The invention discloses a histioplasma capsulatum double-
antibody sandwich
enzyme-linked immunosorbent
assay method, a
reagent and application. The invention relates to the technical field of ELISA (
enzyme-linked immunosorbent
assay). According to the present invention, the
Protein A / G magnetic beads are used to directionally fix the
antibody and ultrasonically assist the
antigen release, such that the
antigen binding site is completely exposed, and the hidden
antigen is released, such that the
test sensitivity is significantly improved, the sensitivity can be improved by 10 times, and the
detection limit is reduced to the lower level (such as 1 ng / mL or even 0.1 ng / mL). Due to the use of the block
copolymer / zwitterionic surfactant, non-specific binding is effectively blocked, background signals are reduced, and the test specificity is improved; the method comprises the following steps: S1,
antibody coating: diluting a captured antibody to a working concentration (generally 1-10 [mu] g / mL) by using a
coating buffer solution, and adding into an ELISA plate hole (100 [mu] L / hole); carrying out overnight incubation at 4 DEG C (or at 37 DEG C for 2h) after plate sealing; and discarding the liquid, and washing with PBST for 3 times (300 [mu] L / hole, and
drying after standing for 1 min each time).
Protein A / G magnetic beads are used for directionally fixing the antibody through an Fc segment.