CN104808001A - Time-resolved fluorescence immunoassay method of content of sH2a in serum, and detection kit thereof - Google Patents
Time-resolved fluorescence immunoassay method of content of sH2a in serum, and detection kit thereof Download PDFInfo
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Abstract
The invention discloses a time-resolved fluorescence immunoassay method of the content of sH2a in serum. The method comprises the following steps: adding an sH2a substandard substance or a serum sample to be detected into an anti-sH2a monoclonal antibody coated solid phase material, incubating, washing, adding a lanthanide element ion labeled anti-sH2a monoclonal antibody capable of being paired to the anti-sH2a monoclonal antibody, incubating, washing, adding a dissociation enhancement liquid, shaking, determining the fluorescence intensity, drafting a standard curve according to the determination result of the sH2a substandard substance, and calculating the content of sH2a in the serum sample to be detected according to the standard curve. The invention also discloses a time-resolved fluorescence immunoassay kit of the content of sH2a in the serum. The detection method and the kit have the advantages of wide linear range, zero background, good stability, high sensitivity, strong specificity, high accuracy and short detection time, can be used for detecting the content of sH2a in hepatopathy patient serum, and is convenient for clinic assisted diagnosis of hepatopathy.
Description
Technical field
The invention belongs to protein detection technology field, relate to a kind of protein quantification detection method and detection kit.
Background technology
SH2a is a kind of solubility secreted protein liver specific expression, and molecular weight is about 35KD.It and another kind are also only in the H2 subunit homology of the asialoglycoprotein receptor protein (ASGPR) of liver cell expression, but research shows, sH2a does not participate in the assembling of ASGRP H2 subunit.The precursor protein of sH2a liver cell expression out after, be sheared removal about 5 amino acid in endoplasmic reticulum and become sH2a, be namely released to extracellular, enter blood circulation.In normal human peripheral blood, sH2a keeps higher level, but the content of sH2a significantly declines in the hepatopaths such as hepatic injury, hepatitis, liver fibrosis, therefore, the information of liver function situation can be obtained by detecting sH2a content in patient's blood, being convenient to the auxiliary diagnosis of the hepatopathys such as clinical hepatic injury, hepatitis, liver fibrosis.
Inventor has invented a kind of sH2a immue quantitative detection reagent box (CN103336125A) in previous research work, system adopts Double antibody sandwich-ELISA quantitatively to detect sH2a, but it is narrower still to there is the range of linearity in this invention, detect background higher, detect the problem such as longer consuming time, need further improvement.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of time-resolved fluoroimmunoassay and detection kit thereof of serum sH2a content, have that the range of linearity is wide, zero background, good stability, highly sensitive, high specificity, the advantage such as accuracy is high, detection time is short.
After deliberation, the invention provides following technical scheme:
1. the time-resolved fluoroimmunoassay of serum sH2a content, comprises the following steps:
A. the preparation of insolubilized antibody
Using after the dilution of anti-sH2a monoclonal antibody as coating buffer bag by solid phase material, coating buffer is removed in washing, then closes solid phase material with the damping fluid containing closed protein as confining liquid, and confining liquid is removed in washing, drying, obtained bag is by the solid phase material of anti-sH2a monoclonal antibody;
B. the preparation of labelled antibody
The anti-sH2a monoclonal antibody that selection can be matched with sH2a monoclonal antibody anti-described in steps A, carries out lanthanide ion mark, obtained labelled antibody;
C. detect
The bag obtained in steps A is by the solid phase material of anti-sH2a monoclonal antibody, add sH2a standard items or test serum sample, hatch, washing solid phase material, add the labelled antibody that step B is obtained again, hatch, washing solid phase material, then add the enhancing liquid that dissociates, concussion, measure fluorescence intensity, according to sH2a standard items measurement result drawing standard curve, calculate the sH2a content in test serum sample according to typical curve.
Further, the time-resolved fluoroimmunoassay of described serum sH2a content comprises the following steps:
A. the preparation of insolubilized antibody
To be 50mM, pH using anti-sH2a monoclonal antibody concentration be after the carbonate buffer solution dilution of 9.6 as coating buffer bag by micro reaction plate, coating buffer is removed in washing, again with containing the PBST of bovine serum albumin(BSA) as confining liquid closed porosity reaction plate, confining liquid is removed in washing, drying, obtained bag is by the micro reaction plate of anti-sH2a monoclonal antibody;
B. the preparation of labelled antibody
The anti-sH2a monoclonal antibody that selection can be matched with sH2a monoclonal antibody anti-described in steps A, carries out trivalent europium ion mark, obtained labelled antibody;
C. detect
SH2a standard items PBS is diluted to variable concentrations, as gradient sH2a standard solution; The labelled antibody reaction buffer that step B is obtained dilutes, obtained labelled antibody working fluid; The bag obtained in steps A is by the micro reaction plate of anti-sH2a monoclonal antibody, add above-mentioned gradient sH2a standard solution or test serum sample, hatch, washing micro reaction plate, add labelled antibody working fluid again, hatch, washing micro reaction plate, add the enhancing liquid that dissociates again, concussion, measure fluorescence intensity with time resolved immuno fluorometric analyser in excitation wavelength 340nm, emission wavelength 614nm, according to gradient sH2a standard solution measurement result drawing standard curve, calculate the sH2a content in test serum sample according to typical curve.
