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This is a continuation-in-part application of U.S. application Ser. No. 454,768, filed Dec. 30,1982, now U.S. Pat. No. 4,520,110, issued May 28, 1985, which is a continuation-in-part application of U.S. Ser. No. 309,169, filed Oct. 6, 1981, now abandoned. 15
BACKGROUND OF THE INVENTION
Field of the Invention
Fluorescent probes are valuable reagents for the analysis and separation of molecules and cells. Some specific 20 examples of their application are: (1) identification and separation of subpopulations of cells in a mixture of cells by the techniques of fluorescence flow cytometry, fluorescence-activated cell sorting, and fluorescence microscopy; (2) determination of the concentration of a 25 substance that binds to a second species (e.g., antigenantibody reactions) in the technique of fluorescence immunoassay; (3) localization of substances in gels and other insoluble supports by the techniques of fluorescence staining. These techniques are described by Her- 30 zenberg et al., "Cellular Immunology," 3rd ed., chapt. 22, Blackwell Scientific Publications, 1978 (fluorescence-activated cell sorting); and by Goldman, "Fluorescence Antibody Methods," Academic Press, New York, 1968 (fluorescence microscopy and fluorescence 35 staining).
When employing fluorescers for the above purposes, there are many constraints on the choice of the fluorescer. One constraint is the absorption and emission characteristics of the fluorescer, since many ligands, 40 receptors, and materials associated with such compounds in the sample in which the compounds are found e.g. blood, urine, cerebrospinal fluid, will fluoresce and interfere with an accurate determination of the fluorescence of the fluorescent label. Another consideration is 45 the ability to conjugate the fluorescer to ligands and receptors and the effect of such conjugation on the fluorescer. In many situations, conjugation to another molecule may result in a substantial change in the fluorescent characteristics of the fluorescer and in some 50 cases, substantially destroy or reduce the quantum efficiency of the fluorescer. A third consideration is the quantum efficiency of the fluorescer. Also of concern is whether the fluorescent molecules will interact with each other when in close proximity, resulting in self- 55 quenching. An additional concern is whether there is non-specific binding of the fluorescer to other compounds or container walls, either by themselves or in conjunction with the compound to which the fluorescer is conjugated. 60
The applicability and value of the methods indicated above are closely tied to the availability of suitable florescent compounds. In particular, there is a need for fluorescent substances that emit in the longer wavelength visible region (yellow to red). Fluorescein, a 65 widely used fluorescent compound, is a useful emitter in the green. However, the conventional red fluorescent label rhodamine has proved to be less effective than
fluorescein. The impact of this deficiency is felt in the area of fluorescence-activated cell sorting. The full potential of this powerful and versatile tool has not yet been realized because of limitations in currently available fluorescent tags. Two and three-parameter fluorescence sorting have not been effectively exploited, largely because of the unavailability of good long wavelength emitting probes.
Other techniques, involving histology, cytology, immunoassays would also enjoy substantial benefits from the use of a fluorescer with a high quantum efficiency, absorption and emission characteristics at longer wavelengths, having simple means for conjugation and being substantially free of non-specific interference.
SUMMARY OF THE INVENTION
Proteins with bilin prosthetic groups are employed as fluorescent labels in systems involving ligand-receptor reactions. The biliproteins are readily conjugated, provide for high quantum efficiency with absorption and emission at long wavelengths in the visible, and enhance the sensitivity and accuracy of methods involving ligand-receptor reactions. The biliproteins may be used individually, in combination, or together with nonproteinaceous fluorescers.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the high pressure liquid chromatograms of phycoerythrin-immunoglobulin conjugate (PE-S-S-IgG) and the reactant precursors thereof, thiolated phycoerythrin (PE-SH) and activated immunoglobulin (IgG-S-S-Pyr).
FIG. 2 shows the fluorescence-activated cell sorter analysis of a cell population containing spleen cells bearing PE-B-A stained anti-IgG immunoglobulin.
