 10min, 15 min, 20 min, 25 min and 30 min;
  • Reading the microplate reader select the plate holes on the microplate, and then add the samples to be tested separately, and read at different wavelengths at 340 nm wavelength (see Figure 8);
  • Antigen antibody preparation :
  • the phage display antigen is used as a detection antigen, and the phage particle concentration is 3.525 ⁇ 10 9 / ⁇ l;
  • Antibody diluted rabbit antiserum 1 with PBS, the dilution factor was 10 times;
  • Antigen and antibody mixing Prepare the antigen and antibody manually and mix thoroughly, set PEG6000, set the concentration to 0% (PBS), 1%, 2%, 3%, 4%, 5% and 6% PEG6000, respectively. Set at 37 ° C, as follows: 0min, 2min, 4min, 6min, 8min, 10min, 15min and 30min;
  • Reading the microplate reader select the plate holes on the microplate, and then add the samples to be tested, and read at different wavelengths at 340 nm wavelength (see Figure 9);
  • the rabbit polyclonal antiserum prepared in Example 6 was precipitated by saturated ammonium sulfate method, dialyzed against PBS, centrifuged at 2000 rpm, centrifuged for 10 minutes, and precipitated, and the supernatant was taken to prepare the prepared polyclonal antibody;
  • the prepared rabbit polyclonal antibody modified M13 phage was collected and used.
  • Antigen antibody preparation :
  • Antigen Take the chicken bursal virus protein VP2 protein as a detection antigen at a concentration of 1500 ng/ml;
  • Antibody taking rabbit polyclonal anti-chemically modified phage particles as an antibody at a concentration of 1 ⁇ 10 8 / ⁇ l;
  • Antigen and antibody mixing Prepare the antigen and antibody manually and mix thoroughly, set PEG6000, set the concentration to 0% (PBS), 1%, 2%, 3%, 4%, 5% and 6% PEG6000, respectively. Set at 37 ° C, as follows: 4 min, 6 min, 8 min, 10 min, 15 min, 30 min and 1 h;
  • Antigen antibody preparation :
  • Antigen Take the bursal virus protein VP2 protein as a detection antigen with a concentration gradient of 111 ng/ml, 333 ng/ml, 1000 ng/ml, 3000 ng/ml and 9000 ng/ml;
  • the antibody is obtained by using rabbit chemically modified phage particles as an antibody at a concentration of 1 ⁇ 10 9 / ⁇ l;
  • Antigen-antibody mixing Prepare the antigen and antibody manually and mix thoroughly, set 3% PEG6000, set at 37 ° C, and test results after 8 minutes;
  • sequence, primers and method steps involved in the next experiment can be referred to CN103509115A.
  • Primers eg, primer design software DNAman
  • Primers were designed based on the sequence of Cystatin C Nanobody 3-2 and 7-4, and Cystatin C Nanobody 3-2, 7-4 genes were cloned and overlapped.
  • the complete genes of the Cystatin C Nanobody 3-2 and 7-4 fusion proteins were obtained, and the designed primers were added with Nhe I and BamHI at both ends of the Cystatin C Nanobody 3-2, 7-4 fusion protein gene. Enzyme cleavage point;
  • the Cystatin C Nanobody 3-2, 7-4 fusion protein gene was ligated to the phage T vector, and the TA-Costatin C Nanobody 3-2, 7-4 fusion protein gene plasmid was obtained and sequenced. After sequencing, the plasmid pCTcon2 (from Addgene) and TA-Costatin C Nanobody 3-2, 7-4 fusion protein gene were digested with Nhe I and BamHI, and separated by gel electrophoresis. Then, the recovered product is ligated with T4 DNA ligase, transformed into E. coli DH5 ⁇ , single plaque is picked, verified by PCR, and sequenced, and the correct strain is stored;
  • the strain DH5 ⁇ containing the recombinant plasmid was amplified, and the recombinant plasmid pCTcon2-cystatin C nanobody 3, 2, 7-4 fusion protein gene was extracted using a plasmid extraction kit (provided by Bioengineering (Shanghai) Co., Ltd.), and concentrated. Plasmid spare;
  • Yeast EBY100 glycerol bacteria (yeast EBY100 from NTCC National Type Culture Collection) was resuscitated by fresh yeast leaching powdered sputum glucose medium (YPD) plate, and then cultured overnight with 5 ml. After about 14 hours, the bacteria were taken and pressed. After diluting 1:100, 70 ml of the bacterial solution was inoculated into YPD to expand the culture, and after 14 hours, the OD 600 value was measured to be about 1.3;
  • Positive clones were selected from MD plates and downstream primers for the Cystatin C Nanobody 3-2, 7-4 fusion protein gene (with "Genes for obtaining Cystatin C Nanobody 3-2, 7-4 fusion protein")
  • the primers used in the procedure are the same) and the upstream primer of pCTcon2 (with "Get Cystatin C Nanobody 3-2, 7-4
  • the gene for the fusion protein is the same as the primer used in the step.
  • the primer is assembled, and the clone is verified by PCR.
  • the clone with the correct result is cultured in MD liquid medium and stored in glycerin.
  • Negative control wild type EBY100
  • positive control yeast with empty pCTcon2 plasmid
  • experimental group yeast transformed with recombinant plasmid
  • the wild type EBY100 was cultured in YPD medium, and the experimental group and the positive control were cultured in SD-CAA medium (Synthetic dextrose medium with casamino acids, containing glucose) at 30 ° C, and cultured until the OD 600 value was between 2 and 5, 4,000 g. Centrifuge, adjust the OD 600 value to 0.5 to 1 with SC-CAA medium (containing galactose), incubate at 30 ° C for 12 hours, 24 hours and 36 hours, take 2OD of yeast per time period, store at 4 ° C ;
  • SD-CAA medium Synthetic dextrose medium with casamino acids, containing glucose
  • yeast cell ELISA Recombinant yeast and wild-type yeast in different time periods were used for yeast cell ELISA.
