CN101706497A - ELISA test kit of human TFF3 - Google Patents

Abstract

The invention relates to a new ELISA test kit of human TFF3, and the key technique is characterized in that the specific anti-TFF3 antibody is prepared by a gene engineering method, an elisa plate coated by the antibody is used as an important part of the kit, and then is assembled with TFF3 multi-antibody, secondary antibody marked by horse radish peroxidase, TFF3 standard, TFF3 positive control, sample diluent, washing liquor, coloration solution and stopping solution into the ELISA test kit of human TFF3; ELISA is carried out by a bi-anti sandwich method to establish a measurement method which can sensitively and rapidly capture TFF3 concentration in human serum or cell and tissue culture, and has the characteristics of high specificity and high stability. The kit is mainly used for clinical research use such as general laboratory or gastrointestinal tract pathology.

Description

The ELISA detection kit of human TFF 3

Technical field

The present invention relates to multiple biopreparate---how anti-the specificity of human TFF 3 protein, anti-TFF3 is, the ELISA detection kit of monoclonal antibody and a kind of people of being used for source TFF3 content detection, is applicable to the detection by quantitative of TFF3 in people's tissue, cell culture or the serum.

Background technology

TFFs (The trefoil factor families) is a trefoil factor family, be a kind of secreted polypeptides that a class is produced mucinous surface epithelial cell, gain the name because its protein all comprises the trilobal cross structure that one or more 3 disulfide bond that formed by inner 6 cysteine residues by 38 to 39 amino acid residues constitute.

The gene of TFFs is positioned on the 21q22.3 chromosome, and the shared zone of the gene of all three protein is total to 55kb in the family.TFFs expresses in a plurality of tissues in human body, but in intestines and stomach the expression maximum.

Among intestinal inflammatory and the carcinoma of prostate patients serum, TFFs content (the Vestergaard EM that obviously rises, et al.Plasmalevels of trefoil factors are increased in patients with advanced prostate cancer.Clin Cancer Res., 2006; 12:807-12).TFFs is considered to safeguard the key factor of mucous membrane surface health, and TFFs content significantly rises during latter's inflammation, thereby its mechanism of action is interpreted as by strengthening the protective capability on the migration raising mucous membrane top layer of cell with mucinous interaction.Though also have research to point out that TFFs content in multiple cases of cancer raises, these protein realize that the mechanism of function is still waiting further investigation.

There are 3 member: TFF1, TFF2 and TFF3 in this family.TFF1 contains 60 amino acid residues, and is relevant with breast cancer; TFF2 contains 106 amino acid residues, and tool is separated the spasm function; TFF3 contains 73 amino acid residues.Wherein, TFF1 and TFF3 comprise the domain that, TFF2 comprise two three leaf configurations, and TFF1 and TFF3 can form homodimer by the cysteine residues that is positioned at c-terminus.

TFF3 also is called intestine trilobate factor (intestinal trefoil factor), because its cloverleaf structure closely, so to proteinase, acid, thermally-stabilised.The about 8kDa of TFF3 monomer molecule amount, the about 15.6kDa of dimer.TFF3 mainly is positioned at the Weishang, and also great expression is in the mucous membrane cell of small intestine and large intestine.The physiological effect of TFF3 is considered to help growth, propagation and the transfer of cancer cell in the enterocyte.The concentration of TFF3 in healthy contributor's serum is 0.7ng/mL to 2ng/mL (Vestergaard EM, et al.Development and Evaluation of an ELISA for Human Trefoil Factor 3.Clin.Chem., 2002; 48:1689-1695).Clinical data shows that the expression of TFF3 can present related (Yamachika T et al.Intestinal Trefoil Factor A Marker of Poor Prognosis in Gastric Carcinoma.Clin.Cancer Res., 2002 with the poor prognosis of cancer of the stomach; 8:1092).In addition, by with mucinous interaction, TFF3 can make the intestines cells of superficial layer avoid the influence of numerous injurious factors.Studies show that in the body of mouse, oral TFF3 can make it avoid the stomach lining injury, resist the drug-induced damage of non-steroid, and the mucous membrane repair function of TFF3 clpp gene deratization is obviously impaired, when their enteron aisles are impaired, dying from wound can't heal, the mechanism of action of TFF3 comprises safeguards epithelial cell, make it avoid apoptosis, perhaps lure border cell (bordering cell) migration into and cover wound, perhaps stop deposition (the Mashimo H that enters epithelial SC system component through wound, et al.ImpairedDefense of Intestinal Mucosa in Mice Lacking Intestinal Trefoil Factor[J] .Science, 1996; 274:262-265.).

