Method for carrying out immunochromatographic assay with dissociating fluorescent tag

FIELD: medicine.

SUBSTANCE: invention refers to medicine and concerns a method for carrying out an immunochromatographic assay with a dissociating fluorescent tag, wherein complexes comprising molecules of an antigen or antigens, specific antibodies and tag molecules are formed on a membrane test strip. After the assay is completed, the tag is extracted from the working membrane of the test strip, and a fluorescent signal is measured in a liquid volume.

EFFECT: method according to the invention provides creating the optimum conditions for tag fluorescence, as well as eliminating the harmful influence of the membrane which is impermeable to decrease an intensity of both exciting and emitted light.

1 dwg, 1 ex


The invention relates to immunology and biotechnology and is a way immunochromatographic analysis dissociable fluorescent label.

Immunochromatographic method for content evaluation of various compounds widely used in the practice of medical diagnosis [Raphael C. Wong l Harley Y. Tse (Editors) (2009) Lateral Flow Immunoassay. Springer, USA].

Lateral flow tests can be implemented in several different ways. Let us consider the General form of the scheme of "sandwich"format chromatography as one of the most frequently used. Sample potentially containing the designated connection, under the action of capillary forces is moved along the test strip. However, it first interacts with the labeled component (usually antibodies against defined connection), then the front of the liquid overcomes area (analysis area) to the immobilized component, linking the designated connection. The degree of binding of the label in the analytical zone and, accordingly, the intensity of staining of the membrane is determined by the concentration of the analyte in the sample. The main advantages of immunochromatographic analysis is the rapidity of results (5-15 min) and the ability besprovodnoy detection. As the label in the vast majority of cases are particles of colloidal gold. [Babaar Ngom & Yancheng Guo & Xiliang Wang & Dingren Bi. (2010) Development and application of lateral flow test strip technology for detection of infectious agents and chemical contaminants: a review // Anal Bioanal Chem 397:1113-1135]. However, if you want to increase the sensitivity analysis using other markers, such as fluorescent dyes [Yoo, Jisun, Jung, Young), Hahn, Jong Hoon, Pyo, Dongjin (2010) 'QUANTITATIVE ANALYSIS OF A PROSTATE-SPECIFIC ANTIGEN IN SERUM USING FLUORESCENCE IMMUNOCHROMATOGRAPHY', Journal of Immunoassay and Immunochemistry, 31:4, 259-265]. Although in this case, the signal requires additional equipment, this approach allows to combine the rapidity of results (due to the use of the principle of chromatography and highly sensitive detection (due to the low detection limit of fluorescent dye).

Compared with organic fluorescent dyes fluorescent lanthanide (Eu3+Sm3+, Tb3+, Dy3+, ...) complexes have the advantage of a long lifetime fluorescence (up to 1000 microseconds), a large Stokowski shift (>250 nm), narrow profile issue, allowing their use in a method microsecond spectroscopy with a time resolution [J. Yuan, G. Wang (2005) Lanthanide complex-based fluorescence label for time-resolved fluorescence bioassay. J. Fluorescence, 15, 559-568].

Fluorescent labels based on lanthanide complexes are successfully used in vysokochuvstvitel resolved in time fluorescent immunologicals the x methods. Due to the fact that the technology of reception of the signal with a time resolution allows easy separation of specific long-lived fluorescent signal marks from the short-lived background noise in the bioassay and get rid of the effects of light scattering (scattering Tindale, Rayleigh and Raman), the limit of detection of the method is significantly increased [J. Yuan, G. Wang (2005) Lanthanide complex-based fluorescence label for time-resolved fluorescence bioassay. J. Fluorescence, 15, 559-568.], [Hemmila I. (1985) Fluoroimmunoassays and immunofluorometric assays. Clin. Chem., 31, 359-370].

The formation of complexes with some organic ligands significantly enhances the fluorescence of lanthanide ions. After light absorption ligand is efficient energy transfer from the excited singlet energy level through the triplet level at the resonant energy level of the metal [Ferrari M, Ozkan M. & Heller, M. J. (2007) BioMEMS and Biomedical Nanotechnology. Volume II: Micro/Nano Technologies for Genomics and Proteomics. - Springer US].

