Immobilization of small molecules on solid matrices: A novel approach to enzyme-linked immunosorbent assay screening for saxitoxin and evaluation of anti-saxitoxin antibodies

• Published in: Toxicon (Oxford) 1996-10, Vol.34 (10), p.1127-1140
• Main Authors: Kralovec, Jaroslav A., Laycock, Maurice V., Richards, Robert, Usleber, Ewald
• Format: Article
• Language: English
• Subjects: alexandrium tamarense; antibodies; Antibodies - analysis; Antibodies - immunology; Biological and medical sciences; bovine serum albumin; dinophyta; enzyme-linked immunosorbent assay; Enzyme-Linked Immunosorbent Assay - methods; Hydrogen-Ion Concentration; immobilization; laboratory equipment; Marine; maxisorp microtitre plates; Medical sciences; neurotoxins; Plant poisons toxicology; Saxitoxin - analysis; Saxitoxin - immunology; Toxicology
• ISSN: 0041-0101; 1879-3150
• DOI: 10.1016/0041-0101(96)00063-3

A novel enzyme-linked immunosorbent assay (ELISA) technology was developed for detecting saxitoxin or evaluation of anti-saxitoxin antibodies, which is based on non-covalent immobilization of ‘free’ saxitoxin to Maxisorp microtitre plates. The effect of pH on immobilization was studied in media with wide-range buffering capacities (piperazine-glycylglycine and barbiturate buffers). Increasing pH resulted in better responses, although this was mainly due to non-specific interactions. At pH 10.0, however, saxitoxin immobilization was quite effective and specific. The same pattern was found under four different conditions; absence vs presence of bovine serum albumin precoating and absence vs presence of 150 mM NaCl. The best results (high specific response) were achieved with bovine serum albumin precoating in the presence of 150 mM NaCl. The method of choice involved precoating Maxisorp with 5 μg/ml albumin followed by addition of 5 μM saxitoxin in 0.01 M piperazine-glycylglycine buffer, pH 10.0. The efficacy of this technology was demonstrated on a polyclonal rabbit anti-saxitoxin antibody and compared with a conventional ELISA of saxitoxin using saxitoxin-bovine serum albumin conjugate as the coating antigen. In the experiments investigating cross-reactivities of various saxitoxin derivatives based on a competitive assay, significantly greater sensitivity was achieved with the novel approach, e.g. 35 pM saxitoxin could be detected (3 × 10 4 times lower concentrations than using the conjugate). The assay works well with mussel tissue homogenates, and because it does not require the use of the covalent saxitoxin-carrier conjugates it offers a simpler alternative to the traditional ELISA for saxitoxin.