  • the primary antibody was anti-c-myc monoclonal antibody
  • the secondary antibody was HRP-labeled goat anti-mouse polyclonal antibody.
  • the cystatin C nanoparticle was verified and obtained.
  • the antibody 3-2, 7-4 fusion protein successfully displayed the sample on yeast.
  • Antigen antibody preparation :
  • Antigen 2 ⁇ g/ml of Cystatin C protein, diluted with Tris-HCl buffer at a ratio of 1:1, 1:2, 1:4, 1:8 and 1:16;
  • Antibody Dilute the No. 2 yeast display clone with Tris-HCl buffer at a concentration of 1 ⁇ 10 5 /ml and 1 ⁇ 0 6 /ml;
  • Antigen and antibody mixing the prepared antigen and antibody are thoroughly mixed, set at room temperature, and tested according to changes in turbidity;

Abstract

一种生物载体及其组合物、在免疫比浊检测技术中的应用。生物载体的表面具有识别并与目标分子结合作用的聚合物。该生物载体制取方便、稳定性强、特异性高，可以大量扩增，降低生产成本；载体表面的抗原或抗体使定量检测更准确。

Description

生物载体及其在检测中的应用 技术领域
本发明涉及一种生物检测载体的应用，尤其涉及一种利用展示技术获得的生物检测载体在免疫比浊检测中的应用。
背景技术
为了辅助临床医生对某类疾病的判断，实现早发现早治疗，对症下药的防治目的，首先需要对生物体各项生理指标进行检测，以作直观的了解。常用的检测方法如：生化检测、微生物检测和特异性抗体检测等，也因此产生了众多针对各种病症的检测产品(也称：试剂盒)。
抗原或抗体的检测属于免疫学检测技术，根据检测标记物的不同，可以将检测方法分为：酶联免疫检测、同位素免疫检测、胶体金免疫检测、荧光免疫检测和化学发光免疫检测等，根据检测方式不同，又可以分为：免疫酶标板、免疫侧向层析试纸和免疫渗滤等方法。除沉淀反应、凝集试验和补体结合试验等可用于特异性抗体检测以外，免疫测定技术已成为一中广泛并常规应用的检测技术，其中酶联免疫吸附(Enzyme-linked immunosorbent assay，ELISA)是目前应用最广泛的检测技术之一。
对于检测多肽或蛋白类分子，限于酶联免疫吸附技术定量准确性差、操作时间长、自动化程度低和干扰大等缺陷，其一般只能用于定性检测。为了实现目标蛋白的定量测定，中国发明专利申请200910194749.3公开一种半定量检测类风湿因子方法，以丙种球蛋白通过物理吸附原理致敏在载体胶乳颗粒上，检测时用不同稀释度的待测血清与致敏胶乳颗粒起凝集反应，实现对类风湿因子的半定量检测。中国发明专利申请201210545717.5公开了一种人胱抑素C化学发光定量检测方法，将磁微粒分离技术和酶促化学发光技术相结合，采用双抗体夹心一步反应法原理，可用于血清、血浆和尿液样本中人胱抑素C的含量测定。该技术中，将FITC抗体共价连接在含羧基的磁微粒的表面制得磁分离试剂。
研究发现，当抗原与抗体在特殊稀释系统中反应而且比例合适(一般规定抗体过量)时，形成的可溶性免疫复合物在稀释系统中的促凝剂(如：聚乙二醇)的作用下，自液相析出，形成微粒。在沉淀反应中形成的复合物微粒具有特殊的光学性质，可用仪器检测，提高了检测的速度、灵敏度和易操作性。这种以抗原和抗体结合动态测定的方法即为免疫比浊检测技术(如：CN1556408A)，又被分为：免疫透射比浊法、免疫散射比浊法和免疫胶乳比浊法。胶乳颗粒增强比浊法(particle-enhanced turbidimetric immunoassay，PETIA)也是近年来出现的一种较为稳定、准确的体液蛋白均相免疫比浊检测方法。
如：中国发明专利申请200510096787.7公开了一种用于免疫比浊定量检测的氨基化高分子微球。将聚苯乙烯制成的种子微粒与氨基化单体进行化学反应，使种子微粒表面接上氨基基团，在交联剂(戊二醛)的作用下与蛋白质交联而成稳定的交联产物。
另如：中国发明专利申请200810084390.X公开了一种检测血清或血浆中胱抑素C的胶乳增强型免疫比浊试剂盒，采用胶乳包被的抗胱抑素C的抗体，胶乳通过免疫反应一层一层聚集形成不溶性免疫颗粒复合物，胶乳的粒径发生改变引起浊度明显变化，用光透射强度或光散射强度检测该变化。
