Pamela Grace D.

Apostol December 10, 2018

PPTH 115

Enzyme-Linked Immunosorbent Assay and Polymerase Chain Reaction

INTRODUCTION

ELISA is one of the commonly used methods for detection and diagnosis of viruses because of

its high sensitivity, fast, economical and efficient with the use of antibodies. The principle behind ELISA

is that target antigen (or antibody) capture in samples using a specific antibody (or antigen), and of target

molecule detection/quantisation using an enzyme reaction with its substrate.

PCR involves the primer mediated enzymatic amplification of DNA. PCR is based on using the

ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template

strand. Primer is needed because DNA polymerase can add a nucleotide only onto a pre-existing 3′-OH

group to add the first nucleotide. DNA polymerase then elongates its 3 end by adding more nucleotides to

generate an extended region of double stranded DNA.

MATERIALS AND METHODOLOGY

Enzyme-Linked Immunosorbent Assay (ELISA)

For this exercise, we performed indirect ELISA. A sap dilution in carbonate coating buffer (1:10)

is prepared. 100 ul was added in the microtiter plates in each well and these were incubated overnight at 4

degrees Celsius. The samples were washed using PBS-T for three times. A blocking solution in 1% skim

milk in PBS – Tween 20 was added, this was done for three times and then washed for another three

times. A diluted antiserum which contains the primary antibody (1:200 dilution) was added, 100 ul was

added per well. The samples were then incubated for 2-4 hours at 37 degrees Celsius. The samples were

then again washed for another three times. Secondary antibody (Enzyme labeled) was added and then

washed for another three times. 100 ul of substrate was added per well. The results were analyzed using

the ELISA reader.

Polymerase Chain Reaction and Agarose Gel Electrophoresis

After the components are prepared, there are three steps for the PCR. First is denaturation in

which involves heating of the mixture to 94 degrees Celsius for 1 minute. The double stranded DNA will

be denatured to single strand because of the breakage of H-bonds. Next step will be annealing in which

the temperature is set at 60 degrees Celsius for 30 seconds. The primer binds to the complementary

sequence of the DNA template. Last step will be extension in which the temperature is set to 72 degrees

Celsius. The polymerase enzyme sequentially adds bases to each 3’ primer extending the DNA sequence

in the 5’ to 3’ direction. Each cycle the dsDNA is amplified into two separate pieces of DNA.

Components Initial Final V2 [14X]

Concentration Concentration

Dream Taq - - 12.5 ul 175

Forward Primer 10 uM 0.2 uM 12.5 ul 7

Reverse Primer 10 uM 0.2 uM 0.5 ul 7

DNA template 50 mg/uM 50 mg 0.5 ul 14

H2O 1 ul 147

TOTAL 25 ul 350

Table 1. Components and the amount used for the Polymerase Chain Reaction

For the gel electrophoresis, prepared DNA was placed in a small piece of parafilm. Loading

buffer is added to each DNA drop and mixed by repeated pipetting. The entire mixture is loaded into a

well in an agarose gel which is immersed in 1 x TAE buffer. The fragments are separated by

electrophoresis at 50 volts until bromophenol blue dye is at the bottom of the gel. The gel was then

removed from apparatus and soaked in ethidium bromide stain solution for 10 minutes. The bands are

then viewed under an ultraviolet box.

 RESULTS AND DISCUSSION

ENZYME-LINKED IMMUNOSORBENT ASSAY

Figure 1. ELISA reading for Cucumber Mosaic Virus in Tomato plant in different time readings.

Highlighted data indicate positive control.

For the first reading (40 minutes), the wells that have a higher reading of 0.230 indicates positive

for ELISA. The next reading (1 hour), the values that indicated positive for the first reading had a higher

OD level and some wells indicated positive for the test. For the last reading (1 hour and 30 minutes), there

are additional wells that indicate positive for the test, including the positive control and the wells that

indicated positive for the first and second reading had a higher OD value.