SINGAPORE POLYTECHNIC SCHOOL OF CHEMICAL AND LIFE SCIENCES

Diploma in Biomedical Science CP2033 Applied Immunology

Experiment 6: ELISA- Detection of antibodies to hepatitis B surface antigen Date: 25th May 2010

Prepared for: Mr Woo Wee Hong

Submitted by: Lee Kah Jing 0818755 Lynette Liew Sin Yee 0818841 Yeo Ying Shan 0818713 Samuel Tong Jia Ming 0818385

DBS/FT/3B/22 2010/11 Semester 1

Principle
1. Labelled Diagram/Flowchart

2. ELISA is a competitive/non-competitive assay? Enzyme-linked immunosorbent assay (ELISA) is both a competitive and non-competitive assay. However, in this experiment, Monolisa Anti-HBs plus ELISA is based on a non-competitive, indirect ELISA in which the microplate is coated with human hepatitis B surface antigen, test samples are then added into the microplate wells where specific antibody binds to the antigen, followed by the addition of an enzymelinked antibody which binds to the specific antibody, then the addition of a substrate solution which is converted by an peroxidase enzyme to form a colored product and finally the addition of a stop solution to stop the reaction. However, for competitive assay, an additional step is required which is the incubation of an unlabelled antibody with the antigen to be measured before adding the antigen-antibody complex to the antigen coated wells of the microplate. Therefore, since this additional step is not being carried out in this experiment and the objective of this experiment is to detect antibodies in the patients serum to hepatitis B surface antigen instead of detecting antigen in the patients sample, this indicates ELISA is based on a non-competitive assay in this experiment.

Results and Discussion

1. Is the results valid? Interpret the results and discuss.

Reagent
Negative Control Calibrator (10 mIU/mL) Calibrator (10 mIU/mL) Calibrator (10 mIU/mL) Positive Control (100 mIU/mL) Patient 1 - Evon Patient 1 - Evon Patient 2- Pei Shan

A450
0.029 0.088 0.146 0.093 0.798 2.203 2.176 1.275

The results are valid because the 3 absorbance values of Calibrator 1 falls within the acceptable range of 0.050 0.200. Mean ODC1 = (0.088 + 0.146 + 0.093) / 3 = 0.109 CO (Cut off value) = Mean ODC1

= 0.109 The CO value is an indication of whether the patients have Hepatitis B antibodies. Samples with absorbance values greater than or equal to the cutoff value are considered reactive, meaning that the antibodies and antigens have showed a positive reaction. On the other hand, samples with absorbance values less than the cutoff value are considered non-reactive, which means that there was reaction between the antigens and antibodies, thus a negative result. According to the absorbance values, it showed that patient 1 and patient 2 both have positive results, with patient 1 having a higher linearity limit.