再如：中国发明专利ZL201010140329.X公开了一种采用胶乳免疫比浊法检测纤维蛋白(原)降解产物(FDP)含量的试剂盒，将酸化的抗人FDP抗体与聚乙烯基苄基氯胶乳颗粒发生化学交联并封闭后形成的抗人FDP抗体胶乳试剂。
又如：中国发明专利申请201210200339.7公开了一种竞争法胶乳颗粒增强免疫比浊BNP检测试剂盒及其制备方法，在胶乳颗粒包被有FGRKMDR-X氨基酸序列，解决了目前夹心法检测BNP难度较高和检测时间长等问题。
基于胶乳颗粒增强比浊法定量检测各种物质的类似技术还有很多，各种技术各有特点。虽有相关技术对胶乳进行了改进(如：CN101819208A)，但共同之处在于必须将具有识别作用的分子(如：蛋白、多肽或抗体)共价或非共价结合于载体颗粒(如：胶乳或磁微粒)。虽然解决了定量检测的问题，简化了操作难度，缩短了检测时间和提高了准确性，但是同样增加了胶乳及其与特定分子结合的制备环节。由此，带来了胶乳检测试剂均一性和稳定性等新问题。
发明内容
本发明的一个目的在于提供一种生物载体，具有在其表面展示的聚合物，该聚合物具有识别并与目标分子结合的作用，适用于免疫比浊检测技术。
本发明的另一个目的在于提供一种生物载体，作为载体颗粒应用于免疫比浊检测产品。
本发明的又一个目的在于提供一种免疫比浊检测组合物，含有一种表面具有聚合物的生物载体。
本发明的再一个目的在于提供一种免疫比浊检测方法，向待测样品中加入含有生物载体的检测试剂，以形成复合物(如：抗原和抗体形成的免疫复合物)，并可通过进行比浊法定量或定性检测。
聚合物是一种由众多原子或原子团主要以共价键结合而成的化合物，尤其是以氨基酸或核糖核苷为单体结合而成的多肽、蛋白质、核酸、抗体或纳米抗体。氨基酸或核糖核苷可直接来自于自然界或通过水解、酶解或化学方式间接获得，其构型不得限定本发明。本领域技术人员所能理解的，这些聚合物还应当包括其它必要的分子修饰或成键，如：但不仅限于糖基化、二硫键和硫修饰等，以及以非共价键，如：但不仅限于氢键和疏水相互作用。
这些聚合物应理解为内源性或外源性的分子，即其作为一个整体来自于生物载体的遗传信息所复制、转录或表达而获得的分子，如：核酸、多肽、蛋白质和抗体及其片段等，也可以是并非完全来自于生物载体的遗传信息所复制、转录或表达的结果，即所谓的具有外源性的属性。这些外源性的聚合物可以通过生物或化学的方法在体外或体内获得、表达或于生物载体表面展示，如：分离、多肽的化学合成、核酸的PCR扩增、噬菌体展示、细菌展示和真菌展示等。在这些聚合物中，优先选择的是含有抗原、抗体及其Fab片段、单链抗体、单域抗体等分子。
本领域技术人员可以理解，抗原和抗体系免疫学上对两种分子的表述，本发明中应理解为是一对能够互相特异性结合的分子，具有识别性。
抗原是能在机体中引起特异性免疫应答的物质。抗原进入机体后，可刺激机体产生抗体和引起细胞免疫。在免疫测定中，抗原是指能与抗体结合的物质。抗原的反应性取决于抗原决定簇(antigenic determinant)，亦称表位(epitope)。一个抗原分子可带有不同的决定簇。此外，还可以通过基因工程或化学连接的方式对表位进行拼接。
本发明所称的抗原还包括但不限于通常认为的半抗原(即只有反应原性，不具免疫原性，hapten)。此外，那些既不具有反应原性，也不具免疫原性的分子(如：重金属)通过与其它分子连接后而形成的半抗原或形成完全抗原而能够被识别的分子也应属于此范畴。
抗体是能与抗原特异性结合的免疫球蛋白(immunoglobulin，Ig)。Ig分五类，即IgG、IgA、IgM、IgD和IgE。与免疫测定有关的Ig主要为IgG和IgM。本发明中，抗体还包括具有识别抗原作用的分子，如：但不限于由Ig衍生而来的抗体Fab段、单链抗体、单域抗体和纳米抗体等也应属于此范畴。
一种本发明纳米抗体的具体实施方式，是基于一个重链可变区组成的单域抗体，是通过克隆缺失轻链的重链抗体(如：来源于骆驼体内)的可变区得到的功能性抗原结合片段，亦称单域抗体。其抗体片段可通过多肽合成的方式获得，也可以通过生物载体表达所克隆的序列而得到。
多肽、蛋白质、抗体、抗体Fab段、单链抗体、单域抗体或纳米抗体的制备，可以通过从自然界生物体中分离、多肽化学合成(Eur.J.Immunol.1994,24,3188-3193；J.Org.Chem.1972,37,3404-3409；多肽合成[P]，北京：科学出版社，1985)、原核微生物(如：大肠杆菌)基因工程菌表达后纯化、真核微生物(如：啤酒酵母、毕赤酵母和乳酸克鲁韦酵母等)基因工程菌表达后纯化或由动物细胞(如：中国仓鼠CHO、仓鼠BHK、鼠骨髓瘤细胞小鼠成纤维细胞、猴CV1细胞和人淋巴细胞等)等进行表达并纯化。
核酸的制备可以通过体外扩增技术获得，这些技术已记载于《分子克隆手册》一书中，也是普通技术人员必须具备和熟练掌握的操作技能。
生物载体如：但不仅限于噬菌体、病毒、细菌、真菌和细胞等，其通常包括遗 传物质和包被。包被应当理解为能将遗传物质包覆于其内，并能在其表面承载遗传物质所表达的聚合物或能将聚合物结合于其表面的一类物质的统称。适宜的包被，如：但不仅限于脂膜、脂质体、细胞膜、细胞壁、细胞器膜、病毒外壳和噬菌体外壳等。
本发明中，通过生物表达系统而将外源性的分子引入生物载体中，并在生物载体表面得以展示。对于这样的展示，本领域技术人员仅需采用常见的展示技术就可实现。生物表达系统应当理解为是一类能携带遗传信息的物质，如：噬菌体展示相关质粒(如：包括但不限于pCantab、pHen和pComb等)、病毒展示相关质粒(如：包括但不限于pBACsurf-1、pLenti6、pAAv和pLV.Des2d.P等)、细菌展示相关质粒(如：包括但不限于pMB172、pINA1317、pETAg43、pTNIM和pBLIM等)和真菌展示相关质粒(如：包括但不限于pCTcon2、pYD1和pKFS等)。
本发明所称的展示还包括采用物理或化学方法将外源性的分子通过吸附、疏水相互作用或偶联等方式标记到生物载体表面。
本发明提供的生物载体还包括聚合体，即若干个生物载体以自动的方式(如：物理吸附)聚集在一起，而形成的集合体。在此情况下，应理解为一种生物自发的自然现象。
本发明提供的生物载体还包括连接体，即采用生物的或化学的连接技术(BioConjugate Chemistry)以人为操作方式实现将一种或若干种生物载体相互连接在一起。在此情况下，应理解为其有别于生物的自然现象，而是在人为干预的情况下实现。这些技术可以从现有已公开的专利/申请、期刊或论文中了解并实施，如：美国化学协会(ACS)提供的期刊BioConjugate Chemistry。
本发明的提供一种免疫比浊检测组合物，采用含有一种表面具有聚合物的生物载体，其它组份与已公开的技术大体相同。该免疫比浊检测组合物用于定量检测待测样品所含的目标分子。
本发明提供的一种免疫比浊检测方法，其特征在于将生物载体作为检测试剂加入待测样品中形成免疫复合物，或再辅以增敏剂、表面活性剂和缓冲液等手段，进而通过比浊法对待测样品的进行定量和定性的检测。
本发明所称的“待测样品”或“试样”源自于受试者或病患的血液、分泌物、组织液、体外培养液或组织等。
本发明所称的“病患”、“病人”和“受试者”指人、野生动物和家畜(Livestock)。野生动物为自然状态下未经人工驯化的动物。家畜是为了提供食物来源而人工饲养的动物，如：猴、狗、鼠、仓鼠、猪、兔、奶牛、水牛、公牛、绵羊、山羊、鹅和鸡等。给予诊断的“患者”优先选择哺乳动物，尤其是人。
本发明提供的一种生物载体，为噬菌体，其外壳上具有抗体。
本发明提供的一种生物载体，为噬菌体，其外壳上具有抗原。
本发明提供的另一种生物载体，为细菌，其上细胞壁具有抗体或抗原。
本发明提供的另一种生物载体，为真菌，其上细胞膜或细胞壁具有抗体或抗原。
本发明针对不同种类的特定分子(抗原或抗体)，可以通过添加目前通用的免疫比浊法相关的增敏剂(如：PEG)、表面活性剂(如：但不仅限于非离子性表面活性剂、阴离子性表面活性剂、阳离子性表面活性剂和两性表面活性剂)、缓冲液(如：磷酸缓冲液、Tris缓冲液、咪唑缓冲液、三乙醇胺·盐酸缓冲液、MES缓冲液、Bis-Tris缓冲液、ADA缓冲液、PIPES缓冲液、Bis-Tris-丙烷缓冲液、ACES缓冲液、MOPS缓冲液、BES缓冲液、TES缓冲液、HEPES缓冲液、HEPPS缓冲液、Tricine缓冲液、Bicine缓冲液和TAPS缓冲液等)或其他试剂对检测时间、检测范围或检测灵敏度进行调整。同时，检测结果也可能会因检测仪器(如：不同光径或光程的比色杯)、检测环境(如：温度和pH值等)不同而略有差异。
本发明技术方案实现的有益效果：
本发明通过在生物载体上展示特定抗原或抗体的方式，改变了目前使用杂交瘤获得抗体的方式，不仅简化了制取的过程，而且所获得的单个生物载体(如：噬菌体)所展示的抗体或抗原均为单克隆。
经验证，本发明生物载体适用于在可见光范围内应用于样品的免疫比浊检测，不仅大大加快检测速度(与ELISA相比)，其在较宽的浓度范围内均表现出良好的线性关系，有利于提高微量物质检测的准确性。
本发明生物载体制备方便，稳定性强、特异性高和易于大规模制取等特点。
附图说明
图1为本发明生物载体用于检测IgY的时间与浓度关系图；
图2为本发明生物载体用于检测无关蛋白BSA的时间与浓度关系图；
图3为本发明生物载体在320nm波长下的免疫比浊检测结果；
图4为本发明生物载体在400nm波长下的免疫比浊检测结果；
图5为本发明生物载体浓度为3.525×105检测血清中抗体结果；
图6为本发明生物载体浓度为3.525×107检测血清中抗体结果；
图7为本发明生物载体配合使用5％PEG4000助剂而获得的浊度变化情况；
图8为本发明生物载体配合使用不同分子量的PEG助剂而获得的浊度变化情况；
图9为本发明生物载体配合使用不同浓度的PEG6000助剂而获得的浊度变化情况；
图10为通过化学法获得的本发明生物载体使用不同浓度的PEG6000助剂而获得的浊度变化情况；
图11为通过化学法获得的本发明生物载体对梯度抗原进行检测而获得的浊度变化情况。
具体实施方式
以下结合附图详细描述本发明的技术方案。本发明实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围中。
