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Automated nephelometric immunoassay II. Its application to the determination of hapten

Authors
Journal
Journal of Immunological Methods
0022-1759
Publisher
Elsevier
Publication Date
Volume
5
Issue
3
Identifiers
DOI: 10.1016/0022-1759(74)90114-8
Disciplines
  • Medicine

Abstract

Abstract The principle of a novel method for the immunological assay of haptens, based on nephelometry, has been tested, using ϵ-dinitrophenyl-lysine and progesterone as model substances. It is proposed to call this method the nephelometric inhibition immunoassay (NINIA). In an automated continous flow system where aliquots of antigen are discontinuosly injected into a constant stream of diluted antiserum, the nephelometric effect of the antigen-antibody complexes formed markedly increases with the concentration of antibody relative to antigen, as well as with the affinity of the antibodies. Haptens inhibiting this reaction can be titrated by measuring the resulting decrease of the nephelometric peaks. In practice, the precipitin reaction is carried out with a ‘developer antigen’, BGG, HGG or ferritin carrying numerous haptenic groups, and antiserum preabsorbed with graded, known, amounts of hapten, or with the unknown sample. Means of increasing the sensitivity include the use of (i) high affinity antibodies, (ii) large, heavily substituted, developer antigens, (iii) 4% polyethylene glycol as the diluent of the antiserum, and (iv) adjusting the proportions of the reactants to near-equivalence. The threshold of the method as applied to progesterone is about 10 ng.

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