CN105486855A - Improved indirect enzyme-linked immunosorbent assay method - Google Patents
Improved indirect enzyme-linked immunosorbent assay method Download PDFInfo
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- CN105486855A CN105486855A CN201510845665.7A CN201510845665A CN105486855A CN 105486855 A CN105486855 A CN 105486855A CN 201510845665 A CN201510845665 A CN 201510845665A CN 105486855 A CN105486855 A CN 105486855A
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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
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
Abstract
The invention discloses an indirect enzyme-linked immunosorbent assay method and provides an indirect enzyme-linked immunosorbent assay method for detecting a sample to be detected through a low-purity antigen. The indirect enzyme-linked immunosorbent assay method comprises 1, adding an antibody capable of bonding to a low-purity antigen into a pore plate and carrying out coating to obtain an antibody-coated pore plate, 2, adding a low-purity antigen into pores of the antibody-coated pore plate and carrying out antigen capture to obtain an antigen capture pore plate and 3, orderly adding a detected sample capable of bonding to the low-purity antigen, a second antibody capable of bonding to the sample to be detected and a chromogenic reaction liquid into the pore plate and reading an OD value of the sample to be detected at specific wavelength so that indirect enzyme-linked immunosorbent assay of the detected sample is realized. An experiment proves that the method improves purity of an antigen to be coated and reduces an antigen coating amount.
Description
Technical field
The present invention relates to enzyme linked immunosorbent assay, particularly relate to indirect enzyme-linked immunosorbent assay.
Background technology
Indirect enzyme-linked immunosorbent assay (ELISA) detects the most frequently used method of antibody, and its principle is utilize the antibody of enzyme labeling to examine antibody to detect being subject to of being combined with solid phase, therefore is called indirect method.Same enzyme mark antiantibody just can be utilized to set up the method detecting corresponding antibodies as long as the advantage of indirect method is conversion envelope antigen.The successful key of indirect method is the purity of antigen.Although sometimes antigen coatedly actual effective result also can be obtained with slightly carrying, purifying should be given as far as possible, to improve the specificity of test.Should note except the impurity reacted with general health human serum that deenergizes especially, such as, be the recombinant antigen of engineering enzyme with E.Coli, as wherein containing E.Coli composition, probably reacts with being subject to the anti-E.Coli antibody in E.Coli infected person anteserum.In addition as contained unrelated protein in antigen, bag also can be affected because of competitive Adsorption by effect.Recombinant protein is due to its physical characteristics (self structure, Acidity of Aikalinity etc.) difference in prokaryotic expression, and some cannot reach expection purity unavoidably, and affects the accuracy of ELISA.
Summary of the invention
An object of the present invention is to provide a kind of indirect enzyme-linked immunosorbent assay utilizing low-purity antigen to detect sample to be tested.
Indirect enzyme-linked immunosorbent assay provided by the invention, comprises the steps:
1) antibody is added orifice plate and carry out bag quilt, obtain coated antibody orifice plate;
Described antibody is the antibody of label in low-purity antigen, and described low-purity antigen is the fusion after albumen is connected with label;
2) described low-purity antigen is added the described each hole of coated antibody orifice plate, carry out antigen capture, obtain capture antigen orifice plate;
3) successively sample to be tested, ELIAS secondary antibody are added orifice plate, reaction, detect OD value, the indirect enzyme-linked immunosorbent assay realizing sample to be tested detects.
In said method, described low-purity antigen is the antigen that purity is less than or equal to 70%.
In said method, described label is HIS label;
Described ELIAS secondary antibody is the Goat anti human IgG of alkali phosphatase enzyme mark.
Above-mentioned method is also the scope of protection of the invention whether detect sample to be tested be application in paraneoplastic pemphigus sample;
Or above-mentioned method is also the scope of protection of the invention whether detect people to be measured be application in Features of Paraneoplastic Pemphigus.
Another object of the present invention is to provide and a kind ofly utilizes low-purity antigen to detect or whether couple candidate detection sample to be tested is the method for paraneoplastic pemphigus sample.