Therefore, the quantitative test to TFF3 helps to disclose its effect in intestines and stomach.

In addition, report is also arranged, TFF3 raises (Gronbaek H, et al.Serum trefoil factors in patients with inflammatory bowel disease.Digestion, 2006 unusually in breast cancer, colon cancer, cancer of the stomach and carcinoma of prostate patients serum; 74:33-39).

The tendency that rises to the gastrointestinal disease generation in view of TFF3 concentration in serum or tissue and cell, also take place relevant simultaneously with prognosis with the pathology of breast cancer, colon cancer, cancer of the stomach and prostate cancer, therefore, strengthen the detection of TFF3 in blood, tissue and cell and have researching value and clinical meaning.

At present, several in the world big ELISA kit manufacturers produce TFF3 albumen or antibody only singlely, and Shang Weiyou produces the report of the ELISA kit of TFF3, and also constantly bring forth new ideas at the result of study of TFF3 albumen in recent years, so in the present invention, use GenBank:AAH17859.1 (BC017859) sequence to be template, by cellular expression TFF3 protein, according to same amino acid sequence, express the TFF3 polypeptide of many group different lengths by chemosynthesis or by biological recombinant cell again, prepare the monoclonal antibody of TFF3 correspondence and how anti-again, use the antibody sandwich ELISA Plate for preparing, TFF3 polypeptide with the front gained is standard items, forms the ELISA kit.

The present invention adopts the double antibodies sandwich method, as the antibody of catching TFF3, has guaranteed the specificity of testing result with specific antibody, has at utmost avoided false positive; How anti-with specificity subsequently as detecting antibody, further got rid of false positive; Add ELIAS secondary antibody again, form the bond of the sandwich shape of one 4 component, add substrate colour developing liquid at last, the above correct signal that obtains is effectively amplified, contrast is parallel standard items that carry out and confidential reference items experiment synchronously, further get rid of reagent or operate miss in the experiment, finally obtain reliable conclusion.

Summary of the invention

The purpose of this invention is to provide people source TFF3 in a kind of detection of biological sample ELISA adsorption analysis method ( ENzyme LInked- IMmuno- SOrbent ASsay, ELISA), this method comprises: (1) contacts sample with the specific antibody (antibodies) of anti-TFF3 of the present invention, form TFF3::TFF3 antibody complex (complex), (2) situation that combines of TFF3 molecule and antibody in the test sample, (3) compare with control sample, determine the content of TFF3 in the biological sample.

In some embodiments, described biological sample comprises human serum, tissue or cell sample extract or lysate.In some embodiments, the content of TFF3 is because of the health status of individuality, perhaps whether canceration and form difference of tissue and cell.

In some embodiments, the specific antibody of anti-TFF3 is monoclonal antibody, polyclonal antibody, chimeric antibody, single-chain antibody, Fab fragment or above one or more potpourri.In some embodiments, this antibody is through mark, and mark can be enzyme, radioactive isotope or fluorophor.In some embodiments, antibody is lower than about 1 * 10 to the binding affinity of the polypeptide except that TFF3 ⁵Ka.

The present invention also provides the TFF3 polypeptide that separates, and this polypeptide comprises 1 or 2 amino acid sequence that is selected from down group: SEQ1 or SEQ 2, see Table 1, and wherein, SEQ 1 is the TFF3 of total length.In some embodiments, the length of this polypeptide is about 73 or be about 146 amino acid whose fragments, or is both potpourri.In some embodiments, this polypeptide or mixtures of polypeptides specifically with anti-TFF3 antibodies.

Table 1: the TFF3 polypeptide of separation

??SEQ?No.	Amino acid sequence
		??SEQ?1	??MLGLVLALLSSSSAEEYVGLSANQCAVPAKDRVDCGYPHVTPKECNNRGC??CFDSRIPGVPWCFKPLQEAECTF
??SEQ?2	??MLGLVLALLSSSSAEEYVGLSANQCAVPAKDRVDCGYPHVTPKECNNRGC??CFDSRIPGVPWCFKPLQEAECTFMLGLVLALLSSSSAEEYVGLSANQCAV??PAKDRVDCGYPHVTPKECNNRGCCFDSRIPGVPWCFKPLQEAECTF

The present invention also provides the TFF3 antibody that separates, and wherein this antibody recognition is corresponding at least one TFF3 sequence area among SEQ 3, SEQ 4, SEQ 5, SEQ6, SEQ 7, SEQ 8, SEQ 9, SEQ 10, SEQ 11, SEQ 12, SEQ 13 or the SEQ 14.Each artificial synthetic polypeptide sees Table 2.