Further, the time-resolved fluoroimmunoassay of described serum sH2a content comprises the following steps:
A. the preparation of insolubilized antibody
To be 0.05mol/L, pH using anti-sH2a monoclonal antibody concentration be 9.6 carbonate buffer solution be diluted to 5 μ g/ml as coating buffer, with 100 μ l/ hole bags by micro reaction plate, 4 DEG C of bags are spent the night, coating buffer is removed in washing, then with containing mass percentage be the PBST of bovine serum albumin(BSA) of 1% as confining liquid, with 100 μ l/ hole closed porosity reaction plates, close 2 hours for 37 DEG C, confining liquid is removed in washing, 37 DEG C of dryings, and obtained bag is by the micro reaction plate of anti-sH2a monoclonal antibody;
B. the preparation of labelled antibody
The anti-sH2a monoclonal antibody that selection can be matched with sH2a monoclonal antibody anti-described in steps A, carries out trivalent europium ion mark, obtained labelled antibody;
C. detect
SH2a standard items PBS is diluted to respectively 0.1,1,10,100,1000,10000ng/ml, as gradient sH2a standard solution, by labelled antibody reaction buffer obtained for step B by volume for 1:5000 dilutes, obtained labelled antibody working fluid, the bag obtained in steps A is by the micro reaction plate of anti-sH2a monoclonal antibody, add above-mentioned gradient sH2a standard solution or test serum sample, 100 μ l/ holes, hatch 1 hour for 37 DEG C, washing micro reaction plate, add labelled antibody working fluid again, 100 μ l/ holes, hatch 1 hour for 37 DEG C, washing micro reaction plate, add the enhancing liquid that dissociates again, 100 μ l/ holes, shake 20 minutes, with time resolved immuno fluorometric analyser in excitation wavelength 340nm, emission wavelength 614nm measures fluorescence intensity, according to gradient sH2a standard solution measurement result drawing standard curve, the sH2a content in test serum sample is calculated according to typical curve.
Further, labelled antibody described in step B is the Tris-HCl damping fluid marking anti-sH2a monoclonal antibody and bovine serum albumin(BSA) containing trivalent europium ion; The standard items of sH2a described in step C are the Tris-HCl damping fluid containing sH2a, bovine serum albumin(BSA) and antiseptic; Reaction buffer described in step C is the Tris-HCl damping fluid containing bovine serum albumin(BSA), ethylenediamine tetraacetic acid, Tween40, Inert dyes and antiseptic; The enhancing liquid that dissociates described in step C is the aqueous solution be made up of triton x-100, glacial acetic acid and sequestrant; Wash all using the Tris-HCl damping fluid containing Tween20 and antiseptic as cleansing solution described in steps A and C.
Further, labelled antibody described in step B is the Tris-HCl damping fluid marking anti-sH2a monoclonal antibody and bovine serum albumin(BSA) containing trivalent europium ion, wherein trivalent europium ion marks the concentration of anti-sH2a monoclonal antibody is 0.1 μ g/ml, the mass percentage concentration of bovine serum albumin(BSA) is the concentration of 0.5%, Tris-HCl damping fluid be 50mM, pH is 7.8; The standard items of sH2a described in step C are the Tris-HCl damping fluid containing sH2a, bovine serum albumin(BSA) and antiseptic, wherein the mass percentage concentration of bovine serum albumin(BSA) is 0.5%, the mass percentage concentration of antiseptic is the concentration of 0.05%, Tris-HCl damping fluid be 50mM, pH is 7.8; Reaction buffer described in step C is the Tris-HCl damping fluid containing bovine serum albumin(BSA), ethylenediamine tetraacetic acid, Tween40, Inert dyes amaranth and antiseptic, wherein the mass percentage concentration of bovine serum albumin(BSA) is 0.5%, the mass percentage concentration of ethylenediamine tetraacetic acid and Tween40 is respectively 0.01%, the mass percentage concentration of Inert dyes is 0.02%, the mass percentage concentration of antiseptic is the concentration of 0.05%, Tris-HCl damping fluid be 50mM, pH is 7.8; The enhancing liquid that dissociates described in step C is the aqueous solution be made up of triton x-100, glacial acetic acid and sequestrant nitrilotriacetic acid, and wherein the mass percentage concentration of triton x-100 is 0.1%, and the mass percentage concentration of glacial acetic acid is 0.01%, and the concentration of sequestrant is 15 μMs; Wash all using the Tris-HCl damping fluid containing Tween20 and antiseptic as cleansing solution described in steps A and C, wherein the mass percentage concentration of Tween20 is 0.01%, the mass percentage concentration of antiseptic is the concentration of 0.05%, Tris-HCl damping fluid be 50mM, pH is 7.8.
