Results

(I) PCR





After PCR, we carried out Gel Electrophoresis. Our J591 gene sequence is ~ 2800 bp. In Figure1, according to the ladder, it suggested there is an obvious banding for the gene of J591 lines between the base pair from 2kb to 3kb. Our genes were successfully amplified.

(II) Ligation





After ligation, we carried out single digestion to produce recombinant plasmid with around 8000-9000 bp. In Figure 2, according to the ladder in the left, it is suggested that there is an obvious banding of the gene of recombinant plasmid between the base pairs between 8kb to 9kb. We successfully insert the target gene to the plasmid.

(III) Antibody concentration





We checked the concentration of the antibody in different samples by measuring the absorbance A260 / A280 using a spectrophotometer. The graph shows that mean antibody concentration under different concentrations of IBTG. 0.4 mM IBTG induced highest protein expression.

(IV) Elisa kit



Elisa kit is a biochemical assay tool used to detect and quantify specific protein, antibody and peptides. We labeled our sample with a type of enzyme called HRP by targeting primary amine groups which is present in IgG antibodies. For other proteins that cannot bind with HRP, the enzyme will be deactivated by the quencher and removed during the first washing. After that we add the conjugated antibody solution to the ELISA plates with antigens coating on it, then substrate. To prevent the binding of any non-specific antibodies to the plate and minimize false-positive results, we washed the plate several times to remove the non-binding protein. The color change occurred due to the reaction between HRP bind to the antibody and substrate and we read the absorbance of the mixture of 450 nm wavelength.

However, the PSMA antigen of 50ug cost HK$2200, as an IGEM team with cost efficiency in mind, we needed to carefully allocate our resources to minimize our expenditure. As the amount of antigen added to each vial of ELISA plate is fixed, ranging from 1-2ug/ml, we chose the minimum concentration of the antigen to limit the consumption of the antigen. Moreover, by using low concentration of PSMA in ELISA kit, it can simulate the condition of prostate cancer in the early stages. We can thus obtain data that are similar to ones collected from an actual patient.

According to the protocol of the ELISA KIT, it is recommended that the antibody level can be determined using 450 nm wavelength checked by the microplate reader. The results were interpreted using SPSS statistical analysis by independent sample T-test. Water was used instead of the J591 antibodies we synthesized as the control. There is a significant difference in the mean value of 450 nm between water and J591 antibody. The mean absorbance for J591 antibody and water is 2.8462 and 0.1102 respectively (P < 0.01). It is found that the mean absorbance was 20 times folder larger in J591.



There is an obvious color difference in the plate for water sample and J591 sample under the ELISA KIT.



(V) Optimization of Antibody Concentration

We use non-linear regression to work out the minimal amount of the concentration to determine the fixed amount of the antigen. In order to determine the 1 ug / ml, the optimal concentration for the antibody is around 2.0 ug / ml from the result shown below. It passes the non-linear regression with p-value (p < 0.05).