Table 2: the TFF3 polypeptide of synthetic

??SEQ?No.	Sequence	Length	Amino acid sequence position through the location
				??SEQ?3	??GLVLALL	??7-mer	??3-9
??SEQ?4	??LGLVLAL	??7-mer	??2-8
				??SEQ?5	??LGLVLALL	??8-mer	??2-9
??SEQ?6	??AVPAKDRVDCG	??11-mer	??26-36
				??SEQ?7	??AVPAKDRVDC	??10-mer	??26-35
??SEQ?8	??AVPAKDRVD	??9-mer	??26-34
				??SEQ?9	??GYPHVTPKECN	??11-mer	??36-46
??SEQ?10	??GYPHVTPKEC	??10-mer	??36-45
				??SEQ?11	??GYPHVTPKE	??9-mer	??36-44
??SEQ?12	??CFKPLQEAECT	??11-mer	??62-72
				??SEQ?13	??CFKPLQEAEC	??10-mer	??62-71
??SEQ?14	??CFKPLQEAE	??9-mer	??62-70

In some embodiments, this antibody produces by the following method:

(1) antibody library of a synthetic TFF3 on bacteriophage;

(2) by this bacteriophage and composition are mixed to come the screening of sample being carried out antibody library, said composition comprises corresponding among SEQ3, SEQ 4, SEQ 5, SEQ 6, SEQ 7, SEQ 8, SEQ 9, SEQ 10, SEQ 11, SEQ 12, SEQ13 or the SEQ 14 at least one;

(3) eluriate (panning) and separate the bacteriophage that combines with composition, wherein the feature of this antibody is, have and the ability that combines corresponding at least one the TFF3 sequence area among SEQ3, SEQ 4, SEQ 5, SEQ 6, SEQ 7, SEQ 8, SEQ 9, SEQ 10, SEQ 11, SEQ 12, SEQ13 or the SEQ 14, its binding affinity is at least 10 ⁸I/;

(4) analyze the gained bacteriophage, and determine that this bacteriophage is combined with the amino acid coding corresponding at least one the TFF3 sequence area among SEQ 3, SEQ 4, SEQ 5, SEQ 6, SEQ7, SEQ 8, SEQ 9, SEQ 10, SEQ 11, SEQ 12, SEQ 13 or the SEQ 14.

In some embodiments, this antibody produces by recombinant host cell, and this recombinant cell is selected from Chinese hamster ovary cell.

In some embodiments, this antibody produces by hybridoma, and this hybridoma merges the back gained by the mouse lymph B cell and the rat bone marrow tumour cell of the secreting specificity antibody of TFF3 polypeptide after as antigen immune.

The present invention also provides some polypeptide fragment SEQ 15, SEQ 16, SEQ 17, SEQ 18, and these fragments are about 23 to 25 amino acid, comprises a sequence among SEQ 5, SEQ 6, SEQ 9, the SEQ 12 at least, sees Table 3.In some embodiments, this fragment is by artificial chemosynthesis; In some embodiments, this fragment produces in recombinant host cell, and these recombinant cells are selected from: Chinese hamster ovary cell or bacterial host cell.In some embodiments, these polypeptide fragments are used for the animal immune of mouse and/or rabbit, and in some embodiments, these polypeptide fragments are used for the constituent of kit standard items.

Table 3: the TFF3 polypeptide fragment of separation

??SEQNo.	Sequence	Length	Amino acid sequence position through the location
				??SEQ?15	??MLGLVLALLSSSSAE??EYVGLSANQC	??25-mer	??1-25
??SEQ?16	??VGLSANQCAVPAKDR??VDCGYPHV	??23-mer	??18-40
				??SEQ?17	??DCGYPHVTPKECNNR??GCCFDSRIP	??24-mer	??34-57
??SEQ?18	??CCFDSRIPGVPWCFK??PLQEAECTF	??24-mer	??50-73

Embodiment

The invention provides TFF3 standard items, antibody and use these reagent to detect the method for TFF3 concentration.TFF3 many tissues and clone as tissue relevant and cell with prostate cancer, breast cancer, oophoroma and colon cancer in differential expression.