2. the time-resolved fluoroimmunoassay detection kit of serum sH2a content, comprises following component: wrap by the solid phase material of anti-sH2a monoclonal antibody, the anti-sH2a monoclonal antibody of lanthanide ion mark, and sH2a standard items; The anti-sH2a monoclonal antibody that the anti-sH2a monoclonal antibody of described solid phase material wrapping quilt can mark with lanthanide ion is matched.
Further, described bag is wrap by the micro reaction plate of anti-sH2a monoclonal antibody by the solid phase material of anti-sH2a monoclonal antibody, and the anti-sH2a monoclonal antibody of described lanthanide ion mark is the Tris-HCl damping fluid marking anti-sH2a monoclonal antibody and bovine serum albumin(BSA) containing trivalent europium ion; Described sH2a standard items are the Tris-HCl damping fluid containing sH2a, bovine serum albumin(BSA) and antiseptic.
Further, the anti-sH2a monoclonal antibody of described lanthanide ion mark is the Tris-HCl damping fluid marking anti-sH2a monoclonal antibody and bovine serum albumin(BSA) containing trivalent europium ion, wherein trivalent europium ion marks the concentration of anti-sH2a monoclonal antibody is 0.1 μ g/ml, the mass percentage concentration of bovine serum albumin(BSA) is the concentration of 0.5%, Tris-HCl damping fluid be 50mM, pH is 7.8; Described sH2a standard items are the Tris-HCl damping fluid containing sH2a, bovine serum albumin(BSA) and antiseptic, wherein the mass percentage concentration of bovine serum albumin(BSA) is 0.5%, the mass percentage concentration of antiseptic is the concentration of 0.05%, Tris-HCl damping fluid be 50mM, pH is 7.8.
Further, the time-resolved fluoroimmunoassay detection kit of described serum sH2a content also comprises reaction buffer, dissociating strengthens liquid and cleansing solution; Described reaction buffer is the Tris-HCl damping fluid containing bovine serum albumin(BSA), ethylenediamine tetraacetic acid, Tween40, Inert dyes and antiseptic; The described enhancing liquid that dissociates is the aqueous solution be made up of triton x-100, glacial acetic acid and sequestrant; Described cleansing solution is the Tris-HCl damping fluid containing Tween20 and antiseptic.
Further, described reaction buffer is the Tris-HCl damping fluid containing bovine serum albumin(BSA), ethylenediamine tetraacetic acid, Tween40, Inert dyes amaranth and antiseptic, wherein the mass percentage concentration of bovine serum albumin(BSA) is 0.5%, the mass percentage concentration of ethylenediamine tetraacetic acid and Tween40 is respectively 0.01%, the mass percentage concentration of Inert dyes is 0.02%, the mass percentage concentration of antiseptic is the concentration of 0.05%, Tris-HCl damping fluid be 50mM, pH is 7.8; The described enhancing liquid that dissociates is the aqueous solution be made up of triton x-100, glacial acetic acid and sequestrant nitrilotriacetic acid, and wherein the mass percentage concentration of triton x-100 is 0.1%, and the mass percentage concentration of glacial acetic acid is 0.01%, and the concentration of sequestrant is 15 μMs; Described cleansing solution is the Tris-HCl damping fluid containing Tween20 and antiseptic, and wherein the mass percentage concentration of Tween20 is 0.01%, and the mass percentage concentration of antiseptic is the concentration of 0.05%, Tris-HCl damping fluid be 50mM, pH is 7.8.
Beneficial effect of the present invention is: the time-resolved fluoroimmunoassay and the detection kit thereof that the invention provides a kind of serum sH2a content, have that the range of linearity is wide, zero background, good stability, highly sensitive, high specificity, the advantage such as accuracy is high, detection time is short, can be used for the hepatopathy patient serum sH2a content detection such as hepatic injury, hepatitis, liver fibrosis, be convenient to the auxiliary diagnosis of the hepatopathys such as clinical hepatic injury, hepatitis, liver fibrosis.
Accompanying drawing explanation
In order to make object of the present invention, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing and being described:
Fig. 1 is the agarose gel electrophoresis figure of sH2a genetic fragment pcr amplification product, and wherein M is DNA molecular amount standard, and 1 and 2 is pcr amplification product.
Fig. 2 is the SDS-PAGE figure of pET28a-sH2a conversion BL21 (DE3) the bacteria lysis supernatant of IPTG induction.
Fig. 3 is that the western blotting of sH2a recombinant protein schemes, the albumen swimming lane of wherein 1,2,3,4,5,6 difference corresponding diagram, 2 six variable concentrations IPTG inductions.
Fig. 4 is the anti-sH2a monoclonal antibody of SDS-PAGE purification Identification.
Fig. 5 is the anti-sH2a monoclonal antibody of western blotting purification Identification.
