WO1997017610A1

WO1997017610A1 - Method for fluorescent labeling of antibody

Info

Publication number: WO1997017610A1
Application number: PCT/US1996/017826
Authority: WO
Inventors: Bryan R. Wilson; R. Tyler Mccabe; Robert E. Raulli; Gary I. Kevelson
Original assignee: Pharmaceutical Discovery Corporation
Priority date: 1995-11-08
Filing date: 1996-11-05
Publication date: 1997-05-15
Also published as: AU7723096A

Abstract

A method has been developed for labeling of antibodies with a fluorescent dye where the antibody is first bound to immobilized antigen, the antibody is labeled with the dye, the unbound dye is removed, and then the antibody is eluted from the antigen. As demonstrated by the examples, antibody immunoreactive with AI was labeled with a fluorescent dye and then used in an assay for AI. The results showed that the antibody prepared by this method had a 20 % increase in specific binding compared to radioimmunoassay (RIA).

Description

METHOD FOR FLUORESCENT LABELING OF ANTIBODY

Background of the Invention

This invention is generally in the field of labeled antibody, and is more specifically directed to a method for labeling an antibody with a dye.

Many immunoassay employ labeled antibodies, including antibodies having radiolabels, dyes, fluorescent molecules, and enzymes or substrates conjugated to the antibodies directly or indirectly via a second molecule such as biotin-avidin or an

immunoreactive with the first antibody

While labels such as radiolabels are so small as to almost invariably not affect binding of the labeled antibodv larger labels can interfere with the antibody binding, lowering binding affimty The most common solution to this problem has been to attach the label to the antibody via a linker, in many cases a second antibody to which the label is actually bound and which is easily added in excess after reaction of the analyze with the first antibody This requires a two step process, however, increasing the reaction cost as well as reagent costs

It is therefore an object of the present invention to provide a method for labeling antibodies with a fluorescent label which does not alter binding affinity of the antibody

It is a further object of the present invention to provide a method for labeling antibodies with a fluorescent label which is simple, rapid and inexpensive

Summary of the Invention

An affinity purification and labeling process that yields an increased dye to protem ratio has been developed This process protects the immunogenic site from steric interference by conjugating the fluorescent moiety to the antibody molecule while the active site is bound to antigen This results in the ability to greatly increase the dye (signal) to protein ratio while maintaining the antibody's affinity. This technology eliminates the need for a secondary antibody and greatly improves the diagnostic sensitivity and accuracy of the assay. This technology also results in the reduction of procedural steps as well as a shortened incubation and assay time. The method involves labeling of antibodies with a fluorescent dye, where the antibody is first bound to immobilized antigen, the antibody is labeled with the dye, the unbound dye is removed, and then the antibody is eluted from the antigen.

As demonstrated by the examples, antibody immunoreactive with angiotensin I ("Al") was labeled with the amine reactive fluorescent dye dichlorotriazinylaminofluorescein I dihydrochloride ( "DTAF"), and then used in an assay for Al. The results showed that the antibody prepared by this method had a greater than 20% increase in specific binding compared to radioimmunoassay (RIA).

Detailed Description of the Invention

Measurement of fluorescence has a number of advantages as compared to measurement of other types of labels. Results can be obtained extremely rapidly, in milliseconds or less, allowing use of the label in following binding over time. In contrast, measuring radioactivity requires reaching equilibrium (e.g. , 60 minutes), then stopping of the reaction (15 to 60 seconds), then a relatively lengthy process (hours to days to months) to make a determination. Other advantages include the ability of some fluorescent labels to fluoresce at different wavelengths with different intensities under different conditions. The latter is useful in determining whether or not the labeled ligand has penetrated into a cell, since the conditions, for example, pH, intracellularly versus extracellularly are quite different. For example, one can also look at lateral mobility, the passage of molecules into and out of cells. This is not possible with radioactive labels. The intensity of some fluorescent labels declines over time after binding, allowing one to measure binding kinetics with one label. One can also use a quenching ligand to reduce intensity, for example, where more than one fluorescent label has been used, to create a three dimensional structural/activity comparison of a receptor conformation.

