Detection of Herpes Simplex Virus (HSV) Type-1 IgG and IgM Antibodies by Enzyme-Linked Immunosorbent Assay (ELISA)

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1 Detection of Herpes Simplex Virus (HSV) Type- IgG and IgM by Enzyme-Linked Immunosorbent Assay (ELISA) JEAN JORDAN, B.S., MT(ASCP) AND MICHAEL W. RYTEL, M.D. Jordan, Jean and Rytel, Michael W.: Detection of herpes simplex virus (HSV) Type- HgG and IgM antibodies by enzyme linked immunosorbent assay (ELISA). Am J Clin Pathol 76: , 9. A sensitive enzyme linked immunosorbent assay (ELISA) is described for. detection of herpes simplex virus (HSV) type, IgG and IgM antibodies. The antigen consisted of a crude extract of HSV infected human foreskin fibroblast cells. Specific horseradish peroxidase conjugated antisera were used to detect total immunoglobulin, IgG and IgM antibodies bound to viral antigen. The substrate was a solution of 5-aminosaiicylic acid and hydrogen peroxide, which yielded a readily visible endpoint. Results obtained by the ELISA method were compared with the micro-complement fixation (CF) method on 36 sera. ELISA was shown to be at least 0-20 fold more sensitive than CF, with a correlation coefficient of (p <.00). HSV antibodies in these sera were mainly IgG, although IgM antibodies could also be detected by ELISA. HSV antibodies were not found in 6 cerebrospinal fluids from.patients without HSV encephalitis (HSVE). ELISA appears to be a rapid, sensitive, and specific method for demonstration of IgG and IgM HSV antibodies. It may have possible application in the diagnosis of HSVE. (Key words: Enzyme linked immunosorbent assay (ELISA); Herpes simplex virus; Total immunoglobulin; IgG and IgM antibodies; Serum and cerebrospinal fluid.) THE enzyme linked immunosorbent assay (ELISA) is more sensitive than many other serologic technics. 2 ELISA also has advantages oyer the comparably sensitive radioimmunoassay (RIA), by detection of antibodies without the use of hazardous radioactive isotopes and the expense of reagents with short shelf lives. Both ELISA and RIA can identify the immunoglobulin class of antibodies studied, such as IgG, IgM, and IgA. In this study, ELISA was used to detect and differentiate IgG and IgM antibodies to herpes simplex virus (HSV) in sera, and to compare serum to cerebrospinal fluid (CSF) ratios of total immunoglobulin antibodies. This was done to assess the possible usefulness of ELISA in the diagnosis of primary and secondary HSV infections, including encephalitis. Received December 30, 90; received revised manuscript and accepted for publication February 26, 9. Address reprint requests to Dr. Rytel: Division of Infectious Diseases, Department of Medicine, The Medical College of Wisconsin; Milwaukee County General Hospital, 700 West Wisconsin Avenue, Milwaukee, Wisconsin Antigen Preparation Division of Infectious Diseases, Department of Medicine, Medical College of Wisconsin, Milwaukee County General Hospital, Milwaukee, Wisconsin Materials and Methods A crude antigen extract of HSV- (Maclntyre) was prepared in human foreskin fibroblast cells (FS-5). After >0% CPE was evident, the flasks were frozen and thawed three times and the media pooled and spun at 00 rpm (60 x g) at 4 C for ten minutes to remove cellular debris. The supernate was carefully decanted off and spun again at 4,000 rpm (30,00 x g) at 4 C overnight to pellet the virus. The supernate was discarded and pellets pooled in phosphate buffered saline (PBS) ph 7.2, breaking up any clumps with a 23 gauge needle and syringe. One milliliter volumes of the extract were pipetted into sterile 35 mm glass petri dishes and inactivated with ultraviolet (UV) light (germicidal lamp (G T5)), by rotating for 0 min at a distance of 3 cm below the light source. Aliquots were quick frozen and stored at -70 C for future use. Control antigen was prepared in the same manner from uninfected FS-5 cells. The TCID 50 of HSV (antigen source) before UV inactivation was 0~'! - 5 /0.05 ml. No live virus was detected after UV irradiation. The optimal dilution of HSV antigen used in this assay was calculated by a checkerboard titration using known positive and negative sera, as determined by CF test. Serum and CSF Specimens Thirty-six serum specimens* on which CF antibody titers were previously determined were examined for total immunoglobulin (Ig), IgG, and IgM antibodies to HSV. Sixteen serum and CSF pairs were assayed for total Ig antibodies only. They were originally * Provided by Milwaukee Health Department Virology Laboratory //000/0467 $00.75 American Society of Clinical Pathologists 467 Downloaded from on 2 January 20