Fig. 6 is the sH2a typical curve of the time-resolved fluoroimmunoassay of serum sH2a content of the present invention.
Fig. 7 is the ROC curve drawn with serum of cirrhosis patients group and normal human serum group measurement result.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in preferred embodiment, usually conveniently condition, or carry out according to the condition that manufacturer advises.
Embodiment 1, the expression of sH2a recombinant protein, Isolation and characterization
With liver cancer tissue cDNA for template, with 5 '-CGGGATCCATGGCCAAGGACTTTCAAGATATCCA-3 ' (SEQ ID No.1) and 5 '-CGGAATTCTCAGGCCACCTCGCCGGT-3 ' (SEQ ID No.2) for primer, pcr amplification sH2a genetic fragment (Fig. 1), after cutting glue recovery purifying, be connected to cloning vector pCR2.1 and carry out order-checking qualification, again by sH2a gene fragment clone correct for Sequence Identification in expression vector pET28a (+), restriction enzyme site is BamH I and EcoRI, obtain recombinant plasmid pET28a-sH2a, this recombinant plasmid is containing 6 × HIS label, be convenient to follow-up protein purification.
By recombinant plasmid pET28a-sH2a transformation of E. coli BL21 (DE3), picking transforms successfully clone, and 37 DEG C are cultured to OD600nm value about 0.6, add 0.2 respectively, 0.5,1mM IPTG, continue to cultivate 3h and induce destination protein expression.After induction terminates, ultrasonic degradation thalline, gets cracking supernatant, purifies with HIS protein purification post, obtains sH2a recombinant protein.
Identify by the activity of western blotting method to sH2a recombinant protein.First, get cracking supernatant and carry out SDS-PAGE, set up the pET28a empty carrier without IPTG induction to transform BL21 (DE3) bacteria lysis supernatant in contrast, result shows simultaneously, destination protein size is 35KD(Fig. 2), conform to sH2a recombinant protein theoretical value, then, transfer printing instrument is utilized by destination protein to be transferred on NC film, used by NC film the phosphate buffer containing 1%BSA to close in 4 DEG C to spend the night, film is washed again with the phosphate buffer containing 0.5%Tween20, add mouse-anti people sH2a antibody again in 37 DEG C of incubated under agitation 2 hours, against murine IgG ELIAS secondary antibody incubated under agitation is added again 1 hour after washing film, finally wash film, ECL develops the color, result shows, obvious specific band (Fig. 3) is had in destination protein corresponding position, and in contrast corresponding position without band, prove that gained sH2a recombinant protein has good biologic activity.
The preparation of embodiment 2, anti-sH2a monoclonal antibody
Get BALB/c female mice 2 in 6 week age, carry out immunity according to following steps:
First immunisation: by sH2a recombinant protein solution and not formula Freund's complete adjuvant by volume 1:1 mix, after fully emulsified, in mouse back subcutaneous point 2 injection 0.1ml(sH2a recombinant protein 30 μ g/ only);
Second time immunity: interval is after 14 days, by sH2a recombinant protein solution and not formula Freund's incomplete adjuvant by volume 1:1 mix, after fully emulsified, inject 0.1ml(sH2a recombinant protein 50 μ g/ altogether in the mouse both sides subcutaneous branch of pars inguinalis);
Third time immunity: interval is after 14 days, by sH2a recombinant protein solution and not formula Freund's incomplete adjuvant by volume 1:1 mix, after fully emulsified, inject 0.1ml(sH2a recombinant protein 50 μ g/ altogether in shoulder rear portion, mouse both sides and subcutaneous abdomen branch);
Final immunization: interval is after 10 days, and tail vein blood ELISA detects serum titer, reaches 1:10000(table 1) booster immunization afterwards, only inject sH2a recombinant protein solution 0.1ml(sH2a recombinant protein 50 μ g/ in mouse peritoneal).
The titration of table 1 Mouse Antisera
Final immunization was taken a blood sample after 3 days, got spleen and carried out Fusion of Cells, utilized HAT Selective agar medium to select hybridoma on 96 porocyte culture plates, and utilized indirect ELISA method to identify the cell producing anti-sH2a antibody.Result obtains the hybridoma cell strain of 9 strain secretion specificity sH2a antibody, called after 1C3D4,1H4G5,2B8C6,2D4H5,3D7F4,4A7D8,4B6C10,4E2F5 and 5C3E4 respectively.Cell fermentation device is utilized to cultivate the hybridoma cell strain obtained, collecting cell culture supernatant, with the anti-sH2a monoclonal antibody of Protein A-Sepharose affinity column separation and purification from cells and supernatant, and carry out SDS-PAGE and western blotting qualification (Fig. 4, Fig. 5).
The pairing screening of embodiment 3, anti-sH2a monoclonal antibody
Be coated in solid phase microwell plate respectively by the anti-sH2a monoclonal antibody of above-mentioned 9 strain purifying, the anti-sH2a monoclonal antibody of all the other except self 8 strain marked with Eu3+ matches between two tests.Result shows, and anti-sH2a monoclonal antibody 2D4H5 and 4A7D8 can match and be used for setting up reaction system (table 2).