As described herein, a method for labelling antibody with a fluorescent dye to yield an antibody with a higher affinity for substrate than the same antibody labelled with a radioactive label has been developed. The method principally involves first binding the antibody to the desired antigen which has been immobilized, then labelling the bound antibody, then washing the antibody to remove unbound label and then to elute the labelled antibody.

The method uses the following reagents. Antibodies Antibodies that can be used as described herein include natural and recombinant antibodies and fragments thereof including at a minimum the variable region. Monoclonal and polyclonal antibodies can be used, depending on the desired specificity of binding. Antibodies are commercially available or can be prepared using standard immunization and recombinant techniques. Fluorescent Dyes DTAF (dichlorotriazinylamino fluorescein I dihydrochloride,

Research Organics. Cleveland, OH), used in the following example, is a preferred dye. The criteria for selecting an appropriate fluorescent label include the reactivity of the dye, the solubility of the dye, and type of fluorescent measurement to be made. For example, the dye may be amine reactive, aldehyde/ketone and cytidine reactive, or sulfhydryl reactive. The solubility of the dye is relevant to the buffering system and mobile phases used in the purification and labeling. The type of fluorescent assay measurement being performed, such as energy transfer, where the emission wavelength of the labeled antibody must be matched to the excitation wavelength of another assay component, is also an important variable. Examples of other suitable fluorescent labels include fluorescein, 5,6-carboxymethyl fluorescein

(5-carboxyfluorescein-N-hydroxysuccinimide ester), Texas red, nitrobenz-2-oxa-l,3-diazol-4-yl (NBD), coumarin, dansyl chloride, and rhodamine (5,6-tetramethyl rhodamine). These can be obtained from a variety of commercial sources, including Molecular Probes, Eugene, OR and Research Organics, Cleveland, Ohio.

Reagents for Coupling Dye to Antibody The antibodies are bound to an appropriate substrate such as CH SEPHAROSE^® 4b (Pharmacia, Uppsala, Sweden), or AFFIGEL^® 10,

(Bio-Rad, Hercules, California), or other chromatography substrates, via the antigen of interest. The antibodies can also be bound to the antigen immobilized to a solid support such as a polystyrene well or test tube, or membranes or films. The antigen of interest is conjugated to the support using standard techniques to form covalent or ionic bonds.

Non-specific antibodies are washed away with a low ionic strength buffer such as 10 mM phosphate buffer pH 7.5. The affinity support with the specific antibodies attached is equilibrated in a buffer equivalent to 50 mM sodium borate, 40 mM sodium chloride, pH 9.0 - 9.4. The following methods are used for coupling dye to bound antibody, and removal of unbound dye.

Method for Coupling and Removal of Unbound Dye In the preferred embodiment, once equilibrium is reached, the dye is introduced to the affinity support with the specific antibodies attached at a concentration of 0.2 to 2 mg ml^"1 in 50 mM sodium borate, 40 mM sodium chloride, pH 9.0 - 9.4. The dye solution is passed through the column at a low flow rate -0 =2E25 to 0.5 ml m ¹) for a mimmum of 40 column volumes and maximum of 400 column volumes (theoretically labeling 100% of the antibodies attached to the column). The unbound dye removed by washing, for example, with 10 column volumes of 10 mM phosphate buffer pH 7.5 followed by a gradient increase to 25% ethylene glycol in 10 mM sodium phosphate which is maintained for 20 column volumes. The column is equilibrated back to 10 mM sodium phosphate pH 7.5.