FIG. 3a shows the visualization of a mixture of agarose beads containing a labeled anti-immunoglobulin by fluorescence microscopy utilizing standard fluorescein emission filter combinations, wherein some beads were labeled with PE-B-A and some beads were labeled with fluorescein-avidin.
FIG. 3b shows the visualization of the same cell population as in FIG. 3a under fluorescence microscopy utilizing a red filter combination.
DESCRIPTION OF THE SPECIFIC
Compositions are provided comprising biliproteins, (the term "biliproteins" is equivalent to the term "phycobiliproteins") conjugated to a member of a specific binding pair, said pair consisting of ligands and receptors. These compositions find use for labeling by non-covalent binding to the complementary member of the specific binding pair. A wide variety of methods involve competitive or non-competitive binding of ligand to receptor for detection, analysis or measurement of the presence of ligand or receptor. Many of these techniques depend upon the presence or absence of fluorescence as a result of non-covalent binding of the labeled member of the specific binding pair with its complementary member.
The conjugates of the subject invention are biliproteins bound either covalently or non-covalently, normally covalently, to a particular ligand or receptor. The biliproteins have a molecular weight of at least about 30,000 d, (d-daltons) more usually at least about
40,000 d, and may be as high as 600,000 or more daltons plement factors, lymphokines mucoproteins, poly sialic
usually not exceeding about 300,000 d. acids, chitin, collagen, keratin, etc.
The biliproteins will normally be comprised of from 2 Depending upon the molecule being labeled, a wide to 3 different subunits, where the subunits may ranged variety of linking groups may be employed for conjufrom about 10,000 to about 60,000 molecular weight. 5 gating the biliprotein to the other molecule. For the The biliproteins are normally employed as obtained in most part, with small molecules, those under 2,000 mote natural form from a wide variety of algae and lecular weight, the functional group of interest for linkcyanobacteria. The presence of the protein in the bili- «ng will be carbonyl, either an aldehyde to provide for proteins provides a wide range of functional groups for reductive animation or a carboxyl, which in conjuncconjugation to proteinaceous and non-proteinaceous 1° ti°n with carbodiimide or as an activated ester e.g. molecules. Functional groups which are present include N-hydroxy succinimide, will form a covalent bond with amino, thio and carboxy. In some instances, it may be amino groups present in the biliprotein; a thio ether desirable to introduce functional groups, particularly or disulfide, where the biliprotein may be modified with thio groups where the biliprotein is to be conjugated to an activated olefin and a mercapto group added or another protein 15 activated mercapto groups joined e.g. Ellman's reagent;
Depending upon the nature of the ligand or receptor isothiocyanate; diazonium; nitrene or carbene. Where to be conjugated, as well as the nature of the biliprotein, biliproteins are conjugated with a protein, various the ratio of the two moieties will vary widely, where functional reagents may be employed, such as dialdethey may be a plurality of biliproteins to one ligand or „n ^ tetrazohum salts, diacids, or the like or alternareceptor or a plurality of ligands or receptors to one 20 tive}Yl °TM 01 both of *e two Proteins involved may be biliprotein. For small molecules, that is, of molecular modified for conjugation to the other protein, for examweight less than 2,000 d, there will generally be on the Ple' a mercfto Sr°uP »ay be present or be introduced average at least one and not more than about 100, usu- ont one P'otem a*d an activated olefin e.g. maleimide „ . ^, u cn * j * U-t » ■ mtroduced onto the other protein, ally not more than about 60 conjugated to a biliprotein. ~ . _ . ... . K, ... ., .„.'. . . . * ■ "i i * u * i Ann 25 There is ample literature for conjugating a wide vanWith larger molecules, that is at least about 2^)00 mo- of ^ to tems. See fQT e| le A. R
lecular weight, more usual y at least about 5,000 molec- Q1 TheV ... Vol. IIA, 3rd Ed., N. Neurath and
ular weight, the ratio of biliproteins o ligand or recep- R L Hm e(J Academic Press> 1_103 (19760 A.
