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. 1970 Jun;18(6):875–881.

Conjugation of fluorescein isothiocyanate to antibodies

II. A reproducible method

T H The, T E W Feltkamp
PMCID: PMC1455722  PMID: 4913804

Abstract

A reproducible method for conjugation of fluorescein isothiocyanate to antibodies, based on previous experiments, is described in detail. The negative electrical charge of antibodies was found to increase by conjugation. This negative charge may cause the non-specific fluorescence of acidophilic tissue components if overlabelled antibodies are used. Even non-specific nuclear fluorescence was observed when the substrate was previously washed in phosphate buffer. This was probably caused by acid extraction. Underlabelled antibodies showed a weak specific fluorescence. Antibodies with molecular fluorescein/protein ratios between 1 and 4 or `optical density ratios' (E4951:E2801) between 0.3 and 0.95 were found to be optimal. However, even antibodies conjugated to an optimal degree may demonstrate non-specific fluorescence if used in high concentrations. Restricted preservation testing of reconstituted lyophilized conjugates revealed a negative effect of exposure to daylight at room temperature.

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Selected References

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

  1. Beutner E. H., Holborow E. J., Johnson G. D. Quantitative studies of immunofluorescent staining. I. Analyses of mixed immunofluorescence. Immunology. 1967 Mar;12(3):327–337. [PMC free article] [PubMed] [Google Scholar]
  2. Brighton W. D., Taylor C. E., Tomlinson A. H., Wilkinson A. E. A provisional specification for fluorescein-labelled antibody against human globulin (IgG). Mon Bull Minist Health Public Health Lab Serv. 1967 Sep;26:179–181. [PubMed] [Google Scholar]
  3. GOLDSTEIN G., SLIZYS I. S., CHASE M. W. Studies on fluorescent antibody staining. I. Non-specific fluorescence with fluorescein-coupled sheep anti-rabbit globulins. J Exp Med. 1961 Jul 1;114:89–110. doi: 10.1084/jem.114.1.89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. HUGHES P. E., LOUIS C. J. Differential staining of normal and neoplastic tissue with fluorescein-egg albumen. Arch Pathol. 1959 Nov;68:508–512. [PubMed] [Google Scholar]
  5. Hebert G. A., Pittman B., Cherry W. B. Factors affecting the degree of nonspecific staining given by fluorescein isothiocyanate labelled globulins. J Immunol. 1967 Jun;98(6):1204–1212. [PubMed] [Google Scholar]
  6. LOUIS C. J. Fluorescein-globulin affinities of the Shope papilloma. Br J Cancer. 1960 Jun;14:216–223. doi: 10.1038/bjc.1960.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. LOUIS C. J. The significance of the cell type in the fluorescein-globulin staining of tissues. Br J Cancer. 1958 Dec;12(4):537–546. doi: 10.1038/bjc.1958.62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. MCDEVITT H. O., PETERS J. H., POLLARD L. W., HARTER J. G., COONS A. H. PURIFICATION AND ANALYSIS OF FLUORESCEIN-LABELED ANTISERA BY COLUMN CHROMATOGRAPHY. J Immunol. 1963 Apr;90:634–642. [PubMed] [Google Scholar]
  9. Pittman B., Herbert G. A., Cherry W. B., Taylor G. C. The quantitation of nonspecific staining as a guide for improvement of fluorescent antibody conjugates. J Immunol. 1967 Jun;98(6):1196–1203. [PubMed] [Google Scholar]
  10. Strauss A. J., Kemp P. G., Jr Serum autoantibodies in myasthenia gravis and thymoma: selective affinity for I-bands of striated muscle as a guide to identification of antigen(s). J Immunol. 1967 Nov;99(5):945–953. [PubMed] [Google Scholar]
  11. Wood B. T., Thompson S. H., Goldstein G. Fluorescent antibody staining. 3. Preparation of fluorescein-isothiocyanate-labeled antibodies. J Immunol. 1965 Aug;95(2):225–229. [PubMed] [Google Scholar]
  12. ten Veen J. H., Feltkamp T. E. Formalinized chicken red cell nuclei as a simple antigen for standardized antinuclear factor determination. Clin Exp Immunol. 1969 Dec;5(6):673–678. [PMC free article] [PubMed] [Google Scholar]
  13. von MAYERSBACH, SCHUBERT G. [Immunohistological methods. III. The unspecific reaction between labelled serum and tissues in the immunohistological technic]. Acta Histochem. 1960 Oct 25;10:44–82. [PubMed] [Google Scholar]

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