CN1209615C - Visual fluorescence immunoassay method for determining biological active substance - Google Patents
Visual fluorescence immunoassay method for determining biological active substance Download PDFInfo
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- CN1209615C CN1209615C CN 02106357 CN02106357A CN1209615C CN 1209615 C CN1209615 C CN 1209615C CN 02106357 CN02106357 CN 02106357 CN 02106357 A CN02106357 A CN 02106357A CN 1209615 C CN1209615 C CN 1209615C
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Abstract
The present invention relates to a visual fluorescence immunity analysis detection method which is used for qualitatively or semi-qualitatively detecting biological active substances. The method comprises the steps: metal ions of the lanthanide series multiply label biologic compatible components participating in a biologic compatible reaction; coacervation particles comprising chelate substances of the lanthanide series and fluorescence enhancement ion chelate substances are formed after the reaction is carried out; the fluorescence emitted by the particles is visually detected by naked eyes under the excitation of ultraviolet light; carrier proteins (such as cow thyroglobulin) or macromolecule polymers are used in the multiple labeling to enhance the labeling number of the metal ions of the lanthanide series in order to enhance the fluorescence intensity so as to be convenient for visual detection; and a biotin-mildew chain compatible avidin system is used as a universal reagent system.
Description
The present invention relates to the method for visual fluorescence immunoassay (VFIA) detection of biological active substance.It comprises with lanthanide metal ion makes multiple labelling, after the reaction of biological affinity, forms the hyperfluorescence coagulated particles and detects with visual fluorescence.
Some material by UV-irradiation after, can send the reflection this substance characteristics fluorescence, utilize this phenomenon to carry out the qualitative or quantitative test of this material, be called fluorescence analysis.Fluorescence analysis has been widely used in many fields such as industrial and agricultural production, medical science, health, biotechnology and scientific research.Qualitative or the quantitative test of fluorescence, the overwhelming majority adopts instrument detecting, judge whether existing of certain fluorescent material and amount with the size of fluorescence intensity, also have the sub-fraction material to judge that with visual detection it exists and general amount only by means of a uviol lamp.The visual fluorescence that a most typical example is exactly a trace uranium is measured.Trace uranium can send tangible yellow-green fluorescence with the bead that NaF or the fusion of NaF+ carbonate generate under UV-irradiation, detect by an unaided eye and can measure 10
-10Gram uranium.Yet, up to now, do not see as yet and only use ultraviolet light and macroscopic visible fluorescence method at the clinical report that in the concentration range bioactivator detected of needing, this is because most of significant clinically bioactivator content in the body fluid of going into is very little, measure them and need use very sensitive method, for example radiommunoassay (RIA), EIA enzyme immunoassay (EIA), chemiluminescence immune assay (CLIA) and fluoroimmunoassay (FIA), these analytical approachs all need precision and expensive determining instrument.All immunoassays all be with antibody as special analytical reagent, be that immune response is a based analyzing method with the special biological affinity reaction of antigen-antibody.React the difference of used label according to its biological affinity of spike, department is divided into above-mentioned various immunoassay again.
Worked out immune analysis method in the past in for many years based on fluoroscopic examination quantitative measurement bioactivator, i.e. fluoroimmunoassay (FIA), this is a kind of with the fluorescent material immunoreactive immune analysis method of thing spike that serves as a mark.In this field, one of most important improvement is to have developed time resolved fluoro-immunoassay (TR-FIA), and the basis of this method is time-resolved fluorescence technology and lanthanide chelate labelling technique (U.S.patent No.4,058732 and 4,374,120).Some lanthanide chelate, as europium, the chelate of terbium and samarium, because of it has very high fluorescence intensity, big Stokes shift and quite long fluorescence lifetime, thus successfully be applied to time resolved fluoro-immunoassay.Carry out when the measurement of label fluorescence can be on it be incorporated into antigen or antibody, also can be before measurement it be carried out after antigen or antibody disintegrate down.For this reason, can add a kind of beta-diketon that contains, the fluorescence enhancement solution of the low pH value of collaborative compound and surfactant is to dissociate lanthanide ion and make it to be transformed into new fluorescence chelate (U.S.Patent No.4,565,790).
US.Patent No.5,316,909 have narrated one improves one's methods, and promptly before fluorescence measurement, lanthanide ion is transformed into the extremely strong coagulated particles of fluorescence, and this particulate had both contained lanthanide chelate and had also contained fluorescence enhancing ion chelate complex.In coagulated particles, strengthened the exciting light energy that ion chelate complex absorbs by fluorescence,, can pass to the lanthanide ion chelate effectively by intermolecular energy transmission, thereby the fluorescence of lanthanide ion chelate has obtained very big enhancing, and this fluorescence enhancing is called as common fluorescence and strengthens.Use fluorescence enhancing altogether,,, still do not reach the visual level that can detect of naked eyes general lanthanide ion mark level though greatly improved fluorescence intensity.
The present invention combines with the lanthanide ion multiple labelling being total to the fluorescence enhancing, greatly improve the fluorescence intensity of the tested label in affinity reaction back from these two aspects, thereby can under ultra violet lamp, come qualitative or semiquantitative determination bioactivator by naked eyes visual detection fluorescence.The objective of the invention is to develop a kind of visual fluorescence immunoassay (VFIA) method of simple, sensitive, convenient and cheap qualitative or semiquantitative determination bioactivator.
The present invention selects Eu for use
3+Or Tb
3+Ion is as the label in the affinity reaction, this is because the chelate of these two ions all has very high fluorescence intensity, quite Chang emission wavelength and big Stokes shift, and have very strong common fluorescent effect, these characteristics all help visual detection.
Use Eu
3+Or Tb
3+The immunoassay of thing of marking can be a state of conflict, also can be non-state of conflict.In non-competing type immunoassay, determinand antigen is incorporated into insolubilized antibody and Eu simultaneously
3+Or Tb
3+Labelled antibody forms the sandwich type immune complex, and the excessive labelled antibody of flush away after the immune response adds fluorescence then and strengthens solution from the labelled antibody Eu that dissociates
3+Or Tb
3+, in solution, form the amount that new its fluorescence intensity of fluorescence chelate is proportional to determined antigen.
The state of conflict immunoassay can be divided into several types again when actual carrying out.In DELFIA type competitive immunoassay, with the antibody sandwich solid phase that can capture anti-analyte antibody.Produce if anti-analyte antibody is a mouse, then insolubilized antibody should be the anti-mouse IgG antibody that is produced by sheep or rabbit.Analyte antigen and Eu
3+Or Tb
3+-labelled antigen is competed the limited binding site on the anti-analyte antibody.Flush away excess reagent after the immune response adds fluorescence enhancement solution, then with Eu
3+Or Tb
3+Ion dissociates to the solution from labelled antibody, forms new fluorescence chelate in solution.Another type competitive immunoassay is to use analyte-carrier protein connector bag by solid phase, analyte antigen and solid phase antigen competition Eu
3+Or Tb
3+The anti-analyte antibody of mark, flush away excess reagent after the immune response, the same then adding fluorescence strengthens solution.In the state of conflict immunoassay, analyte content is changing inversely in the fluorescence intensity of final label to be measured and the sample.
In the present invention, so that can with the naked eye can carry out visual detection, following two means have been adopted in order to improve fluorescence intensity as far as possible simultaneously.