Elution and Washing of Labelled Antibody

The column is washed with a glycine-salt buffer, then washed with a buffered salt solution to remove the labelled antibody. For example, the column is subjected to 4 column volumes of 100 mM glycine, 500 mM sodium chloride pH 2.0 followed by 4 column volumes of 10 mM sodium phosphate pH 7.5 in which the highly specific labeled antibody is eluted and pH is adjusted to 7.5 - 7.8 with tris buffered saline. The labeled antibody is concentrated, for example, to a concentration of between 0.5 and 2 mg ml-1. The dye to protein ratio is calculated by measurement of the absorbance at 280 nm and 495 nm. Example: (ABS₄₉₅ =F6 36,629) = F6 [(ABS₂₈₀ - ( 0.309 = x ABS₄₉₅)) =F6212,000] =3D dye to protein ratio. Methods for measuring fluorescence

Due to the chemical nature of the fluorescent-labeled compounds, treatment of the assay plates and well caps with a chlorinated organopolysiloxane dissolved in hapten is necessary to avoid non-specific reactions. Another material that could be used instead of a chlorinated organopolysiloxane is dimethyldiphenylpolysiloxane.

The excitation and emission spectra of the fluorescent label are measured using commercially available instrumentation. Quantitation of binding is accomplished by creating a standard line relating fluorescence intensity values (counts per second) to known amounts of fluorescent ligand (antigen) in a sample. The amount of fluorescent ligand bound is estimated by linear regression using fluorescence intensity, as described in more detail below.

The present invention will be further understood by reference to the following non-limiting example of a fluorescent labeled antibody and its use in an assay for Al, the decapeptide cleaved by renin from the hepatic alpha-2-globulin carrier protein angiotensinogen (renin substrate). Example 1: Preparation of Fluorescent labeled antibody.

Materials

The following equipment was used: a) FPLC ( Pharmacia, Uppsala, Sweden) b) spectrophotometer, Lambda 2, (Perkin Elmer, Ueberlingen,

GmbH) c) HPLC Millennium system, ( Waters, Milford, MA) d) stirred cell with 30,000 D molecular weight cutoff membranes (Amicon Division W.R. Grace & Company, Beverly, MA) with nitrogen source e) magnetic stir plates

The following reagents were used: a) 10 mM sodium phosphate, pH 7.5-7.6 at 4 o C. b) 25% ethylene glycol in 10 mM sodium phosphate, pH 7.5-7.6 at 4 o C. c) 0.1 M glycine. 0.5 M NaCl, pH 2.0 at 4 o C. d) 50 mM sodium borate, 40 mM NaCl, pH at 4 o C. e) 0.2 M sodium phosphate, 0.2 M NaCl. pH 6.8 Methods The following procedure was used: a) The FPLC XK column (Pharmacia, Uppsala. Sweden) packed with affinity matrix was equilibrated with 20 column volumes of 10 mM sodium phosphate, pH 7.5 at 4 o C. flow rate 2 ml min-1. b) An antibody solution with a concentration of 1 mg ml-1 was circulated through the column at a flow rate of 0.5 ml min-1 for 550 min.

The antibody solution passed through the column at a minimum of two and a maximum of four times. The flow rate was then increased to 2 ml min-1 for an additional 225 min of washing. The column was then washed with 20 column volumes of 10 mM sodium phosphate, pH 7.5 at 4=F8C. flow rate 2 ml min-1. The flow rate was changed to 1 ml min-1. c) The buffer was then changed using a gradient of equal parts of 10 mM sodium phosphate to 50 mM sodium borate, 40 mM NaCl, pH 9.0, over 30 min. The column was then washed with 2 column volumes of 50 mM sodium borate, 40 mM NaCl, pH 9. d) The dye to be coupled to the antibody was then bound by circulating DTAF in 50 mM sodium borate, 40 mM NaCl, pH 9. through the column. The column was then washed with 20 column volumes of 50 mM sodium borate. 40 mM NaCl, pH 9. e) The buffer was changed back to the low ionic strength phosphate buffer using a gradient of equal pans of 50 mM sodium borate, 40 mM NaCl, pH 9 to 10 mM sodium phosphate. pH 7.5. over 30 min. The column was then washed with 20 column volumes of 10 mM sodium phosphate. f) The excess dye was then removed by washing the column with 20 column volumes of 25 % ethylene glycol in 10 mM sodium phosphate, pH 7.5 at 4 o C 2E. The pump was then flushed with O. l M glycine. 0.5 m NaCl, pH 2.0 at 4 o C. and the column washed with 0.1 M glycine, 0.5 M NaCl, pH 2. Four column volume fractions were collected and 3 ml 0.1 M untitrated tris added per collection tube. g) The column was then eluted with four to six column volumes