For dissociative analysis method use the primary complexes polyaminopolycarboxylate of chelators with lanthanides, demonstrating the high stability and solubility in water. Most chelating agents form a lanthanide chelates with a binding constant of order 1015M-1. For conjugation of the label with antibodies in the structure complexone there is a corresponding chemical group, such as, for example, the R, diisopentyl-EDTA or AMINOPHENYL-EDTA. These chelating agents are well suited for dissociative analysis with subsequent increase in fluorescence by adding β-diketonates. Based on the same principle analysis system kits are used (Dissociative-Enhansed Lanthanide Fluorescence Immunoassay). In this system, europium dissociates from complexone (ITC-DTTA-Eu) in acidic medium, and then measured the fluorescence of the chelate Eu with β-diketone-tri-n-octylphosphine in micellar aqueous solution [J. Yuan, G. Wang (2005) Lanthanide complex-based fluorescence label for time-resolved fluorescence bioassay. J. Fluorescence, 15, 559-68].

To register fluorescence of lanthanide complexes using the method of fluorimetry with a temporary permit. After short-term exposure of the sample luminescence of all molecules and the cell (tablet) begins to grow exponentially. Short-lived background fluorescence decreases rapidly and does not interfere with the measurement of the fluorescence of the label. This allows the measurement of extremely long-lived fluorescence of lanthanide labels. Usually spend many cycles of measurements for one sample (within 1 s), which improves the signal-to-noise calculations [Hemmila I. (1985) Fluoroimmunoassays and immunofluorometric assays. Clin. Chem., 31, 359-70].

Despite the above advantages dissociable lanthanide labels, previously, this approach was not used for signal amplification in the lateral flow systems.

If the detection with the persecuted label is not with the membrane surface, and out of solution, volume of solution, you can create optimal conditions for fluorescence labels, as well as to fix the light shielding signal membrane test strips. To do this, applicants are encouraged after conducting a lateral flow analysis to extract related in the analytical zone of the label molecules and hold the instrument activity registration fluorescent label in the volume of extracting solution.

The advantages set forth above is confirmed experimentally and are shown in the following example.


The concentration of monoclonal antibodies HTM81 against recombinant 38 kDa antigen of Mycobacterium tuberculosis.

1. Getting conjugate antibodies with ITC-DTTA-Eu

For conjugation used a set of "kits are used® Eu-Labelling kit 1244-302" manufactured by PerkinElmer™ (Wallac Oy, Finland) containing:

1) Eu-labelling reagent" is 0.2 mg (300 nm) N1-(p-isothiocyanatobenzene)-Diethylenetriamine-N1N2N3N4-leads to compounds, which Eu3+(ITC-DTTA-Eu), freeze-dried.

2) Reinforcing solution "kits are used® Enhancement Solution)) - Triton-X100, acetic acid and chelators.

2 mg of monoclonal antibody (MAB) T C1.81 in 1 ml of 0.1 M carbonate buffer, pH 8.5 (antibodies were transferred into dialysis buffer for 35 min.) was mixed with 0.2 ml of 7.5 mm aqueous solution of Eu-labelling reagent (R-DTTA-Eu, 500-fold excess is for). The mixture was left overnight at +4°C. Purification of the conjugate Mat-ITC-DTTA-Eu carried out by the method of gel filtration using a column of 1.5×28 cm with medium Sephadex G-50, equilibrated with 50 mm Tris-HCl and 0.15 M NaCl and 0.05% NaN3, pH 7.75.

The optical density of the obtained fractions was measured on a spectrophotometer Shimadzu 1202 at a wavelength of 280 nm. To determine the amount of incorporated label aliquots of antibodies were diluted in the amplifying solution in the ratio of 1:10,000 to a final volume of 200 μl in the wells of the microplate, incubated for 2 min, and then recorded the fluorescence on a tablet reader EnSpire 2300 production company PerkinElmer™.