本发明涉及分子生物学实验，如没有特别注明，可参考自《分子克隆》一书(J.萨姆布鲁克、E.F.弗里奇、T.曼尼阿蒂斯著，科学出版社，1994)。该书及其后续出版版本是本领域技术人员在进行与分子生物学相关的实验操作时最常用的具有指导性的参考书籍。此外，根据不同实验目的，本领域技术人员在各种商品化试剂盒(Kit)所附带的操作手册的指导下完成相应的实验或委托专业化的公司进行，如：基因测序、质粒测序和确定分子量等。
本发明实施例其它所用的试剂若未经说明，均购自西格玛－奥德里奇(Sigma－Aldrich)公司或者国药集团化学试剂有限公司。
实施例1 噬菌体纳米抗体库构建
通过抽取7只羊驼血细胞，并分离其淋巴细胞后，设计羊驼纳米抗体引物，通过巢式PCR，克隆羊驼纳米抗体基因，构建噬菌粒，感染细菌后，在辅助噬菌体协助下，包装成展示纳米抗体的噬菌体，成功获得滴度在1011的纳米抗体库。主要内容如下：
涉及的三对引物如表1
表1
Figure PCTCN2014093838-appb-000001
所列引物包括简并引物，其中，R＝A/G，K＝G/T，B＝G/T/C，W＝A/T。
以羊驼淋巴细胞中的mRNA作为模板反转录出来的cDNA作为第一轮PCR的模板，共获得750ng的PCR产物(以引物1为扩增引物获得)。以第一轮PCR产物作为第二轮PCR的模板，共获得1.4μg的PCR产物(以引物2为扩增引物获得)。以第二轮PCR产物作为第三轮PCR的模板，共获得40μg PCR产物(以引物3为扩增引物获得)。然后，以 引物3再一轮PCR之后割胶回收400bp左右的PCR产物。将酶切噬菌粒pCANTAB5E(所展示蛋白与P3蛋白融合)与酶切后PCR产物连接转化，构建噬菌体纳米抗体库。
实施例2 噬菌体展示技术筛选鸡抗体蛋白IgY的纳米抗体
1)第一轮
①称取鸡抗体蛋白IgY 44μg，溶于3ml的碳酸钠-碳酸氢钠包被液(pH＝9.5)中，加入容量为4 ml的Nunc-ImmunoTM MaxisorpTM免疫试管中，4℃过夜。
②Tris-HCl封闭免疫试管的未饱和的蛋白偶联位点。先用偶联液清洗免疫试管3次，尽量去除上清，加入4ml 0.1M Tris-HCl缓冲液(pH 8.0)，室温静置2小时，以封闭活性位点。
③BSA封闭潜在蛋白结合位点，4ml 5％BSA放入免疫试管中，室温缓慢摇动2小时，PBS清洗3次，甩干净。
④1ml噬菌体纳米抗体库液，总共1014个噬菌体加入免疫试管，室温下，混合器缓慢摇晃30分钟，然后室温静置90分钟，PBST清洗免疫试管10次，PBS清洗免疫试管10次，甩干。
⑤噬菌体洗脱。免疫试管中加入600μl的10mMHCl，缓慢摇晃，室温30分钟，然后加入100μl 0.1M Tris-HCl将pH调至7.5，加入2ml新鲜的，OD＝7.0的XL1-Blue菌液，37℃静置50分钟，转移到50ml离心管中，加入5ml 2×TY，37℃摇荡30分钟。
⑥加入1μl的1011个M13K07噬菌体，37℃静置50分钟，3000g，离心5分钟，去掉上清，沉淀溶于50ml的2×TY+AMP+KAN+GLU培养基，30℃过夜。
⑦PEG4000沉淀培养基上清液中噬菌体，14000rpm离心，2ml的1×TE溶解噬菌体，滴度约为1011。
2)第二、三、四、五轮，步骤同第一轮。
3)ELISA检测鸡抗体蛋白IgY的纳米抗体富集度
①抗原包被：天然胱抑素C分别包被ELISA板，包被浓度为1μg/ml。
②BSA封闭；
③每个ELISA孔加入109个不同富集度的纳米抗体噬菌粒，稀释到PBST中，100μl/孔，1小时静置。
④Anti-噬菌体抗体(兔来源)，稀释度1∶5000，100μl/孔，37℃，1小时。
⑤羊抗兔IgG抗体(HRP)，稀释度1∶10000，100μl/孔，37℃，1小时。
4)结果：随着一轮一轮的筛选的进行，能够特异性识鸡抗体蛋白IgY的纳米抗体逐渐被富集。
实施例3 鸡抗体蛋白IgY不同时间的免疫比浊检测
1.抗原抗体准备：
抗原：取一定量的抗原用1×PBS稀释至一定的浓度，然后倍比稀释至所需浓度(以10ng/ml为起始点，3倍倍增)；
抗体：用1×PBS稀释至所需要的浓度(一般109个/μl-1010个/μl)；
2.抗原抗体混合：准备好的抗原抗体手动充分混匀，置37℃，按每小时检测；
3.酶标仪读取：在酶标板上选择板孔，然后分别加入待测样品，进行340nm波长不同时间的读取；
4.结果：由于实验未添加任何增敏剂和表面活性剂，浊度反应较慢，到第7小时开始出现明显变化，并随着时间的增加呈现随浓度增加而增长的趋势(参见图1)。
实施例4 鸡抗体蛋白IgY特异性的免疫比浊检测
实验操作同实施例3，增加一组无关蛋白牛血清白蛋白BSA作为对照，结果发现随时间变化，无关蛋白的浊度未见明显变化(参见图2)。可见，本发明提供的生物载体具有高度的检测特异性，不受其它物质的干扰。
实施例5 鸡抗体蛋白IgY不同波长下的免疫比浊检测
实验操作同实施例3，分别测定波长320nm(参见图3)及400nm(参见图4)条件下浊度质的变化。由此，本发明生物载体可适用于在可见光范围内实现检测。
实施例6 鸡法氏囊病毒蛋白VP2兔多抗制备
鸡法氏囊病毒VP2蛋白质免疫2只兔子，同时1号兔子注射拂氏完全佐剂，2号兔子不注射，每周一次，打三次，在第四周时，少量取血，检测其中的抗体滴度，结果如表2。
表2
Figure PCTCN2014093838-appb-000002
