A homogeneous immunoassay for the mycotoxin T-2 utilizing liposomes, monoclonal antibodies, and complement.
The trichothecene mycotoxin T-2 is a fungal metabolite known to contaminate agricultural products and cause intoxication of humans and animals. We have developed a homogeneous competition inhibition assay for T-2 mycotoxin based on complement-mediated lysis of liposomes. The T-2 mycotoxin was converted to an acid chloride derivative, subsequently coupled to the amino group of phosphatidylethanolamine, and incorporated with the phospholipid into unilamellar liposomes. Carboxyfluorescein, which is self-quenched at high concentrations, was entrapped in the liposomes as a release marker. We used a monoclonal IgG1 antibody specific for T-2 mycotoxin and a polyclonal anti-mouse Ig as a secondary antibody since the anti-T-2 IgG1 does not activate complement. In the absence of free T-2, the liposomes were lysed within 30 min after the addition of complement, releasing carboxyfluorescein into the surrounding buffer. In the presence of free T-2 toxin, the binding of antibodies to the liposomes was reduced, causing a corresponding decrease in lysis. This assay proved to be sensitive to T-2 toxin levels as low as 2 ng, which is 10-fold more sensitive than the present enzyme immunoassay using the same antibodies.
Ligler
F S
FS
Bio/Molecular Engineering Branch, Naval Research Laboratory, Washington, DC 20375-5000.
Bredehorst
R
R
Talebian
A
A
Shriver
L C
LC
Hammer
C F
CF
Sheridan
J P
JP
Vogel
C W
CW
Gaber
B P
BP
D000911:Antibodies, Monoclonal; D003165:Complement System Proteins; D007118:Immunoassay / Q000379:methods*; D008081:Liposomes; D012717:Sesquiterpenes / Q000032:analysis*; D013605:T-2 Toxin / Q000032:analysis*
D000911:Antibodies, Monoclonal; D008081:Liposomes; D012717:Sesquiterpenes; D003165:Complement System Proteins; D013605:T-2 Toxin