tor may vary widely, since a plurality of biliproteins N Gkzer et ^ «chenlica] Modification of Proteins," may be present in the conjugate or a plurality of the 3Q Laborat Techniques in Biochemistry and Molecular
specific binding pair member may be present m the Bhl Vol 4> pRT j T s Work and R Work> eds
conjugate. In addition in some instances, complexes North-Holland Publishing Co. (1975); and K. Peters et
may be formed by covalently conjugatmg a small liquid ^ Am Rev Biochem^ 46) 423-51 (1977), the descrip
to a biliprotem and then forming a specific bmding pair tions of which are incorporated by reference herein, complex with the complementary receptor, where the 35 Examples of commercially available cross-linking rea
receptor may then serve as a ligand or receptor in a gents are disclosed in the Pierce 1981-82 Handbook and
subsequent complex. General Catalog, pp. 161-166, Pierce Chemical Co.,
The ligand may be any compound of interest for Rockford 111.
which there is a complementary receptor. For the most Known linking procedures as described in the above part, the ligands of interest wUl be compounds having 4o publications may be employed. For example, the
physiological activity, either naturally occurring or phycobiliprotein may be reacted with iminothiolane,
synthetic. One group of compounds will have molecu- thereby placing an accessible sulfhydryl group thereon,
lar weights in the range of about 125 to 2,000, more The other component ofthe conjugate may be activated
usually from about 125 to 1,000, and will include a wide by reaction with succinimidylpyridylthiopropionate.
variety of drugs, small polypeptides, vitamins, enzyme 45 Mixture ofthe two prepared components ofthe conju
substrates, coenzymes, pesticides, hormones, lipids, etc. gate results in joining thereof through disulfide bonds.
These compounds for the most part will have at least Alternatively, instead of employing succinimidyl
one heteroatom, normally chalcogen (oxygen or sulfur) pyridylthiopropionate, the protein may be reacted with
or nitrogen and may be aliphalitic, alicyclic, aromatic, m-maleimidobenzoyl N-hydroxysuccinimide ester, and
or heterocyclic or combinations thereof. Illustrative 50 the resulting conjugate combined with the sulfhydryl
compounds include epinephrine, prostaglandins, thy- modified protein to form a thioether.
roxine, estrogen, corticosterone, ecdysone, digitoxin, As previously indicated, instead of having a covalent
aspirin, pencillin, hydrochlorothiazide, quinidine, oxy- bond between the specific binding pair member of inter
tocin, somatostatin, diphenylhydantoin, retinol, vitamin est and the biliprotein, non-covalent bonds may be em
K, cobalamin, biotin and folate. 55 ployed. For example, if one wishes to conjugate a bili
Compounds of greater molecular weight, generally protein to avidin, biotin may be covalently conjugated
being 5,000 or more molecular weight include to the biliprotein through its carboxyl group, and the
poly(amino acids)-polypeptides and proteins-polysac- resulting biotinylated biliprotein combined with avidin,
charides, nucleic acids, and combinations thereof e.g. whereby a biliprotein labeled avidin will result,
glycosaminoglycans, glycoproteins, ribosomes, etc. 60 As already indicated, biliproteins are naturally occur
Illustrative compounds include albumins, globulins, ring compounds which may be found in a wide variety
hemogloblin, surface proteins on cells, such as T- and of sources and even individual sources may have more
B-cells e.g. Leu, Thy, la, tumor specific antigens, a- than one biliprotein.