" differential expression " described herein typically refers to TFF3 protein and expresses with higher or reduced levels, as comparing with the content in normal structure or the cell, in cancerous issue or the cell expression higher or lower be a kind of gene that in colon, mammary gland, prostate and other cancer, has presented differential expression at least about 50%, 1 times, 5 times, 10 times or more .TFF3. these relatively can carry out between two kinds of tissues of two individualities or same individuality, also can carry out at the iuntercellular that derives from same tissue.

Term " about " used herein is meant a certain numerical value+/-10%.

The TFF3 polypeptide

" TFF3 " described herein or " TFF3 polypeptide " is meant protein (polypeptide) or its fragment with people's trefoil factor 3 (GenBank:AAH17859.1) homology, though comprise any sequence in the above-mentioned sequence or the nucleotide sequence difference of gene, because the amino acid sequence of the polypeptide fragment of the degeneracy of genetic code and gained and the TFF3 variant of basically identical still.In the embodiment of some optimization, TFF3 comprises SEQ 1, SEQ 2, SEQ 3, SEQ 4, SEQ 5, SEQ 6, SEQ 7, SEQ 8, SEQ 9, SEQ 10, SEQ 11, SEQ 12, SEQ 13 or SEQ 14.

Term described herein " polypeptide " and " protein " can substitute use mutually, all can refer to the amino acid polymerized form of any length, can be by artificial chemosynthesis, and can also be by the bacterium or the eukaryotic cell expression of reorganization; These amino acid condensates can through as glycosylation modified or without chemistry or bio-modification; Its end contains or does not contain the terminal methionine residues of N.

" TFF3 polypeptide " also refer to the amino acid sequence identity of SEQ 1 at least about at each the seed amino acid polymerized form more than 90%, what its source can be for natural expression or synthetic.Sequence homogeneity for example utilizes default parameters (default parameter) to measure with BLAST algorithm (algorithm).

" TFF3 polypeptide " refers to that also some length are at least the above polypeptide variants of 20 amino acid, these variants comprise peculiar zone of TFF3 albumen or epi-position fragment, especially biological active fragment or with the corresponding fragment of functional domain.

Antibody

This paper term " antibody " uses its implication widely, contained the polyclonal antibody and the monoclonal antibody of assembling fully, and antibody fragment that can conjugated antigen (as, Fab,, F ' (ab) ₂).

" polyclonal antibody " described herein is called for short " how anti-", refers to by repeatedly subcutaneous (sc), intraperitoneal (ip) or intramuscular (im) are injected corresponding antigens and auxiliary agent, the potpourri of the discrete antibody that produces in animal.Term used herein " monoclonal antibody " is called for short " monoclonal antibody ", refers to that the antibody individuality of promptly forming this colony is all identical available from the antibody of the antibody population of homology basically, but not the potpourri of discrete antibody.

Antibody fragment described herein, usually derive by the proteolytic digestion of complete antibody and come (referring to Morimoto K, et al.Single-step purification of F (ab ') 2fragments of mouse monoclonal antibodies (immunoglobulinsG1) by hydrophobic interaction high performance liquid chromatography using TSKgelPhenyl-5PW[J] .J.Biochem.and Biophys.Meth., 1992; 24:107-117.).Simultaneously, these fragments also can directly be produced by recombinant host cell, and for example, this antibody fragment separates in can antibody phage storehouse from above and obtains; F (ab ') ₂Fragment can directly reclaim Fab '-SH fragment earlier from Escherichia coli (E.coli), form (referring to Carter P through chemical coupling again, et al.High level Escherichia coli expression and production of a bivalent humanizedantibody fragment[J] .Bio/Technology, 1992; 10:163～167.).Industry those skilled in the art clearly understand and the generation technique of the antibody fragment that applies unerringly.

