Skip to main content
PMC home page
Applied Microbiology logoLink to Applied Microbiology
. 1964 Jul;12(4):343–348. doi: 10.1128/am.12.4.343-348.1964

Technical Considerations in the Preparation of Fluorescent-Antibody Conjugates

Vester J Lewis 1, Wallis L Jones 1, John B Brooks 1, William B Cherry 1
PMCID: PMC1058130  PMID: 14201087

Abstract

A comparison was made of (NH4)2SO4, HCl, ethodin, and ethanol for fractionation of rabbit antiserum prior to conjugation with fluorescein isothiocyanate. Fractionation with the salt was found to be the method of choice from the standpoints of simplicity and recovery of antibody effective in conjugates prepared from the fractions. Effects of pH, temperature, dye-protein ratio, and molarity and type of buffer upon conjugation were studied. These technical factors were adjusted to produce conjugates for Corynebacterium diphtheriae which possessed higher specific titers than did reagents obtained by previously employed techniques.

Full text

PDF
343

Selected References

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

  1. COONS A. H., KAPLAN M. H. Localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody. J Exp Med. 1950 Jan 1;91(1):1–13. doi: 10.1084/jem.91.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. FIFE E. H., Jr, MUSCHEL L. H. Fluorescent-antibody technic for serodiagnosis of Trypanosoma cruzi infection. Proc Soc Exp Biol Med. 1959 Jul;101(3):540–543. doi: 10.3181/00379727-101-25009. [DOI] [PubMed] [Google Scholar]
  3. FROMMHAGEN L. H., MARTINS M. J. A comparison of fluorescein-labeled gamma-globulins purified by Rivanol and DEAE chromatography. J Immunol. 1963 Jan;90:116–120. [PubMed] [Google Scholar]
  4. FROMMHAGEN L. H., SPENDLOVE R. S. The staining properties of human serum proteins conjugated with purified fluorescein isothiocyanate. J Immunol. 1962 Jul;89:124–131. [PubMed] [Google Scholar]
  5. MARSHALL J. D., EVELAND W. C., SMITH C. W. Superiority of fluorescein isothiocyanate (Riggs) for fluorescent-antibody technic with a modification of its application. Proc Soc Exp Biol Med. 1958 Aug-Sep;98(4):898–900. doi: 10.3181/00379727-98-24222. [DOI] [PubMed] [Google Scholar]
  6. MOODY M. D., JONES W. L. IDENTIFICATION OF CORYNEBACTERIUM DIPHTHERIAE WITH FLUORESCENT ANTIBACTERIAL REAGENTS. J Bacteriol. 1963 Aug;86:285–293. doi: 10.1128/jb.86.2.285-293.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Moody M. D., Siegel A. C., Pittman B., Winter C. C. Fluorescent-Antibody Identification of Group A Streptococci from Throat Swabs. Am J Public Health Nations Health. 1963 Jul;53(7):1083–1092. doi: 10.2105/ajph.53.7.1083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. NETER E., BERTRAM L. F., ARBESMAN C. E. Demonstration of Escherichia coli 055 and 0111 antigens by means of hemagglutination test. Proc Soc Exp Biol Med. 1952 Feb;79(2):255–257. doi: 10.3181/00379727-79-19343. [DOI] [PubMed] [Google Scholar]
  9. RIGGS J. L., SEIWALD R. J., BURCKHALTER J. H., DOWNS C. M., METCALF T. G. Isothiocyanate compounds as fluorescent labeling agents for immune serum. Am J Pathol. 1958 Nov-Dec;34(6):1081–1097. [PMC free article] [PubMed] [Google Scholar]
  10. THOMASON B. M., CHERRY W. B., DAVIS B. R., POMALES-LEBRON A. Rapid presumptive identification of entero-pathogenic Escherichia coli in faecal smears by means of fluorescent antibody. 1. Preparation and testing of reagents. Bull World Health Organ. 1961;25:137–152. [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES