Radioimmunoassay for herpes simplex virus

Radioimmunoassay for herpes simplex virus

Journal of Immunological Methods, 10 (1976) 357--362 357 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands RADIOIMMUNOASS...

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Journal of Immunological Methods, 10 (1976) 357--362


© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands



Department of Laboratory Medicine, University of California School of Medicine, 1001 Portero Avenue and Twenty~econd Street, San Francisco, California, U.S.A. and LEO V. DOS REMEDIOS, PAUL M. WEBER

Department of Nuclear Medicine, Kaiser-Permanente Medical Center, Oakland, California, U.S.A. and LAWRENCE DREW

Division of Virology, Mount Zion Medical Center, San Francisco, California, U.S.A. (Received 13 August 1975, accepted 25 September 1975)

Radioimmunoassay has been used for some time in the detection of the Australia Antigen. In this experiment, radioimmunoassay with Iodine-125 (12sI)permitted rapid detection of the presence of herpes simplex virus, type I, in vitro. This is a convenient technique for the detection of virus, which now requires laborious methods involving observation for the cytopathic effect of virus upon cultured cells.


Current methods of viral detection in man require multiplication of the infecting particle, usually by cell culture, and depend upon the observation of cultured cells for the cytopathic effect of the virus. Since radioimmunoassay has proven to be highly specific and sensitive in detecting the presence of minute quantities of antigens in various procedures (Greenwood et al., 1963; Cart and Tregear, 1967; Garcia et al., 1967; Walsh et al., 1970; Odell and Daughaday, 1971), we explored the feasibility of using viral radioimmunoassay as a direct quantitative method {Ball, 1973}. MATERIALS AND METHODS

Herpes simplex virus antigen (HSV) (strain Mayo 1814, NIH # V346/ 001/015), passed and titrated in rabbit kidney cell monolayer (TCIDs0 103"3/ml), was obtained from the Research Resources Branch of the National

358 Institute of Allergy and Infectious Diseases (National Institutes of Health, Bethesda, Maryland 20014).

Method Purified HSV was radioactively labeled to compete with the virus being assayed. Initially, an extract of HSV was purified from sonicated cell-culture preparation. The HSV then was iodinated with Iodine-125 (~2sI) (Greenw o o d et al., 1963; Garcia et al., 1967; Walsh et al., 1970). These procedures are described in detail, as follows:

HS V p u rifica tion The HSV was purified according to the method described by Chambers et al. (1971). A continuous sucrose gradient was prepared b y introducing 370 ml of 40% sucrose over 300 ml of 60% sucrose in 0.1 M Tris (hydroxy-methyl) amino-methane (tris) hydrochloride at pH 7.0. The gradient was formed by duffusion in a flow-through rotor. The rotor was filled with Tris--hydrochloride buffer, which maintained a slow flow through the rotor as it accelerated to speed. At 28,000 rpm, the HSV suspension was introduced into the rotor, and the rate was adjusted to 3 1/h. Fractions (25 ml) were collected. The effluent was monitored spectrophotometrically at 260 nm. Fractions with relatively high optical density were pooled, and the HSV was recovered by sedimentation for 3 h at 40,000 g in a Spinco ultracentrifuge. Radioactive labeling The HSV extract was iodinated with ~2sI, according to the m e t h o d of Greenwood et al. (1963). To label the HSV with ~2sI, the suspension was brought to r o o m temperature, and 50 pl of the HSV suspension were pipetted into a thoroughly cleaned vial, together with 50 pl of 20 mCi/ml of Na~2SI, and 50 pl of 0.5 M phosphate buffer at pH 7.5 (Garcia et al., 1967). Chloramine T (10 pg) and sodium metabisulfite (100 pl) in aqueous solution (2.4 mg/ml) then were added, after which 200 pl veronal were added. The mixture was separated from the excess iodide by the veronal buffer, and 1 ml fractions were collected. A n t i b o d y against HSV (Mayo 1814) was raised in guinea pigs. Neutralization tests performed against HSV (Mayo 1814, TCIDs0 102 ) had a neutralization titer 1 : 64. Initial assessment o f purification o f virus and specificity o f antibody Immunoreactivity was evaluated by radioimmunodiffusion; 0.2 ml purified HSV was placed in peripheral wells of 1% agarose gel plate. HSV antib o d y was applied to the central wells. The gel was incubated for 24--48 h, washed in saline for 48--72 h, dried, and exposed to Kodak Royal Blue Xray film for 1--6 days for autoradiography. The precipitation band formed contained labeled HSV--antibody com-


plex. The non-precipitated antigen was washed off, and the complex was identified as a band of radioactivity that had migrated toward the central well. The radioactivity of the complexed HSV--antibody was assessed in a 3" internal diameter manual well counter (140, 255 cpm), and the result was compared with that of the unwashed controls (618 cpm). The ratio of the radioactivity of the controls to the radioactivity of the complexes crudely reflected the avidity of the HSV and antibody. Specificity of the antibody was tested by neutralization against herpes virus simiae (B virus, E 2490) and herpes simplex (HF). Mycoplasma and bacterial culture of the HSV and antibody preparations were negative. A solid-phase separation technique (Cart and Tregear, 1967) was used to separate the excess HSV from the HSV--antibody complex. Since the polystyrene tubes were coated with antibody which b o u n d firmly to the tube wall, the complexed HSV could be separated in the solid phase, the free virus remaining in the liquid phase. The tubes were coated with 1 ml of the antibody (5000 : 1) by rotating the tubes for 16 h in a vertical rotator so that the entire tube was wetted by the solution. The tubes then were washed three times with 0.15 M NaC1, and once with a protein solution consisting of 10% aged human plasma and 0.01% merthiolate in 0.15 M NaC1.