fetoprotein, retinol binding protein, C-reactive protein, Examples of phycobiliproteins useful in the present
enzymes, toxins, such as cholera toxin, diphtheria toxin, 65 invention are allophycocyanin, phycocyanin, phycoer
botulinus toxin, snake venom toxins, tetrodotoxin, saxi- ythrin, allophycocyanin B, B-phycoerythrin, phycoery
toxin, lectins, such as concanavalin, wheat germ agglu- throcyanin, and b-phycoerythrin. The structures of
tinin, and soy bean agglutinin, immunoglobulins, com- phycobiliproteins have been studied and their fluores
cent spectral properties are known. See A. N. Glazer, "Photosynthetic Accessory Proteins with Bilin Prosthetic Groups," Biochemistry of Plants, Volume 8, M. D. Hatch and N. K. Boardman, EDS., Academic Press, pp. 51-96 (1981), and A. N. Glazer, "Structure and Evolu- 5 tion of Photosynthetic Accessory Pigment Systems with Special Reference to Phycobiliproteins," The Evolution of Protein Structure and Function, B. S. Sigman and M. A. Brazier, EDS., Academic Press, pp. 221-244 (1980). The spectroscopic properties, including fluores- io cence emission maxima, of some common phycobiliproteins are shown below in Table 1.
C = cyanobacteria; R = red aigae.
2For a given biliprotein, the exact positions of the absorption and emission maxima vary somewhat depending on the organism that serves as the source of the protein and on the method of purification.
Of particular interest are biliproteins having absorption maxima of at least about 450 nm, preferably at least about 500 nm, having Stokes shifts of at least 15 nm, preferably at least about 25 nm, and having fluorescence emission maxima of at least about 500 nm, preferably at 35 least about 550 nm. The subject conjugates may be used in a wide variety of ways, enhancing known methodologies for the detection, diagnosis, measurement and study of antigens, either present as individual molecules, or in more complex organizations, such as viruses, cells, 40 tissue, organelles e.g. plastids, nuclei, etc.
One of the uses of the subject conjugates is in fluorescent staining of cells. The cells may then be observed under a microscope, the presence of the fluorescer being diagnostic of the presence of a specific determi- 45 nant site or the cells may be used in a fluorescence activated cell sorter (FACS). One or more of the biliproteins may be used, where the fluorescence emission maximum of the biliproteins is separated by at least about 15 nm, preferably by at least about 25 nm. Alter- 50 natively, the biliproteins may be used in conjunction with fluorescers other than biliproteins, for examples fluorescein, dansyl, umbelliferone, benzoxadiazoles, pyrenes, rose bengal, etc., where the emission maxima are separated by at least about 15 nm, preferably at least 55 about 25 nm.
By using combinations of fluorescers, one can provide for the detection of subsets of aggregations, such as particular types of cells, strains of organisms, strains of viruses, the natural complexing or interaction of differ- 60 ent proteins or antigens, etc. Combinations of particular interest are combinations of fluorescein with biliproteins capable of being activated with the same laser light source. That is, biliproteins which have absorption maxima in the range of about 450 to 500 nm e.g. phyco- 65 erythrin.
Another use of the subject biliproteins is in immunoassays or competitive protein binding assays, where the
subject biliproteins serve as fluorescent labels. Here, the biliprotein may be conjugated to either a ligand or a receptor, particularly an antibody. While for the most part the antibodies will be IgG, other antibodies such as IgA, IgD, IgE and IgM may also find use. In addition, various naturally occurring receptors may be employed, particularly receptors having high binding specificity, such as avidin. By biotinylating either the receptor, the biliprotein or both, one can link various molecules through avidin. A wide variety of fluorescent assays are known. A few of these assays are illustrated in U.S. Pat. Nos. 3,998,943; 3,985,867, 3,996,345; 4,036,946; 4,067,959; 4,160,016 and 4,166,105, the relevant portions of which are incorporated herein by reference.
The biliproteins have many favorable properties. (1) they have very high absorption coefficients in the longer wavelength visible spectral region; (2) they have high fluorescence quantum yields; (3) they are stable proteins and have good storage stability; (4) they are highly soluble in aqueous solutions; (5) the biliprotein unit can readily be coupled to a wide range of biologically specific molecules; (6) they do not bind nonspecifically to cells. The fluorescence of biliproteinbiomolecule conjugates is more than thirty times as intense as that of fluorescein conjugates, on a molar basis. The long wavelength emitting fluorescent conjugates of the present invention have an additional advantage over shorter wavelength emitters. Most biomolecules in cells and body fluids do not absorb and emit in the red end of the visible spectrum. Consequently, biliprotein conjugates are less subject to interference by endogenous biomolecules than are shorter wavelength emitting conjugates. Furthermore, it is easier to work in the red end of the spectrum rather than in the ultraviolet region because plastic materials do not absorb and emit in the yellow to red spectral region.
