Conjugation of Fluorochromes to Monoclonal Antibodies

Conjugation of Fluorochromes to Monoclonal Antibodies UNIT 4.2 The most widely used application of flow cytometry is the detection of cell surface molecules labeled by monoclonal or polyclonal antibodies conjugated with a fluorochrome. The specificity provided by monoclonal antibodies makes them ideal for use as diagnostic reagents, and therefore the ability to conjugate these proteins with a variety of fluorochromes adds to their flexibility and utility in flow cytometric applications. This unit consists of protocols for tagging monoclonal antibodies with fluorescein, biotin, Texas Red, and phycobiliproteins. In addition, a procedure is included for preparing a PE Texas Red tandem conjugate dye that can then be used to conjugate antibodies. Using these protocols, investigators can label antibodies of their choice with multiple fluorochromes; not only is this cost-effective, but it permits more combinations of antibodies to be used in multicolor flow cytometric applications. The protocols for the conjugation of fluorescein (see Basic Protocol 1), biotin (see Basic Protocol 2), and Texas Red (see Basic Protocol 3) are similar consisting of simple chemistry and dialysis steps as these are all small inorganic molecules. Differences in the labeling procedures depend upon the type of reactive group attached to the fluorochrome: labeling buffers are optimized for the reactive groups, not the fluorochrome itself. Thus, for example, Basic Protocol 1 is for fluorescein with an isothiocyanate reactive group (FITC); other forms of fluorescein are available with succidimyl ester reactive groups, and for these the biotin labeling protocol should be substituted. The protocols for conjugation of phycobiliproteins (see Basic Protocol 4) and preparation of the PE Texas Red tandem conjugate dye (see Basic Protocol 5) are more involved, requiring size separation of products on large gel filtration columns, and more complex chemistries. CAUTION: DMSO, DMF, and THF are hazardous; follow appropriate precautions for handling and disposal when performing these procedures. LABELING ANTIBODY WITH FLUORESCEIN ISOTHIOCYANATE (FITC) Conjugation of fluorescein isothiocyanate (FITC) to purified antibody is an extremely valuable technique for identifying surface molecules using either fluorescence microscopy or flow cytometry. In the procedure that follows, the amino groups of the antibody molecule are coupled with fluorescein derivatives. Following removal of unbound FITC, the fluorochrome/antibody ratio is determined and the labeled antibody is used in the basic and alternate protocols. BASIC PROTOCOL 1 Materials 1 to 2 mg/ml purified monoclonal antibody FITC labeling buffer (prepare 2 weeks before use; see recipe) 5 mg/ml FITC, isomer I, in anhydrous dimethyl sulfoxide (DMSO) Final dialysis buffer (see recipe) Sephadex G-25 column (Pharmacia Biotech PD-10; optional) Dialysis tubing Molecular and Cellular Probes Contributed by Kevin L. Holmes, Larry M. Lantz, and Wesley Russ (1997) 4.2.1-4.2.12 Copyright 1997 by John Wiley & Sons, Inc. 4.2.1

Conjugate FITC and antibody 1. Dialyze purified monoclonal antibody against 500 ml FITC labeling buffer at 4 C with two or three changes over 2 days. Allow 4 hr between buffer changes. This step removes free NH 4 + ions and raises the ph to 9.2. Generally, up to 5 ml antibody can be dialyzed against 500 ml buffer. For discussion of dialysis and a detailed procedure, see Andrew and Titus (1991). 2. Determine antibody concentration based upon A 280. Concentration of antibody (mg/ml) = A 280 0.74 (dilution factor). 3. Add 20 µl of 5 mg/ml FITC in DMSO for each milligram of antibody. Incubate 2 hr at room temperature. Both the dye and organic solvent must be anhydrous; prepare FITC/DMSO solution immediately before use. 4. Remove unbound FITC by dialysis against 500 ml final dialysis buffer at 4 C with two or three changes over 2 days. Alternatively, filter on a Sephadex G-25 column. Determine FITC/antibody ratio 5. Dilute a small volume ( 100 µl) FITC-IgG complex with dialysis buffer so that A 280 = <2.0. 6. Determine and record A 280 and A 492. 7. Calculate protein concentration as follows: mg/ml protein = A where 1.4 is the reciprocal of the FITC-conjugated antibody molar coefficient. 8. Calculate moles of protein: ( A. ) 1.4 280 492 035 mg/ml protein moles protein = 5 1.5 10 A 492 5 moles FITC = 069. 10 where 1.5 10 5 = mol. wt. Ig and 0.69 10 5 = mol. wt. FITC. 9. Determine fluorochrome/protein (F/P) ratio: F/P = moles of FITC moles of protein An F/P of 5 to 6:1 is usually optimal for flow cytometry. Stabilize antibody-dye conjugate 10. Dilute FITC-IgG complex 1:1 with stabilizing buffer. Conjugation of Fluorochromes to Monoclonal Antibodies 4.2.2