Antibody is called as " specificity in conjunction with " with combining of antigen, this means: demonstrate between (1) antigen-antibody in conjunction with active threshold level, significant cross reaction does not take place with known related polypeptide molecule in (2) antigen or antibody.Those of ordinary skill in the art can use the binding affinity of certain technical measurement antibody easily, as analyze by Scatchard (ScatchardG.The attractions of protein for small molecules and ions[J] .Ann.N Y Acad.Sci., 1949; 51:660-672.). antibody of the present invention is higher than at least 1000 times of other TFF3 associated protein with combining of TFF3. in some embodiments, antibody of the present invention not with known relevant peptide molecule specificity combination, for example, the Westemblotting of use standard analyzes, and does not combine with known related polypeptide when they combine with the TFF3 polypeptide. and the exemplary of other analysis well-known in the art comprises: radioimmunoassay (RIA), radioimmunoprecipitation, Enzyme Linked Immunoadsorbent Assay (ELISA), dot blot or inhibition or competition analysis and sandwich assay.

Host cell

The expression construct of coding TFF3 can be imported in the host cell, these host cells can be any suitable protokaryon or eukaryotic.

Expression system in the bacterium comprises Goeddel DV, et al.Direct expression in Escherichia coli of a DNAsequence coding for human growth hormone[J] .Nature, 1979; 281:544-548 and Siebenlist U, et al.E.coli RNA polymerase interacts homologously with two different promoters.Cell, 1980; The system of describing among the 20:269-281.

Expression system in the yeast comprises Kelly JM, et al.Transformation of AspergiIlus niger by the amdSgene of Aspergillus nidulans.EMBO J, 1985; 4:475479 and Yelton MM, et al.Transformation ofAspergillus nidulans by using a trpC plasmid[J] .Proc Natl Acad Sci, 1984; 81:1470-1474, with Gaillardin, C, et al.Integrative transformation of the yeast Yarrowia lipolytica.Curr.Genet., 1985; 10:49-58, and Van den Berg JA, et al.Kluyveromyces as a host for heterologous gene expression:expression and secretion of prochymosin[J] .Biotechnology, 1990; System described in the 8:135-9.

Expression system in the insect host cell comprises " molecular biology of baculoviral " (THE MOLECULARBIOLOGY OF BACULOVIRUSES, W.Doerfler compiles) in, " regulation and control of baculoviral gene expression " (TheRegulation of Baculovirus Gene Expression, Friesen compiles) in, and Smith GE, et al.Modification andsecretion of human interleukin 2 produced in insect cells by a baculovirus expression vector[J] .Proc Natl Acad Sci., 1985; System described in the 82:8404-8408.

Expression system in the mammalian cell comprises Dijkema, R, et al.Cloning and expression of thechromosomal immune interferon gene ofthe rat.EMBO J., 1985; 4:761-767 and Boshart M, et al.A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus.Cell, 1985; 41:521-530, with Barnes D, et al.Methods for growth of cultured cells in serum-freemedium[J] .Anal Biochem., 1980; System described in the 102:255-270.

The expression construct of coding TFF3 is imported host cell, the recombinant cell of construction expression TFF3, employed technology comprise that virus infections, liposome-mediated Fusion of Cells, the transfection of calcium phosphate mediation, electroporation, bioplast merge, the DNA of cationic chemical polymkeric substance mediation shifts and particle gun.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention. be to be understood that, these embodiment only are used to the present invention is described and are not used in restriction the scope of protection of present invention, also not representing following experiment is whole or only experiments of being carried out. not marked concrete experiment condition and method in the following example, usually according to normal condition as chief editors such as J. Sa nurse Brookers, Science Press, 1992, " molecular cloning experiment guide " (second edition); D.L. Spector etc., Science Press, 2001, the condition that " cell experiment guide " advised. we endeavour to ensure the accuracy (for example, quantity, temperature or the like) of data, but need explanation to have some experimental errors and deviation. unless otherwise indicated, mark is meant massfraction, molecular weight is an average molecular mass, and the unit of temperature is ℃, and pressure then is atmospheric pressure or near atmospheric pressure.

Embodiment 1: the expression of TFF3 albumen in the Escherichia coli

(1) prokaryotic expression of 8kDaTFF3 protein fragments: (available from American type culture collection, ATCC) be template, with a pair of specific positive anti-primer (primer 1:TTTT GAATTC ATG CTG GGG CTGGTC CTG GCC with BC017859DNA; Primer 2: TTTT CTCGAG TCA GAA GGT GCA TTC TGC TTC), pcr amplification TFF3 dna fragmentation, with the gained fragment cloning in carrier pET28a (available from German Merck company), transform BL21 (DE3) (available from Merck) competence Escherichia coli, according to the kalamycin resistance screening positive clone.Extract the plasmid DNA of positive colony and identify that obtain and the corresponding to fragment of purpose fragment molecular weight, preliminary judgement is positive.Extract plasmid DNA with the PCR positive colony, check order, the result shows that TFF3 cDNA fragment correctly is cloned into pET28a, is a complete open reading frame with 219bp, with TFF3 gene 100% homology in people source among the GenBank, calculate that its expressed protein molecular weight is 8kDa.

