Skip to main content
PMC home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1983 Aug;18(2):331–343. doi: 10.1128/jcm.18.2.331-343.1983

Production and characterization of monoclonal antibodies specific for a glycosylated polypeptide of human cytomegalovirus.

K S Kim, V J Sapienza, C M Chen, K Wisniewski
PMCID: PMC270801  PMID: 6194174

Abstract

Nine hybrid cell lines producing antibodies specific for cytomegalovirus (CMV) antigen were obtained after fusion of P3/X63-Ag8 myeloma cells with spleen cells from BALB/c mice immunized with CMV complement-fixing antigen. By the immunoblot technique, five of nine antibodies (4D11, 7B4, 7D2, 8E3, and 8E10) were identified as being reactive to a CMV glycosylated polypeptide with molecular weight of 66,000 (GP66). Four other antibodies (1B8, 8E9, 4D2, and 7E2) appeared to be reactive with CMV antigen(s) only if the antigen was not denatured by sodium dodecyl sulfate. These remain unassigned until further studies are done. With the enzyme-linked immunosorbent assay (ELISA), competitive bindings were performed with a constant amount of horseradish peroxidase-conjugated antibody and various concentrations of unconjugated homologous and heterologous antibodies on CMV antigen-coated ELISA wells, and the antigenic determinant specific for each antibody was determined. The nine antibodies could be classified into six different groups, each group reacting with a different epitope or a different region with two or more antigenic determinants which are so close to each other that they cause binding inhibition. They are groups A (4D11), B (7B4, 8E10), C (7D2), D (4D2, 7E2, 8E9), E (8E3), and F (1B8). The extent of competition among antibodies within each group was the same. By using the two antibodies that reacted with different epitopes on GP66, a double-antibody sandwich ELISA method was developed. The method was sensitive enough to detect as little as 50% of the antigen present in one infected cell or 0.000245 U of CMV complement-fixing antigen per test well. Other strains of CMV (David, Kerr, Espilat, C-87, and five clinical isolates) gave positive results, whereas herpes simplex virus types 1 and 2, varicella-zoster virus and Epstein-Barr virus nuclear antigen preparations did not. By the indirect immunofluorescence assay, antibodies 4D11 and 8E3 were able to detect GP66 in the nucleus of CMV-infected F-5000 human embryonic fibroblasts as early as 2 h postinfection and were superior in this respect to the remaining seven antibodies tested. By the double-antibody sandwich ELISA, the presence of GP66 in CMV-infected cells was detected as early as 2 h postinfection.