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Fig. 1. Direct binding of 12 SI-HSV antigen (undiluted virus TCIDs0 10 s/ml). Relation between 12SI-HSV dilution added to antibody-coated tubes (solid line) and uncoated control tubes (dashed line~. Control tubes ranged from 0.09 x 103 cpm (1 X 10 -5 titer) to 1.22 × 103 cpm (1 × 10 ° titer).


The avidity of the virus--antibody complexing and separation of u n b o u n d HSV were tested further by measuring the bound radioactivity residual on the tube wall after the labeled virus had been added, the tube incubated, and the excess HSV washed out. Competition between the labeled and unlabeled HSV for available binding sites on the antibody-coated tubes was then evaluated. RESULTS

To confirm that the antibody was bound to the tube walt, direct HSV-antibody complexing was measured by adding 0.1 ml of 12SI-labeled HSV (TCIDs0 1033/ml then diluted 50 : 1 in labeling technique) to the tubes in logarithmically incremented dilutions. Except that no antibody was added to the control tubes, they otherwise were prepared identically. The cultures were incubated for 4 h at 37°C, and were triply washed with isotonic NaC1. The 12SI-HSV--antibody complex that remained bound on the tube wall was counted in triplicate in a well-type scintillation gamma counter, and was plotted as cpm versus the dilution titer of labeled HSV (fig. 1). Insignificant amounts of labeled HSV were bound in the control tubes. In the tubes containing antibody, the lowest 12 s I-HSV concentration tested was the 10 -6 dilution, which counted 75 × 103 cpm. The counts increased mono-

1000 ~,







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HSV dilution Fig. 2. C o m p e t i t i v e b i n d i n g o f u n l a b e l e d H S V versus 12SI-HSV ( u n d i l u t e d virus T C I D s 0 10 s ml). C o m p e t i t i v e b i n d i n g o f u n l a b e l e d H S V d i l u t i o n c o m p a r e d w i t h b i n d i n g o f a fixed t i t e r o f 12Si_HS V t o a n t i b o d y .


exponentially until the 10 -2 dilution, which counted 1250 × 103 cpm. The vast difference in ,2 SI.HS V binding between the tubes containing the antib o d y and the control tubes {approx. 1000-fold) confirmed that antibody was b o u n d firmly to the tube wall. In the study of competitive binding, 0.1 ml of unlabeled HSV {TCIDs0 103"3/ml) in logarithmically incremented dilutions and 0.1 ml of 12SI-labeled HSV were added to each tube; all tubes had been coated with antibody previously. The tubes were rotated at 4°C for 12 h, and then were washed three times with isotonic NaC1. The ,2SI.HS V b o u n d to the tube wall was counted in triplicate, and radioactivity was plotted against the unlabeled HSV titers. The resulting sigmoid curve resembled the typical competitive binding curve (Odell and Daughaday, 1971) (fig. 2). DISCUSSION

The radioimmunoassay (RIA) for HSV was simple, economical, and reproducible. Extremely small amounts of infecting HSV could be assayed (fig. 2). When diluted, the antibody was sufficient for several hundred HSV assays. Since the binding of the antibody to the tube wall was relatively stable, the tubes could be coated in batches, refrigerated, and stored for future use. The effectiveness of RIA in clinical virology can be inhibited by the presence of patient antibody that interferes with the RIA. Two measures can be adopted to resolve this problem. One approach is to resort to a testing specimen that contains little or no antibody; for example, urine. The detection of virus in urine can be of importance in the diagnosis of several viral diseases, particularly cytomegalovirus disease, in which t h e virus is shed by the renal tubular cells. We chose the HSV as a preliminary virus test model because of the relative ease with which the purified virus and specific antiserum can be obtained. This pilot study presents a relatively new approach to the detection of HSV w i t h o u t tissue culture. It provides a basis for further investigation into the direct assay of the virus, from which a clinically proven method of viral detection might be derived. ACKNOWLEDGEMENTS

We wish to thank Drs. Myron Pollycove and Keith Hadley for their interest and help in this research. We also thank Dr. Robert H o o for his help in the virus assay, Susan Eastwood for her editorial assistance, and Ruth Strauss, Anne Bandoni, Shirley Andrews, and Marilyn McGowan for their assistance in preparing the manuscript. REFERENCES Ball, E.M., 1973, Virology 55,516--520. Catt, K. and G.W. Tregear, 1967, Science 158, 1570--1572.

362 Chambers, R., J.A. Rose, A.S. Rabson, H.E. Bond and W.T. Hall, 1971, Appl. Microbiol. 22, 914--918. Garcia, J.F., J.A. Linfoot, E. Manougian, J.L. Born and J.H. Lawrence, 1967, J. Clin. Endocrinol. Metab. 27, 1395--1402. Greenwood, F.C., W.M. Hunter and J.S. Glover, 1963, Biochem. J. 89, 114--123. Odell, W.D. and W.H. Daughaday, 1971, Principles of Competitive Protein-Binding Assays (J.D. Lippincott, Philadelphia). Walsh, J.H., R. Yalow and S.A. Berson, 1970, J. Infect. Dis.'121,550--554.