LABELING ANTIBODY WITH LONG-ARMED BIOTIN Biotin is a naturally occurring vitamin with a molecular weight of 244 Da and an extremely strong affinity for avidin (K d = 10 to 15 M 1 ). Thus, biotin-labeled antibodies can be detected using commercially available avidin coupled to fluorochromes. Labeling antibodies with biotin provides flexibility by offering a choice of different fluorochromes to be used depending on the needs of the experiment. Moreover, because avidin has four binding sites for biotin and multiple biotin molecules can be conjugated to a single antibody, the fluorescent signal is considerably amplified when biotin/avidin is used, compared to that obtained by direct conjugation of the antibody with the fluorochrome. Because the binding of biotin or the subsequent binding of avidin may induce changes in protein structure, many companies now supply biotin containing a spacer between the protein-binding site and the avidin-binding site (sometimes known as long-armed or spacer biotin). Biotin can also be easily coupled to antibodies via a hydroxysuccinimide ester, usually without disturbing the biological properties of the antibody. The following protocol is for conjugating either IgG or IgM antibodies; alternative information appropriate for the two types of antibodies is indicated in certain steps. Conjugation of IgM antibodies using dialysis buffer at ph 7.5, rather than ph 8.4, provides consistently better labeling, perhaps due to overlabeling og the IgM at higher ph. BASIC PROTOCOL 2 Materials 1 to 2 mg/ml purified monoclonal antibody Succinimide ester labeling buffer or IgM labeling buffer (see recipes) 10 mg/ml long-armed biotin (Zymed) in anhydrous N, N-dimethylformamide (DMF) Dialysis tubing 1. Dialyze 1 to 2 mg/ml purified antibody against 500 ml succinimide ester labeling buffer (for IgG) or IgM labeling buffer (for IgM) at 4 C with two to three changes over 2 days. Allow 4 hr between buffer changes. For discussion of dialysis and a detailed procedure, see Andrew and Titus (1991). 2. Determine protein concentration by measuring A 280. Concentration of antibody (mg/ml) = A 280 dilution factor 0.7 (for IgG) or 0.8 (for IgM). 3. Add 10 µl of 10 mg/ml biotin in DMF for each milligram of antibody. Incubate 1 hr at room temperature. Both the dye and organic solvents must be anhydrous; prepare biotin/dmf solution immediately before use. 4. Remove unbound biotin by dialysis against final dialysis buffer at 4 C as in step 1. Biotin/protein ratio cannot be determined spectrophotometrically, but titration comparison of the same antibody labeled with FITC can indicate whether relabeling is necessary. 5. Dilute biotin-antibody complex solution 1:1 with stabilizing buffer. Molecular and Cellular Probes 4.2.3

BASIC PROTOCOL 3 LABELING WITH TEXAS RED X Texas Red, the sulfonylchloride derivative of sulforhodamine 101, has been used for many years in dual-laser multiparameter flow cytometry. However, directly labeling antibodies with this dye can be difficult, depending upon the class of the antibody and host species (Titus et al., 1982). Concentrations required to achieve adequate dye/protein ratios often precipitate the antibody-dye conjugates. The recent development of the modified Texas Red X succinimidyl ester has greatly improved Texas Red labeling, allowing a greater range of antibodies to be labeled with substantially less precipitation of antibody-dye conjugates. The procedure is similar to the protocol for biotin labeling, with the modifications detailed below. Materials 1 to 2 mg/ml purified monoclonal antibody Succinimide ester labeling buffer (see recipe) 5 mg/ml Texas Red X succinimidyl ester (Molecular Probes) in N,N-dimethylformamide (DMF) Final dialysis buffer (see recipe) Stabilizing buffer (see recipe) Dialysis tubing Sephadex G-25 column (Pharmacia Biotech; optional) 1. Dialyze purified monoclonal antibody against 500 ml succinimide ester labeling buffer at 4 C with two or three changes over 2 days. Allow 4 hr between buffer changes. For discussion of dialysis and a detailed procedure, see Andrew and Titus (1991). 