(2) evaluation of 8kDaTFF3 protein fragments: the e. coli bl21 that has transformed recombinant plasmid after inducing with microorganism collection, ultrasonic treatment, mycoprotein shows through SDS-PAGE protein electrophoresis and coomassie dyeing, near newly-increased band 8kDa, identify through Western blotting, can with the antibody generation strong positive reaction of TFF3, illustrate that the TFF3 albumen of this 8kDa has stronger immunogenicity.

(3) preparation and the purifying of the TFF3 protein fragments of 8kDa: through cultivation, IPTG induce, express, use Ni in a large number ²⁺Affinity column (available from Pharmacia) purifying has the TFF3 albumen of 6 * His mark, obtains a large amount of 8kDa TFF3 protein fragments.

Embodiment 2: MONOCLONAL ANTIBODIES SPECIFIC FOR

TFF3 with acquisition among the embodiment 1 is an example, with TFF3 is the antigen immune mouse, obtain the lymph B cell of secretion specific monoclonal antibody, the latter and rat bone marrow tumour cell merge hybridoma, directly from supernatant, separate the monoclonal antibody of anti-TFF3 after the cellular incubation, or hybridoma directly is seeded to mouse peritoneal obtains monoclonal antibody.The latter's process is exemplified below:

(4) animal immune of TFF3 albumen: TFF3 protein fragments that will about 8kDa mixes with freund adjuvant fully and after fully emulsified, immunized mice age is 3 to 4 of the BALB/c in 8～12 weeks healthy mices (available from Animal Experimental Study center, Hubei Province), hypodermic injection, each immunity was spaced apart for 2 weeks, reach immunity thereafter for the second time and change non-complete freund adjuvant and TFF3 hypodermic injection into, be higher than 20000: 1 until serum titer.

(5) preparation of hybridoma: back 4 days of last immunity, separate mice spleen cell, (available from China typical culture collection center, CCTCC) merge with 4: 1 ratio, feeder cells are from the mouse peritoneal cell with the Sp2/0 myeloma cell strain of exponential phase under the PEG existence condition.Cell after the fusion is selected high antibody-secreting hole to contain the nutrient culture media screening of HAT behind limiting dilution, with hole inner cell clone, the ELISA that carries out antigen-specific measures, and selects high secretion sexual cell strain enlarged culture or frozen.

(6) monoclonal antibody preparation: the hybridoma cell strain that will secrete specific monoclonal antibody carries out the mouse peritoneal inoculation, collects ascites, crosslinked affinity column (available from the Pharmacia company) purified monoclonal antibody of usefulness staphylococcal protein A after waiting to produce obvious ascites.Antibody is stored among the PBS of the 0.01mol/L, the pH7.4 that contain 55% glycerine, 0.1%BHA and 0.01% thimerosal.

Embodiment 3: polyclonal preparation

(1) animal immune: anesthesia 3-4 monthly age, 3 to 4 of the healthy Japan large ear rabbit (available from Animal Experimental Study center, Hubei Province) of body weight more than 1.7Kg, used the DNA expression vector of coding human TFF 3 to carry out immunity at O days and the 28th day, expression vector is referring to " expression system in the mammalian cell " in the preamble " host cell ". particularly, for the 1st time and the 2nd immunity, DNA expression vector to injection 0.6mg in every rabbit muscle, and the injection site is used gentle electric current momently, and muscle cell absorbs DNA. then in this position to stimulate, gave rabbit muscle interior injection recombined human TFF3 albumen such as SEQ 1 with booster immunization in every month, recombinant protein is fully emulsified in incomplete freund adjuvant.

(2) serum obtains: each immunity after 14 days from rabbit the arteria auricularis blood sample collection, leave standstill after 30 minutes centrifugal, 3000rpm, 5 minutes, supernatant is serum.With the tiring of the antibody response determining reorganization human TFF 3 albumen is produced, be higher than 20000: 1 with ELISA test determination rabbit anteserum until serum titer.

