Disease Investigation in Biomedics - Essay Example

The difference is made clear through the drawing of a standard curve, by making use of concentrations that are known; as belonging to antibodies of Rabbit IgG.

Method:

Materials, preparation of plates, and methods used in Practical 1 (weeks 1 & 2):

Materials used:

• Coating buffer: phosphate buffer saline (PBS)
• Wash buffer: 0.05% Tween 20® in PBS, pH 7,4.
• Diluent: PBS
• Blocking solution: 1 % (v/v) bovine serum albumin (BSA) in PBS
• Antigen: rabbit IgG
• Coating antibody: mouse monoclonal anti-rabbit IgG (dilution to be used determined on weeks 1 and 2)
• Detection antibody: Goat anti-rabbit IgG- Peroxidase conjugated (dilution to be used determined on weeks 1 and 2).
• Colour reagent (3,5,3’,5’ tetramethyl benzidine (TMB; SureBlue, KPL)
• Stop solution (ClH 1M)

Plates were already prepared as follows:

1. Adding 200ul of Rabbit IgG antigen to the first row (A1-A12) of the microtiter plate; see the yellow color picture illustration below.
2. Secondly, add 100ul of coating buffer to the remaining rows; see the light blue figure below.
3. Transfer 100ul rabbit IgG antigen through serial dilution as in the figure below; from A to G.
4. To keep the (H) column a blank, 100 ul rabbit IgG antigen was done away with after column (G)
5. Incubation was done on different amounts of rabbit IgG. Microtiter plates were then cleaned by use of washing buffer, three times; and washed again after blocking them with blocking buffer. They then were refrigerated and ready for use in the practical.

Practical 1 (weeks 1 & 2):

• Monoclonal antibody (left half of the plate)
• For the monoclonal antibodies, the left half of the plate got used. As in the illustration below in a picture with blue color; in the first column, 200ul of monoclonal mouse anti-rabbit IgG was added. 100ul of diluents buffer PBS was then filled to columns 2 and 6, for the monoclonal antibodies as in the below illustration.
• Then, 100ul of mouse anti-rabbit got transferred from A1 to A6 in order to have the sample diluted, and from well A6 100ul of mouse anti-rabbit IgG antibody was thrown away. A repeat of the process was done on the rest of the rows.
• The plate was then incubated at room temperature for 30 minutes.
• The plate was washed with Phosphate Buffer Saline three times; which only applies to the left half. It is necessary to cover the remaining half of the plate, using a sticker.
• Polyclonal antibody titration (right half)
• The polyclonal part was uncovered and onto columns 8 to 12, 100ul of diluents buffer PBS was added, and 200ul of goat anti-rabbit IgG-HRP was to column number 7.
• As in the monoclonal step, the preparation of polyclonal Ab dilutions was then done.
• As shown in the below illustration, to all the wells in the left half, 100ul goat anti-mouse IgG-HRP(fixed dilution 1/5000) was added.
• The next step was then having the plate incubated for 30 minutes at room temperature.
• In order to be sure that the wells had no bubbles, using a washing buffer, the plate was washed three times.
• The plate was then protected from light until color developed.
• To stop the reaction, 50 ml of 1 M was added to the wells.
• To read the microtiter plate, a spectrophotometer at (450 nm) can be used, and the graph is therefore drawn.

Practical 2 (week 15 & 16)     

Method:

• The plate coated with mouse anti-rabbit IgG monoclonal antibody was washed, blocked, and washed for a second time, after incubation overnight; and 100ul of PBS was added to the 1 and 2 columns.
• Secondly, unknown sample X(100ul) was added to A3 and A4.
• To columns, B3 and B4, 100ul of the unknown sample (Y) was added, and A1 and A2 were filled with 200ul of rabbit IgG (2ug/ul).
• By transferring 100ul from the first well in column 1 (A1) to (G1), serial dilution was done and the mixture after G1 was thrown away; the same was done to the second column too.
• With the plate covered with a sticker, it was incubated for 30 minutes at room temperature and washed with (PBS/Tween).
• To all the wells with samples, 100ul of goat anti-rabbit IgG-HRP was added.
• The plate was then for 30 minutes incubated and washed thrice using (PSB/Tween )
• TMB substrate (100 ul/well) was added and then the plate was hidden from the light until the color developed.
• Finally, the reaction stopped by the addition of 1 M HCL (50 µl/well) and absorbance read at 450nm.