10 mM sodium phosphate buffer, pH 7.5 at 4 o C, while adding 3 ml 0.1 M untitrated Tris to the first tube, 1.5 ml to the second tube and 0.5 ml to the third tube. This was repeated six times to ensure all conjugated antibodies were recovered. The column was then washed with 20 column volumes of 10 mM sodium phosphate, pH 7.5 at 4 o C . and the system flushed with 20% ethanol. h) The labeled antibody was concentrated from the pooled sodium phosphate fraction, adjusting the pH to 7.4 to 7.8. The concentrated antibody was added to a cell for exchanging buffers to 50 mM sodium phosphate, 137 mM NaCl, pH 7.5 or 50 mM Tris, 137 mM NaCl, pH 7.5. These buffers may or may not have NaN₃ or other preservative added to them. i) The glycine fraction was collected in the event that the purification failed. Once the concentrated glycine strip is evaluated by HPLC and determined not to contain antibody it is discarded. j) The ratio of dye to protein is calculated from the ratio of absorbance in the ultraviolet range (280 nm is indicative of protein concentration) versus the visible range (495 nm is wavelength to measure dye concentration). The purity of the labeled antibody is then determined by HPLC.

Example 2: Measurement of Al concentration in a patient sample using fluorescently labeled antibody.

Reagents in Kit

The kit includes an Al-Photodiagnostic Fluorescent-Labeled Antibody (excitation and emission maxima of 495 nm and 517 nm), a set of seven angiotensin I (Al) standards ranging from 8 to 1 ,024 pg Al in 10 μl, two calibration standards (TSB, 100% of the total specific binding; NSB, non specific binding), Al-Photodiagnostic Solid Support, Al generation reagents, 96-well plates for incubation and filtration, buffer, and a detailed protocol. The kit is designed to test 192 wells. Assay Plate Preparation The assay uses a 96-well microtiter plate for assay incubation.

Due to the chemical nature of the fluorescent-labeled compounds, treatment of the plates and well caps with chlorinated organopolysiloxane dissolved in hapten is necessary. This procedure creates results that are representative of the specific antibody antigen interaction rather than the non-specific interactions that result without the treatment. Principles of the Test

The PDC Al-Photodiagnostic is based on competitive inhibition of fluorescent-labeled antibody binding to solid-phase-immobilized Al (Al-Photodiagnostic Solid Support) using free Al from patient plasma samples. In the presence of free Al from the patient plasma sample, the Al Photodiagnostic Fluorescent-Labeled Antibody is displaced in a concentration dependent manner from the Al-Photodiagnostic Solid Support. Subsequently, Al Photodiagnostic Fluorescent-Labeled Antibody binds to the free Al from the patient plasma sample. The amount of fluorescent-labeled antibody displaced from the Al Photodiagnostic Solid Suppon by free Al in the patient plasma sample can then be quantified from the standard curve. PRA is estimated from the determined Al value in the patient plasma sample. Protocol

Al Photodiagnostic standards, controls, or "Al generated" patient plasma samples (10 μl) are combined in 96-well plates (or si conized microfuge tubes) containing the Al-Photodiagnostic Solid Suppon and Al Photodiagnostic Fluorescent-Labeled Antibody The assay volume is allowed to reach equilibrium by invemng and rotating the suspension for 90 minutes at room temperature (RT). The bound complex of Al Photodiagnostic Fluorescent-Labeled Antibody/AI Photodiagnostic Solid Suppon is separated trom all other assay components by filtration through a filter membrane located on the bottom of the 95-well plate (or by centrifugation if the assay is performed using microfuge tube format). Fluorescence intensity of the samples is measured using spectrofluorometry A standard curve is plotted and the quantity of Al (pg) in the patient sample is determined trom the standard curve The Vc Specific Binding is calculated for each point in the standard curve and each patient sample by using the following equation: % Specific Binding = B/B₀ x 100, where B is the fluorescence intensity value (counts per second, cps) for each standard or patient sample, and B₀ is the fluorescence intensity value (cps) for the TSB (Total Specific Binding). The NSB (Nonspecific Binding) value (cps) is subtracted from each cps value for standards, controls, and Al-generated patient plasma samples. The % Specific Binding on the y-axis is plotted in relation to the log Al (pg) on the x-axis. Al levels in the patient plasma samples are determined by non-linear regression analysis using the standard curve. The mean Al value (pg) for each triplicate set of patient plasma samples is then calculated. PRA is estimated from the determined Al value in the patient plasma sample. Comparison of the values obtained with the fluorescently labeled antibody as compared with results obtained using the same unlabeled antibody with a radioactive labeled peptide show that the fluorescently labeled antibody prepared as described in Example 1 has a 20% greater specific activity as compared with the radiolabelled antibody. Correlation between PDC AI-PhotodiagnosticTM versus radioimmunoassay performed by the Cornell Medical Center Hypertension Laboratory (NYC) yielded a slope of 0.955 =F1 0.04 and a correlation (r) = 3D 0.971 for 41 samples.

Claims

We claim:

1. A method for labeling an antibody with a fluorescent label having increased affinity for antigen as compared with the antibody labelled with a radiolabel comprising binding the antibody to immobilized antigen, covalently coupling the fluorescent label to the antibody, washing the uncoupled fluorescent label from the labelled antibody, and eluting the labelled antibody from the immobilized antigen.

2. The method of claim 1 further comprising calculating the dye to protein ratio.

3. The method of claim 1 wherein the antibody is polyclonal.

4. The method of claim 1 wherein the antibody is monoclonal.

5. The method of claim 1 wherein the antibody is immunoreactive with angiotensin.

6. The method of claim 1 wherein the dye is selected from the group consisting of dichlorotriazinylaminofluorescein I dihydrochloride, fluorescein, 5,6-carboxymethyl fluorescein, Texas red. nitrobenz-2-oxa-1.3-diazol-4-yl, coumarin, dansyl chloride, and 5,6-tetramethyl rhodamine.

7. A kit for quantitating antigen in a sample comprising antibody labelled with a radiolabel comprising a fluorescently labelled antibody prepared by binding the antibody to immobilized antigen, covalently coupling the fluorescent label to the antibody, washing the uncoupled fluorescent label from the labelled antibody, and eluting the labelled antibody from the immobilized antigen, antigen in known quantities, and buffer in which the antigen is bound by the labelled antibody.

8. The kit of claim 7 further comprising means for measuring fluorescence.

9. The kit of claim 7 further comprising means for reacting antigen in a sample with the labelled antibody.

10. The kit of claim 7 wherein the antibody is polyclonal.

11. The kit of claim 7 wherein the antibody is monoclonal.

12. The kit of claim 7 wherein the antibody is immunoreactive with angiotensin and the antigen is angiotensin.

13. The kit of claim 7 wherein the dye is selected from the group consisting of dichlorotriazinylaminofluorescein I dihydrochloride, fluorescein, 5,6-carboxymethyl fluorescein, Texas red, nitrobenz-2-oxa-l ,3-diazol-4-yl, coumarin, dansyl chloride, and

5 , 6-tetramethy 1 rhodamine .

14. The kit of claim 12 wherein the dye is dichlorotriaziny lamino fluorescein I dihydrochloride.