Method provided by the invention, comprises the steps:
1) antibody is added orifice plate and carry out bag quilt, obtain coated antibody orifice plate;
Described antibody is the antibody of label in low-purity antigen, and described low-purity antigen is the fusion after albumen is connected with label;
2) described low-purity antigen is added the described each hole of coated antibody orifice plate, carry out antigen capture, obtain capture antigen orifice plate;
3) successively sample to be tested, ELIAS secondary antibody, chromogenic reaction liquid are added orifice plate, read sample to be tested OD value at specific wavelength, according to described sample to be tested OD value and blank OD value, determine whether sample to be tested is paraneoplastic pemphigus sample;
Described low-purity antigen is the fusion after bag spot albumen or its truncate or its subunit are connected with label, and the purity of described low-purity antigen is less than or equal to 70%.
In said method,
Described according to described sample to be tested OD value and blank OD value, determine whether sample to be tested is that paraneoplastic pemphigus sample comprises the steps: to calculate cut-off value according to the OD value Medcalc software of described detect aperture and described blank control wells; If the OD value of sample to be tested is greater than cut-off value, then sample to be tested is or candidate is Features of Paraneoplastic Pemphigus; If the OD value of sample to be tested is not more than cut-off value, then sample to be tested be not or candidate for Features of Paraneoplastic Pemphigus.
In said method,
The amino acid sequence of described low-purity antigen is sequence 2;
Described sample to be tested is in vitro serum;
Described specific wavelength is 405nm;
Described cut-off value is 0.031.
In said method,
Described antibody is the anti-his tag antibody in mouse source;
Described two resist the Goat anti human IgG for alkali phosphatase enzyme mark.
In said method,
Described bag by rear often add a kind of material hatched after (except chromogenic reaction) all to carry out washing the step of plate.
In said method,
Described mouse source anti-his tag antibody adds orifice plate with the form in mouse source anti-his tag antibody solution (solvent is for confining liquid);
The concentration of described mouse source anti-his tag antibody solution is 2.5ug/ml; The application of sample amount 50 microlitres/hole of the anti-his tag antibody in described mouse source;
Low-purity antigen shown in described sequence 2 adds orifice plate with the form of the low-purity antigenic solution (solvent is for confining liquid) shown in sequence 2;
The concentration of the low-purity antigenic solution shown in described sequence 2 is 4.375ug/ml; The application of sample amount 50 microlitres/hole of the low-purity antigenic solution shown in described sequence 2;
The Goat anti human IgG of described alkali phosphatase enzyme mark adds orifice plate with the form of the Goat anti human IgG solution (solvent is for confining liquid) of alkali phosphatase enzyme mark;
The concentration of the Goat anti human IgG solution of described alkali phosphatase enzyme mark is 0.167ug/ml; The application of sample amount 50 microlitres/hole of the Goat anti human IgG solution of described alkali phosphatase enzyme mark; It is described that to wash cleansing solution that plate adopts be pH value be 7.5 concentration is the PBST of 0.02M;
The confining liquid of described closed employing is that skimmed milk power to be added pH value be 7.5 concentration is the solution obtained in the PBST of 0.02M, makes the mass percentage of skimmed milk power be 5%.
Experiment of the present invention demonstrates, method of the present invention is wrapped by the antibody acted on it by specific immune response before low-purity is antigen coated, low-purity antigen is caught by this specific antibody, realizes the object improving antigen purity, reduce antigen coated required content simultaneously; The antigen that found through experiments the purity 60% of this modification method bag quilt is compared than the antigen assay result of the purity 60% of conventional indirect method bag quilt, have higher susceptibility and specificity, and difference has statistical significance.
Embodiment
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.MaxiSorp96 orifice plate (ThermoFisher467466), anti-6 histidines (his) mouse monoclonal antibody (the biological company limited HT501-01 of full formula gold), 0.05MPH9.6 carbonate buffer solution (CBS), skimmed milk power (BD232100), containing the PH7.5 phosphate buffer (PBST) of 0.05%Tween20, test is with containing histidine-tagged recombinant protein, testing sample, detect the specificity ELIAS secondary antibody (lifetechnologyiesG-21060) of antibody in testing sample, EL-P-NPP chromogenic reagent box (sangonbiotechPW014).
