PROCEDURE NO. 40515

IMMUNOFLUORESCENCE ASSAY OF HYBRIDOMA SUPERNATANTS

J. Michael Mullins and Kathryn M. Wolf

Department of Biology, The Catholic University of America, Washington, D.C. 20064

SUMMARY: Methods are described for pefforming immunofluorescence assay using cultured mammalian cells as target specimens. This procedure uses slides that allow processing of 12 specimens as a unit. Avidin-biotin is employed to provide increased sensitivity.

Key words: hybridoma; monoclonal antibodies; immunofluorescence assay.

I. INTRODUCTION

Speed, convenience, and sensitivity have made solid phase assays, such as enzyme-linked immuno- sorbent assay (ELISA), the popular choice for testing hybridoma supernatants for antibody activity (1). By contrast, immunofluorescence (IF) is tedious and time consuming. Repetitive pipetting steps are required for transfer of individual culture supernatants onto target specimens, and each specimen taust be examined under the microscope. Additionally, IF is less sensi- tive than ELISA (9).

Nonetheless, IF assay offers definite advantages in some circumstances. If the antigen of interest is loca- lized in a distinct cell structure or region, microscopic examination will quickly reveal whether antibody binding is to the structure in question. This is particu- larly important wherc immunization was done with a crude mixturc of antigens, making it likely that anti- bodies of a wide range of specificities will be found when hybridomas are produced. In addition, if anti- bodies are eventually to be utilized for IF, it is essen- tial that an IF assay be employed before extensive cloning and antibody production are undertaken. Epi- topes accessible to antibody binding on an antigen molecule adsorbed to a solid phase may not be acces- sible in the intact cell; polymer formation or other mo- lecular interactions may resuh in this situation. Epi- topes may also be altered by the fixatives generally required for IF, eliminating antibody binding (5,7).

We routinely screen all our hybridoma supernatants by IF. To ease the problems of handling large numbers of specimens, target cells are grown on commercially available slides which have a thin epoxy coating that leaves 12 circular (5 mm diameter) openings of glass exposed. Twelve specimens can thus be processed as a unit. Advantage has also been taken of the increased sensitivity afforded by the use of avidin-biotin systems (3,6). Maximum brightness of the fluorescence image is important where antibody titers may be low or an- tigen present in minimal concentrations, or both.

II. MATERIALS

A. Equipment

1. Fluorescence microscope, E. Leitz 1 LM LUX microscope Pleomopak 2.3 epi-illuminator with I2-fiher block Mercury arc lamp 6 3 × [numerical aperture (N.A.) 1.3] fluo- rescence objective (cat. no. 519474)

2. 37 ° C incubator

3. Watet bath

B. Chemicals

Gelvatol, no. 20-30, Monsanto 2 Glycerol, no. G-33, Fisher 3 HC1, no. A-144, Fisher a KC1, no. P-217, Fisher a KH2PQ, no. P-285, Fisher z Methanol, no. A-412, Fisher 3 NaC1, no. S-671, Fisher 3 Na2HP04, no. S-374, Fisher a Trizma base, no. T-1503, Sigma 3

C. Solutions

1. Prepare phosphate buffered saline (PBS) as follows: 8.0 g NaC1, 0.2 g KC1, 0.9 g NaH2P04, 0.2 g KH2PO 4 dissolved in dH20 to 1.0 liter.

2. Immunofluorescence mounting m~edium as pre- pared according to published methods (7), but substituting Gelvatol for the now unavailable polyvinyl alcohol, Elvanol. Briefly, to 3.0 g gly- cerol in a 50-ml eonical centrifuge tube, add 1.2 g Gelvatol, stirring to mix completely but avoiding spreading the Gelvatol on the sides of the tube. Add 3 ml dH20, stir to mix and allow to stand 4 h at room temperature. Add 6 ml 0.2 M tris buffer (for 100 tal, 2.42 g Trizma base, pH to 8.5 with 1 N HC1) and ineubate 10 min in

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MULLINS AND WOLF - - PROCEDURE NO. 40515

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a 50 ° C watet bath with intermittent stirring to dissolve the Gelvatol. Centrifuge at 1500 x g for 15 min. to elarify, al iquot and store at - 20 ° C.

