Reagent-in-film (RIF): A simplified reagent delivery system

ANALYTICAL

BIOCHEMISTRY

Reagent-in-Film DONALD Marine

95, 549-553 (19791

(RIF): A Simplified W. RENN, Colloids

Division,

Reagent

Delivery

ERIC EVANS, AND W. PRESTON FMC

Corporation.

Rockland.

Maine

System

BRAWN 04841

Received July 19, 1978 Water-soluble or dispersible reagents, including antigens, antibodies, enzymes. substrates, stains, antibiotics, or nutrient media, can be incorporated into supported or unsupported hydrocolloid films, dried, and stored in this form until ready for use. The “reagent-in-film” can then be placed in contact with a water-containing medium such as agarose, agar, polyacrylamide, gelatin, cellulose acetate, or paper, whereupon the reagent and its hydrocolloid carrier are transferred rapidly and essentially quantitatively into the medium.

Methods generally used for applying reagents to or into preformed media include soaking the media in solutions of reagent, applying reagent solutions to the surface of the media, or applying a piece of paper, cellulose acetate, or some other solid matrix containing the reagent in either dry or solution form. In many cases, reconstitution of dry reagents is necessary, an excess of often expensive reagents is required to assure sufficient penetration, and the reagent transfer is not complete. Supported or unsupported films of mixtures of the reagents and hydrocolloids (for aqueous systems), prepared and stored until needed, overcome these problems. When placed on the surface of a water-containing matrix (gel, film, tissue, etc.), the hydrocolloid first absorbs surface water; then concentration gradient differences provide the driving force which rapidly carries the reagent into the aqueous system. Although somewhat dependent on hydrocolloid concentration, reagent application by RIFI favors direct penetration without lateral diffusion.

This technique was first described in U. S. Patent 3,975,162 (1). However, the basic procedure has been modified to simplify the film preparation and additional applications have been developed. A few selected applications are described and illustrated in this paper to demonstrate the reagent-infilm principle. MATERIALS,

METHODS, AND RESULTS

LDH lsoenzyme

Visualization

The LDH substrate RIF was prepared by dissolving Dade Tetraform reconstitutable powder (Lot DHC- 110) in 3 ml of the buffer (Lot DHS216A), mixing with an equal volume of 4% dextran R (Pharmachem) in distilled water, spreading 0.3 ml evenly on the hydrophilic side of each piece of 76 x 76mm GelBond (Marine Colloids Div., FMC Corp., Rockland, Maine), and drying at 30°C in a Blue M forced-air oven for 10 min. The reagent-containing films were stored in a tightly closed polyethylene bag at 4°C until used. Different samples of human plasma were separated using a Coming-AC1 Universal ’ Abbreviations used: RIF, reagent-in-film; LDH, lactic dehydrogenase; Ig, immunoglobulin; (r,-AT, CQ- agarose film (Lot 0022478) with the Corning EP chamber-power supply combination acantitrypsin; EIA, electroimmunoassay; IEP, immunoelectrophoresis. cording to the manufacturer’s instructions. 549

0003~2697179/080549-05$02.0010 CopyrIght 0 1979 by Academc Press. Inc. All rtghts of reprodudwn in any form reserved.

550

RENN, EVANS,

AND BRAWN

Electrophoresis of human serum was done as described in the section on the LDH procedure. Upon completion of the run, the agarose film was overlaid with the amido black RIFs, which were removed after 2 min. Destaining could be accomplished by the filter paper overlay-pressure method (2), using 5% acetic acid (as), or a combination of these methods. Immunofxation RIF-a,-Antitrypsin (a,-AT) Pi Typing

