We are developing an antigen capture ELISA using monoclonal antibodies
(Mab).
The methods are here;
1. The first Mab (capture Ab; in carbonate buffer, pH 9.6) are added
onto microtitre plates (Costar; Cat No. 3590). Incubated for overnight
at 4oC.
2. The plates were blocked with 3% BSA (Sigma, A4503) in TBST for 2 h at
37oC.
3. The plates were washed three times and added diluted antigen solution
and TBST (negative control).
Incubated for 2 h at 37oC.
4. The plates were washed seven times and added biotinylated Mab
(Detector Ab) in 0.1% BSA-PBST.
Incubated for 2 h at 37oC.
5. The plates were washed seven times and added Streptavidin alkaline
phosphatase conjugate (DAKO, D0396) solution (1:10,000 diluted in 0.1%
BSA-PBST). Incubated for 30 min at 37oC.
6. The plates were washed seven times and added substrate solution.
Incubated for 40 min at RT.
7. And added amplifier and incubated at RT for 10 min.
8. Stop the reaction and read the OD at 490 nm.
We used cyclic amplification system of Self (J Imm Methods, 76;389) at
the ELISA.
This works fine except that we get a high background. Not only that, we
get a high background if we just add negative control (TBST). So, what
is happening is that the detector Mabs (biotinylated) are cross react to
the capture Mabs coated on the wells. The capture and detector Mab are
the same Mab purified using Protein A column from ascitic fluids. Does
anyone have an explanation for this? Even better, can anyone suggest a
way to overcome it?
It is commonly stated that biotin has many binding sites for one
antibody molecule and thus amplifies the reaction (i.e. if many
avidin-En. are linked through one biotin to an antibody, it may lead
signal amplification). In practice, is it possible?
I would like to know the difference (signal noise ratio, sensitivity,
etc.) between direct conjugation of enzyme to Ab and biotin-Ab through
Streptavidin-enzyme system.
Please respond to either this posting or e-mail any answers you might
have directly to me at reattack at chollian.net
Any help would be appreciated. Thanks, Jang
Won-Jong Jang, Ph D.
Department of Microbiology
Seoul National University, College of Medicine
28, Yongon-Dong, Chongno-Gu, Seoul 110-799, Korea
Tel. (Lab.) 02) 740-8312
(Home) 02) 996-4551
Fax: 02) 743-0881
E-mail: reattack at chollian.net, reattack at plaza.snu.ac.kr