心脏抽血，制备血清，加入叠氮钠，4℃冰箱保存备用。
实施例7 鸡法氏囊病毒蛋白VP2片段的噬菌体展示(基因工程法)
首先，将鸡法氏囊病毒蛋白VP2基因片段与噬菌粒载体pCANTAB5E-8(携带噬菌体g8基因)连接；连接产物转化入TG1化学转化感受态菌；
接着，将已经转化入包含目的基因质粒的TG1，接种到10ml 2×TY中，加入氨苄抗性， 摇菌至OD600＝0.4-0.6，加入M13K07(约1011个噬菌体)放置1h，4500rpm室温离心10分钟，弃上清，沉淀重悬于50ml新鲜的2×TY培养基中，加Amp至100μg/ml，Kan至50μg/ml，葡萄糖加至1％，30℃，200rpm摇过夜；
最后，将过夜菌转移到无菌50ml离心管内，4800rpm，4℃离心20分钟，上清液转移到新管中，按照1/5的体积比例加入含有40％PEG 4000的2.5M NaCl液，强烈震动后冰浴30分钟。4,800rpm，4℃离心20分钟，弃上清，沉淀重悬于1ml无菌PBS缓冲液中，然后再于12,000rpm，室温离心5分钟，取上清，获得展示鸡法氏囊病毒蛋白VP2片段的噬菌体。
实施例8 通过噬菌体展示抗原检测血清中抗体
1.抗原抗体准备：
抗原：取高低2个浓度的噬菌体展示抗原作为检测，噬菌体颗粒浓度为3.525×105个/μl和3.525×107个/μl；
抗体：用1×PBS稀释1号兔抗血清，稀释倍数为10、100、1,000、10,000、100,000和1,000,000；
2.抗原抗体混合：准备好的抗原抗体手动充分混匀，置37℃，按每小时检测；
3.酶标仪读取：在酶标板上选择板孔，然后分别加入所要检测的样，进行340nm波长不同时间的读取；
4.结果：尽管实验未添加任何增敏剂和表面活性剂，浊度反应较慢，但高、低浓度抗原出现浊度变化的时间不一样，低浓度(3.525×105)抗原检测，出现浊度时间要快(4-5小时)，高浓度(3.525×107)抗原检测，出现浊度时间要慢(16-17小时)，但在某一区段总体呈现一致的趋势(线性)，如：低浓度(3.525×105个/μl)抗原检测，在1∶10、1∶100、1∶1,000和1∶10,000范围内表现线性关系(参见图5)。再如：高浓度(3.525×107个/μl)抗原检测，在1∶10、1∶100、1∶1,000和1∶10,000范围内表现线性关系(参见图6)。
实施例9 免疫比浊法中助剂的应用——添加5％PEG4,000
1.抗原抗体准备：
抗原：取噬菌体展示抗原作为检测抗原，噬菌体颗粒浓度为3.525×109个/μl；
抗体：①用1×PBS稀释1号兔抗血清，稀释倍数为10倍；②以无关蛋白BSA作为对照组，以观察检测的特异性以及5％PEG是否对特异性检测构成影响；
2.抗原抗体混合：准备好的抗原抗体手动充分混匀，设置添加或不添加5％PEG4000实验组，置37℃，按如下时间检测：0.5h、1h、1.5h、2h、3h、4h、5h和6h；
3.酶标仪读取：在酶标板上选择板孔，然后分别加入待测试样，340nm波长进行不同时间的读取(参见图7)；
4.结果：①加入5％PEG后，抗原抗体复合物形成速度明显加快，在30分钟时已经超过不加PEG组，1小时的差异则显得更为显著，在1.5小时处到达峰值；②不加 PEG的血清试样组，浊度形成显得较为缓慢，且表现出不稳定，但随时间延长，有增加的趋势；③非特异性对照组，无论加或不加PEG，其浊度没有明显变化；④在本实验中，添加5％PEG对特异性没有影响。
实施例10 观察不同分子量PEG对比浊形成的影响
1.抗原抗体准备：
抗原：取噬菌体展示抗原作为检测抗原，噬菌体颗粒浓度为3.525×109个/μl；
抗体：用1×PBS稀释1号兔抗血清，稀释倍数为10倍；
2.抗原抗体混合：准备好的抗原抗体手动充分混匀，设置添加均为5％的PEG4000，PEG6000、PEG10000和PEG20000四种不同分子量的PEG，置37℃，按如下时间检测：5min、10min、15min、20min、25min和30min；
3.酶标仪读取：在酶标板上选择板孔，然后分别加入待测试样，340nm波长进行不同时间的读取(参见图8)；
4.结果：显示大分子量具有明显增强浊度的效果，尤其是PEG6000相较PEG4000效果最为明显，而PEG6000、PEG10000和PEG20000之间的浊度差异则并不显著。
实施例11 观察不同百分比PEG6000对比浊形成的影响
1.抗原抗体准备：
抗原：取噬菌体展示抗原作为检测抗原，噬菌体颗粒浓度为3.525×109个/μl；
抗体：用1×PBS稀释1号兔抗血清，稀释倍数为10倍；
2.抗原抗体混合：准备好的抗原抗体手动充分混匀，设置添加PEG6000，设置浓度为分别为0％(PBS)、1％、2％、3％、4％、5％和6％PEG6000，置37℃，按如下时间检测：0min、2min、4min、6min、8min、10min、15min和30min；
3.酶标仪读取：在酶标板上选择板孔，然后分别加入待测试样，340nm波长进行不同时间的读取(参见图9)；