Suitable RIFs were prepared by mixing I vol of 4% dextran R (aq) with 2 vol of the IgG fraction of goat antiserum to human alantitrypsin (Atlantic Antibodies, Westbrook, Maine, Lot AAT-SFD-005-SP). Agarose gel electrophoresis of human serum generally followed the method of Ritchie and Smith (3). Gels were prepared using 10 ml of hot solution of SeaKem ME agarose (Marine Colloids, Lot 73207) in FIG. 1. Collage of RIF examples. (a) LDH isoM, pH 5.1 sodium acetate buffer, evenly 0.05 enzyme visualization. (b) al-Antitrypsin immunofixadistributed on the hydrophilic side of a 7.5 tion. (c) Electroimmunoassay (single). (d) Electroimmunoassay (multiple). (e) Immunoelectrophoresis. x 15-cm piece of GelBond. Plates to be stored for later use were covered with Electrophoresis was stopped at 35 min, the other pieces of GelBond with the hydrofilm removed from the chamber, and the philic side facing the gel, wrapped in Saran LDH-RIFs were cut to cover two channels Wrap, and placed in a tightly closed polyeach and applied to contact the agarose ethylene bag at 4°C. gel surface evenly and exclude air pockets. After electrophoresis for 3 h at 20 mA The agarose gel-RIF “sandwich” then was constant current, the agarose gel was overplaced in a covered plastic box and incubated laid with an (u~-AT-RIF and placed in a at 37°C for 1 h, although after 15 min the covered plastic box for 1 h. The backing was purple LDH bands were clearly visible (Fig. then removed and the agarose film washed la). The RIF plastic backings were removed overnight with stirring, in 0.85% NaCl, 0.02 and the agarose film placed in a stirred solu- NaN, (aq), then 1 h in distilled water. After tion of 5% acetic acid (aq) for 15 min to set drying at 60°C in the forced-air oven, the the bands. The plate was then dried at 60°C film was stained using 0.1% Coomassie blue in a Blue M forced-air oven for 20 min. R (Sigma, Lot 31C-0230) in 1:l 10% acetic acid (aq)-ethanol, excess stain removed with Total Serum Protein Visualization this solvent mixture, and the plate dried. The multiple cw,-antitrypsin bands were Amido black 10B protein stain RIFs were clearly visible (Fig. lb). prepared from a solution of 2% dextran T70 (Pharmacia) or dextran R (Pharmachem), Radial Immunodiffusion 4.5% citric acid (Pfizer, anhydrous), and Using the method described in the section 0.25% amido black 10B (Coming, Lot on immunofixation, RIFs were prepared us9122177) in distilled water.

REAGENTS-IN-FILMS

5.51

ing rabbit anti-human IgG (Behring, Lot 2410A). Agarose plates were prepared by pouring 6 ml of an 0.8% solution of SeaKem HGT(P) agarose (Marine Colloids, Lot 146334) in 0.85% NaCl, 0.02% NaN, (aq) evenly on the hydrophilic side of 76 x 76 mm GelBond 7 and allowing to gel. Circular wells (1.5 mm) were cut in the gel and removed by aspiration. An anti-IgG-RIF was carefully placed on one gel to exclude air bubbles. After 10 min, the RIF backing was removed, each of the wells inoculated with 2 ~1 of human plasma, and the gel placed in a humid chamber overnight at room temperature. Circular precipitin zones were seen (Fig. 2). Essentially equivalent in diameter, these zones were enhanced by soaking 5 min in a 0.15% phosphomolybdic acid solution (4). Electroimmunoassay

(EIA)

Single. Goat anti-human C3 antiserum FIG. 2. Radial immunodifbsion plate using RIF. (Atlantic Antibodies, Lot G31-34) was used to prepare RIFs as described pre- pH 8.6 Beckman B-2 barbital buffer. Five milliliters were used for each 76 x 76-mm viously, except polyvinylpyrollidone (GAF, Lot 2GA-PVP-225- 1) was used as the hydro- piece of GelBond. The agarose gel plate was inoculated colloid carrier. The EIA procedure used was that described by Renn and Evans with human plasma by using a Drummond tube previously (5) except that instead of incorporating the IO-p1 glass microcapillary dipped into the plasma. The capillary was antibodies in the gel, they were applied by placing the RIF on the gel for 10 min thrust into the gel then withdrawn, during before inoculating the wells with serum. which time about 1 ~1 of the plasma was point approxiWell-formed rockets were observed (Fig. deposited. An application lc). Similar results were obtained using an mately ‘1.5 cm from each edge in the lower anti-IgA-RIF prepared from goat anti- left-hand corner was chosen. A similar human IgA (Atlantic Antibodies, Lot G31- technique was used to apply a spot of Gelman RBY indicator dye about 0.5 cm 36) and Polyethylene Glycol 4000 (Baker, below the plasma application site. Filter Lot 2-1890). paper (S&S Grade 470) was cut and used Multiple (“‘2”“). Anti-whole human serum-RIFs, 76 x 76 mm, were prepared for the wicks. A Shandon EP chamber as described previously under immunocontaining 0.0375 M, pH 8.6 Beckman B-2 fixation, using rabbit and anti-whole human barbital buffer and a Buchler regulated serum (Behring, Lot 2055T) and water- power supply was used for the EP run. Separation of the plasma protein was acsoluble polyacrylamide, Dow NP-10, puricomplished at 15 mA constant current fied by Marine Colloids (Lot 146732B). Agarose gel plates were prepared using (170- 140 V) for 30 min. The wicks were removed and a 40 x 76-mm strip of the anti0.8% SeaKem HGT(P) agarose (Marine applied evenly to Colloids, Lot 146334) dissolved in 0.0375 M, whole human serum-RIF

