Labeled luciferase antibody and a method thereof, a method of performing analysis on specific binding and set for use in the analysis of the specific binding |
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IPC classes for russian patent Labeled luciferase antibody and a method thereof, a method of performing analysis on specific binding and set for use in the analysis of the specific binding (RU 2199125):
 
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(57) Abstract: The invention relates to biotechnology, relates to a method of conjugating luciferase with chemical particle, in particular antibody, providing (a) mixing luciferase with one or more components, such as D-luciferin, magnesium ions and ATP, and (b) carrying out the reaction of covalent binding between luciferase and linking reagent using covalently bonding agent, where D - luciferin, magnesium ions and/or adenosine triphosphate present in a quantity sufficient to protect the luciferase activity from inhibition of covalently binding agent. Stage (a) is performed by mixing luciferase with its substrates, thus it is preferable if the magnesium and ATP are present in the form of a complex of ATP with magnesium (Mg2+- ATP) with optional D-luciferine. In the second aspect of the present invention relates to a labeled luciferase antibody representing the antibody, conjugated with an active Firefly luciferase method of the present invention. The invention concerns a kit for use in the analysis of specific binding to the antigen and the way it is implemented is seeking to method ringing chemical materials, in particular biological materials for chemical analysis, specifically biological analyses, and more specific tests for specific binding, such as immunoassay and analysis by the method of hybridization probing, and to enable the labels to the products specific amplification, for example, using polymerase chain reaction (PCR) analyses of a specific type. The cleavage reaction of luciferin (see below equation 1) - mediated luciferase, derived from the Firefly has a high quantitative output and stable light that allows you to detect the enzyme even at very low concentrations using relatively simple equipment (see McCapra. Potential application of bioluminescence and chemiluminescence in Turner et al. (Edit.) Biosensors fundamentals and applications: Oxford University Press, (1988): 617-37). We developed many methods using luciferase as an indirect tag (see Wannlund and DeLuca, "Bioluminescence immunoassays: Use of luciferase antigen conjugates for determination of methoxylate and DNP in Deluca & McElroy (Edits). Bioluminescence and Chemiluminescence: Basic chemistry and analytical applications. London: Academic Press, (1981): 693-696; Geiger & Miska, Bioluminescence anhanced anzyme immunoassay: a New ultrasensitive detection system for enzyme immunoassay, Clin. Chem. Clin. Biochem. J. (1987) 25, 31-38; and Murakami et al., Development of a bioluminescent detection system using adenylate kinase and is his invention, it was observed, what the analysis scheme can be simplified, while maintaining the sensitivity analysis, through the use of luciferase as a direct label, but it is necessary that such linking luciferase with analytical binding agents did not lead to inactivation of enzymatic activity, which is known to be very unstable. Standard covalently linking reagents such as glutaraldehyde, SMCC and SMPB quickly and irreversibly inhibit luciferase activity. Luciferase occurring, for example, from Photinus pyralis (American Firefly), contains four cysteine residue (see, Wet et al. (1987) Molecular and Cellular Biology, 7, 725-737), two of which are located near the active site or are part of it (see, Deluca & McElroy (1978), Methods in Enzymology, 57, 3-15). The authors of the present invention, it was found that the binding covalently svyazuyushaya reagents with these residues may cause the observed inactivation of the enzyme, and therefore, developed a method of bonding a luciferase with such analytical agents, which protect the enzyme from irreversible inhibition. Thus, in its first aspect the present invention relates to a method of conjugating Lucifer antigen, or nucleic acid, and even more specifically with the antibody; and specified the method comprises (a) mixing luciferase with one or more components, namely D-luciferine, magnesium ions, and ATP, so that the concentration of magnesium ions and ATP was more than 0.2 mmol/l and 0.05 mmol/l, respectively; and (b) carrying out the reaction of covalent binding of luciferase and chemical molecules using covalently binding reagent. Stage (a) is carried out preferably by mixing luciferase with its substrates in solution; however, it is preferable that the magnesium and ATP was present in the form of a complex of ATP with magnesium (MD2+APR) optionally together with D-luciferine. Preferably, in the reaction were present either MD2+APR, or D-luciferin. If MD2+ATP and luciferin are present together, then it is preferable to exclude from the reaction mixture the oxygen. In its second aspect the