The following examples are offered by way of illustration and not by way of limitation.
As an example of a fluorescent conjugate of the invention, a phycoerythrin-immunoglobulin conjugate was prepared. Thiolated phycoerythrin (PE-SH) was prepared by the addition of 2-iminothiolane to phycoerythrin. Activated immunoglobulin (IgG-S-S-Pyr) containing 2-pyridyl disulfide groups was prepared by the addition of N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP). The fluorescent conjugate (PE-S-S-IgG) was then formed by mixing PE-SH with IgG-S-S-Pyr. The product was analyzed by high pressure liquid chromatography (HPLC) on a Varian G3000SW column. This gel filtration column separates molecules primarily according to their hydrodynamic radii. PE-SH elutes 12 minutes after injection and IgG-S-S-Pyr elutes at about 13 minutes. See FIG. 1 showing the HPLC data. The reaction product PE-S-S-IgG emerges from the column at 8.5 minutes, much sooner than either reactant because the conjugate is larger than either component. The fluorescence emission of a 0.5 ml sample of this conjugate could readily be detected at a phycoerythrin conjugate concentration of less than 10_10M.
A second example of the joining of a phycobiliprotein to another biomolecule is provided by the synthesis of a
phycoerythrin-avidin conjugate. Avidin was activated by the addition of m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS). The ester group of MBS reacted with nucleophiles on avidin. Sulfhydryl groups on thiolated phycoerythrin then reacted with maleimide 5 groups on activated avidin molecules. Uncombined avidin was removed from the reaction mixture by chromatography on carboxymethyl-Sephadex.
EXAMPLE 3 1Q
A third example of the joining of a phycobiliprotein to another biomolecule is provided by an alternative route for the synthesis of a phycoerythrin-avidin conjugate. Biotinylated phycoerythrin was prepared by reacting phycoerythrin with the N-hydroxysuccinimide 15 ester of biotin. Avidin was added to biotinylated phycoerythrin to form a phycoerythrin-biotin-avidin conjugate (PE-B-A). Excess avidin was removed by gel filtration. PE-B-A, which binds very tightly to biotinylated molecules, was then used as a fluorescent stain in a 20 fluorescence-activated cell sorting experiment. Biotinylated monoclonal antibody having specific affinity for immunoglobulin D (IgD) was added to a mixture of spleen cells. This monoclonal antibody combines with IgD molecules, which are present on the surface of 25 about 40% of spleen cells. Excess antibody was removed by washing. PE-B-A was then added to this mixture of cells. The avidin unit of this highly fluorescent conjugate combined with biotin groups on cell surfaces bearing anti-IgG immunoglobulin. The fluores- 30 cence-activated cell sorter analysis of this cell population is shown in FIG. 2. The fluorescence intensity of cells labeled by the phycoerythrin conjugate is comparable to that obtained with a fluorescein conjugate in a parallel experiment. This finding demonstrates that 35 phycobiliprotein conjugates are effective long wavelength fluorescent labels for fluorescence analyses of cells.
The phycoerythrin-biotin-avidin conjugate described above was also used to fluorescent-stain beads containing an antigen. Biotinylated monoclonal antibody having specific affinity for a target immunoglobulin was added to agarose beads (insoluble matrices) containing covalently attached target antigen. These beads were washed and PE-B-A was then added. Beads labeled with this fluorescent phycobiliprotein conjugate were examined by fluorescence microscopy. The labeled beads appeared yellow when viewed with a standard filter combination designed for fluorescein emission. With longer wavelength filters, the labeled beads appeared orange-red. A mixture of fluorescein-avidin labeled beads and PE-B-A labeled beads were also examined by fluorescence microscopy. The PE-B-A labeled beads could readily be distinguished from the fluorescein labeled beads because they were yellow rather than green (FIG. 3a) using fluorescein optics. With a longer wavelength set of filters, only the PE-B-A beads were brightly stained, in this case orange-red (FIG. 3b). These experiments show that phycobiliproteinbiomolecule conjugates are effective fluorescent stains for fluorescence microscopy.