(3) many anti-purifying: the method that adopts chromatography obtains the total IgG component by albumin A post separation of serum component, and affinity column (available from the Pharmacia) purifying of the TFF3 albumen of using total length more crosslinked in advance obtains the how anti-of anti-TFF3.Antibody behind the purifying is stored among the PBS of the 0.01mol/L, the pH7.4 that contain 55% glycerine, 0.1%BHA and 0.01% thimerosal, and many anti-final concentrations are 1mg/mL.

The composition and the preparation of embodiment 4:ELISA kit

Kit comprises: how anti-TFF3 standard items, TFF3 positive control sample, TFF3 are, biological sample dilution, cleansing solution, ELIAS secondary antibody (how anti-the goat-anti rabbit of HRP mark is), substrate colour developing liquid, stop buffer, ELISA Plate.Specify as follows:

(1) TFF3 standard items

The 0.01mol/L PBS solution of 73 amino acid whose TFF3 albumen of total length (as SEQ 1), pH7.4, contain 55% glycerine, 0.1%BHA and 0.01% thimerosal, the TFF3 final concentration is 240 μ g/mL, uses biological sample diluted to 2400,1200,600,300,150, six gradients of 75pg/mL during use.

(2) TFF3 positive control sample

The PBS solution of 0.01mol/L, the pH7.4 of the TFF3 albumen of total length contains 50% glycerine, 10% human serum, 0.5%BSA, 0.1%BHA and 0.01% thimerosal, and the final concentration of TFF3 is 5 μ g/mL, does dilution in 1: 10000 with the biological sample dilution during use.

(3) how anti-TFF3 is

For many anti-PBS solution, contain 55% glycerine, 0.1%BHA and 0.01% thimerosal, pH7.4, many anti-final concentrations are 1mg/mL, do dilution in 1: 5000 with sample diluent during use.

(4) biological sample dilution

0.01mol/L PBS for the pH7.4 that contains 8% calf serum, 1%Trito X-100,0.01% thimerosal and 1% human IgG.

(5) cleansing solution

Be the PBST:NaCl 8.0g of pH7.4, concentration 0.01mol/L, NaH ₂PO ₄2H ₂O 0.3g, Na ₂HPO ₄12H ₂O2.9g, Tween 200.5g, thimerosal 0.1g adds distilled water to 1000mL, transfers pH to 7.4.

(6) ELIAS secondary antibody

How anti-for the goat-anti rabbit of HRP mark, available from U.S. R﹠D systems, do dilution in 1: 10000 with the biological sample dilution during use.

(7) substrate colour developing liquid

Be the TMB system, available from U.S. SIGMA.

(8) stop buffer

Be 2mol/L H ₂SO ₄Solution.

(9) ELISA Plate

Be the ELISA Plate that antibody embedding in advance and serum seal, air-dry final vacuum packs on superclean bench, takes out in sealing bag during use, and remaining enzyme mark bar is put back in the sealing bag in 4 ℃ of preservations.

Implement 5: the source of biomaterial

Biological sample can derive from the lysate or the extract of human blood or tissue, cell culture.Following mask body is prepared as example with the lysate of PC3 (Human Prostate Cancer Cells) and is illustrated.

(1) 1640 nutrient culture media adds 10%FBS and cultivate PC3 in the T75 plastic bottle, and cell covers culture surface 85% when above, and collecting cell is centrifugal afterwards with the PBS washed cell of precooling 2 times;

(2) add cell extraction buffer (Cell Extraction Buffer), cell extraction buffer addition is decided according to the amount of cell, and both proportionate relationships are 1 * 10 ⁶Individual cell adds the cell extraction buffer of 1mL;

(3) be mixed between or up and down, ice bath was transferred in the microcentrifugal tube after 30 minutes, 13000rpm, 4 ℃ centrifugal 10 minutes, get the protein extract that supernatant is cell pyrolysis liquid or cell.Cell pyrolysis liquid can be stored in-80 ℃, simultaneously, should avoid repeatedly freeze thawing circulation in use.

The cell extraction buffer is formed: 10mM Tris, 100mM NaCl, 1mM EDTA, 1mM EGTA, 1mM NaF, 20mM Na4P2O7,2mM Na3VO4,1%Triton X-100,10%glycerol, 0.1%SDS, 0.5%deoxycholate, 1mM PMSF, protease inhibitor cocktail (available from Sigma).It should be noted that before PMSF and protease inhibitors use to add that other composition can be mixed with solution in advance, places 4 ℃ can deposit 15 days, can deposit 6 months for-20 ℃.