Supplies

1. Tissue culture

a. Ham's F12 medium, no. 12-423-54, Flow Labs 5

b. Fetal bovine serum, no. 200-6140, GIBCO 6

c. Petr i d ish , 100 × 15-mm plas t i c , no. 08-757-12, Fisher 3

d. P r i n t e d m i c r o s c o p e s l i de , no. 00012 , Carlson Scientific 7

e. PtK1 Cells, no. CCL 35, American Type Culture Colleetion 8

2. Immunological reagents

a. Biotinylated antimouse IgG, no. BA-2000, Veetor Labs 9 (no. BA-4000 for antirat IgG)

b. F luoresce in eonjugated to Avidin D, no. A-2001, Veetor Labs 9

3. General

a. A d j u s t a b l e m i e r o p i p e t t e r , no. P - 2 0 0 , Rainin lo

b. Pipetter tips, no. RC20, Rainin lo

e. Cover s l ips , 24 × 60 mm, no. 1, no. 12-545M, Fisher 3

d. Coplin jars, no. 08-816, Fishera

e. Fi l ter paper , 9 cm no. 1, no. 09-805D, Fisher 3

f. Moist ehamber, eonsisting of two to three pieees of the filter paper placed in the 100- mm petri dish and lightly moistened with dH20

g. 50 ml Polypropylene , eonieal eentrifuge tubes, no. 05-538-60, Fisher 3

I I I . P R O C E D U R E

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A. Preparation of slide cultures

1. To a 100-mm petri dish containing a sterile (au- toclaved) printed slide and 15 ml Ham's F12 medium (5% serum) add 2 × 105 PtK l cells from an actively growing culture.

2. Incubate at 37 ° C in a 5% CO2 atmosphere for a minimum of 24 h before performing the assay.

B. Immunofluorescence assay

1. Hybridoma cuhures are ready for testing when individual colonies occupy approximately 30% of the growth surface in a weil of a 96-well plate and when the cuhu re medium has become slightly acidic.

2. Chill absolute methanol to - 20 ° C in the freezer COnlpartment of a standard refrigerator.

3. Decant the culture medium from the petri dish; the slide will be held in place by capillarity. Holding the dish at a slight angle, pour 15 to 20 ml of - 2 0 ° C methanol so that it quiekly runs over and covers the cells.

4. Immediately cover the plate and place it in the - 20 ° C freezer for 6 to 10 min fixation.

5. Remove the plate from the freezer, decant the methanol, and add 20 ml room temperature PBS to rehydrate the cells. These steps should be performed rapidly to prevent drying of the cells.

6. Wash the slide with an additional change of PBS.

7. Remove the slide ti'om the PBS and drain lightly on papcr towels. This should remove excess PBS without drying the cells.

8. Place the slide cell side up in the moist chamber and, using strips of filter paper, dry the slide between the "wells" leaving a small amount of PBS over eaeh well. A diamond-tipped marker may be used at this point to scratch a label into the epoxide layer of the slide.

9. In a laminar flow hood, using sterile tips on the micropipetter, withdraw 30 I, zl of cuhure super- natant from eaeh cu l ture to be tes ted and transfer to a weil on the slide. Careful transfer will leave the supernatant as a rounded drop over the well.

10. Cover the chamber and incubate for 45 min at 37 ° C.

11. Wash the slide by placing it in a Coplin jar filled with PBS and dipping the slide in and out five times. Repeat with three additional jars of PBS.

12. Repeat steps 7 and 8.

13. Pipette 15 ~1 of biotinylated antimouse IgG, di- luted 1:30 in PBS, onto each well.

14. Cover and incubate for 30 min at 37 ° C.

15. Using fresh PBS repeat step 11, followed by steps 7 and 8.

16. Pipette 15 I, zl of the fluorescein-avidin conju- gate, diluted 1:30 in PBS, onto eaeh weil.

17. Cover and ineubate for 30 min at 37 ° C.

18. Using fresh PBS, repeat step 11, followed by steps 7 and 8.

19. Pipette 10 p~l of IF mounting medium onto eaeh weil. Carefully place a 24 x 60-mm cover slip over the wells so that the mounting medium will spread without leaving bubbles over the eells.

20. Plaee the slide in the dark for 1 to 2 h at room temperature to allow the mounting medium to gel. (Afterwards the slides may be stored at re-

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frigerator temperatures, in the dark, for months to years).

Exarnine with the fluorescenee microscope. Note that the epoxy layer on the slides fluoresces; it has a granular appearanee that ean be used as an aid to attaining tbcus, a useful feature for exam- ining wells with little-to-no antibody binding where finding focus ean be difficuh.

DISCUSSION

Examination of the processed specimens will reveal sites of antibody binding as yellow-green fluorescent regions eontrasted againsl a dark baekground. Where no specifie antibody binding has occurred in a spec- imen, a diffuse, very weak fluorescenee will usually be seen throughout the eells. Should this nonspeeific, background fluoreseence be tbund to be exeessive it may be possible to reduee it eonsiderably with a t~w extra steps. Before step 9, 15 IM of goat nonimmune immunoglobulins (50 ng/ml in PBS) should be applied to eaeh well, followed by 20-min incubation at 37 ° C and washing with PBS. It should also be noted that although the biotinylated antibody employed is an an- timouse IgG, it will reeognize the antibody light ehains shared by IgG and IgM antibodies and so deteet both antibody elasses.

