Journal of’ Virologicul Methods, 40 (1992) 175-I 82 c) 1992 Elsevier Science Publishers B.V. / All rights VIRMET

175 reserved

/ 0166-0934/92/$05.00

01406

Monoclonal

anti-idiotypes for the rapid detection of human cytomegalovirus

E.S. Tackaberry”,‘,

J. Hamel”,

Y. Larosea,

R. Kuhl” and B.R. Brodeu@’

“Nationul Lahoratory,fiw Immunology, Laboratory Cenfrr for Disease Control, Ofta,*.a, Ontario (Cunadu), hDepartment qf‘ Microbiology and Immunolog)~, University of Ottavva, Ottrnl.u, Ontario (Canadu) and ’ ADI Diagnostics Inc., Rexdale, Onturio (Canadu) (Accepted

12 June

1992)

Summary A novel enzyme-linked immunosorbent assay (ELISA) was developed for human cytomegalovirus (HCMV) utilizing a monoclonal anti-idiotype specific for CMVBI, an antibody to HCMV. Samples of HCMV were measured by their inhibition of the binding of CMVBI to anti-idiotype. The ELISA detected HCMV in a concentration-dependent manner from 20 to 0.6 x 10’ PFU/ml, with 50% inhibition at approx. 3 x lo3 PFU/ml. These data demonstrate the potential of anti-idiotype antibodies as the basis of simple and rapid diagnostic tests for infectious agents. Idiotype; Anti-idiotype;

Human cytomegalovirus;

Immunoassay

Introduction Human cytomegalovirus (HCMV) is a major cause of intrauterine viral infection resulting in CNS damage to newborns, and may also cause lifethreatening disease in immunocompromised patients. Although various tests for identifying active HCMV infection are available, none is ideal. For example, it is widely recognized that the most reliable diagnosis is achieved by direct viral culture from clinical specimens, but traditional tube culture may take several weeks. This has led to the recent development of a variety of other Corres~o~~dencc (0: B.R. Brodeur, National Control. Health Protection Building (#7), Canada.

Laboratory for Immunology, Rm. 181. Tunney’s Pasture,

Laboratory Centre for Disease Ottawa. Ontario, KIA OL2.

tests including shell vial culture, solid-phase enzyme immunoassays, and PCR (reviewed in Ho, 1991). While these are much faster than traditional culture, each assay is limited by certain disadvantages of sensitivity and/or specificity (Ho, 1991). Moreover, at present, not all of these methods can easily be incorporated into a routine laboratory. Thus, there remains significant interest in providing an improved test for the rapid diagnosis of active HCMV infection. Anti-idiotype (anti-id) antibodies are made against the unique set of determinants (idiotopes) present on the hypervariable region of an immunoglobulin molecule. As proposed by Jerne’s network theory (Jerne, 1974). and now supported by evidence from many laboratories, including our own (Brodeur et al., 1991; Hamel and Brodeur, 1990; Hiernaux, 198X; Kohler et al., 1989; Nisonoff, 1991) some of these anti-id antibodies may functionally mimic the conformation of the original antigen, representing its ‘internal image’. and will thus be able to substitute for the antigen in its binding to antibody. Despite considerable interest in anti-id. there have been. to our knowledge. no reports in the literature of anti-id antibodies being exploited for the improved diagnosis of infectious diseases. In the current study we postulated that anti-id monoclonal antibodies (mAbs) could be engineered to an antibody to HCMV, and could then be used as the basis of a unique immunoassay foi rapid viral detection. We reasoned that if viral antigen was first allowed to bind to anti-HCMV antibody, then subsequent interaction between the anti-HCMV antibody and its complementary anti-id would be inhibited. As a first step towards achieving this goal we describe herein the generation of an anti-id mAb and its subsequent use in the development of an enzyme-linked immunosorbent assay (ELISA) for measuring a laboratory strain of HCMV.

Materials and Methods Antihocf~~ to HCMV

CMVBl is an anti-HCMV mouse mAb (IgG2a, kappa) made previously in our laboratory (Rossier et al., 1987). It is specific for gB, an immunodominant glycoprotein complex of 55/130 kDa, expressed on the surface of HCMV, and is highly neutralizing for HCMV infectivity in vitro (ascitic fluid titre > 1.50000 by standard plaque reduction assay). CMVBl has no reactivity with other human herpesviruses (HSV-1, HSV-2, EBV or VZV), but recognized 24 of 24 clinical isolates obtained from patients in different regions of Canada over a five-year period. CMVBl was purified by affinity chromatography over protein A Sepharose (Pharmacia, Montreal, Quebec) as previously described (Hamel and Brodeur, 1990). The F(ab’)7 fragments were generated by digestion with pepsin (Parham, 1983) and subsequently isolated by passage over protein A Sepharose. For use in the anti-id ELISA, CMVBl was labelled with biotinX-NHS (Calbiochem, San Diego, CA) using a biotinprotein molar ratio of

177

approx.

1O:l (Harlow

and Lane, 1988).

Anti-id mAb-8C9 The protocol used in our laboratory for producing anti-id mAbs has been reported elsewhere (Brodeur et al., 1991; Hamel and Brodeur, 1990). Briefly, syngeneic (Balb/c) mice were immunized with purified CMVBl conjugated to the carrier protein keyhole limpet hemocyanin (KLH, Sigma, St. Louis, MO), and the immune splenocytes were used to generate hybridomas (Brodeur et al., 1985). Supernatants of clones were screened for anti-id reactivity by an ELISA which measured their binding to immobilized F(ab’)z of CMVBl, as detected by enzyme-conjugated anti-mouse Fc-specific IgG (Cappel/Organon Teknika, Durham, NC and Jackson ImmunoResearch Laboratories, West Grove, PA). An unrelated control anti-id (mAb AHb-23) has been described elsewhere (Hamel and Brodeur, 1990; Brodeur et al., 1992). The anti-id mAb-8C9 selected for use in the current study is an IgG2a, kappa. It was purified from ascitic fluid by ammonium sulphate precipitation and affinity chromatography over protein A Sepharose, and stored at -20°C until use. For experiments in which the ability of mAb-8C9 to block interaction between CMVBl and HCMV was tested, the anti-id was pre-incubated with CMVBl for 90 min at room temperature, whereupon residual CMVBl reactivity was determined either for binding to HCMV-infected cells by indirect immunofluorescent assay (IFA), or for viral neutralizing activity by plaque reduction assay. Both of these techniques have been described previously (Larose et al., 1991). Where specified, a pool of five different anti-id mAbs was used. The pool consisted of mAb-8C9 plus four others directed against CMVBl. Virus The prototype HCMV strain AD1 69 was obtained from the Laboratory Centre for Disease Control, Ottawa, Ontario, and was propagated in human foreskin Iibroblasts (Larose et al., 199 1). Supernatant was harvested from cells exhibiting extensive cytopathic effect, claritied of cellular debris by centrifugation at 1000 x g for 10 min, and stored at -70°C until use. The concentration of infectious virus in our stock was 2.2 x lo5 PFU/ml, as determined by plaque titration assay (Larose et al., 1991). Prior to use in the anti-id ELISA the virus was inactivated by heating at 95°C for 10 min. This step was included in order to minimize handling of infectious virus, after a comparison of the ELISA with inactivated vs. non-inactivated HCMV revealed that the assay was unaffected by treating the virus in this manner. Since HCMV is shed into urine during active clinical infection, virus samples for our experiments were prepared by spiking 100 ~1 HCMV (or medium, as a control) into 1 ml normal urine. Preliminary investigations had shown that normal urine itself did not affect the assay. Routinely, 4.4 ml of sample were used for each test. After removal of cellular debris, the samples were centrifuged for 30 min at 10000 x g

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(Eppendorf model ,~l of 0.5% (w/v) (PBS) containing antibody mixture below.

5415C) and the pelleted virus immediately resuspended in 70 b ovine serum albumin (BSA) in phosphate-buffered saline biotinylated CMVBI at 12.5 ng/ml. This virus/biotinylated was then used in the anti-id ELISA for HCMV, as described

Anti-id ELISA for HCMV Microtitre plates (Linbro/Titertek, ICN Biomedicals, Mississauga, Ontario) were coated with 0.5 ,ug/well purified anti-id in PBS (pH 7.2) overnight at room temperature. After washing with PBS/Tween-20 (0.02% v/v) the plates were blocked for 30 min with 1% (w/v) BSA/PBS. Fifty ,~l of virus/biotinylated antibody mixture (or control/biotinylated antibody mixture) which had been incubated together for 90 min at 37°C was then added to the blocked wells. After 20 min at room temperature the wells were washed, followed by addition of alkaline phosphatase-conjugated avidin (Cappel/Organon Teknika, Durham, NC) in 3% (w/v) BSAjPBS for 60 min at 37°C. Phosphatase substrate (pnitrophenyl phosphate, Sigma) in 10% (v/v) diethanolamine, (pH 9.6) was added and absorbance at 410 nm measured spectrophotometrically after 60 min (Dynatech MR7000 plate reader). All assays were done in triplicate. Virus present in the sample was quantitated by its inhibition of the binding of biotinylated CMVBl to immobilized anti-id, relative to the control, using the formula: [(Ahlo control sample - A4i0 virus sample)/ild10 control sample] x 100%.