2 46 JORDAN AND RYTEL A.J.C.P. October 9 :92 :4096 :204 ELISA l:,024 HSVAb titer :- FIG.. Relationship of herpes simplex virus complement-fixation antibody titers to antibody titers by ELISA. Results on 36 sera are depicted. * : t Y/ i <l: - r*.7s2 p-<.ooi <l: : :6 :32 : CF HSV Ab titer submitted because a diagnosis of HSV infection was suspected or proven. The source of the 6 simultaneous serum and CSF pairs was our consulting service. The diagnoses on these patients were: ) Latent syphilis-two, 2) coccidioidomycosis meningitis-one, 3) cryptococcal meningitis-one, 4) aseptic meningitisnine, 5) serum hepatitis-one, 6) herpes labialis-one, and 7) HSV-2 meningitis-one. All specimens were stored at -70 C until assayed. IgG and IgM antibody titers were also determined on the 36 sera before and after reduction with 2-mercapto-ethanol (2-ME).t This was done to help establish the specificity of the ELISA for IgM antibodies to HSV. The procedure was previously described in detail by Minor and colleagues. Briefly nine volumes of whole serum were mixed with one volume of 0.25 M 2-ME in PBS ph 7.2 and the mixture incubated two hours at 37 C. Control sera contained one volume of PBS instead of 2-ME, and were incubated in the same manner. Conjugates Horseradish peroxidase conjugated IgG fraction goat antihuman Ig, IgG, and IgMt were used in this t Sigma Chemical Company, St. Louis, Missouri. X Cappel Laboratories, Inc., Ccchranville, Pennsylvania. assay. Checkerboard titrations were done to determine optimal dilutions for each of the conjugates. Conjugates were stored in concentrated form in small aliquots at -20 C and diluted in 0.05 M PBS with 0.05% tween 20 ph 7. immediately before use. Substrate Preparation The substrate, 5-aminosalicylic acid (5-AS) was prepared fresh before each assay. Eighty milligrams 5-AS was dissolved in 00 ml hot (70 F) distilled water and filtered through # Whatman filter paper.h Immediately before use, the ph of the solution was increased to 6.0 with N NaOH. Nine volumes of 5-AS were then combined with one volume 0.05% hydrogen peroxide for the working substrate solution. Microplate Sensitization A : dilution of antigen was made in bicarbonate buffer ph 9.6 and 50 /u. (for total Ig and IgG) or 200 l±\ (for IgM) was added to each well of a standard polystyrene "U" bottom microtiter plate.** Plates were sensitized overnight at 4 C by passive adsorption, Aldrich Chemical Company, Milwaukee, Wisconsin. H W & R Balston Ltd., England. ** Dynatech Laboratories, Inc., Alexandria, Virginia. Downloaded from on 2 January 20

3 Vol. 76 No. 4 BRIEF SCIENTIFIC REPORTS 469 Table. Titers of Various HSV Antibody Classes Determined by ELISA, and the Effect of 2-ME Treatment IgG IgM Total Ig IgG reduced Reduced No Specimen No :204 :204 :204 : :204 :4096 :92 : : :204 : : :204 :40% : :204.40% :204 : : : : :92 :92 :92 : : : : : : :204 : : : :204 :204 :204 : : : : : : IgM. : : : : : : : : : : : : : : : : : :6 :32 <l:6 :6 :6 <I:6 and then washed three times (three minutes per wash) in tween saline (0.05% tween 20) and blotted dry. Similar volumes as described above, of a 0% formalin solution in PBS were added to each well and incubated ten minutes at 25 C. Washings were repeated as described above. Wells were then filled with a % bovine serum albumin fraction V in PBStt and incubated two hours at 37 C. After three additional washings, plates were stored at -70 C until use. ELISA Procedure For total Ig and IgG HSV antibody determinations, 50 fx\ test samples of serial twofold dilutions of sera (:^:,536) and CSF (: * :92) were made in 0.0 M PBS with 0.05% tween 20 ph 7.0 and added to wells previously sensitized with HSV antigen. Plates were incubated one hour at 37 C and then washed three times (3 min each wash) with tween saline. The optimal dilution of conjugate, specific for tt Sigma Chemical Co., St. Louis, Missouri. the antibody being measured, was made in 0.05 M PBS with 0.05% tween 20 ph 7., and 50 fj.