2. Determine antibody concentration based upon A 280 and adjust to 1 to 2 mg/ml. Concentration of antibody (mg/ml) = A 280 0.7 dilution factor. 3. Add 5 µl of 5 mg/ml Texas Red X in DMF for each milligram of antibody. Incubate 1 hr at room temperature. Both the dye and organic solvents must be anhydrous; prepare Texas Red X/DMF solution immediately before use. 4. Remove unbound Texas Red X by dialysis at 4 C as in step 1, but using final dialysis buffer. Alternatively, filter on a Sephadex G-25 column. 5. Remove any precipitated antibody by centrifuging 3 min at 10,000 g. 6. Determine Texas Red/antibody ratio by measuring A 596 /A 280. A ratio of 0.5 to 0.7 usually gives the best results and probably represents two to three Texas Red molecules bound per antibody, based upon a molar extinction coefficient for antibody bound to Texas Red of 8.4 10 4 M 1 at 596 nm (Titus et al., 1982). 7. Dilute Texas Red Ig complex solution 1:1 with stabilizing buffer. BASIC PROTOCOL 4 Conjugation of Fluorochromes to Monoclonal Antibodies LABELING ANTIBODY WITH PHYCOBILIPROTEINS Coupling phycobiliproteins such as phycoerythrin (PE) and allophycocyanin (APC) to antibodies is more difficult than labeling with FITC or biotin. A sulfhydryl-maleimide linkage is used to couple the antibody to the phycobiliprotein. The unbound antibody and phycobiliprotein are then separated by size on a gel filtration column. The procedure described here is for PE-antibody coupling. The step for APC coupling is identical except where noted. 4.2.4

Materials 10 to 25 mg/ml phycoerythrin (PE; purchased as suspension in buffered ammonium sulfate solution) Coupling buffer, ph 5.5 and 7.5 (see recipe) Sulfhydryl addition reagent: N-succinimidyl-S-acetylthioacetate (SATA; Calbiochem; store under nitrogen after opening) Dimethylformamide (DMF) Nitrogen Deacetylation buffer (see recipe) Heterobifunctional cross-linker: γ-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS; Calbiochem; store under nitrogen after opening) Tetrahydrofuran (THF) 0.1 mg/ml cysteine Running buffer, degassed (see recipe) Dialysis tubing AcA 34 column (IBF Biotechnics) Sephacryl S-200 column (Pharmacia Biotech; optional) Prepare the PE-SATA conjugate 1. Dialyze PE against 500 ml coupling buffer, ph 7.5, at 4 C with two or three changes over 2 days. Use sufficient PE to give a PE/IgG (w:w) ratio of 3:1 and allow 4 hr between buffer changes. For discussion of dialysis and a detailed procedure, see Andrew and Titus (1991). The precise concentration of PE must be determined by spectrophotometric measurements at A 596 and the concentration adjusted with coupling buffer to fall within the indicated range. 2. Dilute SATA to 1 mg/ml in DMF. 3. Add 10 µl diluted SATA solution for each milligram of PE to be labeled. Incubate 30 min at room temperature. 4. Dialyze PE-SATA conjugate in 500 ml coupling buffer, ph 7.5, at 4 C with two or three changes to remove unreacted SATA. Store at 4 C for later use. Label the antibody and isolate the conjugate 5. Dialyze purified antibody in 500 ml coupling buffer, ph 7.5, as for FITC labeling (see Basic Protocol 1, step 1) to a final IgG concentration of 1 mg/ml. 6. Dilute GMBS to 2 mg/ml (7.14 mm) in THF. 7. Deacetylate PE-SATA conjugate from step 4 by adding 100 µl deacetylation buffer for each milliliter of PE-SATA. Incubate 1 hr at room temperature. 8. Add 10 µl diluted GMBS solution for each milligram of antibody to be labeled. Incubate 30 min at room temperature. 9. Wash one Sephadex G-25 column for each 2.0 ml IgG-GMBS conjugate solution to be loaded by adding 10 ml coupling buffer (ph 5.5) per column. Load 2.0 ml IgG-GMBS solution onto washed column. Monitor eluate spectrophotometrically using a 280-nm filter and collect the portion represented by the first peak. Proceed immediately to step 10. The first peak is the GMBS-labeled antibody. The second peak is free GMBS and should be discarded. Molecular and Cellular Probes 4.2.5

