title: Anti-lipid A antibodies in childhood arthritis: methods of immobilization affect quantitation and crossreactivity measured by ELISA.
abstract: OBJECTIVE
  To find an optimal method to study antibodies reactive with monophosphoryl lipid A characteristic of oligoarticular arthritis in children.
  
  
  METHODS
  ELISA using 3 different methods of immobilization were compared, in (1) HCO3 buffer, pH 9.6; (2) HCl, pH 2.0; and (3) methanol. Competitive inhibition studies were carried out to quantitate relative avidity of cross reactions with suspected autoantigens.
  
  
  RESULTS
  Sera from healthy children reacted significantly more strongly with monophosphoryl lipid A after immobilization in acid or in methanol than in a basic buffer. Sera from children with oligoarticular arthritis reacted more strongly than normal sera with the basic buffer method and even more strongly with the methanol method, but were not distinguishable from normal sera with the acid method. Results with individual oligoarticular sera correlated from method to method, but results with normal sera did not. Collagen types I and II, cardiolipin, and denatured DNA can block the anti-monophosphoryl lipid A reactivity to varying degrees on plates prepared with basic buffer, but only collagen type I and DNA block reactivity on plates prepared with methanol.
  
  
  CONCLUSIONS
  The epitope on monophosphoryl lipid A recognized by oligoarticular sera is differentially affected by the method of immobilization. The crossreactivity of the anti-monophosphoryl lipid A antibody in this disease is confirmed.
authors.affiliation: Division of Pediatric Rheumatology, Lucile Salter Packard Children's Hospital, Stanford, CA, USA.
authors.forename: J J
authors.initials: JJ
authors.lastname: Miller
authors.forename: S
authors.initials: S
authors.lastname: Zhu
authors.forename: R L
authors.initials: RL
authors.lastname: Smith
mesh_terms: D000906:Antibodies / Q000032:analysis*; D001171:Arthritis, Juvenile / Q000276:immunology*; D002021:Buffers; D002413:Cations, Divalent / Q000494:pharmacology; D002648:Child; D002675:Child, Preschool; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007223:Infant; D008050:Lipid A / Q000031:analogs & derivatives* / Q000276:immunology; D009994:Osmolar Concentration
chemical_list: D000906:Antibodies; D002021:Buffers; D002413:Cations, Divalent; D008050:Lipid A; C048436:monophosphoryl lipid A