The SuperSignal ELISA Femto Maximum Sensitivity Substrate from Pierce Biotechnology is an enhanced chemiluminescent substrate developed for luminometer-based applications. This substrate is specific for peroxidase labels (e.g. horseradish peroxidase) and can be used in ELISA procedures that employ luminometer detection. It also can be used in many other immunobased detection applications, whether using glass slides, membranes, or polystyrene plates. We developed an array-based ELISA system for the simultaneous detection of hepatitis viral antigens and antibodies in human serum (both HBV and HCV).
Each well of the microplate was pre-spotted with antibodies or antigens specific to HBV or HCV. Non-bound proteins were removed and the HRP-labeled detection antibodies/antigens were added, binding to a second site on the target proteins. After the excess detection proteins were removed, the SuperSignal ELISA Femto Chemiluminescent Substrate was added. The HRP used in the prepared sample reacts with the SuperSignal ELISA Femto Chemiluminescent Substrate producing a luminescent signal that can be detected using a cooled CCD camera. The intensity of the signal produced is proportional to the amount of each HBV surface antigen or human anti-HCV antibody in the standard or sample.
We used a dispensing arrayer, Bio-Dot’s AD5000, and spotted 2 x 2 dots into a standard 96-well polystyrene plate. The dots (which contained either Hepatitis B or Hepatitis C viral antigens) were stored at 4ºC overnight and then the plate was blocked using BSA. Duplicates of the reaction mix were prepared and sealed after combining with dilute serum and plasma samples, followed by preparation of standards. The samples were incubated for 1 h at 37ºC with shaking and then thoroughly washed (5 times) with PBS buffer. Either HRP-labeled HBsAg antibody or mouse-anti-human secondary antibody was added to each well and the plate was re-sealed and incubated for 30 min at 37ºC with shaking. Following incubation, the plate was thoroughly washed again and the prepared SuperSignal Substrate (stable peroxide plus SuperSignal Luminol Enhancer) was added to each well.
Charged coupling device (CCD) cameras use sensor technology to provide improved detection of chemiluminescent signals. By cooling the CCD sensor using a thermoelectric device such as a single- or multi-stage peltier device, the CCD operating temperature is reduced and sensitivity is increased. For our array system, the assay is performed, SuperSignal ELISA Femto Maximum Sensitivity Substrate is added and the plate is placed under the CCD camera in a light-tight environment. The CCD chip is exposed to the image over 10 seconds, 20 seconds and 30 seconds. A digitized image is acquired, displayed on a monitor and the image can then be saved as a tiff file for analysis with array software.
Because this array system utilizes chemiluminescent detection, it offers approximately a 4-fold increase in sensitivity than traditional colorimetric ELISAs. We can detect about 0.0625ng/ml of Human Hepatitis B Surface Antigen using this array based system, while traditional colorimetric ELISA’s limit is 0.5ng/ml. Researchers looking for greater sensitivity in their ELISAs or any other solution-based assay are now able to use chemiluminescent substrates for enzyme detection and quantification.
Mu Feng, MD
Beijing BGI-GBI Biotech Co., Ltd.
Beijing Genomics Institute
China