Solid phase radioimmunoassay for plant viruses

VIROLOGY

66, 516-620 (1973)

Short Solid

Phase

Communications

Radioimmunoassay ELLEN

Department

of Plant Pathology,

for

Plant

Viruses’

M. BALL

University Accepted

of Nebraska,

June

Lincoln,

Nebraska

68603

26, 1973

In a radioimmunoassay procedure, adapted to the antigenic analysis of plant viruses, antibody was irreversibly attached to the walls of disposable polystyrene tubes. In the direct test, labeled antigen was effectively assayed in a range of lo-100 rig/ml. In the indirect test, unlabeled antigen from 0.6 to 5.Opg/ml and a constant amount of labeled antigen were added sequentially to antibody-coated tubes. The latter procedure was applied effectively to serological differentiation of virus strains and of coat-protein and intact virus rods.

The use of radioimmunoassay in the past 34 years has increased phenomenally, especially in research with hormones and immunoglobulins. The principle advantage of radioimmunoassay is its sensitivity in the nanogram or picogram range, but the sensitivity is dependent upon effective procedures for separating antibody-bound, labeled antigens from free, unreacted antigen by carefully controlled precipitation (1,2). The solid-phase radioimmunoassay procedure circumvents most of these separation problems. Catt and coworkers (S-6) described a procedure in which disposable polystyrene or polypropylene tubes or poly (tetrafluorethylene) discs were irreversibly coated with antibody and used directly to measure bound radioactively labeled antigen; the unbound labeled antigen was removed merely by washing the solidphase. A sensitive

inhibition

test

was

described

in

which antibody sites were blocked by incremental amounts of unlabeled antigen prior to the addition of a constant amount of labeled antigen. Herein the solid-phase radio1 Published with the approval of the Director as Paper No. 3582, Journal Series, Nebraska Agricultural Experiment Station. Research reported was conducted under Project No. 21-3 from a grant by the National Science Foundation.

immunoassay test has been adapted to the antigenic analysis of 5 purified plant viruses. Purified preparations of tobacco mosaic virus (TMV), southern bean mosaic virus (SBMV), wheat streak mosaic virus (WSMV), and barley stripe mosaic virus (BSMV), obtained from M. K. Brakke and W. G. Langenberg, and cowpea mosaic virus (CPMV), prepared by C. Niblett, were measured spectrophotometrically and used to immunize rabbits by giving 3 intramuscular injections of 3-5 mg of virus emulsified in Freunds incomplete adjuvant at 5-7-day intervals and by giving 1 intravenous injection of 1 mg of virus 2 days after the second adjuvant injection. Serum was collected by standard methods and stored at -20°C. All antisera had titers of at least 1: 1024 with 0.5-1.0 mg/ml of antigen in the microprecipitin test. Immune gamma globulin (IgG) was separated from serum by 2 consecutive precipitations at 33-40 % cold saturated ammonium sulfate, pH 7. Aliquots of 0.250.5 ml IgG were sealed in small test tubes and stored at -20°C. Antigens were labeled with lz51 by the method of Bale et al. (‘i’), except that the iodine monochloride solution was injected into the protein-Y mixture using a pipet fitted with a suct.ion bulb.

516 Copyright All rights

@ 1973 by Academic Press, Inc. of reproduction in mu’ form reserved.

SHORT

*vi17

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The IgG was diluted in 0.05 M sodium carbonate-bicarborlate buffer, pH 9.5-10.0, and added to l-ml disposable polystyrene centrifuge tubes (Fisher Scientific, 4-97% 145). The tubes were rotated, end-over-end, at 8 rpm on a tissue culture rotator held at about 60” from horizontal for an overnight period at 4°C. Excess antibody was eliminated by washing the tubes with saline (0.15 2-3 times. The t’ubcs wcrc filled 111 NaCl) wit,11 protein buffer, i.e., 2 ‘% normal rabbit serum (NRS) or 0.1 ‘i;, bovine serum albumin (BSA) in saline containing 0.025 ‘To sodium azide, and stored at 4°C. In the direct test, labeled antigen n-as added in the appropriate conccnbration scrics to a triplicate set of drained tubes. These were rotated on the tissue culture rot,ator as described for antibody coating. The tubes were count’cd to dctcrmine t,hr total amount of antigen added in each sample, then washed at least 3 times xvith salirw and recounted. In t’he indirect test, unlabclccl antigen was added in increasing amounk in an appropriate buffer to roakd tubes which wrc incubated at’ 37°C for 2 3 hr, refrigerated for 4 hr, then charged direct,ly with 0.1 ml of lab&d aruigcn, rotated overnight at, 4”CI, washed at] least ?, times with saline, and counted. Radioact)ivit?~ was asswsed in a \\-cll-typo gamma scintillation counter. The average of counts for :3 samples was plotted against the amount of antigen added, labeled antigen in the direct test, and unlab&~d in the indirect t,est. 111 cxpcriments to determine the amount, of antibody required to saturate sites on the p~~lystyrcnc~ tubes, dilutions of IgG from 1 : 100 to 1 :X,000 gave maximum, conslant cwunts lwr minute. but higher dilutions resulted in dccrcawd counts per minute. Normal rabbit gamma globulin in the same dilution range gave a minimum con&ant, counts per minute with labeled antigen, the same as observed in tubes with too little 1gG. For routine coating of tubes, IgG was used at 1: 500. Airlock formation preserncd difficult’ics in wetting the plastic tubes \vith IgG dilution greater than 1 : 1000. The coating solution could bo used repeatedly (6-7 times), since only small amounts of total antibody absorbed to the plastic. Specific immune gamma globulin prepared by elution