unbound PE and antibody 1:1 OD 280 > 1:1 Fraction Figure 4.2.1 Elution profile of phycoerythrin-labeled IgG from a gel filtration column. The initial peaks have greater than a 1:1 ratio of dye bound to antibody, and are not used. Optimal material is found in the center fractions designated 1:1. The trailing peaks contain unbound dye and unconjugated antibodies, and are discarded. Couple PE-SATA and IgG-GMBS conjugates 10. Mix deacetylated PE-SATA conjugate from step 7 with IgG-GMBS conjugate from step 9 immediately after isolating the latter. Incubate 2 hr at room temperature. Use a 3:1 ratio of PE/IgG for optimum yield, but use a 2:1 ratio of allophycocyanin/igg. 11. Quench residual maleimide groups by adding 0.1 mg/ml cysteine to twice the antibody concentration. For example, add 25 ìl of 0.1 mg/ml cysteine (570 ìm) per milligram of IgG. 12. Separate PE-IgG conjugate from unconjugated PE and free IgG using an AcA 34 column. Sample volume loaded onto the column should be between 0.5% and 4% of total column bed volume. Pour an appropriately sized column, using degassed running buffer, according to manufacturer s directions. Due to slow packing and running rates, it generally requires one night to pack a column and an additional night to isolate the sample. Therefore it is advisable to pack the column before labeling. 13. Load sample onto column and run column at manufacturer s suggested rates. Collect fractions 1 20 the column volume. Two well-separated red bands will appear on the column and several peaks will appear on the column A 280 printouts. The first peaks are PE-IgG conjugates with more than one PE per IgG. The peak with one PE per IgG will appear immediately before the largest peak consisting of unconjugated PE and IgG. See Figure 4.2.1 for sample results. Confirm number of PE per IgG using flow cytometry techniques or spectrophotometrically using A 596 /A 280 ratios. Best results have come from using the one-pe-per-ig conjugate. BASIC PROTOCOL 5 Conjugation of Fluorochromes to Monoclonal Antibodies 4.2.6 CONJUGATION OF TEXAS RED TO R-PHYCOERYTHRIN TO PRODUCE AN ENERGY TRANSFER FLUOROCHROME An advantage of flow cytometric analysis is the ability to distinguish functionally significant populations of cells. The need to further subdivide these populations using antibody probes against known cell surface antigens requires increasingly complex multicolor analyses. Fluorochromes excitable with a single excitation source and possessing emission wavelengths distinct enough to be detected separately are needed to distinguish the different antigens. A highly effective way to achieve this purpose is the use of energy-transfer fluorochromes, which allow three- and four-color single-excitation

flow cytometry. One of the more useful energy-transfer fluorochromes is the conjugate of R-PE and Texas Red, whose preparation and use is detailed in this protocol. The emission from R-PE overlaps with the absorption of Texas Red, allowing energy transfer if the two molecules are placed within a limiting distance from each other (Glazer and Stryer, 1983). Materials 10 to 50 mg R-phycoerythrin (R-PE; purchased as suspension in buffered ammonium sulfate solution) R-PE dialysis buffer (prepared within 2 days of use; see recipe) Conjugation buffers A and B (see recipes) Texas Red sulfonyl chloride (Molecular Probes) N,N-Dimethylformamide (DMF) Glycine (ultrapure or ACS grade) 0.5 M hydroxylamine HCl, ph 7.2 (prepared as for deacetylation buffer, but without EDTA; see recipe) Equilibration buffer (see recipe for HIC column buffers) First-wash buffer (see recipe for HIC column buffers) Elution buffers A and B (see recipe for HIC column buffers) Dialysis tubing Sephadex G-50 fine columns, one with ~5 ml capacity and one larger (e.g., 50 ml capacity for labeling 10 mg of R-PE) Fraction collector HIC (hydrophobic interaction column) TSK-Gel Toyopearl Butyl 650M (e.g., 25 ml capacity for labeling 10 mg of R-PE) Gradient maker with 200-ml capacity UV monitor and chart recorder Magnetic stir plate 10- to 15-ml glass test tube Flea (small stir-bar) Peristaltic pump Centrifuge and appropriate tubes (e.g., Beckman