2. Determining the amount of antibodies labeled ITC-DTTA-Eu, with the use of amplification fluorescence

In the method used chelate Eu3+with N1(p-isocyanatobenzyl)dietilen triamine-N1N2N3N3-tetraoxane acid (ITC-DTTA-Eu) as aiming reagent. To the drug labeled antibody or conjugate antibodies with KZ added a solution of β-diketone (2-afterdepolarization β-NTA) and slightly amplifying the fluorescence of the solution (pH 3,2) containing trioctylphosphine (TORO) and Triton X-100. Ion Eu3+of the labeled agent is passed into the solution and contacted with the ligand. The solution was turned into a highly fluorescent micellar solution containing Eu(β-ITA)3(TORO)2chelate, f is uorescence which was measured by the method of fluorometry with a temporal resolution on the device Wallac 1234 Fluorimetr (Perkin Elmer, Finland) [Ferrari M., Ozkan M., Heller, M. J. (2007) BioMEMS and Biomedical Nanotechnology. Volume II: Micro/Nano Technologies for Genomics and Proteomics. - Springer US]. Registration settings fluorescence of Eu3+: delay time 0.1 MS, frequency flash 1 MS, the time of signal reading 0.4 MS, excitation at 340 nm and registration of fluorescence at 615 nm.

Preparation of calibration graph to determine the amount of bound peroxidase label on the antibody molecule. Labeled antibodies (2-20 ál, the concentration of 1-100 μg/ml) were diluted in the amplifying solution to a final volume of 200 μl in the wells collapsible transparent microplate from the set of "kits are used® Eu-Labelling kit 1244-302", incubated 10 min at room temperature, then measured the fluorescence. The content of antibodies in the fractions and the amount of incorporated label in the received drugs labeled antibodies was calculated by the following formulas:


where A=10000 - dilution factor;

B=1000000 - signal standard preparation 1 nm Eu3+;

C1(mAndt)= A280-0,008C(Eu3+)1,34(mg/ml);


where 1,34 - absorption of 1 mg/ml antibody at 280 nm, the 160,000 - molecular weight IgG, 0,008 - absorption of 1 μm chelate complex of europium at 280 nm.

The ratio of the number of bound peroxidase label to the antibody was 0.57.

3. Construction of calibration curve for determining the concentration of labeled antibodies immobilized on the lateral flow membrane, fluorescence Eu3+

Antibodies were applied to the working membrane CNPC-SN12 L2-P25 (pore size 15 μm) "mdi Easypack" company "Advanced Microdevices" (India). Then cut off the portion of the membrane to the immobilized antibodies and were placed in Eppendorf, was filled with 100 ál Eppendorf amplifying solution "kits are used® Enhancement Solution, incubated for 2 min, were collected aliquots of 50 µl and registered fluo is ascencio on a tablet reader EnSpire 2300 production company PerkinElmer™.

The calibration curve for the detection of antibodies shown in the drawing.

The limit of detection according to current recommendations [Burdon D., Markov, B. N. (1975) fundamentals of Metrology, 2nd ed., M], [Charykov A. K. (1984) Mathematical processing of results of chemical analysis, L.] was defined as the concentration giving a signal value equal to the background signal (640 units) plus 3 standard deviations of the background signal (3·197 units). The limit of detection antibody was 0.9 ng/ml, which corresponds to the limit of detection labels 3,1·10-12M with regard to the ratio of the amount of bound peroxidase label to the antibody.

4. Determination of limit of detection of colloidal gold as a label in the lateral flow assay (for comparison of limits of detection of two labels)

On the lateral flow membrane CNPC-SN12 L2-P25 inflicted solutions, representing a series of dilutions of particles of colloidal gold size 25 nm. Then scanned the image of the test strip and aziraphale, quantitatively assessing the intensity of the color of colloidal gold on the membrane with the help of the program "Nonlinear Dynamics TotalLab TL120 v2009". As in the case of a fluorescent label, the limit of detection was defined as the concentration of the label, giving the intensity of staining at three standard deviations above background signal. The limit of detection of colloidal gold amounted to 1.3·10-10M, h is about (at a ratio of 1 antibody at a 1-particle colloidal gold) corresponds to the limit of detection antibodies, equal to 20 ng/ml.

Thus, the limit of detection of antibodies using dissociable fluorescent label more than 20 times lower than when using traditional label is colloidal gold (0,9 ng/ml and 20 ng/ml, respectively).


A drawing. The calibration graph to determine the concentration of labeled antibodies immobilized on the lateral flow membrane, fluorescence Eu3+.

The method of conducting lateral flow analysis with dissociative fluorescent label, in which the membrane test strip to form complexes, which are composed of the molecules of the antigen or antigens that are specific to them antibodies, and molecules of the label, characterized in that after analysis of the label is extracted from the working membrane test strip and a fluorescent signal is measured in the liquid volume.


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