The carbonate buffer solution (CBS) of pH value to be 9.6 concentration be 0.05M: 1.59g sodium carbonate (Beijing Chemical Plant S0148) and 2.93g sodium bicarbonate (Beijing Chemical Plant S0037), be dissolved in 1L deionized water (self-control of millipore water machine), 0.45um filter (milliporeSLHV033RB) filters, adjust ph, to obtain pH value be 9.6 concentration is the CBS of 0.05M.
The PBST of pH value to be 7.5 concentration be 0.02M: PBS pulvis (Beijing biotech firm of Zhong Shan Golden Bridge ZLI-9062) and Tween20 (Amresco0777) are dissolved in 2L deionized water, the concentration 0.02M of PBS pulvis, the volumn concentration of Tween20 is 0.05%, adjust ph, to obtain pH value be 7.5 concentration is the PBST of 0.02M.
Confining liquid: skimmed milk power to be added above-mentioned pH value be 7.5 concentration is the solution obtained in the PBST of 0.02M, makes the mass percentage of skimmed milk power be 5%.
Embodiment 1, indirect enzyme-linked immunosorbent assay
One, the preparation of antigen EVPL-N1
The amino acid sequence of EVPL-N1 albumen is sequence 2 6-146 amino acids, and the nucleotides sequence of its encoding gene is classified as sequence 1 16-438 position nucleotide.
1, the structure of recombinant vector
By sequence
in tableeVPL-N1 protein coding gene shown in sequence 1 16-438 position nucleotide (for N1 gene) inserts pEASY-E2 expression vector (the biological company limited CE201-01 of full formula gold) and carries out flush end connection, the recombinant vector obtained, called after pEASY-E2-EVPL-N1 realizes MGIGP and the his label coexpression on EVPL-N1 albumen and carrier, obtains the EVPL-N1 that recombinates.
The amino acid sequence of restructuring EVPL-N1 is sequence 2, and 1-5 position is the MGIGP on carrier, and 6-146 position is EVPL-N1 albumen, and 147-158 position is his label;
The nucleotides sequence of the encoding gene of restructuring EVPL-N1 is classified as sequence 1, and 1-15 position is the MGIGP code nucleic acid on carrier, and 16-438 position is EVPL-N1 protein coding gene, and 439-477 position is his label coding gene.
2, the competent structure of recombinant clone
Recombinant vector pEASY-E2-EVPL-N1 is imported in Escherichia coli Trans1-T1 (the biological company limited CD5010-01 of full formula gold), obtain recombinant bacterium Trans1-T1-EVPL-N1.
Extract the plasmid of recombinant bacterium and send to order-checking, plasmid be pEASY-E2-EVPL-N1 be positive recombinant plasmid.
3, EVPL-N1 protein expression
By above-mentioned positive recombinant plasmid transformed to expressing competent cell Transetta (DE3) ChemicallyCompetentCell (the biological company limited CD801-01 of full formula gold), solid-state LB shakes bacterium after picking monoclonal, and packing is frozen.Competent cell is expressed in recovery, after incubated overnight, 1:50 is seeded to liquid LB, 37 DEG C, 200rpm/min, shakes to OD600=0.6, adds the derivant IPTG (Isopropyl-β-D-thiogalactopyranoside of final concentration 0.05mM, the biological company limited GF101-01 of full formula gold), 37 DEG C, 200rpm/min, induces 5 hours.4 DEG C, the centrifugal 10min of 10000rpm collects thalline, and resuspended to the binding buffer liquid (50mMNaH containing protease inhibitors (Roche CompletetabletsEDTA-free, EASYpack04693132001)
2pO4,300mMNaCl, PH7.4) in, obtain thallus suspension liquid.
4, purification of Recombinant EVPL-N1 albumen
With Ultrasonic Cell Disruptor cracking (300W power in thallus suspension liquid ice bath, ultrasonic 5s stops 10s, continue 20min) thalline, 4 DEG C, 14000g, 20min is centrifugal, get supernatant 0.45um frit, add imidazoles, make its final concentration be 10mM/L, use affinity chromatography technology purifying, supernatant will be got out and add 4 DEG C, nickel post (Ni-NTAHisBindResins Merck & Co., Inc. 70666-3, flow velocity 1ml/min), 100rpm, shake 1 hour, containing the wash buffer (50mMNaH of 15mM imidazoles
2pO4,300mMNaCl, PH8.0,15mM imidazoles) rinsing, the elution buffer (50mMNaH of 250mM imidazoles
2pO4,300mMNaCl, PH8.0,250mM imidazoles) wash-out destination protein.
