Okeh. Kita sambung lagi pasal tajuk sebelum ini ya? ^_^
Kalau bosan duduk diam-diam.
Mula-mula tarik nafas dalam-dalam, lepas perlahan-lahan.
Senyum panjang-panjang sambil jerit:
Today I would like to explain how we can use the cute and amazing graph up there. Ya, that’s the graph from our AMG note.
Do you understand what the graphs means?
Me too! “-_-
Ops. Before that, please understand the concept of sandwich ELISA.
Let’s slowly try to understand the graph by reading the journal. I’ve read it and would like to share with you. I hope this is true, not another ajaran sesat from me.
[hoping that reciting basmallah clearly will not lead me to deviation]
Firstly is about the graph…
The graph is a calibration graph that has been developed in ELISA [A pretty girl from Edensor].
Why we need to build the graph?
This is simply because we’ll use it to refer our results i.e the absorbance O.D [optical density] to the graph. Once we got it [the same amout of O.D] then we’ll know the concentration of antigen that we got.
Remember, the one that we want to test in this experiment is the antigen and antigen can be anything, like what i’ve told you, but in this case we’re referring to the heterologous protein from plants.
Do you understand?
Next is the complicated part; the steps to build up that cute and amazing graph.
There are many steps to go through, but i would like to stress on three important steps named first, second and third pilot ELISA.
These three ‘pilots’ have their own objectives.
First PILOT-a young and handsome pilot from Maldives. Try to imagine Ablho: Determine the optimal set of primary and secondary antibody concentration.
Second PILOT-tall pilot with funny face: Determine accurately the linear range of the standard curve, using the established one from the first PILOT
Third PILOT-this pilot has a huge pistol to make sure everything is fine: To check wheter the plant components interfere with the binding of the antigen to the primary antibody and if inteference occurs, at which concentration of total protein. Also to determine which concentration of total protein give background signal to ELISA.
The important tips to tackle him are:
1. You need four different primary and four different secondary antibodies
2. Three types of controls: a) no antigen b) no antigen and secondary antibody c) no primary antibody
Then, you’ll get the optimal concentrations for these two antibodies. Please remember that you need to consider the performance (O.D value for a particular atigen concentration) and the cost/availability of the antibodies. To get a highly sensitive ELISA, please make sure that it is easy to differentiate between the lowest and the highest detectable concentration i.e fourfold higher. Secondly, make sure that the ratio of signal: background is high for the lowest detectable antigen concentration. Thirdly, make sure that the amount of secondary antibody is in excess once compared to the antigen to make sre the assay is quantitative. Next, please check the controls if they ‘make’ noises/ background-> will interfere the accurate readings.
It’s simple to remember him. Just use the tips to tackle Pilot No. 1. Next, use twofold serial dilutions of antigens and you can determine the linear range. Remember that, though this pilot is not as handsome as the pilot no. 1, he is very important because any quatitative determinations of the antigen concentration need him.
Mr. Pilot no. 3, aged 55 years old, surely makes you remember your father at home. He is the one that protect you, give you motivation to face this AMG exam and the one and always loves you…
To check if anything goes wrong, first and foremost, you need to test:
the concentration of antigens at the highest, midpoint and lowest concentration of linear graph+ blocking buffer
constant amount of antigen+ blocking buffer +variable total protein of untransformed plant
variable amount untransformed plant + blocking buffer
Then, check if the signal is significantly lower in the presence of certain amount of plant proteins (untransformed), then this amount of proteins interferes with the binding of antigen to the primary antibody. Get the interference concentration.
Also, please check if the analogous dilution series of untransformed plant results in background.
At last, you can draw the graph now!
Seems weird? Why there’re two curves?
The graph shows two curves of A and B which mean secondary antibodies are in excess and secondary antibodies are in limited amount respectively. The slope of curve B is smaller and the plateae region of high antigen concentration is lower. The region between (a) and (b) shows the linear range, the one below (a) is where the background happened though it is enzymatic-independent reaction and lastly the region of upper (b) is where the primary antibody is in limited amount compared to the antigen.
Pity me don’t have the twit twit thing and FB T,T