4.结果：添加PEG6000在6％的浓度时，立即呈现肉眼可见的浑浊，显示浓度太高。其他结果显示，随着PEG6000浓度的增加，具有明显增强浊度的效果。尤其是PEG6000浓度在4％和5％时，其浊度表现明显，吸光度区间适宜，并且最快在8分钟的时即已出现峰值，适合于作为助剂应用于本发明生物载体的免疫比浊检测。
实施例12 鸡法氏囊病毒蛋白兔多抗的噬菌体展示(化学法)
1.取实施例6所制备的兔多抗血清，饱和硫酸铵法沉淀后，PBS透析，透析液2000rpm，10min离心，去沉淀，取上清，为所制备的多抗；
2.将100μl炔基NHS(50mg/ml)与M13噬菌体溶液(10mg/ml)1ml在10mM，pH＝7.8的磷酸缓冲溶液中混合震荡反应，整个反应处于4℃下，过夜，反应产生的不溶物在12,000g的转速下离心，取上清液，透析除去未反应的炔基 NHS；
3.将兔多抗与炔基修饰过的M13溶液(4mg/ml，1ml)混合，CuSO4以及NaAsc作为催化剂加入，在pH＝8Tris缓冲溶液中室温反应12个小时；
4.收集制备出的兔多抗修饰的M13噬菌体，备用。
实施例13 化学法噬菌体展示兔多抗的免疫比浊
1.抗原抗体准备：
抗原：取鸡法氏囊病毒蛋白VP2蛋白作为检测抗原，浓度为1500ng/ml；
抗体：取兔多抗化学修饰的噬菌体颗粒为抗体，浓度为1×108个/μl；
2.抗原抗体混合：准备好的抗原抗体手动充分混匀，设置添加PEG6000，设置浓度为分别为0％(PBS)、1％、2％、3％、4％、5％和6％PEG6000，置37℃，按如下时间检测：4min、6min、8min、10min、15min、30min和1h；
3.酶标仪读取：在酶标板上选择板孔，然后分别加入待测试样，340nm波长进行不同时间的读取(参见图10)；
4.结果：显示PEG6000浓度在3％时(PEG6000的最佳浓度)，其浊度表现明显，并且最快在8分钟时即已出现峰值。
实施例14 化学法噬菌体展示兔多抗的免疫比浊—抗原梯度检测
1.抗原抗体准备：
抗原：取鸡法氏囊病毒蛋白VP2蛋白作为检测抗原，浓度梯度为111ng/ml、333ng/ml、1000ng/ml、3000ng/ml和9000ng/ml；
抗体：取兔多抗化学修饰的噬菌体颗粒为抗体，浓度为1×109个/μl；
2.抗原抗体混合：准备好的抗原抗体手动充分混匀，设置添加3％PEG6000，置37℃，8分钟后检测结果；
3.酶标仪读取：在酶标板上选择板孔，然后分别加入待测试样，340nm波长进行不同时间的读取(参见图11)；
4.结果：显示PEG6000浓度在3％时，其浊度表现良好，并且在8分钟时检测的结果在浓度111-3000ng/ml间具有良好的线性表现。
实施例15 胱抑素C纳米抗体融合蛋白的酵母展示
本实施例如下实验所涉及序列、引物和方法步骤可以参考CN103509115A。
1.获得胱抑素C纳米抗体3-2、7-4融合蛋白的基因
根据胱抑素C纳米抗体3-2、7-4基因序列，设计引物(如：引物设计软件DNAman)，克隆胱抑素C纳米抗体3-2、7-4基因，并重叠(overlap)连接，获得胱抑素C纳米抗体3-2和7-4融合蛋白完整基因，且所设计引物在胱抑素C纳米抗体3-2、7-4融合蛋白基因两端加上Nhe I和BamHI两个酶切位 点；
抗体；
2.构建重组质粒
将获得胱抑素C纳米抗体3-2、7-4融合蛋白基因连接到噬菌体T载体上，得TA-胱抑素C纳米抗体3-2、7-4融合蛋白基因质粒，并测序。测序正确后将pCTcon2质粒(来自Addgene)和TA-胱抑素C纳米抗体3-2、7-4融合蛋白基因质粒在Nhe I和BamHI两个酶切进行双酶切，凝胶电泳分离并回收，然后将回收产物用T4 DNA连接酶连接，转化到大肠杆菌DH5α中，挑取单菌斑，PCR验证，并测序，测序正确的菌株保存；
3.重组质粒提取
扩增含有重组质粒的菌株DH5α，利用质粒提取试剂盒(生工生物工程(上海)股份有限公司提供)提取重组质粒pCTcon2-胱抑素C纳米抗体3-2、7-4融合蛋白基因，浓缩质粒备用；
4.酵母EBY100电转感受态的制备
(1)酵母EBY100甘油菌(酵母EBY100来自NTCC国家典型培养物保藏中心)经新鲜酵母浸出粉胨葡萄糖培养基(YPD)平板复苏后，取5ml培养过夜，经14小时左右后，取菌并按1∶100稀释后，取70ml菌液接种于YPD扩大培养，14小时后测得OD600值约为1.3；
(2)将山梨醇、去离子水(ddH2O)和菌液均冰浴；
(3)菌液1700g离心5min，用100ml冰的ddH2O洗涤，离心，再用50ml冰的ddH2O洗涤离心，然后用4ml山梨醇洗涤离心，离心获得的酵母溶解于300μl冰山梨醇；每50μl或100μl分装，保存到-80℃；
5.酵母EBY100电转感受态的制备
(1)取50μl分装菌液，加入约3μl的重组质粒(5μg)，吹匀，置0.1CM电转杯，冰浴静置5min；
(2)于1.5kv、25μF和200Ω条件下电击；
(3)立即加入1ml冰浴的山梨醇，静止10min。
(4)立即将其转移至10ml EP管中，加入1ml YPD培养基，30℃摇1小时，复苏EBY100；
(5)然后离心，加入1M冰山梨醇，混匀得到转化液；