tabletop with 50-ml conical tubes) Spectrophotometer with quartz cuvettes 1. Dialyze 10 to 50 mg R-phycoerythrin (R-PE) against 500 ml R-PE dialysis buffer with two or three changes over 2 days at 4 C. Allow 4 hr between buffer changes. Protect R-PE from light by covering containers with foil during dialysis and in all subsequent steps when practical. For discussion of dialysis and a detailed procedure, see Andrew and Titus (1991). R-PE concentration should be in the range of 20 to 30 mg/ml. 2. Determine the total amount of R-PE: determine the concentration of R-PE, and then measure the total volume of R-PE solution. Concentration of R-PE (mg/ml) = A 565 0.122 dilution factor. mg R PE = R PE volume (ml). 3. Decant the R-PE into a 10- to 20-ml glass test tube suspended in an ice bath on a magnetic stir plate. Mix gently with a flea. 4. While stirring, add dropwise sufficient conjugation buffer A to equal 20% the volume of R-PE. 5. While stirring, add dropwise sufficient conjugation buffer B to equal 25% the volume of R-PE. Molecular and Cellular Probes 4.2.7

Conjugation of Fluorochromes to Monoclonal Antibodies 4.2.8 6. Calculate quantity of Texas Red sulfonyl chloride to use to give a 37:1 Texas Red/PE conjugation ratio. mg Texas Red required = (mg R-PE/240,000 mg/mmol) 37 (625 mg/mmol). 7. Increase the mixing rate of the R-PE solution to a very rapid rate. 8. Dissolve twice the amount of Texas Red required (as determined in step 6) in DMF. Mix in an open glass tube or vial on a vortex until dissolved. 9. Immediately add the dissolved Texas Red to the R-PE solution mixing in the ice bath. 10. Increase the mixing to the most rapid rate possible and maintain for 2 to 3 min, then reduce to the normal mixing rate. 11. After 10 min, remove 20 µl of the solution and load it on a Sephadex G-50 column containing 5 ml resin. Collect the first peak and measure A 565 and A 596. If the A 565 /A 596 ratio is between 2.3 and 3.3, proceed to the next step. If the ratio is <2.3, the R-PE is overlabeled with Texas Red. Discard overlabeled conjugate and start again at step 1 using a lower Texas Red/PE ratio (see step 6). If the ratio is >3.3, more Texas Red needs to be reacted with the R-PE and the process repeated. 12. While continuing to mix, add 5 mg solid glycine per mg Texas Red (determined in step 6) to quench the reaction. From step 2, it should take ~3 hr for the conjugation and 2 hr for chromatography. After glycine is added, the product is stable 5 days at 4 C. 13. Once the glycine is dissolved, add a volume of 0.5 M hydroxylamine HCl, ph 7.2, equal to 10% of the mixing R-PE. Measure the volume. 14. Pass the resulting solution over a Sephadex G-50 fine column equilibrated with R-PE dialysis buffer, using 25 ml resin per ml solution volume. At this point the product may be stored 5 days at 4 C. 15. Collect the first peak and dialyze overnight against equilibration buffer at 4 C. 16. Centrifuge 20 min at 900 g, 4 C. Remove the supernatant using a pipet, being careful not to disturb the pellet. Calculate the concentration of R-PE in the R-PE Texas Red complex as in step 2. The concentration prior to starting the next step should be 4 mg/ml; adjust if necessary using equilibration buffer. 17. Equilibrate a HIC Toyopearl butyl column in equilibration buffer, using 1.5 ml of resin per mg of R-PE Texas Red complex. Flow rate should be 1 to 3 ml/min. 18. Load the dye solution onto the column and wash with 2 column volumes of equilibration buffer. 19. Wash the column with 10 column volumes first-wash buffer, at a flow rate of 1 to 3 ml/min. 20. Connect a UV monitor to the column. Run a 1:2 gradient of elution buffer A to elution buffer B, using 15 ml elution buffer A and 30 ml of elution buffer B for each 1.5 ml of column resin. Collect fractions equal to one-tenth the column volume. A broad peak of weakly colored fractions will precede a higher region with intense color trailing off to fractions with less color. The fractions in the middle are optimal for conjugation to antibodies. From step 16 to this point takes ~4 hours. The product may be stored up to 15 days at 4 C. To store longer, add sodium azide to 1% final concentration. The sodium azide will require removal by dialysis prior to conjugation.