Certain parts of the procedure mav be varied to ad- vantage for partieular projeets. We have fixed cells with cold methanol, whieh provides good fixation for a variety of antigens and also permeabilizes the ce]l membrane, allowing imnmnoglobulins aecess to the eell interior {7). A variety of other fixatives has been used for IF. Certain fixation protoeols may offer supe- rior results with partieular types of antigens (5,7). A researeher would thus be well advised to search the literature to determine what fixation might be superior for a given antigen.

Use of the 12 "weil" slides makes the IF assay laster and more eonvenient. Convenience would be flHther increased if the wells on the slide were spaeed at the same distance as the wells on a 96-well euhure plate; this would allow the transfer of several samples of cul- ture supernatant at a time using a Titertek 5 or similar multiehannel pipetter. Such slides eould be custom or- dered from the manufacturer 7 or an equivalent substi- tute eould be produced direetly in the laboratoD~ (8).

Our proeedure speeified PtK I eells as the target specimens. We have chosen this cell line for its utility to eytoskeletal work, but other adherent cell lines may obviously be used. Published methods are also avail- able for attaching nonadherent cells or particles to slides or cover slips (7),

A problem eommonly encountered in viewing and

photographing lE preparat ions is that of photo- bleaching, the progressive loss of fluoreseenee inten- sity with exposure of the specimen to the exciting illu- mination. Where rapid photobleaehing is a problem for aeeurate assessment of the assays it is possible to em- ploy mounting media with substances that retard bleaching (2,4). The use of rhodamine as the fluor- ochrome, in place of fluorescein, will also be useful. Rhodamine offers somewhat less intensity than fluore- stein, hut photobleaches less rapidly (2). Use of rho- damine requires an appropriate filter system in the epi-illuminator.

Fluorescence microscope systems, similar to the Leitz equipment listed here, are available from several mieroscope manufaeturers, The ehoice of an appro- priate objeetive lens is a eritieal faetor in assembling a system, and deserves note. The intensity of the flno- rescence image is generally weak. For adequate obser- vations the image should be as bright as possible. Be- «ause image intensity varies with the square of the ob- jective's N.A., an oil or water immersion objeetive of N.A. 1.0 to 1.4 is preferred. Ideally, the lens should be of a design speeifieally for fluoreseenee or at least of a relatively simple optical design. Complex optics with many elements will reduce image intensity and eontrast, lmage intensity will also be reduced by the phase ring if a phase contrast objective is used.

V. References

1. Engvall, E. Enzyme irnmunoassay, ELISA and EMIT. In: Vunakis. H. V.: Langoee, J. J.. eds. Methods in enzymology. New York: A«- ademi« Press; 1980:70:419--'139.

2. Giloh, tt.; Sadat, J. W. Fluores«ence nfieros«opy: reduced photo- blea~hing of rhodamine and fluorescein protein conjugates bv n- propyl gallate. S«ien«e 217:1252-1204: 1982.

3. Hsu. S.-M.: Raine, L: Fanger. H. [;se of avidin-biotin-peroxidase «onlplex IABC)in immunopemxidase tet hniques. J. Histo«hem. Cy- to«hem. 29:577-580; 1981.

4. Johnson. G. D.; Davidson, R. S.: McNamee, K. C. et al. Fading of immunofluorescen«e during mi«roscopy: a study of the phenomenon and its remedy. J. lmmunol. Methods 55:231-242: 1982.

5. McBeath, E.; Fujiwara. K. Improved fixation for immunofluoreseen«e rnieroseopy using light-a(tivated 1,3,5-triazido-2,:l,6-trinitroben- zene {TTBI. J. Cell Biol. 99:2061-2073; 1984.

6. Nerurbar. L. S.: Ja(ob, A. J.; Madden, D. L. el al. Deteetion of gen- ital herpes simplex infections by a tissue eulture-fluorescent-anti- body le«hnique with biolin-avidin, J. Clin. Mierobiol. 17:14~154; 1983.

7. Osl»orn, M; Weber, K. Immunofluoreseence and immunocytoehemieal pro«edures with affinity purified antibodies: tut~mlin. Methods Cell Biol. 24:97-132: 1982.

8. Rudd. R. J.; Saftbrd, M., Jr. Reusable tissue eulture growth ehambers. J. Tissue Cuh. Methods 8:95-96: 1983.

9. Sehnurs, A. H. W. M.; VanWeemen, B. K. Enzyme-immunoassay. Clini«a Chimi«a Acta 81:1-10; 1977.

E. Leitz, Inc., Roekleigh, NJ 2 Monsanto Plasties and Resins, St. Louis, MO :» Fisher Scientific Co.. Piusburgh, PA '~ Sigma Chemieal Co., St. Louis, MO :; Flow Laboratories, Inc., M«Lean, VA

~' GIBCO, Grand Island, NY 7 Carlson Seientifie, Inc., Peotone, IL

Ameri«an Type Cuhm'e Colleetion, Rockville, MD '~ Ve«tor Laboratories, Ine., Burlingame, CA "~ Rainin Instrument Co., Woburn, MA

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