present invention relates to labeled chemical molecule constituting the chemical molecule, conjugate with active Firefly luciferase in accordance with the method of the present invention. Pie in the analysis of specific binding, preferably, the antibody, antigen or nucleic acid. If the binding agent is a nucleic acid, it is preferable that this was the oligonucleotide, but can also be used polynucleotide or nuke or primer is used as a hybridization probe or primer for chain elongation, such as PCR primer. The most preferred molecule is an antibody, as taken earlier attempts of antibody binding to luciferase resulted in almost complete inactivation. The main advantage of obtaining labeled luciferase chemical molecules, in particular labeled luciferase antibodies is the ability to perform analysis with capture antibodies on the fiber. In one preferred variant of this analysis of the antibody for the target antigen immobilized on the light guide, for example an optical fiber mounted in such a way that incident light signal is transmitted after passing through a light on photoelectric measuring device, such as a photomultiplier tube; the designated antigen in a liquid sample applied to the specified fiber; and a second antibody, labeled with luciferase, put in the solution in contact with the Holy to the immobilized antibody. To determine the presence and/or amount of the captured antigen is necessary only D-luciferin and mg2+-ATP in the solution to be in contact with the surface of the light guide, which is associated with the complex antigen-antibody, and then to measure the amount of light emitted, transmitted to the photomultiplier measuring device such as a photomultiplier. In this way luminometric analysis can be performed with higher sensitivity and with multiple specificity, if you use multiple optical fibers, each of which is able to capture different antigens and if each of these optical fibers placed in a separate chamber for the sample so that it can simultaneously conduct several different immunoassays by adding in the same stage several different antibodies labeled with luciferase. An alternative charge-coupled device (CCD) or an equivalent device, such as a diode matrix or the photomultiplier can be used for simultaneous monitoring of a number of discrete areas on the surface 1 or 2 spatial detector arrays, each with different immobilized antibodies specific to different the receipt. Similarly, the use of such fibers or charge-coupled devices in which the immobilized antigens or antibodies against immunoglobulins specific for the antibody to the target, allows the analysis of specific antibodies by competitive binding, where the number of labeled luciferase antibodies bound to the antigen on the fiber will be reduced while adding competing antibodies in a sample. After removal of the sample and labeled with luciferase antibodies after treatment of the fiber with a solution of D-luciferin and mg2+-Asia-Pacific-substrate the amount of light detected by the photomultiplier tube, can be correlated with the amount of antibody in the sample, which is specific to the immobilized antigen or antibody against immunoglobulin. It should be noted that if you use the fibers on the surface which are associated with oligonucleotide probes (see method described in the application WO 9306241, the authors of the present invention) and the oligonucleotides were labeled with luciferase, can be carried out analysis of oligonucleotides and polynucleotides similar to the analysis using the complex antigen-antibody as the temporal analysis of antigens and nucleic acids from the same sample. The main advantage of the implementation analysis using labeled luciferase material, communicating on the surface of the light guide, such as a planar waveguide or an optical fiber (there may be several) is that in this analysis the separation of the reactants from the desired sample, i.e., the associated sample is not critical, since the detector registers mainly of the light, which is generated at the few hundred nanometers of the surface of the light guide, and not the light that is generated in the bulk solution. Thus, the analysis of this type (usually referred to as analysis type "sharp decline") does not require washing stages or successive addition of reagents, and therefore it can be implemented very quickly and not necessarily in the fluid flow using a flow-through cuvette. Labeled agents of the present invention, and the method of their production and use, as clearly illustrated in the following examples with reference to the drawings, but it should be noted that these examples should not be construed as limiting the invention. In Fig.1 presents a graph of the dependence of luminescence from time to time, Piestany MD2+ATP, as described in example 1. In Fig. 2 presents graphs of the dependence of luminescence from a quantity of ricin, derived from the analysis carried out as described in example 1. The top graph obtained using 1/100-breeding conjugate lgG-luciferase, and the bottom graph obtained using 1/1000-breeding