552

FIG. 3. Antibiotic

RENN, EVANS,

sensitivity assay using RIF.

the surface, just covering the sample application point and parallel to the bottom of the gel. After 5 min, the RIF backing was removed, the plate was turned 90”, new wicks were used, and electrophoresis was continued at 15 mA for 40 min. The albumin rocket and several of the other peaks were clearly visible and were used as a check for completeness of the run. To remove the excess antiserum, the gel was placed in stirred 0.85% NaCl, 0.02% NaN3 (aq) overnight, then in stirred distilled water for 1 h. The gel was dried at 60°C in a Blue M forced-air oven, then stained with Coomassie Blue R as described in the single EIA section. A typical multiple rocket pattern was obtained (Fig. Id). Similar results were obtained using RIFs prepared with Pharmathem dextran R as the hydrocolloid carrier. Immunoelectrophoresis

(IEP; No Troughs)

Agarose gels and anti-whole human serum RIFs with dextran R were prepared as de-

AND BRAWN

scribed under Electroimmunoassay and used for these experiments. Two methods of sample application were tried with equivalent results: precut 1.5-mm-diameter, aspirated wells, and the no-well, capillary tube application method described for multiple EIA. Three sample application points, 3 cm from the top edge of the 76 x 76-mm gel, were used with approximately 1 ~1 of human plasma being deposited in the uncut gel and 1.5 yl in the wells. S&S Grade 470 filter paper was cut and used for the wicks. A Shandon EP chamber containing 0.0375 M, pH 8.6 Beckman B-2 barbital buffer and a Buchler regulated power supply were used for the IEP run. Gelman RBY dye applied near the edge with a capillary was used to follow progress of the run which was made at a constant current setting of 15 mA per plate (170- 140 V) for 35 min. The wicks were removed and 2 x 76 mm strips of the anti-whole human serumRIFs were placed equidistant between the sample application points. After 5 min, the RIF plastic backings were removed and the gels placed in a humid chamber at room temperature. After about 2 h, the albumin precipitation arcs were visible with the others developing overnight. Excess antiserum was removed and the IEP patterns stained as described in the single EIA section. Useful IEP patterns were obtained although the geometry and concentrations of reactants remain to be optimized (Fig. le). Antibiotic

Sensitivity

Assays

An unidentified, fast-growing microorganism from a throat culture was incorporated in nutrient agar in a petri dish. Tetracycline RIFs were prepared by cutting 75-mm-diameter circles or doughnuts of GelBond and putting on these, around the periphery, lo-p1 spots of various concentrations of the antibiotic (Wyeth tetracycline, 250-mg capsules), dissolved in 2% dextran R (Pharmachem) or T-70 (Pharmacia). Total amounts of the tetracycline in

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REAGENTS-IN-FILMS

the spots varied from 0 (control) to 0.05 mg, at fivefold dilutions. These were dried at 30°C in a Blue M forced-air oven. For the antibiotic sensitivity assay, the RIF was placed evenly on the inoculated agar plate. After 5 min, the plastic backing was removed, using the sticky side of a small piece of masking tape, and the agar plate incubated overnight at 37°C. After 1 h, the inhibition zones were clearly visible (Fig. 3). When the plastic backing was left in place during incubation, slower growth was observed with the isolated organism. No inhibition was observed with the dextran control.

nutrient media storage and transfer, gradient buffer introduction, histological staining, and simplified diagnostic test systerns. These experiments and the results obtained clearly demonstrate the practical versatility of the reagent-in-film method of reagent storage and delivery. RIF techniques are not limited to aqueous systerns and the principle can easily be adapted to automation.

DISCUSSION

22, 497. 3. Ritchie, R. F., and Smith, R. (1976) C/in. Chem. 22, 1735. Renn, W., and Evans, E. (1976) Anal. Bio4. chew.D. 64, 620. 5 Renn, D. W.,and Evans, E. (1976)AnaI. B&hem.

In addition to the examples described, applications for which the RIF technique is useful include: intermediate gel electrophoresis techniques for tumor antigen identification (6), counterelectrophoresis,

REFERENCES

I Renn, D. W. (1976) U. S. Patent 3,975,162. ’ Corresponding patents in other countries in various stages of allowance.

? L. Ritchie, R. F., and Smith, R. (1976) Chin. Chem.

’

71, 588.

6. Saravis, C. A. (1978) Personal communication.