(6)分别取100μl、200μl和700μl转化液，分别涂布10cm的最小葡萄糖培养基(Minimal Dextrose Medium，且不含亮氨酸，MD)平板，30℃培养；
(同时做一组阴性对照，转化的是空载的pCTcon2质粒)
6.筛选获得阳性克隆，并鉴定
从MD平板上挑选阳性克隆，并用胱抑素C纳米抗体3-2、7-4融合蛋白基因的下游引物(与“获得胱抑素C纳米抗体3-2、7-4融合蛋白的基因”步骤所用引物相同)和pCTcon2的上游引物(与“获得胱抑素C纳米抗体3-2、7-4 融合蛋白的基因”步骤所用引物相同)组成对拉引物，PCR验证，验证结果正确的克隆，用MD液体培养基培养，保存于甘油中；
7.酵母诱导表达胱抑素C纳米抗体3-2、7-4融合蛋白的鉴定
阴性对照：野生型EBY100，阳性对照：空载pCTcon2质粒的酵母，实验组：转化重组质粒的酵母；
野生型的EBY100用YPD培养基培养，实验组和阳性对照利用SD-CAA培养基(Synthetic dextrose medium with casamino acids，含葡萄糖)30℃培养，培养至OD600值在2～5之间，4,000g离心，将酵母用SC-CAA培养基(含半乳糖)调整OD600值至0.5～1之间，30℃培养12小时、24小时和36小时，每个时间段取2OD的酵母，4℃保存；
将不同时间段的重组酵母和野生型酵母，做酵母细胞ELISA实验，一抗为anti-c-myc鼠单抗，二抗为HRP标记的羊抗鼠多抗，验证并获得胱抑素C纳米抗体3-2、7-4融合蛋白在酵母上成功展示试样。
实施例16 通过酵母展示纳米抗体检测胱抑素C蛋白
1.抗原抗体准备：
抗原：取胱抑素C蛋白2μg/ml，用Tris-HCl缓冲液按比例1∶1、1∶2、1∶4、1∶8和1∶16进行稀释；
抗体：用Tris-HCl缓冲液稀释2号酵母展示克隆，稀释颗粒浓度为1×105个/ml和1×06个/ml；
2.抗原抗体混合：制备的抗原和抗体充分混匀，置室温，根据浊度变化进行检测；
3.酶标仪读取：在酶标板上选择板孔，然后分别加入待测试样，340nm波长进行不同时间的读取；
4.结果：实验未添加任何增敏剂和表面活性剂，但与噬菌体展示相比较，酵母展示浊度反应较快，抗原抗体混合后，在不到10分钟的时间内，高浓度即可出现肉眼可见的浑浊，且随着时间延长，浊度会出现下降，具体结果参见表3。
表3
Figure PCTCN2014093838-appb-000003
Figure PCTCN2014093838-appb-000004
Figure PCTCN2014093838-appb-000005

Claims (18)

  1. 一种生物载体在免疫比浊检测技术中的应用，所述的生物载体表面具有识别并与目标分子结合作用的聚合物。
  2. 一种生物载体在免疫比浊检测产品中的应用，以所述的生物载体作为载体颗粒，其表面具有识别并与目标分子结合作用的聚合物。
  3. 根据权利要求1或2所述的生物载体的用途，其特征在于所述的聚合物包括氨基酸、多肽和蛋白质之一种或几种。
  4. 根据权利要求1或2所述的生物载体的用途，其特征在于所述的聚合物为抗原和抗体之一种或几种。
  5. 根据权利要求1或2所述的生物载体的用途，其特征在于所述的生物载体选自于噬菌体、病毒、细菌和真菌之一种或几种。
  6. 根据权利要求1或2所述的生物载体的用途，其特征在于所述的生物载体为由噬菌体、病毒、细菌和真菌之一种或几种形成的聚合体和连接体。
  7. 一种免疫比浊检测组合物，其特征在于含有一种生物载体，所述的生物载体表面具有识别并与目标分子结合作用的聚合物。
  8. 根据权利要求7所述的免疫比浊检测组合物，其特征在于所述的免疫比浊检测组合物为检测试剂盒。
  9. 根据权利要求7所述的免疫比浊检测组合物，其特征在于所述的聚合物包括氨基酸。
  10. 根据权利要求7所述的免疫比浊检测组合物，其特征在于所述的聚合物为抗原和抗体之一种或几种。
  11. 根据权利要求7所述的免疫比浊检测组合物，其特征在于所述的生物载体选自于噬菌体、病毒、细菌和真菌之一种或几种。
  12. 根据权利要求7所述的免疫比浊检测组合物，其特征在于所述的生物载体为由噬菌体、病毒、细菌和真菌之一种或几种形成的聚合体和连接体。
  13. 一种免疫比浊检测方法，其特征在于将生物载体作为检测试剂加入待测样品中形成复合物，所述的生物载体表面具有识别并与目标分子结合作用的聚合物。
  14. 根据权利要求13所述的免疫比浊检测方法，其特征在于所述的生物载体为由噬菌体、病毒、细菌和真菌之一种或几种形成的聚合体和连接体。
  15. 根据权利要求13所述的免疫比浊检测方法，其特征在于所述的生物载体为由噬菌体、病毒、细菌和真菌之一种或几种。
  16. 根据权利要求13所述的免疫比浊检测方法，其特征在于还加入增敏剂、表面活性剂和缓冲液之一种或几种。
  17. 根据权利要求13所述的免疫比浊检测方法，其特征在于所述的聚合物为抗原和抗体之一种或几种。
  18. 根据权利要求13所述的免疫比浊检测方法，其特征在于所述的聚合物包括氨基酸。
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