conjugate. Equation 1:The reaction involving luciferase Firefly: < / BR> LH2- AMP.E + O2--> L + CO2+ AMP + light where LH2- luciferin; E - luciferase; AMP - amp, FF1- inorganic pyrophosphate; and L - oxyluciferin. Example 1 For all measurements of light emission were used luminometer Multiliteand polystyrene 3,5-ml tubes (Biotrace Bridgent UK). The Firefly luciferase (L-5256), DTT, BSA (Fraction V), ATP and ricin were obtained from Sigma (l, UK); D-luciferin was obtained from Fluka (Gillingham, UK), and sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (sulfo-SMCC) was obtained from Pierce & Warriner (Chester, UK). Sheep antibodies against ricin was produced by standard methods. Other reagents were of analytical purity. As a substrate for luciferase used in the (adenosine triphosphate), 2 mmol/l EDD, 2 mmol/l DTT (dithiothreitol), 0.2% BSA (bovine serum albumin) and 2 gmol/l of sodium pyrophosphate. Fresh substrate were prepared daily from mother solutions. Analysis of luciferase activity was performed by adding 210-6l the analyzed sample to 20010-6l substrate (described above) in polystyrene 3.5 ml/tube and subsequent measurement of luminescence after a 5-second delay by integrating the data over a period of time of 10 sec. Method of substrate protection was as follows: Luciferase (4.2 gmol/l) was mixed with sulfo-SMCC (630 gmol/l) in 10 mmol/l HEPES buffer (pH of 7.75), and the mixture was incubated for 30 minutes at room temperature. Through the calculated time intervals, the sample was removed from the reaction mixture and 100-fold diluted in buffer, and then measured the luciferase activity of the sample. Then to the mixture was added with different concentrations MD2+-ATF or D-luciferin and traced their effect on luciferase activity in order to determine the concentrations of substrate, the most effective protection from inhibition. Covalent binding of sulfo-SMCC: sheep IgG antibodies against ricin was restored with the release of thiol groups possesse Warriner). Then prepare a solution of 4 mg of luciferase in 1 ml of phosphate buffer (pH 7.0) containing 0.5 mmol/l ATP, and 2.5 mmol/l of magnesium sulfate. After this was added 50 μl of 20 mmol/l sulfo-SMCC in phosphate buffer (pH 6.0) and the resulting mixture is incubated for 30 minutes at room temperature. Activated luciferase was purified using column for desalting with GF-5 Pierce, after which the activated luciferase and restored IgG were combined in a ratio of 1:1 in phosphate buffer (pH 7.0) containing 0.25 mmol/l EDD, 0.5 mmol/l ATP, and 2.5 mmol/l of magnesium sulfate. The reaction was carried out for 30 minutes at room temperature and then stopped by adding 10 μl of 1 mol/l cysteine for 20 minutes, after which the conjugate luciferase-antibody was purified on a desalting column with GF-5 Pierce. The obtained product was stored at 4oWith in sodium phosphate buffer (pH 7.0) containing 0.25 mmol/l etc, until its use. The residual activity of conjugated luciferase was evaluated using ELISA, as described below. Bioluminescent analysis of ELISA on the tubes: Polystyrene tubes were senzibilizirani overnight at 4oWith 100 Microlitre of ricin in carbonate-bicarbonate buffer is practical solution (PBs) and after blocking of the tubes 200 Microlitre 1% BSA was added 100 μl of labeled antibody, and then incubated for 1 hour at 37oC. After the tubes washed five times in PBs containing 5% tween-20. Then to each tube was added 200 μl of the substrate and immediately thereafter was measured by luminescence. Results: the Results obtained from the experiment on the substrate protection of the enzyme using MD2+ATP is shown in Fig.1, which shows a graph of percent residual luciferase activity measured using a luminometer, from time to time; and the specified graph was constructed by removing samples (3 repeat for each point) from the reaction mixture, which contained 0,6310-6mol/l sulfo-SMCC; 4,210-6mol/l of luciferase and mg2+-ATP. The graph shows the curves obtained for the reaction in the absence MD2+or ATP for the reaction using 0.03 mmol/l ATP, and 0.2 mmol/l MD2+; 0.25 mmol/l ATP, and 2 mmol/l of Mg2+; 0.25 mmol/l ATP, and 2.5 mmol/L. MD2+. It was also evaluated the protective effect of D-luciferin (not shown) and the result of this analysis, it was found that D-luciferin contributes to some of the conservation of luciferase activity, but to a much lesser extent than MD2+ATP. The maximum tested concentration of D-Luc the imposition of the initial luciferase activity, while using MD2+ATF has provided more than 40% saving activity. The results of bioluminescent analysis by ELISA of the tubes shown in Fig. 2. Polystyrene tubes were used to measure the luminous radiation is carried out directly in the luminometer Multilitebut for these purposes can also be used tablets for micrometrology and luminometer plates. The results testified to the production of active conjugate "luciferase-antibody and showed that the antibody retains its binding properties. Example 2 Sheep polyclonal antibody against ricin covalently linked with an optical fiber connected to a photomultiplier, and