Method for homogeneous spotting of antibodies on membranes: application to the sensitive detection of ochratoxin A
IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata
View Archive Info| Field | Value | |
| Title |
Method for homogeneous spotting of antibodies on membranes:
application to the sensitive detection of ochratoxin A
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| Creator |
Saha, Debjani
Acharya, Debopam Dhar, Tarun K |
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| Subject |
Cancer Biology and Inflammatory Disorder Division
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| Description |
Membrane-based dot immunoassays are now
widely used in almost every branch of biology and
medicine. However, the quality of the immobilized antigen
or antibody spots on the membranes was found to be highly
operator-dependent and spotting by conventional methods
often leads to heterogeneous spot morphologies and
deposition inconsistencies. To circumvent these problems,
a spotting method has been developed which is based on
focussed absorption of an applied antibody solution
through an aqueous network of capillary channels formed
between the membrane and a wetted absorbent body. The
method does not require any equipment for creating
vacuum and according to assay requirements highly
homogeneous spots of uniform size, in the range of 0.8-
to 9-mm diameter, can be obtained by varying the volume
of the applied antibody solution. Spot intensities were
sufficiently high even at high antibody dilutions. Immobilization
of anti-ochratoxin A (anti-OA) antibody by this
method gave 2-fold increased sensitivity in a competitive
assay of the toxin compared to conventional spotting
methods. The calculated CV of the colour intensity for
spots of different sizes (0.8 to 9 mm) was between 4.5 and
1%. Application of this spotting technique has been
demonstrated for detection of OA in wine and coffee
samples with the elimination of matrix interferences in the
same immunoassay system. This was achieved by selective
removal of nonspecific interfering substances from the
sample extract during the assay. The detection limit of OA
in wine (1 μg L−1) and coffee (2.5 μg kg−1) obtained by the
present new method is superior to values reported recently.
Thus, the present new method will be highly useful for
improved performance of membrane-based immunoassays
in almost every branch of biology and medicine.
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| Date |
2006
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| Type |
Article
PeerReviewed |
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| Format |
application/pdf
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| Identifier |
http://www.eprints.iicb.res.in/407/1/ANALYTICAL_AND_BIOANALYTICAL_CHEMISTRY%2C385(_5)%2C_847%2D854_[66].pdf
Saha, Debjani and Acharya, Debopam and Dhar, Tarun K (2006) Method for homogeneous spotting of antibodies on membranes: application to the sensitive detection of ochratoxin A. Anal Bioanal Chem, 385 (5). pp. 847-854. |
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| Relation |
htpp://dx.doi.org/10.1007/s00216-006-0484-4
http://www.eprints.iicb.res.in/407/ |
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