It is 18.0KD restructuring EVPL-N1 albumen that the eluent of collection 2 times of column volumes is molecular mass.
Restructuring EVPL-N1 albumen is carried out SDS-PAGE electrophoresis, and the molecular mass of destination protein EVPL-N1 is 18.0KD; Binding buffer liquid 100 times of EVPL-N1 albumen volumes, 2 removing imidazoles of dialysing under 4 DEG C of conditions, with BSA standard items (Pierce
tMbovineSerumAlbuminStandardAmpules, 23209,2mg/mL) be contrast, through overstain glue, scanned photograph, in the purity of Bandscan software analysis destination protein.BCA method detects protein concentration simultaneously.
The purity of result restructuring EVPL-N1 is 60%; The concentration of restructuring EVPL-N1 is 3.5mg/ml.
Two, for detecting the indirect enzyme-linked immunosorbent assay of low-purity antigen
The purity of restructuring EVPL-N1 is 60%, concentration is 3.5mg/ml.
Sample to be tested is the serum sample of 27 routine Features of Paraneoplastic Pemphigus (make a definite diagnosis, and patient knows the inside story, its serum can in conjunction with bag spot albumen) and the serum sample of 20 routine normal healthy peoples.
Sample to be tested is detected with indirect enzyme-linked immunosorbent assay, specific as follows:
1, coated antibody: with the CBS of pH value to be 9.6 concentration the be 0.05M anti-his tag antibody (TransgenBiotechHT501-01) in mouse source according to volume ratio to be 1:400 dilute concentration be 1mg/ml, obtain coated antibody, add 50 microlitre coated antibody 2.5ug/ml to 96 orifice plates according to every hole, 4 DEG C are spent the night;
The PBST (containing 0.05%Tween-20) of secondary daily 300 microlitre pH value to be 7.5 concentration be 0.02M washes plate three times, each 1 minute;
Button dry hole plate, adds 200 microlitre confining liquids and closes, room temperature, 1 hour;
Again wash plate (the same), button dry hole plate, obtains coated antibody orifice plate;
2, capture antigen: be that 1:800 dilutes that purity prepared by embodiment 1 is 60%, concentration is the restructuring EVPL-N1 of 3.5mg/ml according to volume ratio with confining liquid, obtains diluting antigen,
Dilution antigen is added above-mentioned coated antibody orifice plate, 37 DEG C, 1 hour according to every hole 50 microlitre 4.375ug/ml;
Wash plate (the same), button dry hole plate, obtains capture antigen orifice plate;
3, add sample to be tested: with confining liquid according to volume ratio be 1:100 dilute sample to be tested, obtain dilute sample to be tested,
Dilution sample to be tested is added above-mentioned capture antigen orifice plate, 37 DEG C, 1 hour according to every hole 100 microlitre;
Wash plate 4 times, each 1 minute, button dry hole plate, obtains adding sample to be tested orifice plate;
4, add two to resist: with the Goat anti human IgG bis-anti-(Lifetechnologies81-7122) of confining liquid according to volume ratio to be 1:6000 dilute concentration be 1mg/ml alkali phosphatase enzyme mark, obtain dilution two anti-;
Anti-for dilution two to add according to every hole 50 microlitre 0.167ug/ml is above-mentionedly added sample to be tested orifice plate, 37 DEG C, 1 hour;
Wash plate 4 times, each 1 minute, button dry hole plate, obtains adding two anti-orifice plates;
5,100 microliters of chromogenic liquid are added (with deionized water according to volume ratio 1:4 dilution analysis liquid (the biological C510014-0500 of raw work) according to every hole under light protected environment, namely the analytic liquid that 10mg dry powder p-NPP in kit is dissolved in 10ml diluted is mixed with nitrite ion) in two anti-orifice plates, room temperature added 50 microliter of stop solution (the biological C510014-0500 of raw work) according to every hole after 30 minutes.Result is read under the wavelength 405nm that chromogenic reagent box is recommended.