the region of the optical fiber, covalently linked to the antibody, were placed in a chamber into which you can pour and pour, if necessary, different solutions. Then carried out the analysis with the next cycle: (i) in the specified cell was added the substrate solution containing D-luciferin so that any present luciferase caused light emission; (ii) substrate solution was replaced with the test sample, sod is Uchenye luciferase; (iv) a solution of sheep antibodies against ricin was replaced with a substrate solution containing D-luciferin; (v) substrate solution was replaced with recovery buffer. Analysis in accordance with the above scheme allows to determine the amount of ricin in the sample by measuring the increase of the signal at the output of the photomultiplier in stage (iv) compared with the previous signal and estimate the amount of ricin using a standard curve constructed from the data, increasing the signal to known amounts of ricin. The method of conjugation luciferase and labeled chemical molecules described in this application can be used for luciferase described in WO 9525798. Similarly this method can also be used for other luciferase. 1. The method of obtaining labeled luciferase antibodies, including conjugation of Firefly luciferase with an antibody by (a) mixing the indicated luciferase either (i) with D-luciferine, or (ii) with magnesium ions and adenosine triphosphate (ATP), or (iii) with D-luciferine, magnesium ions and ATP, so that the concentration of magnesium ions and ATP when they are present, were respectively 0.2 to 2.5 mmol/ the use of covalently binding reagent. 2. The method according to p. 1, wherein the luciferase is mixed at the stage of (a) with D-luciferine, magnesium ions and ATP, and in which oxygen is excluded from the reaction of covalent binding (b). 3. The method according to p. 1 or 2, characterized in that stage (a) is carried out by mixing the indicated luciferase, D-luciferine, magnesium ions and/or ATP in solution. 4. The method according to PP. 1, 2 or 3, characterized in that as these magnesium ions and ATP are present in the form of marriageregistration (Mg2+ATP). 5. The method according to any of paragraphs.1-4, characterized in that stage (a) is carried out using 0.2 mmol/l or more ATP per 46-6mol/l of luciferase. 6. The method according to any of paragraphs.1-5, characterized in that stage (a) is carried out in the presence of 2 mmol/l of magnesium ions. 7. The method according to any of paragraphs.1-6, characterized in that the covalent binding reagent is glutaraldehyde, Succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) and/or Succinimidyl-4-(p-maleimidomethyl)butyrate (SMPB). 8. The method according to any of paragraphs.1-7, characterized in that stage (b) is carried out using covalent binding reagent after mixing luciferase reagent (i), (ii) or (iii).th antibody, representing antibody conjugated with Firefly luciferase way on PP.1-9. 11. The method of analysis for specific binding, including the stage of binding of labeled luciferase antibodies specified in paragraph 10, with the antigen. 12. The method according to p. 11, wherein the presence and/or amount of chemical particles target is determined by its specific binding to labeled luciferase antibody under item 10, further processing of the associated particles D-luciferine in the conditions under which the luciferase enzyme D-luciferin and causes the emission of light, and make the number any light emission from the presence of specified chemical species target. 13. The method according to p. 11 or 12, characterized in that the chemical particle-target is captured antigen and specific to the target immobilized antibody before binding with the labeled luciferase antibody under item 10. 14. The method according to p. 13, wherein the immobilized antibody kongugiruut with the surface of the fiber or speedmachine matrix, giving light to the photoelectric measuring device, such as a photomultiplier. 15.different topics the photomultiplier used for simultaneous monitoring of a number of discrete areas on the surface 1 or 2 spatial detector arrays, each region has different immobilized antibodies or antigens that are specific for different antigens or antibodies, respectively, so that the presence of a specific antigen can be detected by the position of the detected light radiation. 17. The method according to p. 15, characterized in that the photomultiplier is a charge-coupled device (CCD). 18. Set for use in the analysis of specific binding on p. 11, characterized in that it contains the antibody, which is conjugated with an active Firefly luciferase method according to any one of paragraphs.1-10, and D-luciferin. 19. Set under item 18, characterized in that the activity of the conjugated luciferase Firefly is 10% or more of the activity of Firefly luciferase on the basis of which was obtained labeled antibody. 20. Set under item 18, characterized in that it contains the fiber or speedmachine matrix, which immobilized antibodies.
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