The screening (double-antibody method wraps by 2D4H5) of table 2sH2a pairing monoclonal antibody
Embodiment 4, the time-resolved fluoroimmunoassay of serum sH2a content and the structure of detection kit thereof
1, the structure of the time-resolved fluoroimmunoassay of serum sH2a content
(1) determination of antibody best effort concentration is detected
Mouse-anti people sH2a monoclonal antibody 2D4H5 carbonate buffer solution (concentration is 50mM, pH is 9.6) is diluted to 5 μ g/ml, with 100 μ l/ hole bags by 96 orifice plates, 4 DEG C of bags are spent the night, with cleansing solution (for containing 0.01wt%Tween20 and 0.05wt% antiseptic, concentration to be 50mM, pH be 7.8 Tris-HCl damping fluid, lower same) wash plate 2 times, add the PBST containing 1wt%BSA, 100 μ l/ holes, close 2 hours for 37 DEG C, wash plate 2 times, 37 DEG C of oven dry, obtained bag is by mouse-anti people sH2a monoclonal antibody 2D4H5(5 μ g/ml) 96 orifice plates, by sH2a standard items (for containing sH2a, 0.5wt%BSA and 0.05wt% antiseptic, concentration to be 50mM, pH be 7.8 Tirs-HCl damping fluid, lower together) be diluted to 500ng/ml with PBS, obtain sH2a standard solution, the mouse-anti people sH2a monoclonal antibody 4A7D8(marked by trivalent europium ion is for marking mouse-anti people sH2a monoclonal antibody 4A7D8 and 0.5wt%BSA containing 0.1 μ g/ml trivalent europium ion, concentration is 50mM, pH is the Tris-HCl damping fluid of 7.8, lower same) use reaction buffer (for containing 0.5wt%BSA, 0.01wt%EDTA, 0.01wt%Tween40, 0.02wt% Inert dyes amaranth and 0.05wt% antiseptic, concentration is 50mM, pH is the Tris-HCl damping fluid of 7.8, lower same) be respectively 1:400 by volume, 1:1000, 1:2000, 1:4000, 1:5000, 1:8000, 1:10000 dilutes, the labelled antibody working fluid of obtained variable concentrations, bag by mouse-anti people sH2a monoclonal antibody 2D4H5(5 μ g/ml) 96 orifice plates in, add sH2a standard solution, 100 μ l/ holes, 37 DEG C of incubations 1 hour, plate is washed 3 times with cleansing solution, add the labelled antibody working fluid of variable concentrations respectively, 100 μ l/ holes, 37 DEG C of incubations 1 hour, plate is washed 5 times with cleansing solution, adding dissociates strengthens liquid (by 0.1wt% triton x-100, the aqueous solution of 0.01wt% glacial acetic acid and 15 μMs of sequestrant nitrilotriacetic acid compositions, lower same), 100 μ l/ holes, shake 20 minutes, with time resolved immuno fluorometric analyser in excitation wavelength 340nm, emission wavelength 614nm measures fluorescence intensity.Result shows, and the best effort concentration detecting the mouse-anti people sH2a monoclonal antibody 4A7D8 of antibody and rare-earth europium ion mark is 1:5000(table 3).
Table 3 detects the determination of antibody best effort concentration
(2) determination of the tested antigen range of linearity
Mouse-anti people sH2a monoclonal antibody 2D4H5 carbonate coating buffer is diluted to 5 μ g/ml, with 100 μ l/ hole bags by 96 orifice plates, 4 DEG C of bags are spent the night, wash plate 2 times with cleansing solution, add the PBST containing 1wt%BSA, 100 μ l/ holes, close 2 hours for 37 DEG C, wash plate 2 times, 37 DEG C of oven dry, obtained bag is by mouse-anti people sH2a monoclonal antibody 2D4H5(5 μ g/ml) 96 orifice plates; SH2a standard items PBS is diluted to variable concentrations (being respectively 0.1,1,5,10,50,100,500,1000,10000ng/ml), respectively as gradient sH2a standard solution; The mouse-anti people sH2a monoclonal antibody 4A7D8 reaction buffer marked by trivalent europium ion is 1:5000 dilution by volume, obtained labelled antibody working fluid; Bag by mouse-anti people sH2a monoclonal antibody 2D4H5(5 μ g/ml) 96 orifice plates in, add gradient sH2a standard solution, 100 μ l/ holes, 37 DEG C of incubations 1 hour, wash plate 3 times with cleansing solution, add labelled antibody working fluid, 100 μ l/ holes, 37 DEG C of incubations 1 hour, wash plate 5 times with cleansing solution, add the enhancing liquid that dissociates, 100 μ l/ holes, shake 20 minutes, measure fluorescence intensity with time resolved immuno fluorometric analyser in excitation wavelength 340nm, emission wavelength 614nm.Result shows, and the range of linearity of tested antigen and sH2a is 0.1-10000ng/ml(Fig. 6).