First means are that common fluorescence strengthens (U.S.Patent No.5,316,909).Use this means, lanthanide ion is transformed into peculiar form--the coagulated particles of sending out hyperfluorescence, in this particulate, not only contain fluorescigenic lanthanide chelate, and contain a kind of ion chelate complex that can strengthen fluorescence.By coming from the intermolecular NE BY ENERGY TRANSFER of excitation energy, the fluorescence intensity of lanthanide chelate can improve 2～3 orders of magnitude.Compare with adopting best direct fluorescence enhancement solution (strengthening liquid as DELFIA), fluorescence intensity can improve 50～70 times.Second means is multiple labellings of lanthanide ion.Only adopting under the situation that fluorescence strengthens altogether, the fluorescence intensity level of label is still not high enough, can not make visual detection to most of bioactivators in the clinical concentration scope.The lanthanide ion reference numerals that improves on each antibody (or antigen) molecule will improve the overall fluorescent strength level.Learn that by calculating and observation adopting under the situation that fluorescence strengthens altogether, for satisfying the visual detection requirement, each antibody molecule IgG goes up the lanthanide ion reference numerals must be more than 150.When adopting conventional method with the direct mark lgG of lanthanide ion, ratio Eu
3+(or Tb
3+)/lgG surpasses 25, will cause the decline of antibody activity, and biotin (B)-mould streptavidin (SA) system has become immunoassay and DNA is hybridized one of the most useful instrument in field.SA has powerful especially binding ability to B, and its affinity constant Ka is up to 10
15M
-1And SA itself is also extremely stable, under a lot of quite exacting terms, also can keep active to the combination of B.Though in this system, always have amplification coefficient to exist, want on each IgG molecule, to introduce a plurality of biotin molecules and use Eu by the biotinylation process
3+Or Tb
3+The mould streptavidin of mark and obtaining surpasses 150 lanthanide ion reference numerals and remains difficulty.Can solve this difficulty by means of macromolecule carrier protein or macromolecule polyalcohol.Bovine thyroglobulin (TG) once was used as the aglucon 4 of europium, 7-two (chlorine sulfophenyl)-1, and 10-phenanthroline-2, the carrier protein of 9-dicarboxylic acid (BCPDA) can reach 160 BCPDA by mark on a TG molecule.We find that TG and bovine serum albumin(BSA) (BSA) also can be used as the carrier of lanthanide ion.When with right-isothiocyano benzyl-DTTA during, can obtain being not less than 60 Eu as difunctional connection reagent
3+(or Tb
3+)/TG grammol connects ratio.With the antibody biotinylation, in general each antibody molecule can connect 10 biotin molecules.Therefore, connect with 1: 1 mole ratio, then can obtain being not less than 600 Eu if SA is connected in TG
3+(or Tb
3+)/IgG ratio.For connecting SA to TG, can use classical protein-crosslinking agent such as glutaraldehyde and carbodiimide etc., also can use different in nature bi-functional cross-linking agent and thiosuccimide 4-(N-maleimide methyl) cyclohexane-1-carboxylate etc.
Above-mentioned two means are combined, be applied to VFIA simultaneously, then the fluorescence of label has obtained very big enhancing and can reach VDL, and only adopts one of them, because the fluorescence of label is still not really strong, can not obtain satisfied visual detection effect.
VFIA can carry out in the hole of microtitre lath, the hole antibody sandwich.After the immune response, add fluorescence enhancement solution altogether, Eu
3+Or Tb
3+Ion dissociates down and forms the hyperfluorescence coagulated particles from solid phase.Under ultra violet lamp in visualization hole, dark place the fluorescence of solution.Uviol lamp can be made desk-top, also can make portablely, and as commercially available detect counterfeit money machine, its emission wavelength all can at 300nm～400nm.Visualization can be carried out in the darkroom, but also can natural light be covered with a black market, makes an environment with dark light, and the latter has bigger convenience and dirigibility.The fluorescence intensity of the fluorescence intensity of unknown sample and standard model can be obtained qualitative or semiquantitative result after relatively.The sensitivity of this method is better than other visual technology, as alcohol coagulation test, and immune chromatograph and enzyme immunoassay etc.
The needed whole checkout equipments of VFIA only are a simple uviol lamp and a black cloth, so this method not only can be used for various hospitals and clinic, and can be used for the work oneself of family detection diagnosis.
Below the invention will be further elaborated and explanation by two examples.
Example one
Detect hTSH in neonate's bleeding of the umbilicus or the Heel blood with the visual fluorescence immunoassay qualitative
Adopt double antibody sandwich method to detect, one of antibody solidifies in the inwall of sample panel aperture, and another antibody biotinylation has label Eu
3+Big molecule TG link to each other with SA, in immune response, form insolubilized antibody-hTSH-antibody-B-SA-TG-Eu
3+Compound is after the immune response, with Eu
3+Dissociating enters solution, Eu in solution
3+Form the coagulated particles that to launch hyperfluorescence with other components that add, under UV-irradiation, carry out the naked eyes visual detection.
The bag quilt of monoclonal anti-hTSH antibody: adding 200 μ l concentration in the aperture of sample panel is monoclonal anti-hTSH antibody-solutions of 5 μ g/ml, and bag is the carbonate buffer solution of pH9.5 by medium.Put room temperature following 16 hours, after the washing, the 0.1%BSA that every hole adds 300 μ l seals, and room temperature was placed 3 hours, inhales deblocking liquid, puts 4 ℃ of preservations.