100

0.01 : : : : : : : : : 1

4

16

64

256



1024

Reciprocal of Anti-id Dilution

’ Concentration of CMVBl (pg/mL)

Fig. I. Primary screening to identify anti-id specific for CMVBI. (A) ELISA measuring anti-id mAb in hybridoma culture supernatant by its binding to the immobilized F(ab’)z fragments of purified CMVBI; (+) mAb-8C9, (0) AHb-23, an unrelated control anti-id mAb. (B) Inhibition of the binding in (A) by preincubation of mAb-SC9 with different concentrations of purified soluble CMVBI.

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Results and Discussion Primary screening experiments for anti-id mAbs were designed to establish whether antibodies specific for CMVBl had been generated. As shown in Fig. 1, the supernatant of mAb-8C9 exhibited a high degree of binding to the F(ab’)* fragments of CMVBl up to a dilution of 1:128, with further dilution resulting in a dose-responsive decrease of binding (Fig. 1A). The isotype-matched control anti-id mAb, AHb-23, showed no binding to CMVBl (Fig. 1A). Moreover, when wells were coated with the F(ab’)* fragments of an unrelated IgG2a mAb, no binding of mAb-8C9 was ever observed (data not shown). The results presented in Fig. 1B show that purified soluble CMVBl at concentrations of 10 pg/ml (or more) was able to inhibit the interaction between mAb-8C9 and CMVBl by over 90%. Three other anti-HCMV mAbs with different epitope specificities did not inhibit the binding of mAb-8C9 to CMVBl; and CMVBl had no effect on the interaction of control id and anti-id in a parallel system (data not shown). Taken together, these data clearly identified mAb-8C9 as an anti-id that was specifically directed against CMVB 1, and suggested that a high binding affinity existed between the two mAbs. The next experiments examined whether mAb-SC9 could block the interaction between CMVBl and viral antigen. First, we showed that preincubation of mAb-8C9 with the F(ab’)* fragments of purified CMVBl completely blocked binding of CMVBl to HCMV-infected cells, as detected by IFA. Second, when mAb-8C9 was pre-incubated with CMVBl, subsequent viral neutralizing activity of CMVBl was inhibited by lOO%, as detected by plaque reduction assay. Medium and an unrelated anti-id mAb had little or no effect when used as controls in either assay system. The clear ability of mAb8C9 to block the anti-HCMV reactivity of CMVBl suggested that this anti-id might be directed against paratope-related sites on CMVBI (i.e., those sites involved with its binding to antigen), and, therefore, might be appropriate for development of the anti-id ELISA. Initial anti-id ELISA experiments compared coating microtitre wells with either mAb-8C9 or a pool of live different anti-id mAbs, to see if one or the other would prove advantageous. The data in Table 1 show that there was over 90% inhibition of CMVBl binding to either mAb-8C9 or the pooled anti-id, when HCMV at 20 x lo3 PFU/ml was included in the assay; and significantly TABLE 1 Percent inhibition Virus concentration (PFU/ml x 103) 20

5

of CMVBI binding to anti-id mAbs by HCMV Anti-id mAbs coating microtitre

wells

mAb-8C9

Pool”

91 f 1 48 + 5

92 + 1 53 k 6

“A pool of five different anti-id mAbs (including mAb-8C9), each directed against CMVBI. n=3. mean + SD.

._

20 -

Virus Concentration

(PFWmL x 103)

Fig. 2. Dose-response curve for measuring HCMV. Virus samples were diluted 2-fold and tested by the anti-id ELISA. Measurement of viral antigen was based on its ability to inhibit the binding of anti-id mAb8C9 to CMVBI. relative to a control. (np 3, mean f SD.)