Full text

PDF
331

Images in this article

336Image
on p.336
337Image
on p.337

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alford C. A., Jr, Stagno S., Reynolds D. W. Diagnosis of chronic perinatal infections. Am J Dis Child. 1975 Apr;129(4):455–463. doi: 10.1001/archpedi.1975.02120410043014. [DOI] [PubMed] [Google Scholar]
  2. Betts R. F., Freeman R. B., Douglas R. G., Jr, Talley T. E. Clinical manifestations of renal allograft derived primary cytomegalovirus infection. Am J Dis Child. 1977 Jul;131(7):759–763. doi: 10.1001/archpedi.1977.02120200041010. [DOI] [PubMed] [Google Scholar]
  3. Breschkin A. M., Ahern J., White D. O. Antigenic determinants of influenza virus hemagglutinin. VIII. Topography of the antigenic regions of influenza virus hemagglutinin determined by competitive radioimmunoassay with monoclonal antibodies. Virology. 1981 Aug;113(1):130–140. doi: 10.1016/0042-6822(81)90142-2. [DOI] [PubMed] [Google Scholar]
  4. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  5. Drew W. L., Conant M. A., Miner R. C., Huang E. S., Ziegler J. L., Groundwater J. R., Gullett J. H., Volberding P., Abrams D. I., Mintz L. Cytomegalovirus and Kaposi's sarcoma in young homosexual men. Lancet. 1982 Jul 17;2(8290):125–127. doi: 10.1016/s0140-6736(82)91092-3. [DOI] [PubMed] [Google Scholar]
  6. Drew W. L., Mintz L., Miner R. C., Sands M., Ketterer B. Prevalence of cytomegalovirus infection in homosexual men. J Infect Dis. 1981 Feb;143(2):188–192. doi: 10.1093/infdis/143.2.188. [DOI] [PubMed] [Google Scholar]
  7. Goldstein L. C., McDougall J., Hackman R., Meyers J. D., Thomas E. D., Nowinski R. C. Monoclonal antibodies to cytomegalovirus: rapid identification of clinical isolates and preliminary use in diagnosis of cytomegalovirus pneumonia. Infect Immun. 1982 Oct;38(1):273–281. doi: 10.1128/iai.38.1.273-281.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Greenberg H., McAuliffe V., Valdesuso J., Wyatt R., Flores J., Kalica A., Hoshino Y., Singh N. Serological analysis of the subgroup protein of rotavirus, using monoclonal antibodies. Infect Immun. 1983 Jan;39(1):91–99. doi: 10.1128/iai.39.1.91-99.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Griffiths P. D., Stagno S., Pass R. F., Smith R. J., Alford C. A., Jr Congenital cytomegalovirus infection: diagnostic and prognostic significance of the detection of specific immunoglobulin M antibodies in cord serum. Pediatrics. 1982 May;69(5):544–549. [PubMed] [Google Scholar]
  10. Hanshaw J. B. Congenital cytomegalovirus infection: a fifteen year perspective. J Infect Dis. 1971 May;123(5):555–561. doi: 10.1093/infdis/123.5.555. [DOI] [PubMed] [Google Scholar]
  11. Hanshaw J. B., Scheiner A. P., Moxley A. W., Gaev L., Abel V., Scheiner B. School failure and deafness after "silent" congenital cytomegalovirus infection. N Engl J Med. 1976 Aug 26;295(9):468–470. doi: 10.1056/NEJM197608262950902. [DOI] [PubMed] [Google Scholar]
  12. Kim K. S., Moon H. M., Sapienza V. J., Carp R. I. Complement-fixing antigen of human cytomegaloviruses. J Infect Dis. 1977 Feb;135(2):281–288. doi: 10.1093/infdis/135.2.281. [DOI] [PubMed] [Google Scholar]
  13. Kim K. S., Sapienza V. J., Carp R. I., Moon H. M. Analysis of structural polypeptides of purified human cytomegalovirus. J Virol. 1976 Dec;20(3):604–611. doi: 10.1128/jvi.20.3.604-611.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Melish M. E., Hanshaw J. B. Congenital cytomegalovirus infection. Developmental progress of infants detected by routine screening. Am J Dis Child. 1973 Aug;126(2):190–194. doi: 10.1001/archpedi.1973.02110190168011. [DOI] [PubMed] [Google Scholar]
  15. Pereira L., Hoffman M., Cremer N. Electrophoretic analysis of polypeptides immune precipitated from cytomegalovirus-infected cell extracts by human sera. Infect Immun. 1982 Jun;36(3):933–942. doi: 10.1128/iai.36.3.933-942.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Pereira L., Hoffman M., Gallo D., Cremer N. Monoclonal antibodies to human cytomegalovirus: three surface membrane proteins with unique immunological and electrophoretic properties specify cross-reactive determinants. Infect Immun. 1982 Jun;36(3):924–932. doi: 10.1128/iai.36.3.924-932.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Pereira L., Stagno S., Hoffman M., Volanakis J. E. Cytomegalovirus-infected cell polypeptides immune-precipitated by sera from children with congenital and perinatal infections. Infect Immun. 1983 Jan;39(1):100–108. doi: 10.1128/iai.39.1.100-108.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Prince A. M., Szmuness W., Millian S. J., David D. S. A serologic study of cytomegalovirus infections associated with blood transfusions. N Engl J Med. 1971 May 20;284(20):1125–1131. doi: 10.1056/NEJM197105202842004. [DOI] [PubMed] [Google Scholar]
  19. Reynolds D. W., Stagno S., Stubbs K. G., Dahle A. J., Livingston M. M., Saxon S. S., Alford C. A. Inapparent congenital cytomegalovirus infection with elevated cord IgM levels. Casual relation with auditory and mental deficiency. N Engl J Med. 1974 Feb 7;290(6):291–296. doi: 10.1056/NEJM197402072900601. [DOI] [PubMed] [Google Scholar]
  20. Sarov I., Abady I. The morphogenesis of human cytomegalovirus. Isolation and polypeptide characterization of cytomegalovirions and dense bodies. Virology. 1975 Aug;66(2):464–473. doi: 10.1016/0042-6822(75)90218-4. [DOI] [PubMed] [Google Scholar]
  21. Stagno S., Reynolds D. W., Amos C. S., Dahle A. J., McCollister F. P., Mohindra I., Ermocilla R., Alford C. A. Auditory and visual defects resulting from symptomatic and subclinical congenital cytomegaloviral and toxoplasma infections. Pediatrics. 1977 May;59(5):669–678. [PubMed] [Google Scholar]
  22. Starr J. G., Bart R. D., Jr, Gold E. Inapparent congenital cytomegalovirus infection. Clinical and epidemiologic characteristics in early infancy. N Engl J Med. 1970 May 7;282(19):1075–1078. doi: 10.1056/NEJM197005072821905. [DOI] [PubMed] [Google Scholar]
  23. Stern H., Tucker S. M. Prospective study of cytomegalovirus infection in pregnancy. Br Med J. 1973 May 5;2(5861):268–270. doi: 10.1136/bmj.2.5861.268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Stinski M. F. Sequence of protein synthesis in cells infected by human cytomegalovirus: early and late virus-induced polypeptides. J Virol. 1978 Jun;26(3):686–701. doi: 10.1128/jvi.26.3.686-701.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Togashi T., Orvell C., Vartdal F., Norrby E. Production of antibodies against measles virions by use of the mouse hybridoma technique. Arch Virol. 1981;67(2):149–157. doi: 10.1007/BF01318598. [DOI] [PubMed] [Google Scholar]
  26. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Waner J. L. Partial characterization of a soluble antigen preparation from cells infected with human cytomegalovirus: properties of antisera prepared to the antigen. J Immunol. 1975 May;114(5):1454–1457. [PubMed] [Google Scholar]
  28. Waner J. L., Weller T. H., Kevy S. V. Patterns of cytomegaloviral complement-fixing antibody activity: a longitudinal study of blood donors. J Infect Dis. 1973 May;127(5):538–543. doi: 10.1093/infdis/127.5.538. [DOI] [PubMed] [Google Scholar]
  29. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  30. Wentworth B. B., French L. Plaque assay of cytomegalovirus strains of human origin. Proc Soc Exp Biol Med. 1970 Nov;135(2):253–258. doi: 10.3181/00379727-135-35031. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES