ELISA of IgG antibody to oxidized low-density lipoprotein: effects of blocking buffer and method of data expression.
We describe an ELISA for serum IgG antibodies against malondialdehyde-modified low-density lipoprotein (mLDL). Optimal antigen concentration, serum dilution, and dilution of enzyme-conjugated second antibody were 25 mg/L, 1:250, and 1:5000, respectively, when 5 g/L human serum albumin was used for blocking. When data were expressed as mLDL/LDL (the ratio of IgG binding to mLDL vs LDL), within-run and between-run CVs were 7.0% and 8.9%, respectively. Antibody concentrations expressed as mLDL/LDL or as mLDL-LDL (the difference between IgG binding to mLDL and to LDL) were higher in women with systemic lupus erythematosus (n = 20) than in controls (n = 20) (P < 0.001). With bovine serum albumin or Superblock blocking buffers, only the mLDL-LDL data were significant. Thus, the choice of blocking agent and the method of data expression should be carefully considered when assaying IgG antibodies against mLDL.
Craig
W Y
WY
Foundation for Blood Research, Scarborough, ME 04070-0190.
Poulin
S E
SE
Nelson
C P
CP
Ritchie
R F
RF
D000293:Adolescent; D000328:Adult; D000368:Aged; D000941:Antigens / Q000097:blood; D001323:Autoantibodies / Q000097:blood*; D002021:Buffers; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood*; D015227:Lipid Peroxidation; D008077:Lipoproteins, LDL / Q000097:blood / Q000276:immunology*; D008180:Lupus Erythematosus, Systemic / Q000276:immunology; D008315:Malondialdehyde / Q000494:pharmacology; D008875:Middle Aged; D010084:Oxidation-Reduction; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000494:pharmacology; D017392:Thiobarbituric Acid Reactive Substances / Q000378:metabolism
D000941:Antigens; D001323:Autoantibodies; D002021:Buffers; D007074:Immunoglobulin G; D008077:Lipoproteins, LDL; D012709:Serum Albumin; D017392:Thiobarbituric Acid Reactive Substances; D008315:Malondialdehyde