The biotinylation of monoclonal anti-hTSH antibody: be used for biotinylated monoclonal anti-hTSH antibody and used antibody different with bag, both have different specific antigen binding sites on the hTSH molecule.Biotinylation carries out in the carbonate buffer solution of the 0.1mol/l of pH9.5.Biotinylation reagent adopts N-hydroxy-succinamide active bio element (NHS-Biotin), and reagent dosage is: antibody/NHS-Biotin=2/1 (weight).Both mix, and put 4 ℃ and spend the night.Second day, use the SephadexG50 gel column, biotinylated antibody is separated 4 ℃ of preservations from excessive N HS-Biotin.
The mark of bovine thyroglobulin (TG): at the carbonate buffer solution of pH9.5 with 300 times of grammol excessive N
1-(right-isothiocyano)-Diethylenetriamine-N
1, N
2, N
3, N
3The europium chelate of-tetraacethyl carries out mark to TG, and room temperature incubation 16 hours uses solvent resistant column TG-Eu
3+From free label reagent, separate.
Mark being connected of bovine thyroglobulin and mould streptavidin (SA): use protein-crosslinking agent carbodiimide (EDC) with TG-Eu
3+Be connected in SA.With SA, TG-Eu
3+With EDC by in 1: 5: 30 water-soluble solution of mole ratio, slowly stirred 1 hour, with Sephadex G-200 gel column with SA-TG-Eu
3+From unreacted SA and EDC, separate, in 4 ℃ of preservations.This gel column can not separate SA-TG-Eu
3+And TG-Eu
3+, confirm that by experiment this separation is unnecessary, because a large amount of TG-Eu
3+Existence do not make background fluorescence that tangible rising is arranged.
The preparation of fluorescence enhancement solution altogether: fluorescence enhancement solution is used for Eu altogether
3+Dissociate to and also form the coagulated particles of sending out hyperfluorescence in the solution.It is made up of dissociate part Ea and enhancing part Eb, and it consists of Ea:30 μ M TTA, 1.5 μ M Y
3+, 0.06% (W/V) triton x-100 and 20% ethanol, solution is regulated pH to 3.2 with acetate; Eb:0.4mM 1, the non-quinoline of coughing up of 10-, 0.2M Tris.
The preparation of negative and positive control sample: the preparation of negative control sample is to get normal health human blood 50 μ l, vertically drips on the filter paper of horizontal positioned, places at least 2 hours, so that the blood cake drying is put 4 ℃ of preservations.The preparation of positive control sample is to get the some milliliters of normal health human blood, the centrifugal blood plasma that makes separates with blood cell, abandon upper plasma, add equivalent physiological saline, slowly stirring makes and mixes, centrifugal once more, so the washing blood cell is 6 times, adds physiological saline and standard hTSH at last to original volume, makes that hTSH content is 20 μ U/ml in the final whole blood, get 50 μ l after stirring and drip on filter paper.The positive control of the same preparation sample.Yin and yang attribute control sample all places 4 ℃ of preservations.
Sample collection: it is when the neonate is born that the umbilical cord blood sample is collected, and directly bleeding of the umbilicus is dripped on filter paper, and horizontal positioned made its drying at least in 2 hours.It is collection in 3-5 days after the neonate is born that Heel blood is collected, and filter paper is once leaned against the puncture metapedes with hemorrhage place, and making blood form diameter is the blood cake of 2cm, and horizontal positioned at least 2 hours makes its drying, 4 ℃ of preservations.
Visual fluorescence immunoassay process: added dried blood scraps of paper sample (the negative or positive control sample of diameter 3mm in the sample panel aperture of monoclonal anti-hTSH antibody at bag, or testing sample), 50ng biotinylation monoclonal anti-hTSH antibody, (contain 9g/l NaCl, 0.05%NaN at 200 μ l
3, 0.5%BSA is in the pH7.7 0.05M Tris-HCl test damping fluid of 0.05% N of globulin and 0.01% polysorbate40), incubation is for the first time made in room temperature vibration 4 hours, takes out the scraps of paper and inhales and remove incubation liquid, wash 3 times after, every hole adding 200ng SA-TG-Eu
3+(in 200 μ l test damping fluid), incubation is for the second time made in room temperature vibration 1 hour, wash 6 times after, every hole adds 200 μ l Ea, vibrates 3 minutes with the Eu that dissociates
3+Ion, every then hole add 20 μ l Eb again, vibrate after 7 minutes, and under uviol lamp, comparison fluorescence detects by an unaided eye.This fluorescence is red, if the fluorescence intensity of sample equals or exceeds the fluorescence intensity of positive control sample, judges that then sample is positive, otherwise negative.
