JOURNAL OF CLINICAL MICROBIOLOGY, June 1976, p. 637-639 Vol. 3, No. 6 Copyright © 1976 American Society for Microbiology Printed in U.S.A. Detection of Virus-Specific Immunoglobulins Using a Doubly Labeled Fluorescein- 125I Antibody A. J. PARKINSON* AND J. KALMAKOFF Department ofMicrobiology, University of Otago, Dunedin, New Zealand Received for publication 17 February 1976 Commercially prepared fluorescein-labeled antihuman antibodies were la- beled with 125I and used to compare specific herpes simplex virus antibody titers as determined by indirect fluorescent antibody and radioimmunoassay tech- niques. Total virus-specific immunoglobulin and virus-specific immunoglobulin G titers did not vary by more than one twofold dilution when compared by the two methods. Efforts are being made to develop a reliable calf serum, penicillin (100 U/ml) streptomycin radioimmunoassay (RIA) for the detection of (100 ,ug/ml), and 0.1% bicarbonate, were in- virus-specific immunoglobulins, acceptable for fected with the isolated virus. Uninoculated use in diagnostic serology (1, 2, 5-8). The estab- monolayers were maintained as controls. When lishment of a satisfactory RIA depends on the infected monolayers showed 75% cytopathic use of antibody with both a high avidity and effect, both inoculated and uninoculated cells selectivity for the material to be assayed (4). were dispersed, using 0.015% ethylenediamine- Consequently, a major obstacle to using RIA tetraacetic acid. Both cellular suspensions were routinely is the necessity of preparing specific standardized to contain 2.5 x 105 cells/ml in high-titer antibody against human immuno- phosphate-buffered saline. Using 0.025-ml vol- globulins (IgG, IgM, and IgA). This usually umes, infected and uninfected cells were ap- requires animal-holding facilities, use of time- plied to an acetone-resistant, transparent, flex- consuming animal immunization schedules, ible plastic (Melonex 0; Imperial Chemical In- and expensive antibody purification and evalu- dustries Ltd.). The drops, each covering a uni- ation procedures. form area of 5 mm in diameter, were rapidly Although commercial antihuman immuno- dried and fixed with acetone for 10 min at room globulins are available, these sera are primar- temperature. ily prepared for immunodiffusion or immunoe- Fluorescein-labeled antihuman immunoglob- lectrophoretic techniques. Since the indirect ulin and antihuman IgG (sheep; Wellcome fluorescent antibody method (IFA) closely par- Foundation Ltd.) were labeled with 1251 (Amer- allels many RIA procedures (5-7), we investi- sham Radiochemical Centre) using the chlora- gated the use of commercially available fluo- mine T method of Hunter and Greenwood (3). rescein-labeled antihuman immunoglobulin for Free '25I was separated from the conjugated labeling with 125I. These antibodies are specifi- protein by gel filtration, using a Sephadex G-50 cally prepared for fluorescent antibody staining column (1.5 by 30 cm). The Fl-25I doubly la- from high-titer antisera demonstrating maxi- beled immunoglobulins were recovered by the mum reactivity against the appropriate class of addition of 0.235 g of solid (NH4)2SO4 per ml to immunoglobulin. Using double-labeled fluores- the protein fractions. Specific activities of 105 to cein-'251 (Fl-'251) antibodies, we were able to 2.0 x 105 counts/min per Ag ofprotein were rou- detect virus-specific immunoglobulins and di- tinely obtained. The Fl-125I-labeled antihuman rectly compare the IFA and equivalent RIA immunoglobulin and antihuman IgG were used systems. at a concentration of 196 ,ug of protein per ml We used herpes simplex type 1 virus as a for both IFA and RIA determinations. This con- model system. The virus was isolated from a centration of Fl-'25I-labeled antibody produced patient who had suffered an acute upper respi- maximum fluorescence and radioactivity in in- ratory illness. Serial serum samples were taken fected cells, with negligible background reac- from 4 days after the onset of symptoms to 90 tivity in uninfected controls. Both the specific days post-illness. fluorescence and radioactivity could be reduced Monolayers of monkey embryo kidney cells to background proportions by blocking the reac- were propagated in complete Eagle basal me- tion with either specific herpes simplex virus dium (GIBCO), supplemented with 10% fetal antisera (rabbit; Center for Disease Control) or 637 638 NOTES J. CLIN. MICROBIOL. noniodinated fluorescein-labeled antibody. By radioactive counting, the virus-specific Virus-specific antibody in each serum sample antibody titers were determined in a strictly was determined, first, by using noniodinated quantitative manner. The radioactivity bound fluorescein-labeled immunoglobulins and, sec- to infected fixed cells at each serum dilution ond, by IFA and RIA using the Fl-'25I-labeled was statistically compared to the radioactivity antihuman immunoglobulin preparations. Two- bound to the uninfected cell controls (Fig. 1). fold dilutions of human serum were made in The titers of both virus-specific immunoglobu- 0.1% fetal bovine gamma globulin in phos- lin and virus-specific IgG obtained by RIA phate-buffered saline, and 0.025 ml ofeach dilu- match closely those of the IFA method. This is tion was applied to infected and uninfected cells consistent with results obtained by other work- fixed to Melonex 0 strips. After incubation for ers (6). 