(3) structure of the time-resolved fluoroimmunoassay of serum sH2a content
Determine that the step of the time-resolved fluoroimmunoassay of serum sH2a content is as follows:
Mouse-anti people sH2a monoclonal antibody 2D4H5 carbonate coating buffer is diluted to 5 μ g/ml, with 100 μ l/ hole bags by 96 orifice plates, 4 DEG C of bags are spent the night, wash plate 2 times with cleansing solution, add the PBST containing 1wt%BSA, 100 μ l/ holes, close 2 hours for 37 DEG C, wash plate 2 times, 37 DEG C of oven dry, obtained bag is by mouse-anti people sH2a monoclonal antibody 2D4H5(5 μ g/ml) 96 orifice plates, sH2a standard items PBS is diluted to variable concentrations (being respectively 0.1,1,10,100,1000,10000ng/ml), respectively as gradient sH2a standard solution, the mouse-anti people sH2a monoclonal antibody 4A7D8 reaction buffer marked by trivalent europium ion is 1:5000 dilution by volume, obtained labelled antibody working fluid, bag by mouse-anti people sH2a monoclonal antibody 2D4H5(5 μ g/ml) 96 orifice plates in, add gradient sH2a standard solution, 100 μ l/ holes, 37 DEG C of incubations 1 hour, plate is washed 3 times with cleansing solution, add labelled antibody working fluid, 100 μ l/ holes, 37 DEG C of incubations 1 hour, plate is washed 5 times with cleansing solution, add the enhancing liquid that dissociates, 100 μ l/ holes, shake 20 minutes, with time resolved immuno fluorometric analyser in excitation wavelength 340nm, emission wavelength 614nm measures fluorescence intensity, according to the measurement result drawing standard curve of gradient sH2a standard solution, sH2a concentration in blood serum sample is calculated according to typical curve.
2, the structure of the time-resolved fluoroimmunoassay detection kit of serum sH2a content
According to the time-resolved fluoroimmunoassay of serum sH2a content, determine the composed as follows of detection kit:
Embodiment 5, the time-resolved fluoroimmunoassay of serum sH2a content and the application of detection kit thereof
Blood serum sample 100 example of liver cirrhosis patient is collected from Shanghai Huashan Hospital; Normal blood donation personnel blood serum sample 100 example is collected from blood station.Use time-resolved fluoroimmunoassay and the detection kit thereof of serum sH2a content of the present invention, detect the sH2a content in liver cirrhosis patient and normal human serum respectively.Result shows, and the sH2a content in normal human serum is about 300ng/ml; SH2a content in serum of cirrhosis patients is significantly lower than the sH2a content in normal human serum.Serum of cirrhosis patients group and normal human serum group measurement result are analyzed, draw ROC curve, display is 300ng/ml for distinguishing truncation points (cut-off point) value of liver cirrhosis patient and normal person, ROC area under curve is 0.845, diagnostic sensitivity is 75%, and specificity is 95.7%(Fig. 7).
What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.
Claims (10)
1. the time-resolved fluoroimmunoassay of serum sH2a content, is characterized in that, comprises the following steps:
A. the preparation of insolubilized antibody
Using after the dilution of anti-sH2a monoclonal antibody as coating buffer bag by solid phase material, coating buffer is removed in washing, then closes solid phase material with the damping fluid containing closed protein as confining liquid, and confining liquid is removed in washing, drying, obtained bag is by the solid phase material of anti-sH2a monoclonal antibody;
B. the preparation of labelled antibody
The anti-sH2a monoclonal antibody that selection can be matched with sH2a monoclonal antibody anti-described in steps A, carries out lanthanide ion mark, obtained labelled antibody;
C. detect
The bag obtained in steps A is by the solid phase material of anti-sH2a monoclonal antibody, add sH2a standard items or test serum sample, hatch, washing solid phase material, add the labelled antibody that step B is obtained again, hatch, washing solid phase material, then add the enhancing liquid that dissociates, concussion, measure fluorescence intensity, according to sH2a standard items measurement result drawing standard curve, calculate the sH2a content in test serum sample according to typical curve.