less inhibition (48% and 53%) when HCMV at 5 x 10” PFU/ml was used. No binding of CMVBI to an unrelated control anti-id mAb was observed. From these data we concluded that: (i) the ELISA could, in fact, detect HCMV, and; (ii) the sens’t’ 1 1~1 ‘ty of the assay was not improved by using the pooled anti-id, compared to mAb-8C9 alone. As a result, only mAb-8C9 was used for coating wells in subsequent assays. We next investigated the concentration of virus for which a linear doseresponse curve could be generated, and examined the sensitivity of the assay. Results of representative experiments are illustrated in Fig. 2. When 2-fold dilutions of the HCMV samples were made, there was a concentrationdependent reduction in the amount of viral antigen measured. The assay was linear over the range tested, 20 to 0.6 x lo3 PFU/ml. Moreover, if 50°/1, inhibition is defined as positive detection, then the assay sensitivity is approximately 3 x lo3 PFU,!ml. These viral concentrations are relevant to those found in the urine of patients with active infection, which range from about lo* to 10h PFUiml (Feldman, 1968). Given these encouraging results, we are now pursuing evaluation of the anti-id ELISA with clinical specimens. In summary, we have generated an anti-id mAb cSC9) against CMVBl (an anti-HCMV mAb), that specifically inhibits the binding between CMVBl and viral antigen. This anti-id mAb was used to develop a novel ELISA that effectively detects HCMV (strain AD169) in a concentration-dependent manner to a sensitivity of approximately 3 x IO’ PFUiml. Our data not only demonstrate the promise of this assay for measuring HCMV, but also reveal the potential of anti-idiotype antibodies as a means of developing simple, rapid diagnostic tests for many infectious agents.

181

Acknowledgements E.T. was supported Council of Canada.

by a Studentship

Award

from the Medical

Research

References Brodeur, B.R., Larose, Y., Tsang, P., Hamel, J., Ashton, F. and Ryan, A. (1985) Protection against infection with Neisseriu meningitidis group B serotype 2b by passive immunization with serotypespecific monoclonal antibody. Infect. Immun. 50, 510-516. Brodeur, B.R., Faucher, S., O’Shaughnessy, M.V. and Hamel, J. (1991) Monoclonal idiotypic and anti-idiotypic antibodies to human immunodeficiency virus type I envelope glycoprotein. J. Gen. Virol. 72, 51-58. Brodeur, B.R., Hamel, J., Martin, D. and Chong, P. (1992) Synthetic peptides and anti-idiotypic antibodies as immunogens for the induction of antibody response to H. irfi’uenzae serotype b. J. Infect. Dis. 165, suppl. I. Feldman, R.A. (1968) Cytomegaloviruses in stored urine specimens. J. Pediatrics 73, 61 l-614. Hamel, J. and Brodeur, B.R. (1990) Induction of an immune response to the porin of H. irzjluenzae type b by monoclonal anti-idiotypic antibodies. Microb. Pathogenesis 9, 81-93. Harlow, E. and Lane, D. (Eds), (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 340-341. Hiernaux, J.R. (1988) Idiotypic vaccines and infectious diseases. Infect. Immun. 56, 140771413. Ho, M. (1991) Virological diagnosis and infections in cells and tissues. In: M. Ho (Ed), Cytomegalovirus: Biology and Infection. 2nd Edn. Plenum Medical Book CO., New York, pp. 75599. Jerne, N.K. (1974) Towards a network theory of the immune system. Ann. Immunol. 125~. 373.~389. Kohler, H., Kaveri, S., Kieber-Emmons, T., Morrow, W.J.W., Muller, S. and Raychaudhuri, S. (1989) Idiotypic networks and nature of molecular mimicry: an overview. In J.V. Abelson and M.I. Simon (Eds.), Methods Enzymolagy: antibodies, antigens, and molecular mimicry, Vol. 178, Academic Press, San Diego, pp. 3335. Larose, Y., Tackaberry, ES. and Brodeur, B.R. (1991) Human monoclonal antibodies to cytomegalovirus recognize viral epitopes on the surface of virus-infected cells. Hum. Antibody Hybridomas 2, 65-73. Nisonoff, A. (1991) Idiotypes: concepts and applications. J. Immunol. 147, 2429-2438. Parham, P. (1983) On the fragmentation of monoclonal IgGl, IgG2a and IgG2b from BALB/c mice. J. Immunol. 131, 289552902. Rossier, E., Dimock, K., Taylor, D., Larose, Y., Phipps, P.H. and Brodeur, B.R. (1987) Sensitivity and specificity of enzyme immunotiltration and DNA hybridization for the detection of HCMVinfected cells. J. Virol. Methods 15. 1099120.

Monoclonal anti-idiotypes for the rapid detection of human cytomegalovirus.

A novel enzyme-linked immunosorbent assay (ELISA) was developed for human cytomegalovirus (HCMV) utilizing a monoclonal anti-idiotype specific for CMV...
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