6, calculate: Index=sample readings OD405-blank control wells reading OD405.
Result
as table 1shown in.
table 1
Through Medcalc software analysis, cut-off value is 0.031.
If the OD value of sample to be tested is greater than 0.031, then sample to be tested is or candidate is Features of Paraneoplastic Pemphigus;
If the OD value of sample to be tested is not more than 0.031, then sample to be tested be not or candidate for Features of Paraneoplastic Pemphigus;
Sensitivity=true positives number/(true positives number+false negative number) * 100%;
Specificity=true negative number/(true negative number+false positive number) * 100%;
The above results shows, adopt restructuring EVPL-N1 as detectable antigens, ELISA detects, 27 examples are actual in having the OD value of 3 examples to be less than 0.031 in Features of Paraneoplastic Pemphigus, be detected as non-Features of Paraneoplastic Pemphigus, in the routine Healthy Volunteers of detection sensitivity 92.59% (25/27), 50 of this method, whole OD value is not more than 0.031, detecting is not Features of Paraneoplastic Pemphigus, specificity 100%.
Therefore, method sensitivity of the present invention and specificity all high.
Comparative example 1, conventional method detect low-purity antigen
Detect the serum sample of 27 routine Features of Paraneoplastic Pemphigus (make a definite diagnosis, and patient knowing the inside story) in embodiment 1 and the serum sample of 20 routine normal healthy peoples with the following method:
1, envelope antigen: be the restructuring EVPL-N1 that purity prepared by dilution embodiment 1 is 60%, concentration is 3.5mg/ml according to volume ratio 1:100 with the CBS of PH9.6, add 96 orifice plates according to every hole 50 microlitre, 4 DEG C are spent the night;
The PBST of secondary daily 300 microlitre pH value to be 7.5 concentration be 0.02M washes plate three times, each 1 minute.
Button dry hole plate, adds 200 microlitre confining liquids and closes, room temperature, 1 hour;
Again wash plate (the same), button dry hole plate, obtains envelope antigen orifice plate;
2, add sample to be tested: with confining liquid according to volume ratio be 1:100 dilute sample to be tested, obtain dilute sample to be tested,
Dilution sample to be tested is added above-mentioned antigen coated orifice plate, 37 DEG C, 1 hour according to every hole 100 microlitre;
Wash plate 4 times, each 1 minute, button dry hole plate, obtains adding sample to be tested orifice plate;
3, add two to resist: the Goat anti human IgG bis-anti-(Lifetechnologies81-7122) with confining liquid according to volume ratio being 1:6000 dilution alkali phosphatase enzyme mark, obtains dilution two anti-(original content is 1mg/ml);
Above-mentioned capture antigen orifice plate is added, 37 DEG C, 1 hour according to every hole 50 microlitre by anti-for dilution two;
Wash plate 4 times, each 1 minute, button dry hole plate, obtains adding two anti-orifice plates;
4, develop the color: add in two anti-orifice plates according to every hole 100 microliter amount by chromogenic reaction liquid with under light protected environment in embodiment, room temperature added stop buffer according to every hole 50 microlitre after 30 minutes.Result is read under the wavelength 405nm that chromogenic reagent box is recommended.
5, calculate: Index=sample readings OD405-blank control wells reading OD405.
Comparative example result
Result
as table 2shown in.
table 2
Comparative example cutoff value is 0.136, and sensitivity is 77.78% (21/27), specificity 80%.
Whether variant in diagnosis for comparing two kinds of methods further, in Medcalc software, statistical analysis is carried out to the ROC curve of two methods, show below obtaining
3-
table 5result.Wherein P value=0.01 (95%CI=0.0306to0.225), difference has statistical significance (p<0.05).
table 3be that two method ROC curves compare
aDeLongetal.,1988
bAUC±1.96SE
Note: AUC: area under curve, SE: standard error, CI: fiducial interval
table 4for comparing between two of ROC curve
cDeLongetal.,1988
Note: AUC: area under curve, SE: standard error, CI: fiducial interval.
Claims (9)
1. utilize low-purity antigen to detect an indirect enzyme-linked immunosorbent assay for sample to be tested, comprise the steps:
1) antibody is added orifice plate and carry out bag quilt, obtain coated antibody orifice plate;
Described antibody is the antibody of label in low-purity antigen, and described low-purity antigen is the fusion after albumen is connected with label;
2) described low-purity antigen is added the described each hole of coated antibody orifice plate, carry out antigen capture, obtain capture antigen orifice plate;
3) successively sample to be tested, ELIAS secondary antibody are added orifice plate, reaction, detect OD value, the indirect enzyme-linked immunosorbent assay realizing sample to be tested detects.
2. method according to claim 1, is characterized in that: described low-purity antigen is the antigen that purity is less than or equal to 70%.
3. method according to claim 1 and 2, is characterized in that: described label is HIS label;
Described ELIAS secondary antibody is the Goat anti human IgG of alkali phosphatase enzyme mark.
4. in claim 1-3, whether arbitrary described method is application in paraneoplastic pemphigus sample detecting sample to be tested;
Or whether arbitrary described method is application in Features of Paraneoplastic Pemphigus detecting people to be measured in claim 1-3.
5. utilize low-purity antigen to detect or whether couple candidate detection sample to be tested is the method for paraneoplastic pemphigus sample, comprise the steps:
1) antibody is added orifice plate and carry out bag quilt, obtain coated antibody orifice plate;
Described antibody is the antibody of label in low-purity antigen, and described low-purity antigen is the fusion after albumen is connected with label;
2) described low-purity antigen is added the described each hole of coated antibody orifice plate, carry out antigen capture, obtain capture antigen orifice plate;
3) successively sample to be tested, ELIAS secondary antibody, chromogenic reaction liquid are added orifice plate, read sample to be tested OD value at specific wavelength, according to described sample to be tested OD value and blank OD value, determine whether sample to be tested is paraneoplastic pemphigus sample;
Described low-purity antigen is the fusion after bag spot albumen or its truncate or its subunit are connected with label, and the purity of described low-purity antigen is less than or equal to 70%.
6. method according to claim 5, is characterized in that:
Described according to described sample to be tested OD value and blank OD value, determine whether sample to be tested is that paraneoplastic pemphigus sample comprises the steps: to calculate cut-off value according to the OD value Medcalc software of described detect aperture and described blank control wells; If the OD value of sample to be tested is greater than cut-off value, then sample to be tested is or candidate is Features of Paraneoplastic Pemphigus; If the OD value of sample to be tested is not more than cut-off value, then sample to be tested be not or candidate for Features of Paraneoplastic Pemphigus.
7. method according to claim 6, is characterized in that:
The amino acid sequence of described low-purity antigen is sequence 2;
Described sample to be tested is in vitro serum;
Described specific wavelength is 405nm;
Described cut-off value is 0.031.
8., according to described method arbitrary in claim 5-7, it is characterized in that:
Described antibody is the anti-his tag antibody in mouse source;
Described two resist the Goat anti human IgG for alkali phosphatase enzyme mark.
9., according to the arbitrary described method of claim 5-8, it is characterized in that:
Described mouse source anti-his tag antibody adds orifice plate with the form of mouse source anti-his tag antibody solution;
The concentration of described mouse source anti-his tag antibody solution is 2.5ug/ml; The application of sample amount 50 microlitres/hole of the anti-his tag antibody in described mouse source;
Low-purity antigen shown in described sequence 2 adds orifice plate with the form of the low-purity antigenic solution shown in sequence 2;
The concentration of the low-purity antigenic solution shown in described sequence 2 is 4.375ug/ml; The application of sample amount 50 microlitres/hole of the low-purity antigenic solution shown in described sequence 2;
The Goat anti human IgG of described alkali phosphatase enzyme mark adds orifice plate with the form of the Goat anti human IgG solution of alkali phosphatase enzyme mark;
The concentration of the Goat anti human IgG solution of described alkali phosphatase enzyme mark is 0.167ug/ml; The application of sample amount 50 microlitres/hole of the Goat anti human IgG solution of described alkali phosphatase enzyme mark.
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| CN104931685A (en) * | 2015-06-09 | 2015-09-23 | 天津医科大学 | Luminescence immune detection method based on recombinant antigen carrying His tag |
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