2. the time-resolved fluoroimmunoassay of serum sH2a content as claimed in claim 1, is characterized in that, comprise the following steps:
A. the preparation of insolubilized antibody
To be 50mM, pH using anti-sH2a monoclonal antibody concentration be after the carbonate buffer solution dilution of 9.6 as coating buffer bag by micro reaction plate, coating buffer is removed in washing, again with containing the PBST of bovine serum albumin(BSA) as confining liquid closed porosity reaction plate, confining liquid is removed in washing, drying, obtained bag is by the micro reaction plate of anti-sH2a monoclonal antibody;
B. the preparation of labelled antibody
The anti-sH2a monoclonal antibody that selection can be matched with sH2a monoclonal antibody anti-described in steps A, carries out trivalent europium ion mark, obtained labelled antibody;
C. detect
SH2a standard items PBS is diluted to variable concentrations, as gradient sH2a standard solution; The labelled antibody reaction buffer that step B is obtained dilutes, obtained labelled antibody working fluid; The bag obtained in steps A is by the micro reaction plate of anti-sH2a monoclonal antibody, add above-mentioned gradient sH2a standard solution or test serum sample, hatch, washing micro reaction plate, add labelled antibody working fluid again, hatch, washing micro reaction plate, add the enhancing liquid that dissociates again, concussion, measure fluorescence intensity with time resolved immuno fluorometric analyser in excitation wavelength 340nm, emission wavelength 614nm, according to gradient sH2a standard solution measurement result drawing standard curve, calculate the sH2a content in test serum sample according to typical curve.
3. the time-resolved fluoroimmunoassay of serum sH2a content as claimed in claim 2, is characterized in that, comprise the following steps:
A. the preparation of insolubilized antibody
To be 0.05mol/L, pH using anti-sH2a monoclonal antibody concentration be 9.6 carbonate buffer solution be diluted to 5 μ g/ml as coating buffer, with 100 μ l/ hole bags by micro reaction plate, 4 DEG C of bags are spent the night, coating buffer is removed in washing, then with containing mass percentage be the PBST of bovine serum albumin(BSA) of 1% as confining liquid, with 100 μ l/ hole closed porosity reaction plates, close 2 hours for 37 DEG C, confining liquid is removed in washing, 37 DEG C of dryings, and obtained bag is by the micro reaction plate of anti-sH2a monoclonal antibody;
B. the preparation of labelled antibody
The anti-sH2a monoclonal antibody that selection can be matched with sH2a monoclonal antibody anti-described in steps A, carries out trivalent europium ion mark, obtained labelled antibody;
C. detect
SH2a standard items PBS is diluted to respectively 0.1,1,10,100,1000,10000ng/ml, as gradient sH2a standard solution, by labelled antibody reaction buffer obtained for step B by volume for 1:5000 dilutes, obtained labelled antibody working fluid, the bag obtained in steps A is by the micro reaction plate of anti-sH2a monoclonal antibody, add above-mentioned gradient sH2a standard solution or test serum sample, 100 μ l/ holes, hatch 1 hour for 37 DEG C, washing micro reaction plate, add labelled antibody working fluid again, 100 μ l/ holes, hatch 1 hour for 37 DEG C, washing micro reaction plate, add the enhancing liquid that dissociates again, 100 μ l/ holes, shake 20 minutes, with time resolved immuno fluorometric analyser in excitation wavelength 340nm, emission wavelength 614nm measures fluorescence intensity, according to gradient sH2a standard solution measurement result drawing standard curve, the sH2a content in test serum sample is calculated according to typical curve.
4. the time-resolved fluoroimmunoassay of serum sH2a content as claimed in claim 2 or claim 3, it is characterized in that, labelled antibody described in step B is the Tris-HCl damping fluid marking anti-sH2a monoclonal antibody and bovine serum albumin(BSA) containing trivalent europium ion; The standard items of sH2a described in step C are the Tris-HCl damping fluid containing sH2a, bovine serum albumin(BSA) and antiseptic; Reaction buffer described in step C is the Tris-HCl damping fluid containing bovine serum albumin(BSA), ethylenediamine tetraacetic acid, Tween40, Inert dyes amaranth and antiseptic; The enhancing liquid that dissociates described in step C is the aqueous solution be made up of triton x-100, glacial acetic acid and sequestrant nitrilotriacetic acid; Wash all using the Tris-HCl damping fluid containing Tween20 and antiseptic as cleansing solution described in steps A and C.
5. the time-resolved fluoroimmunoassay of serum sH2a content as claimed in claim 4, it is characterized in that, labelled antibody described in step B is the Tris-HCl damping fluid marking anti-sH2a monoclonal antibody and bovine serum albumin(BSA) containing trivalent europium ion, wherein trivalent europium ion marks the concentration of anti-sH2a monoclonal antibody is 0.1 μ g/ml, the mass percentage concentration of bovine serum albumin(BSA) is the concentration of 0.5%, Tris-HCl damping fluid be 50mM, pH is 7.8; The standard items of sH2a described in step C are the Tris-HCl damping fluid containing sH2a, bovine serum albumin(BSA) and antiseptic, wherein the mass percentage concentration of bovine serum albumin(BSA) is 0.5%, the mass percentage concentration of antiseptic is the concentration of 0.05%, Tris-HCl damping fluid be 50mM, pH is 7.8; Reaction buffer described in step C is the Tris-HCl damping fluid containing bovine serum albumin(BSA), ethylenediamine tetraacetic acid, Tween40, Inert dyes amaranth and antiseptic, wherein the mass percentage concentration of bovine serum albumin(BSA) is 0.5%, the mass percentage concentration of ethylenediamine tetraacetic acid and Tween40 is respectively 0.01%, the mass percentage concentration of Inert dyes is 0.02%, the mass percentage concentration of antiseptic is the concentration of 0.05%, Tris-HCl damping fluid be 50mM, pH is 7.8; The enhancing liquid that dissociates described in step C is the aqueous solution be made up of triton x-100, glacial acetic acid and sequestrant nitrilotriacetic acid, and wherein the mass percentage concentration of triton x-100 is 0.1%, and the mass percentage concentration of glacial acetic acid is 0.01%, and the concentration of sequestrant is 15 μMs; Wash all using the Tris-HCl damping fluid containing Tween20 and antiseptic as cleansing solution described in steps A and C, wherein the mass percentage concentration of Tween20 is 0.01%, the mass percentage concentration of antiseptic is the concentration of 0.05%, Tris-HCl damping fluid be 50mM, pH is 7.8.