Preparation of Phycobiliproteins
R-phycoerythrin was purified from red algae, Gastroclonium coulteri (Rhodymeniales), which were col
lected from Stillwater Cove, Monterey Peninsula, CA. The fresh algal tissue was washed with distilled water, suspended in 50 mM sodium-phosphate buffer at pH 7.0, and blended for 3 min at the highest speed setting of an Osterizer blender. The homogenate was filtered through several layers of cheese cloth and residual particulate matter removed by low speed centrifugation. The supernatant was brought to 60% of saturation with solid ... All of the above steps were carried out at 4° C. The precipitate was collected by centrifugation, resuspended in 60% of saturation (NKUhSC^in 50 mM sodium phosphate, pH 7.0, and slurried with DEAE-cellulose (microgranular; Whatman, Inc., Chemical Separation Div., Clifton, NJ). The slurry was packed into a column. The column was developed stepwise with decreasing concentrations of (NH^SCMn 50 mM sodium phosphate, pH 7.0, down to 10% of saturation. At that point elution was completed with 200 mM sodium phosphate, pH 7.0. The phycoerythrin eluted between 10% saturation ... mM sodium phosphate, pH 7.0 and 200 mM sodium phosphate, pH 7.0. The eluate was concentrated by ... precipitation, re-dissolved in 50 mM sodium phosphate, pH 7.0 and ... added to 10% of saturation at 4° C. The protein crystallized under these conditions upon standing at 4° C.
Synechococcus 6301 C-phycocyanin (Glazer and Fang, Biol. Chem. (1973) 248: 65-662) Anabaena variabilis allophycocyanin (Bryant et al, Arch. Microbiol. (1976) 110: 61-75) and B-phycoerythrin (Glazer and Hixson, J. Biol. Chem. (1977) 252: 32-42) were prepared as described in the references cited.
Preparation of Phycoerythrin-Avidin
A 50 Jul aliquot of 1 mg/ml N-hydroxysuccinimidobiotin (Sigma Chemical Co., St. Louis, MO or Biosearch, San Rafael, CA) in dimethylsulfoxide was added to 1 ml of 2.7 mg/ml R-phycoerythrin (or B-phycoerythrin) in 50 mM sodium phosphate, pH 7.5 to give a reagent/phycoerythrin molar ratio of 13. The use of avidin and biotin in labeling studies has been described previously (Green, Adv. Protein Chem. (1975) 29: 85-133; Heitzmann and Richards, PNAS USA (1974) 71: 3537-3541). After 90 min at room temperature, the reaction was quenched by the addition of 10 jxl of 100 mM glycylglycine, pH 7.5 and then dialyzed for 3 d at 4° C. against 50 mM sodium phosphate, pH 6.8 1 ml of this mixture of biotinylated phycoerythrin (Biot-PE) and unmodified phycoerythrin was added slowly with stirring to 1 ml of 5 mg/ml avidin in the same buffer. The molar ratio of tetrameric avidin to phycoerythrin was 20. This mixture of phycoerythrin-avidin conjugates (PE-avidin), phycoerythrin, and avidin was fractionated by high-pressure liquid chromatography.
Preparation of Phycoerythrin-Immunoglobulin G
Thiolated phycoerythrin was prepared by the addition of 600 jul of 15.5 mg/ml iminothiolane hydrochloride (Sigma Chemical Co.) (Jue et al., Biochemistry (1978) 17: 5399-5406) to 1.2 ml of 3.6 mg/ml R-phycoerythrin in 125 mM sodium phosphate, pH 6.8. After 90 min at room temperature, the reaction mixture was dialyzed overnight at 4° C. against 50 mM sodium phos