15 min at room temperature, the strips were In Table 1 we demonstrate the differentiation washed free of unbound antibody with phos- of virus-specific IgG from total virus-specific phate-buffered saline and dried. Fluorescein- or antibody. Using iodinated preparations ofother Fl-'251-labeled antihuman antibody (0.025 ml) class-specific, fluorescein-labeled, antihuman was then added to each fixed-cell area, and the immunoglobulins (antihuman IgM and antihu- strips were incubated for 30 min. Unreacted la- man IgA) and appropriate optimization proce- beled antibody was removed by washing in phosphate-buffered saline. Each strip was mounted on a glass slide, fixed-cell area upper- TABLE 1. Reciprocal antibody titer to herpes simplex most, and covered with a cover slip, using phos- virus as determined by IFA staining and RIA phate-buffered glycerol (pH 8.0) as a mounting Days Virus-specific immuno- Virus-specific IgG after globulin medium. Both infected and uninfected cells infec- were examined for fluorescence using dark- tion Fla Fl-'25I RIAb Fl Fl-"251 RIA ground microscopy with an ultraviolet lighting 4 16 32 128 8 8 32 system. The IFA titration end point was taken 10 512 256 512 256 128 256 as the highest serum dilution showing specific 20 512 256 1,024 128 128 512 fluorescence in infected cells. After fluorescence 30 256 256 512 128 128 256 microscopy, the Melonex strips were washed 90 N1TJ 256 512 NT 32 64 free of glycerol, and each cell area was cut out in vials for the detec- a Fl, Noniodinated antihuman antibody. and placed scintillation Results from Fig. 1. tion ofradioactivity. An arbitrary virus-specific c NT, Not tested. antibody titer was determined as that dilution of serum at which the radioactivity bound to in- fected cells exceeded the two-standard devia- tion level of the radioactivity of the negative controls. The use of 0.1% bovine gamma globulin as a diluent for serum and labeled antibody was found to be effective in reducing the amount of background radioactivity and in markedly re- ducing nonspecific fluorescence. Acetone fixa- cpm tion of infected and uninfected cells to Melonex x-lo- O permitted both fluorescence microscopy and radioactive counting to be carried out on the same fixed-cell area. Titration end points ob- tained by IFA or RIA could then be directly compared. The antibody reactivity of Fl-'251-labeled an- tihuman immunoglobulin was compared with 8 16 32 64 128 256 512 1024 2048496 noniodinated fluorescein-labeled antihuman reciprocal of serum dilution immunoglobulin by the IFA method (Table 1). Titers of virus-specific total immunoglobulin FIG. 1. Detection of herpes simplex virus-specific and did not more antibody by RIA. The virus-specific immunoglobulin virus-specific IgG vary by titer in the patient's serum was determined by indi- than one twofold dilution, demonstrating that rect RIA using fluorescein-'25I doubly labeled anti- iodination of fluorescein-labeled antibodies to human immunoglobulin. The radioactivity bound to high specific radioactivities (105 to 2.0 x 105 uninfected cells at any serum dilution was deter- counts/min per ug) does not alter their immu- mined, and the mean and 2 standard deviations nological reactivity. were calculated. VOL. 3, 1976 NOTES 639 dures, it should be possible to measure virus- plex viral antibodies in human sera. J. Clin. Micro- biol. 2:410-418. specific IgM and IgA. 2. Green, R. L., R. W. Scales, and S. J. Kraus. 1974. In this study the use of relatively large Radioimmunoassay on polycarbonate membranes: a amounts of Fl-'25I-labeled antibody (196 ,g/ml) sensitive and simplified method for the detection and was necessary to ensure optimum fluorescence quantitation of antibody. Appl. Microbiol. 27:475- and to enable a direct comparison to be made 479. 3. Hunter, W. M., and F. C. Greenwood. 1962. Prepara- between the IFA and RIA methods. However, tion of 131I labeled human growth hormone of high subsequent experiments indicate virus-specific specific activity. Nature (London) 194:495-496. antibody may be determined by the RIA proce- 4. Hum, B. A. L., and J. Landon. 1971. Antisera for anti- radioimmunoassay, p. 121. In K. E. Kirkham and W. dure alone, using much less Fl-'25I-labeled M. Hunter (ed.), Radioimmunoassay methods. Liv- body (40 ug/ml; unpublished data). Fl-'251-la- ingstone, Edinburgh. beled antibody may provide an economical 5. Hutchinson, H. D., and D. W. Ziegler. 1972. Simplified source of class-specific antihuman immuno- radioimmunoassay for diagnostic serology. Appl. Mi- globulin for the routine detection of virus-spe- crobiol. 24:742-749. 6. Hutchinson, H. D., D. W. Ziegler, and P. M. Georino. cific antibody.