Embodiment 6: TFF3 Determination on content in the human serum

Selecting 1000 gram centrifugal force, centrifugal 15 minutes condition for use preceding 30 minutes, can allow its grumeleuse to the blood sample centrifugal treating.Resulting blood serum sample is analyzed immediately; If can not use at once, sample can be 2-8 ℃ of preservation 7 days ,-20 ℃ of 4 weeks of preservation.Should be noted that the whole blood component should handle as potential danger, avoid pollution simultaneously sample in the reagent bottle.It is as follows that ELISA detects step:

(1) blood serum sample to be checked, standard sample, positive control sample are placed on room temperature temperature balance in advance after, with the biological sample dilution blood serum sample is done 1/2nd, 1/4th and 1/8th dilutions; Standard items are diluted to 2400,1200,600,300,150, six gradients of 75pg/mL; The positive control dilution is done dilution in 1: 10000; Simultaneously, directly select the negative contrast of biological sample dilution for use;

(2) above various kinds is added in the ELISA Plate micropore successively, 2 repeated experiments of each sample were hatched 1 hour for 37 ℃, and TFF3 will be with the capture antibody of surface of solid phase carriers---the antibody that is coated in advance in the hole combines.

(3) through 3 PBST washing, will dilute again in the how anti-adding micropore of 5000 times of anti-TFF3, hatch for the second time, hatch 1 hour for 37 ℃, many anti-detection antibody of playing the part of, and with hatch for the first time in captive TFF3 combine.

(4) how anti-unnecessary of flush away add ELIAS secondary antibody, hatched 40 minutes for 37 ℃, and in hatching for the third time, how anti-in ELIAS secondary antibody and the previous step---detect antibody and combine, the bond of the sandwich shape of one 4 component of formation.

(5) the unconjugated ELIAS secondary antibody of flush away, the substrate 3,3 of adding enzyme ', 5,5 '-(tetramethyl benzidine, TMB), liquid changes into blueness to tetramethyl benzidine in the hole under the catalysis of peroxidase, adds stop buffer, changes into yellow.

(6) content of TFF3 is proportionate in the depth of color and the sample.Measure absorbance (O.D. value) with microplate reader under the 450nm wavelength, the logarithm made from standard items concentration is that X-axis, light absorption value are the typical curve of Y-axis.

(7) TFF3 concentration in the calculation sample: if the measured value of positive control sample and sign value (5 μ g/mL) are compared, its coefficient of variation is less than 15%, illustrate that the mensuration process is reliable, can calculate TFF3 concentration in institute's test sample product according to the typical curve of the rapid gained of previous step.

Although the present invention is described with reference to above concrete embodiment, those skilled in the art should be understood that and can make a lot of changes to it under the prerequisite that does not deviate from spirit of the present invention and scope, also can be replaced with equivalent, to adapt to specific situation, material, material composition, process, program step or each step, all such modifications are all within the scope of the appended claims.

Claims (7)

1. the purposes of ELISA detection kit in the TFF3 content detection of human TFF 3.

2. purposes as claimed in claim 1 is characterized in that, the ELISA that uses the double antibodies sandwich method to carry out TFF3 content detects.

3. purposes as claimed in claim 1 is characterized in that, the reagent of ELISA detection kit is formed the specific monoclonal antibody that comprises TFF3 polypeptide, anti-TFF3 and many anti-, ELIAS secondary antibody, biological sample dilution, cleansing solution, colour developing liquid, stop buffer.

4. as each described purposes in claim 1 and 2, it is characterized in that institute's detected object is human blood, blood plasma, serum, tissue and cell.

5. purposes as claimed in claim 1 is characterized in that, working the molecule of catching TFF3 in the biological sample on the ELISA Plate is the specific antibody of anti-TFF3, comprises monoclonal antibody and how anti-.

6. purposes as claimed in claim 1 is characterized in that, used standard items are the TFF3 polypeptide, corresponding to any one sequence among SEQ 1, SEQ 15, SEQ 16, SEQ 17 and the SEQ 18.

7. purposes as claimed in claim 1 is characterized in that, described detection comprise gained standard items curve and positive control sample compared after, result and the standard items curve with biological sample compares again.