21. For conjugation to antibodies, determine the concentration of the R-PE Texas Red dye as for R-PE. On the same day as conjugation, centrifuge the solution 20 min at 600 g, 4 C; then conjugate using the procedure described for labeling antibody with phycobiliproteins (see Basic Protocol 4). REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX. Conjugation buffers Conjugation buffer A (1.4 M sodium sulfate): Dissolve 20 g sodium sulfate in 100 ml of R-PE dialysis buffer (see recipe). Adjust ph to 7.2 with 1 M KOH. Conjugation buffer B (1 M potassium borate, ph 9.8): Prepare a 1 M solution of boric acid (H 3 BO 3 ) in water. Adjust ph to 9.8 with 8 M KOH. The solid borate will slowly go into solution and lower the ph; slight adjustments with addition of KOH will allow the borate to dissolve completely. Coupling buffer 0.1 M Na 2 HPO 4 7H 2 O 0.1 M NaCl 1 mm EDTA Adjust ph to 7.5 or 5.5 with concentrated HCl Deacetylation buffer Dissolve 3.47 g hydroxylamine (mono HCl; 0.5 M final) and 0.73 g EDTA (anhydrous free acid; 0.025 M final) in 50 ml water and adjust ph to 7.5 with solid anhydrous disodium hydrogen phosphate. Add water to 100 ml final volume. Final dialysis buffer 0.1 M Tris Cl, ph 7.4 0.1% (w/v) NaN 3 0.2 M NaCl Adjust ph to 7.4 with 5 M NaOH Store at 4 C FITC labeling buffer 0.05 M boric acid (H 3 BO 3 ) 0.2 M NaCl Adjust ph to 9.2 with 5 M NaOH Store at 4 C HIC column buffers Equilibration buffer 100 mm K 2 HPO 4 2 mm EDTA 200 mm sodium sulfate Adjust ph to 7.2 with 1 M KOH First-wash buffer 100 mm K 2 HPO 4 2 mm EDTA 135 mm sodium sulfate Adjust ph to 7.2 with 1 M KOH Elution buffer A 100 mm K 2 HPO 4 2 mm EDTA 70 mm sodium sulfate Adjust ph to 7.2 with 1 M KOH Elution buffer B 100 mm K 2 HPO 4 2 mm EDTA Adjust ph to 7.2 with 1 M KOH Molecular and Cellular Probes 4.2.9 Supplement 1

IgM labeling buffer 0.1 M Na 2 HPO 4 7H 2 O 0.15 M NaCl Adjust ph to 7.5 with concentrated HCl Store at room temperature R-PE dialysis buffer 100 mm K 2 HPO 4 2 mm EDTA Adjust ph to 7.2 with 1 M KOH Store at room temperature This buffer may be used for up to one week. Running buffer 81.82 g NaCl 4 ml glycerol Dissolve in 3.8 liters phosphate-buffered saline (PBS; APPENDIX 2A) Adjust ph to 7.5 with concentrated HCl Add PBS to 4 liters To degas buffer, place room temperature buffer in an Erlenmeyer flask equipped with a one-hole stopper and tubing (alternatively, a side-arm vacuum flask and stopper may be used). Apply vacuum through the tubing (or side-arm flask) while stirring buffer vigorously. Sample is degassed when no more bubbles rise out of solution. Stabilizing buffer Hanks balanced salt solution (HBSS) without phenol red (APPENDIX 2A) 0.1% (w/v) NaN 3 5.0% (w/v) bovine serum albumin (BSA; fraction V) Store at 4 C Succinimide ester labeling buffer 0.1 M NaHCO 3 0.1 M NaCl Adjust ph to 8.4 with concentrated HCl Store at room temperature Conjugation of Fluorochromes to Monoclonal Antibodies 4.2.10 Supplement 1 COMMENTARY Background Information The choice of the dye to conjugate with an antibody is dependent upon several factors, including the time the investigator is willing to invest in conjugation, the available excitation source(s), whether the conjugate will be used in combination with other dyes, and the density of the antigen that is being detected. Labeling procedures with fluorescein isothiocyanate (FITC), biotin, and Texas Red are easy to perform, whereas labeling procedures with phycobiliproteins such as phycoerythrin (PE) and allophycocyanin (APC) and with phycoerythrin Texas Red (PE Texas Red) are more difficult and time consuming, taking several days to complete (Brinkley, 1992). In addition, FITC, biotin, and Texas Red can be efficiently conjugated to small amounts (e.g., 1 mg) of purified antibody, but phycobiliprotein and PE Texas Red conjugation require larger amounts of purified antibody and give a lower final yield. FITC, PE and PE Texas Red are all excitable by 488 nm argon lasers, but Texas Red is excitable by argon-dye (rhodamine 6G) or krypton (operating at 568 nm). For detection of surface antigens with low density, phycobiliproteins or tandem conjugate dyes offer better signal-to-noise ratios than FITC or Texas Red, because of their large quantum yields and extinction coefficients. Methods for conjugating with phycobiliproteins using sulfhydryl-maleimide linkages are presented (Duncan et al., 1981; Tanimori et al., 1983), although other linkages can be used (Kitagawa et al., 1981; Hashida et al., 1984; Blattler et al., 1985; Kronick, 1988).