of antibody at low pH from an antigen arttibody precipitate was used in some experiments for a more precise measuw of antigen binding capacity A prot’ein comaining buffer \vas required in the solid-phase assay test to block protein adsorbing sites on the plast,ic not, covcred by antibody. In tests with solutions consisting of saline and 0.025 5;) sodium azidc as base, plus tither 0.1% HEX, 23 or 1O?;8 SRS, 0.2% hemoglobin, or 1, 2> 5, 10, and 20’; normal sow serum to stow IgG coakd tubes, 2 ?S NRS or 0.1 ‘,b BSA buffers \\‘crc found to be suitable for periods up to 6 rr~on~hs of storage. Tests made of tubes stored for S months showed a GO-70 ‘:; drop in counts per minute compatrcd l,o counts lwr niirrutc~ in freshly coated tubes. Hy direct assay, a linear relationship was shown between the logarithm of coums per minute and of ant,igcn conccntmt~ion from 1.6 to 1000 ng,/ml, wit’h CPMV (Fig. 1.) An analysis of total radioactivit,y added ((I the

40 200 10005000 NG

ANTIGEN/ML

FIG. 1. Jhrect, test assay of t.ubes coated with CPMV-IgG, stored in O.lC,; BSA buf%‘cr, and cxposcd t.o fivefold dilution series, beginnirlg wit,h 5 rg/ml of labeled CPMV antigen in BSA bluffer. (O-0) [““I]CF’MV + CPM\‘-I~G, c- --0 = [1zPI]CPMV + N1:S gamma ~lobldin.

518

SHORT

COMMUNICATIONS TABLE

RESULTS

OF INHIBITION IN

Diluent unlabeled labeled

PROTEIN

CONTENT

-

of a@ ag

TESTS

OF TUBES OF THE

DILUENT

Expt I 2% NRS Saline Saline Z-ESNRS

- Saline Saline -_

COATED

1 WITH

FOR

SBMV-IgG

UNLABELED

Expt II 5% NRS 10% NRS @aiG 2%

2% NRS

--

CPm (X 1w

CPm (X

WHEN AND

FOR

VARIATIONS

2% NRS 5% NRS

109

cpm

M.&DE

ANTIGEN

Expt Saline ~~~ 5% NRS

WERE

LABELED

III Saline 10% NRS

2% NRS 10% NRS

109

(X

Inhibiting antigen Gcdml) 0 1 2 3 4 5 $$ Inhibitionb Controlc

17.8 17.7 16.2 14.7 13.7 10.9

18.3 17.1 16.0 15.6 15.1 14.1

17.3 11.9 10.2 9.9 9.5 8.2

17.9 15.1 14.9 14.3 12.3 11.0

38.8

23.0

52.6

38.5

-

18.2 18.0 16.2 14.7 14.5 12.4

17.9 17.5 16.9 16.0 15.5 12.4

31.9

30.7

11.8 9.1 7.0 5.9 5.5 5.3

7.8 7.1 5.9 5.1 5.6 4.2

7.2 6.4 5.6

6.9 6.2 5.6

5.4 5.0

5.2 4.7

55.1

46.2

30.0

31.9

-I-

.-

30.8

a ag, antigen. b Percentage inhibition c Control: Total cpm antigen.

26.6

= (cpm Opg/ml added in 0.002

30.3

- cpm 5 pg/ml)/(cpm ml [12SI]SBMV/ml

tubes and the amount adsorbed by antibody (after complete washing) revealed little unbound antigen, i.e., less than 10 % difference in counts per minute before and after washing. Adsorption of labeled CPMV to normal rabbit gamma globulin was negligible, e.g., 186, 246, and 767 cpm with 0.04, 0.20, and 1.00 Erg/ml of labeled antigen, respectively. All of the viruses tested gave a linear relationship between counts per minute and concentration of antigen added in the range of lo-100 rig/ml. The viruses which tended to aggregate or denature on storage did show the presence of variable amount of unabsorbed antigen. One TMV preparation, stored at 4°C for 5 months prior to labeling, had unabsorbed antigen even at 20 rig/ml, but none was detected with 10 rig/ml of labeled antigen. The effect of protein in the diluent for labeled and unlabeled antigen was determined by the indirect test using sequential addition of the two antigens (8). After a suitable incubation period, the labeled antigen solution replaced the total volume of the unlabeled antigen in these experiments. Data from 3 experiments with SBMV (Table 1)