\ added to each well. Incubation and washings were repeated as described above. Then 50 fx\ aliquots of freshly prepared enzyme substrate were added to each well and incubated one hour at 25 C, protected from the light. The endpoint was read as the highest dilution showing color development. The ELISA to detect HSV IgM antibody differed in two ways from the above procedure. First, 200 /u. volumes were used instead of 50 fil, and secondly, two hour incubation for serum and conjugate replaced the one hour incubation time. This was done because of the lower concentration of IgM antibodies. Substrate incubation remained the same. Controls included wells which were free of antigen and of antibody. In addition, in certain runs, FS-5 tissue culture extracts were used as antigen controls. This was done to rule out the effect of nonspecific absorbance of reagents employed in the assay. No nonspecific absorbance to control antigen occurred at serum dilutions of : and CSF dilutions of :. Downloaded from on 2 January 20

4 470 JORDAN AND RYTEL A.J.C.P. October 9 Table 2. Comparison of HSV Antibody Titers by ELISA in Sera and Corresponding CSF Pt. No. Initials Serum Titer CSF Titer i.e. S.G. L.H. C.K. A.R. R.H. G.T. D.C. K.L. D.O. D.P. S.Z. H.M. MR. K.T. P.Z CF Test CF antibody to HSV was assayed by the standard microtiter technic for complement fixation. 0 M RF Test The presence of rheumatoid factor (RF) was checked by the Rheumaton test kit*i according to the instructs Performed by Dr. G. Sedmak at the Virology Laboratory, Milwaukee Health Department. Wampole Laboratories, Dist., Division of Carter-Wallace Inc., Cranbury, New Jersey. Patient tions given by the manufacturer. This was done only on specimens which had a titer of IgM antibodies to HSV of >:, to exclude the possibility of false positive results being due to the presence of IgM RF. All specimens tested were negative for RF. Results HSV Antibody Determinations by ELISA HSV by ELISA in Serum and Corresponding CSF The comparison of results obtained by ELISA to those by the micro-cf test, is depicted in Fig.. Repeated measures regression analysis was used to compare ELISA to the micrp-cf test in deriving the slope of the line, and calculating the correlation coefficient. A significant degree of correlation between the two procedures was obtained (r = 0.752; p <.00). In our hands, ELISA was approximately 0-20 fold more sensitive than the CF test. Titers of various HSV antibody classes determined by ELISA, and the effect of treatment with 2-ME are shown in Table. There was a good correlation between total Ig and IgG antibodies (r = 0.0 p <.00). IgM titers were much lower or were undetectable at the screening dilution of :. Only 2 of 36 sera tested had detectable IgM antibody titers. Treatment with 2-ME resulted in no significant fall in titers of IgG antibodies (p >.). In fact 3 of 36 titers were exactly unchanged. Since these titers were determined ICSF fgglljg^l <0wC> on orp^a o^o^or<x<>xox<^o. -ia^j< I I I I I I I I AgAb FIG. 2. Herpes simplex virus antibody titers by ELISA in serum and corresponding CSF. Dilutions Controls Downloaded from on 2 January 20

5 Vol. 76 No. 4 BRIEF SCIENTIFIC REPORTS 47 before and after treatment, this also illustrates the reproducibility of the method. All IgM antibody titers, which were detectable in 2 patients' sera, (i.e., >) showed a drop in titer (p <.00), confirming that ELISA assay can indeed distinguish between IgG and IgM antibodies to HSV. Comparison of HSV antibody titers in 6 paired serum and CSF specimens is shown in Table 2. None of these patients had herpes encephalitis, but were affected with a wide variety of CNS disorders (see Materials and Methods). Of the sixteen patients 5 had HSV antibody titers in their CSF of < :. Patient one had a CSF titer of :6. This particular patient with chronic coccidioidomycosis meningitis, has two ventricular reservoirs for antifungal therapy, and two ventriculo-artial shunts for relief of hydrocephalus. It is likely that the HSV antibody titer in her CSF represents contamination of CSF with serum. The results of simultaneous assays or serum/csf pairs from four patients are depicted in Fig. 2. The figure also provides an example of the ELISA employed in this study, and illustrates the sharpness of the titration end-points. Discussion ELISA had been successfully employed by Gilman and Docherty in detection of antibodies to HSV. 2 These authors, employing slightly different reagents and methodology, found ELISA to be approximately 200 times more sensitive than the micro-cf test. The results of this study are similar. The application of ELISA has been expanded beyond that of Gilman and Docherty, in demonstrating that it can detect both IgG and IgM antibodies to HSV. The importance of detection of IgM antibodies lies in the fact that their specific identification could be useful in establishing whether the HSV infection was primary or secondary." Detection of IgM HSV antibodies could also be of help in the diagnosis of herpes simplex virus encephalitis (HSVE) in cases where the infection was primary. 