6. the time-resolved fluoroimmunoassay detection kit of serum sH2a content, is characterized in that, comprises following component: wrap by the solid phase material of anti-sH2a monoclonal antibody, the anti-sH2a monoclonal antibody of lanthanide ion mark, and sH2a standard items; The anti-sH2a monoclonal antibody that the anti-sH2a monoclonal antibody of described solid phase material wrapping quilt can mark with lanthanide ion is matched.
7. the time-resolved fluoroimmunoassay detection kit of serum sH2a content as claimed in claim 6, it is characterized in that, described bag is wrap by the micro reaction plate of anti-sH2a monoclonal antibody by the solid phase material of anti-sH2a monoclonal antibody, and the anti-sH2a monoclonal antibody of described lanthanide ion mark is the Tris-HCl damping fluid marking anti-sH2a monoclonal antibody and bovine serum albumin(BSA) containing trivalent europium ion; Described sH2a standard items are the Tris-HCl damping fluid containing sH2a, bovine serum albumin(BSA) and antiseptic.
8. the time-resolved fluoroimmunoassay detection kit of serum sH2a content as claimed in claim 7, it is characterized in that, the anti-sH2a monoclonal antibody of described lanthanide ion mark is the Tris-HCl damping fluid marking anti-sH2a monoclonal antibody and bovine serum albumin(BSA) containing trivalent europium ion, wherein trivalent europium ion marks the concentration of anti-sH2a monoclonal antibody is 0.1 μ g/ml, the mass percentage concentration of bovine serum albumin(BSA) is the concentration of 0.5%, Tris-HCl damping fluid be 50mM, pH is 7.8; Described sH2a standard items are the Tris-HCl damping fluid containing sH2a, bovine serum albumin(BSA) and antiseptic, wherein the mass percentage concentration of bovine serum albumin(BSA) is 0.5%, the mass percentage concentration of antiseptic is the concentration of 0.05%, Tris-HCl damping fluid be 50mM, pH is 7.8.
9. the time-resolved fluoroimmunoassay detection kit of the serum sH2a content as described in any one of claim 6-8, is characterized in that, also comprise reaction buffer, dissociating strengthens liquid and cleansing solution; Described reaction buffer is the Tris-HCl damping fluid containing bovine serum albumin(BSA), ethylenediamine tetraacetic acid, Tween40, Inert dyes and antiseptic; The described enhancing liquid that dissociates is the aqueous solution be made up of triton x-100, glacial acetic acid and sequestrant; Described cleansing solution is the Tris-HCl damping fluid containing Tween20 and antiseptic.
10. the time-resolved fluoroimmunoassay detection kit of serum sH2a content as claimed in claim 9, it is characterized in that, described reaction buffer is for containing bovine serum albumin(BSA), ethylenediamine tetraacetic acid, Tween40, the Tris-HCl damping fluid of Inert dyes amaranth and antiseptic, wherein the mass percentage concentration of bovine serum albumin(BSA) is 0.5%, the mass percentage concentration of ethylenediamine tetraacetic acid and Tween40 is respectively 0.01%, the mass percentage concentration of Inert dyes is 0.02%, the mass percentage concentration of antiseptic is 0.05%, the concentration of Tris-HCl damping fluid is 50mM, pH is 7.8, the described enhancing liquid that dissociates is the aqueous solution be made up of triton x-100, glacial acetic acid and sequestrant nitrilotriacetic acid, and wherein the mass percentage concentration of triton x-100 is 0.1%, and the mass percentage concentration of glacial acetic acid is 0.01%, and the concentration of sequestrant is 15 μMs, described cleansing solution is the Tris-HCl damping fluid containing Tween20 and antiseptic, and wherein the mass percentage concentration of Tween20 is 0.01%, and the mass percentage concentration of antiseptic is the concentration of 0.05%, Tris-HCl damping fluid be 50mM, pH is 7.8.
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| CN113917162A (en) * | 2021-12-14 | 2022-01-11 | 江苏为真生物医药技术股份有限公司 | Application of asialoglycoprotein receptor fragment sH2a as marker |
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