Critical Parameters and Troubleshooting Preparation and storage of reagents Although the protocols for labeling antibodies with fluorochromes and biotin are simple, results are highly dependent upon the quality of reagents used. The organic solvents (DMF and DMSO) and the dye powders must be anhydrous. For this reason it is recommended that dyes be purchased in small amounts and stored in a desiccator. Organic solvents can be purchased packed under nitrogen in syringe vials. Solutions of FITC, Texas Red X, and biotin should be prepared just prior to use. It is recommended that FITC labeling buffer and coupling buffer be made <2 weeks before use. In immunophenotypic analysis, IgG antibodies can bind to Fc receptors regardless of their antigen specificity. This problem can be minimized by ultracentrifugation (e.g., using a Beckman Airfuge). IgG-FITC, -biotin, or Texas Red conjugates may be airfuged 15 min at 100,000 g to remove aggregates, and retitered for optimal dilution. For IgG-PE, -PE Texas Red, or all IgM conjugates, centrifuge 5 min at 12,000 g to remove aggregates prior to retitration. It is essential that the Texas Red sulfonylchloride ester be added to the rapidly mixing R-PE quickly, as the ester is reactive for only a few minutes. There will be some R-PE Texas Red complexes remaining on the hydrophobic interaction column. This material is not suitable for conjugation to antibodies. When attempting to label small amounts of protein ( 0.5 mg), it is advisable to use an apparatus such as the Pierce Microdialyzer to avoid loss or dilution of antibody. It is not advisable to use a Sephadex G-25 column for separation of labeled antibody from unbound fluorochrome, as this will cause considerable dilution of small-volume samples. Because Texas Red is hydrophobic, the optimal method for separation of Texas Red-X labeled antibody from the fluorochrome is a Sephadex G-25 column. Antibody-fluorochrome conjugates should be stored at 4 C, protected from light. Dilution of antibody-fluorochrome (or -biotin) conjugates with stabilizing buffer greatly increases shelf life by preventing aggregation of conjugates. Conjugates may be stored 1 year or longer at 4 C, although individual antibodies may vary. For longer storage, most antibodies (diluted in stablilizing buffer) may be dispensed in small aliquots and frozen at 70 C. Small volumes should be pretested for stability after freeze-thaw. Antibodies should be frozen only once. Antibody-fluorochrome (-biotin) conjugates can be filter-sterilized as necessary, using a sterile syringe filter equipped with a 0.22-µm low-protein-binding membrane. Deterioration of antibody-fluorochrome (-biotin) conjugates may be indicated by visible precipitation, or by loss of fluorescent signal in standardized flow cytometric analyses. Deterioration of PE antibody Texas Red conjugates may be indicated by the inability to perform sufficient electronic compensation of PE Texas Red signal from the PE detector on the flow cytometer. This may arise from uncoupling of Texas Red from PE, resulting in decreased efficiency of energy transfer, and therefore more PE emmission. Final dialysis buffer contains sodium azide, which when dried at high concentrations may spontaneously combust. Sodium azide containing solutions are highly toxic, and should be disposed of by dilution with large quantities of water. Optimization of fluorescence A high fluorochrome (or biotin)/protein ratio improves fluorescent signal-to-noise ratio in flow cytometric analysis. The amounts of fluorochrome (or biotin) to be used per milligram of antibody cited in the protocols are guidelines only. Because of inherent differences in monoclonal antibodies, it may be necessary to label several batches of antibody, varying the amount of fluorochrome (or biotin) used above and below those suggested amounts in order to achieve optimal labeling. If the efficiency of the energy transfer fluorochrome is poor, then the wash step prior to the elution gradient should be lengthened. A fluorescent spectrophotometer can determine the point when the dye complex is eluting off the column very accurately. The ratio of emission at 575 nm to emission at 613 nm when the eluant was excited at 488 nm is representative of the efficiency of the transfer process. Anticipated Results Labeling of antibodies with either FITC or biotin generally results in excellent yield and very little loss of protein (<5%). Texas Red labeling, on the other hand, generally results in a considerable loss of protein (10% to 20%) presumably due to overlabeling that can be visualized as a precipitate after labeling. It is advisable to isolate and discard this precipitate by centrifugation as described. Molecular and Cellular Probes 4.2.11

