Solid-phase adsorption method for removing undesired antibodies from polyclonal antiserum

K. Köves, A. Arimura

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

We have developed a novel and simple method, requiring only a small amount of antigen, for removal of undesired antibodies from antiserum. The method was established using a well-characterized antiserum against rat luteinizing hormone (anti-rLH). Wells of polystyrene tissue culture plates were coated with rat LH (rLH). Anti-rLH diluted 1:3000 was added to rLH-coated wells and shaken to remove LH antibodies. Control anti-rLH was treated in a similar manner in non-rLH-coated wells. Both antisera were tested by immunocytochemistry on rat pituitaries. Antiserum from rLH-coated wells stained no cells, whereas the control serum stained cells that were morphologically typical of LH cells. The effectiveness of this antibody removal was also confirmed in a modified ELISA. In another experiment, anti-rLH and anti-hTSHβ sera were mixed. The final dilution of both antisera was 1:10,000. Anti-rLH was removed by the purification method described. Completeness of antibody removal was confirmed by a double-immunohistochemical staining of rat pituitary in which sections were first stained by the PAP method and then stained with an immunofluorescence procedure after elution of the first antigen-antibody complex. The mixed antiserum incubated in rLH-coated wells did not stain LH cells. There was no co-localization between the LH immunopositivity demonstrated by an anti-rLH serum using immunofluorescence and cells immunostained with the purified antiserum using the PAP method. As indicated in ELISA, the titer of the TSHβ antiserum was not decreased compared to that of the untreated, mixed control antiserum, and the LH antibodies were eliminated by the treatment. This new purification method has four distinct advantages: (a) antiserum is not treated chemically; (b) it requires only a small amount of antigen compared with the amount required for affinity chromatography; (c) neither the undesired antigen-antibody complex(es) nor an excess amount of antigen is present in the purified antiserum; and (d) removal of undesired antibodies can be monitored by ELISA.

Original languageEnglish
Pages (from-to)903-908
Number of pages6
JournalJournal of Histochemistry and Cytochemistry
Volume37
Issue number6
Publication statusPublished - 1989

Fingerprint

Adsorption
Immune Sera
Antibodies
Luteinizing Hormone
Gonadotrophs
Enzyme-Linked Immunosorbent Assay
Antigen-Antibody Complex
Antigens
Fluorescent Antibody Technique
Serum
Polystyrenes
Affinity Chromatography
Coloring Agents
Immunohistochemistry
Staining and Labeling

ASJC Scopus subject areas

  • Cell Biology
  • Anatomy

Cite this

Solid-phase adsorption method for removing undesired antibodies from polyclonal antiserum. / Köves, K.; Arimura, A.

In: Journal of Histochemistry and Cytochemistry, Vol. 37, No. 6, 1989, p. 903-908.

Research output: Contribution to journalArticle

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