At present, in each country of the whole world that is comprising China, whether be that first low (relevant with inborn dementia) is carried out examination and become the postnatal routine inspection of neonate to the neonate to measure in neonate's bleeding of the umbilicus or the Heel blood hTSH content.Currently used inspection method RIA, EIA, FIA etc. all need adopt expensive high-performance instrument, because this item inspection needs very high sensitivity.The present invention is only by a uviol lamp that price is very cheap, use the VFIA method can carry out this inspection, this VFIA that illustrates that not only the present invention proposes has extra high sensitivity, for the low examination of neonate's first provides simple, convenient, a cheap method, this will make contributions for the prenatal and postnatal care of China and other developing countries simultaneously.
Example two
With hAFP in the visual fluorescence immunoassay semiquantitative determination human blood
The bag quilt of monoclonal anti-hAFP antibody, the biotinylation of monoclonal anti-hAFP antibody, the mark of TG, TG-Eu
3+The configuration that is total to fluorescence enhancement solution with the connection of SA is all identical with example one.
The preparation of the dried blood scraps of paper of hAFP standard sample: collect some normal health human bloods and neonate's bleeding of the umbilicus, respectively get a little centrifuging and go out serum,, be converted into hAFP content in the whole blood again with its hAFP content of DELFIA hAFP kit measurement.According to the survey data, two kinds of blood are mixed by different volume ratios, making the concentration of mixing hAFP in the blood is 5,20,80, and 320U/ml respectively gets 50 μ l and vertically drips on the filter paper of horizontal positioned, places at least 2 hours, and dry back is in 4 ℃ of preservations.
Sample collection: extracting vein blood or ear blood or refer to that blood 50 μ l vertically drip on filter paper, to place at least 2 hours, dry back is in 4 ℃ of preservations.
Visual fluorescence immunoassay process: substantially with example one, making first and second incubations all is at room temperature to vibrate 1 hour.When observing fluorescence, unknown sample and 4 standard samples can be compared one by one, see that itself and which standard fluorescence intensity are approaching, or between two standards, can sxemiquantitative record unknown sample content, if content meets or exceeds 20U/ml, then be judged to be positive, the suggestion patient goes to hospital to do further to check.
Liver cancer is one of higher cancer of frequent clinically at present appearance and grade of malignancy, and the early detection early treatment is even more important.It is the extraordinary approach that reaches early detection that hAFP level in vulnerable crowd (as hepatitis B patient) the mensuration blood is carried out examination, and the VFIA method that the present invention proposes provides simple, convenient, a cheap and practicable method for this extensive examination.
Claims (2)
1. a visual fluorescence immunoassay detection method that is used for qualitative or semiquantitative determination bioactivator is characterized in that it comprises the following steps:
(1) participate in the biological affinity component of biological affinity reaction with the lanthanide metal ion multiple labelling, adopt carrier protein or macromolecule polyalcohol improving reference numerals in the multiple labelling of lanthanide ion, biotin-mould streptavidin system is as the common reagent system.
(2) the mark component is participated in biological affinity reaction;
(3) after affinity reaction was finished, the lanthanide ion and make it to form the coagulated particles of hyperfluorescence of dissociating, this hyperfluorescence coagulated particles were to form through fluorescence enhancing method altogether, and it had both contained lanthanide chelate, also contained fluorescence and strengthened ion chelate complex
(4) under the exciting of ultraviolet light, the fluorescence that sends by particulate of visual detection with the naked eye.
2. according to claim 1, lanthanide metal ion is Eu
3+Or Tb
3+
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| US7340957B2 (en) | 2004-07-29 | 2008-03-11 | Los Alamos National Security, Llc | Ultrasonic analyte concentration and application in flow cytometry |
| US8266951B2 (en) | 2007-12-19 | 2012-09-18 | Los Alamos National Security, Llc | Particle analysis in an acoustic cytometer |
| CN103901010A (en) * | 2014-04-21 | 2014-07-02 | 牡丹江友搏药业股份有限公司 | Biological activity testing method of Chinese medicine preparation containing component of leech or earthworm |
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