For the preparation of the energy transfer fluorochrome, the final amount of usable product is ~25% of the amount of phycoerythrin initially used. For the conjugation of phycoerythrin to antibody, the final amount of usable product is 25% of the amount of antibody used. Time Considerations The labeling of purified antibodies with FITC, biotin, and Texas Red-X will take 3 days. These procedures require relatively small amounts of hands-on time (2 to 4 hr). The majority of time is required for dialysis steps. Sephadex G-25 columns can be used in place of dialysis, but fractionation with a column will usually result in more protein loss and dilution than dialysis. A good stopping point in any of these procedures is after the start of any dialysis step. Labeling of antibodies with phycobiliproteins is more labor-intensive than the FITC, biotin, and Texas Red-X protocols. This procedure will take ~5 to 7 days. Most of this time is required for dialysis, column preparation, and fractionation. Good stopping points are after the start of any dialysis step, after the PE-SATA conjugation (Basic Protocol 4, step 4), and after residual maleimide groups are quenched with cysteine (Basic Protocol 4, step 11). For the preparation of the energy transfer fluorochrome, time to complete the various steps are listed within the protocol. The handson time required is ~10 hr; 3 to 4 days are necessary to complete the dialysis steps. Literature Cited Andrew, S.M. and Titus, J.A. 1991. Dialysis and concentration of protein solutions. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeck, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. A.3H.1-A.3H.2. John Wiley and Sons, New York. Blattler, W.A., Kuenzi, B.S., Lambert, J.M., and Senter, P.D. 1985. New heterobifunctional protein cross-linking reagent that forms an acidlabile link. Biochemistry 24:1517-1524. Brinkley, M. 1992. A brief survey of methods for preparing protein conjugates with dyes, haptens, and cross-linking reagents. Bioconjugate Chem. 3:2-13. Duncan, R.J.S., Weston, P.D., and Wrigglesworth, R. 1981. A new reagent which may be used to introduce sulfydryl groups into proteins, and its use in the preparation of conjugates for immunoassay. Anal. Biochem. 132:68-73. Glazer, A.N. and Stryer, L. 1983. Fluorescent tandem phycobiliprotein conjugates. Biophys. J. 43:383-386. Hashida, S., Imagawa, M., Inoue, S., Ruan, K-H., and Ishikawa, E. 1984. More useful maleimide compounds for the conjugation of Fab to horseradish peroxidase through thiol groups in the hinge. J. Appl. Biochem. 6:56-63. Kitagawa, T., Shimozono, T., Aikawa, T., Yoshida, T., and Nishimura, H. 1981. Preparation and characterization of hetero-bifunctional crosslinking reagents for protein modification. Chem. Pharm. Bull. (Tokyo) 29:1130-1135. Kronick, M.N. 1988. Phycobiliproteins as labels in immunoassay. In Nonisotopic Immunoassay. (T.T. Ngo, ed.) pp. 163-185. Plenum Press, NY. Tanimori, H., Ishikawa, F., and Kitagawa, T. 1983. A sandwich enzyme immunoassay of rabbit immunoglobulin G with an enzyme labeling method and a new solid support. J. Immunol. Methods 62:123-131. Titus, J.A., Haugland, R., Sharrow, S.O., and Segal, D.M. 1982. Texas Red, a hydrophilic, red-emitting fluorophore for use with fluorescein in dual parameter flow microfluorometric and fluorescence microscopic studies. J. Immunol. Methods 50:193-204. Key Reference Brinkley, 1992. See above. Wong, S.S. 1991. Chemistry of Protein Conjugation and Cross-Linking. CRC Press, Inc. A good reference book for both protein-protein coupling and reactive group chemistry Contributed by Kevin L. Holmes and Larry M. Lantz National Institute of Allergy and Infectious Diseases Bethesda, Maryland Wesley Russ Kirkegaard & Perry Laboratories Gaithersburg, Maryland Conjugation of Fluorochromes to Monoclonal Antibodies 4.2.12