20.2

Opg/ml) X 100. carrier buffer after

20.4

removal

of unlabeled

showed that less blocking and less percentage inhibition occurred when protein was added to the diluent containing the unlabeled antigen as compared to unlabeled antigen in saline only. Similar results were obtained in experiments with WSMV in which the unlabeled antigen was added in Tris-citrate buffer, saline, or neutral phosphate-buffered saline (PBS) and the labeled antigen in the same diluents with and without 2% NRS. In every case, the best results were obtained with unlabeled antigen added in saline or PBS and labeled antigen in 2 % NRS buffer. The indirect test was used to differentiate strains of TMV and of BSMV and to study the relationship between coat protein and intact TMV rods. Three strains of TMV, the type strain AC-3 (American Type Culture Collection, Catalog and Registry of Plant Viruses, 2nd Edit., 1958), mild dark green strain, and the mild strain (9, IO), were added in increments to tubes coated with type strain IgG. Absorption was measured by the addition of 1.2 pg of labeled type strain antigen per 0.1 ml per tube. Differences between adsorption of dark green or mild strains and the type strain were clearly

SHORT

519

COMMUNICATIONS

15 -

:-

IO -

0

4 /1g TMV/ML

50 pg

I UNLABELED

I 2

1 3

ANTIGEN/ML

1 4 PBS

FIG. 2. Indirect assay of strains of TMV. Comparison of adsorption of type, mild, and mild dark green strains to tubes coated with type strain IgG and subsequently saturated with labeled type strain ant,igen. Treatment: unlabeled antigen added, then incubation 37°C (2 hr) and 4’C (6 hr), labeled antigen added, rotated 4’C overnight.

evident (Fig. 2). Unlabeled strains of BSI\IIV, t,he type strain, Argentine mild strain, North Dakota 18 strain, in concentrations from 2 t’o 8 pg/ml, were adsorbed on type strain IgG coated t.ubes and tested with type strain labeled antigen. All strains closely resembled the homologous type strain reaction. The indirect, assay test showed significant differences in the adsorption of coat protein antigen to intact virus-IgG as compared to t’ha adsorption of whole virus (Fig. 3). Tho TP\‘IV virions purified by repeat’ed (3 t’imes) precipitation with polyethylene glycol (PEG) would not bind 1251 and could not be used for labeled antigen for radioimmunoassay. This antigen preparation apparcntly contained adsorbed PEG, which could, however, be removed by density gradient, centrifugation to give TMV which would accept a label. Though the T&IV-PEG could not bc labeled, it’ bound to antibody t h(t same as antigen prepared by n’azHP0, extraction, charcoal clarification, and diffrrcntial ccntrifugation (Fig. 3).

FIG. 3. Indirect assay of TMV (0) and coatprot,ein (0) antigens adsorbed t.o tubes coated with TMV-IgC; and tested with 0.63 pg of labeled TMV antigen. Average of total counts per minute added per tube = 294,466. (0 j TMY prepared only by repeated polyethylene glyrol precipitation (see text).

The radioimmunoassay t,est was cxasy to perform, economical if radioactive material was available for other studies, required extremely small amounts of reactants, and was highly sensitive for distinguishing antigcnic propcrtic>s of plant, viruses. SCKNOWLEDC%ENTS I thank l)r. W. C. Langenberg

for labeling

all

antigens and express gratitude to t,he Department of Biochemistry and Nutrit,ion, University of Nebraska, for unlimited use of the gamma srintillation counter. RE:FEKENCES 1.

DAUGHADAY,

“Principles Assays” cds.), pp. 1971.

W. H., and JACOBS, L. S., of Competitive Proteirl-Binding (W. D. Ode11 and W. II. Daughaday, 303-324, Lippincott, Philadelphia,

In

W. M., and GANGULI, I’. C.. In “KadioimmunoassayMet~hods” (K. 1’. Kirkham and W. M. Hunter, eds.), pp. 243.-257. Chlwchill Livingston, London, 1971. 8’. CATT, K. J., XIALL, H. D., and TREGEAR, (+. W., Am. J. Exp. Bid. Med. Sci. 43, 702706 (1967). 2. HUNTER,

SHORT 4. CATT, K., NIALL, H. D., J. Lab. C&n. Med. 70, 6. CATT, K., and TREGEAR, 1570-1572 (1967). 6. CATT, K. J., NIALL, H. and BURGER, H. G., J. 121-126 (1968). 7. BALE, W. F., HELMKAMP, Izzo, M. J., GOODLAND,

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and TREGEAR, G. W., 820430 (1967). G. W., Science 158, D., TREGEAR, G. W., Clin. Endocrinol. 28, R. W., DAVIS, T. P., R. L., CONTREVAS,

M. A., and SPAR, I. L., Proc. Sot. Exp. Biol. Med. 122, 407-414 (1966). 8. WIDE, L., In “Radioimmunoassay Methods” K. B. Kirkham and W. M. Hunter, eds.), pp. 405-412. Churchill Livingston, London, 1971. 9. BALL, E. M., Arch. Biochem. Biophys. 114, 547-556 (1966). 10. MCKINNEY, H. H., Plant Disease Rep. 59, QlQ922 (1968).