4 Many authorities feel that HSVE generally represents a primary rather than a secondary infection. 3 This is suggested by the fact that there is a history of recurrent herpes labialis in only 20% of the cases of HSVE." At the present time, the only way to diagnose HSVE with certainty is by brain biopsy. 5 Virus is seldom, if ever, isolated from the CSF. Lerner and associates have attempted to diagnose HSVE by serologic means.,i,? They have shown, for example, that detection of complement-dependent neutralizing antibodies in the CSF, correlated with the presence of HSVE. They have shown further, in cases of HSVE, that a rise of passive hemagglutination (PHA) or immune adherence hemagglutination (IAHA) antibody titers in the CSF, resulted in a serum to CSF titer ratio of <20. This ratio was seen in all eight patients with biopsy proven HSVE, and in only four of 2 control patients. We have developed preliminary methodology employing ELISA to utilize this approach. ELISA has the advantage of not only permitting estimation of a serum to CSF antibody titer ratio, but also of direct identification of IgM antibodies in the CSF. Since IgM antibodies in the CSF are thought to be due to local antibody production, (IgM not being able to penetrate blood-brain barrier), detection of such antibodies in the CSF could be diagnostic of HSVE. We are, however, awaiting the opportunity to confirm this on a biopsy proven case of HSVE. Future studies are planned to compare the results of brain biopsy and ELISA, in patients with suspected HSVE. Note added in proof: We have recently studied two patients, whose results support validity of our assay. One had HSVE and the other was a newborn delivered by Caesarean section from a mother with active herpes genitalis. In the HSVE patient, the serum total Ig antibody titer was :, and the CSF antibody :92, thus, the CSF/S antibody ratio was :, i.e., < :20. The newborn's (who did not develop disseminated HSV infection), serum IgM antibody was :. Acknowledgment. Dr. G. Sedmak (Milwaukee Health Department, Virology Laboratories) provided serum specimens and CF data. References. Draper NR, Smith H: Applied regression analysis, New York, John Wiley and Sons, Inc., 966, pp Gilman SC, Docherty JJ: Detection of antibodies specific for herpes simplex virus in human sera by the enzyme linked immunosorbent assay. J Infect Dis 36:S26-S293, Juel-Jensen BE, Maccallum FO: Herpes simplex, varicella and zoster, Philadelphia, J.B. Lippincott Company, 972, p Kurtz JB: Specific IgG and IgM antibody responses in herpessimplex-virus infections. J Med Microbiol 7:333-34, Lauter CB: Herpes simplex encephalitis. A great clinical challenge. Ann Intern Med 92:696-69, Lerner AM, Bailey EJ, Nolan DC: Complement-requiring neutralizing antibodies in Herpesvirus hominis encephalitis. J Immunol 04:607-65, Levine DP, Lauter CB, Lerner AM. Simultaneous serum and CSF antibodies in herpes simplex virus encephalitis. J Am Med Assoc 240: , 97. Minor TE, Helstrom PB, Nelson DB, et al: Counterimmunoelectrophoresis test for immunoglobulin M antibodies to group B Coxsackie-virus. J Clin Microbiol 9: , Notkins AL, et al: Workshop on the treatment and prevention of herpes simplex virus infections. J Infect Dis 27:7-9, Rowls WE: Herpes simplex virus types one and two and Herpesvirus simiae. Diagnostic procedures for viral, rickettsial, and chlamydial infections, Edited by EH Lenette and MT Schmidt, Fifth edition, Washington, D.C., American Public Health Association, 979, pp Skaug K, Tjotta E: Diagnosis of recent herpes simplex infections. Acta Pathol Microbiol Scand. [B] 2:323-32, Voller A, Bidwell DE, Bartlett A: Enzyme immunoassays in diagnostic medicine. Theory and practice. Bull WHO 53: 55-65, 976 Downloaded from on 2 January 20