1) Obtain ELISA plate (this is a special plate which absorbs protein). To 1/2 the wells you will add the buffer and protein (ex. GP160) and the other half of the plate will serve as a control which will contain the buffer only. 

GP160 (0.75 ug/ml)                                                                        PBS Control                Date Initials

(2) Pipet 2.5 ml of PBS into a conical tube. (PBS is a saline solution 0.9%). It is made we add tablets and ddH2O.

(3) Vortex the protein (GP160) and add 18.75 ul (this gives a final concentration is 0.75 ug/ml since we started with a 100 ug/ml stock of the protein and our total volume is 2.5 ml. See ) of it to the conical tube. 

(4) Pour contents of conical tube into reservoir and use multi pipet to add 50 ul of each well for 1st half of plate. 

(5) Place plate on shaker to get contents uniform.

(6) Pour PBS into reservoir and pipet 50 ul of the PBS onto the right hand side of the plate with multi-pipette. Shake

(7) Place seran wrap over plate 

(8) incubate at 37.5 for 1 hour (the heat will increase rate of protein binding)

(9) Prepare Blocking buffer (50 ml 1x PBS + 0.5g BSA (powder), mix by shaking and add 50 ul of 1% Tween solution)

(10) Take plate out of incubator and use microplate autowasher to wash it  (0.45% NaCL + 0.05% Tween 20 in deionized water) (set washing to rinse, then start)

(11) Use blocking tray marked “blocking buffer”, pour in blocking solution, and use multip-pipet to pipette 200 ul of blocking solution into each well (the blocking buffer is necessary because protein binding is not uniform. There are empty spaces where our antibody to be used next can bind. We want to block these sites)

(12) Place seran wrap over plate and incubate for 1 hour at 37c (or if overnight incubate at 4c)

Preparation of Antibodies

(1) Wash the blocked plate 4 times with washing buffer. (put other plate back in machine at end.)

(2) Create the ELISA worksheet below. We will be using an antibody (ID6) from a hybridoma here which secretes a mouse monoclonal antibody against an epitope on the gp 160 envelope glycoprotein of HIV-1. As a control, we will be using a murine antibody (IGG) secreted by a hybridoma unrelated to any HIV-1 proteins but of the same isotype of ID6. 

Prepare dilutions of the 2 antibodies. Write the dilution factors in the ELISA chart as below. Neat=no dilution, 10= a factor of 10 and so on. So set up 2 sets of 12 eppendorf tubes. 1 set will belong to the ID6 dilutions and the other set to the control. 

ID6        __            Control Ab                            __        ID6                                __        control Ab

The way to set up the dilution is to put 500 ul of solution in the first tube of each set and 450 ul of solution in each of the other tubes. Then take 50 ul out of the first tube, and place it into the second, then 50 ul out of that 2nd and place it in the 3rd and so on. Question: how much dilution buffer and how much Ab should we add? See 

To first ID6 Tube, add 421.4 ml dilution buffer and 375 ml dilution buffer  to first control tube and 450 ul to all other 11tubes (2sets)

To first ID6 tube add 78.6 ul of ID6 and vortex        add 125 ul of the IGG to 1st tube of control and vortex

Take 50 ul of each tube and move to the next. vortex each tube after transfer. (you can do 2 tubes at at time, ie., 1st set ID6 and then to control.

(3) Place 100 ul of the appropriate solution into each well. (ie., Take 100 out of the neat tube for ID6 and add it to well A1, then another 100 ul to well B1, then 100 ul to well A6, then 100 ul to well B7 and repeat this down the line, then repeat all this for the control).  Shake the plate on a shaker. wrap it up in seran wrap and place at 37C (or at 4c if leaving overnight) for 30-40 min.

Preparation of Secondary Antibody for Detection 

(1) Wash plates with washing buffer (can use the blocking solution) 8 to 10 times.

(2) Prepare goat anti-mouse IgG peroxidase (this is the secondary antibody used for detection) diluted 1:10,000 in the dilution buffer. (50 ml buffer + 5 ul conjugated ab). Pur buffer into reservoir and use multiple pipet to add 100 ul to each well on plate.  Incubate at 37 for 30-45 min. 

(3) Wash plate with washing buffer 8-10 times (use maching, “wash” “start”)

Prepare Substrate

(1) take steril flash and draw line up to the 100 ul on it and add Nonopure water which has been filtered (use filter under hood, filter water twice the second time hook filter up to vacuum to pull water through)

(2) add one tablet phosphate-citrate (need to open protective capsule and pour in)

(3) shake flask with hands until dissolved

(4) Pour 20 ml of that solution into fat conical tube and add substrate tablet (tetramethylbenzidine).

(5) pour solution into steril trough. using multi-pipette dispense 100 ul into each well. Cover plate from light and incubate for about 10-20 min until a blue color appears which incidates substrate reacting with the peroxidate enzyme conjugate)

(6) stop the reaction by adding sulfuric acid (pour some sulfuric acid into trough and add 25 ul of 2M sulfuric acid to each well. Place plates on shaker.

(7) read the optical density (OD) at 450 nm on plate reader/spectrophotometer. (start of wells should be facing in toward machine) [1) turn on reader and printer, 2) put blank cover in reader, hit blank, 405 and read. 3) put sample plate in and hit optical, 450 and read.

(8) Plot results on a graph. (using excel, enter data from all wells from 1st half of plate (gp160) as 1 separate column, in 2nd column enter OD data from all the wells from control, In third column calculate the specific OD which will be (Column 1 – column 2). In the next column on excel, calculate the mean of the 2 wells for each dilution (remember, each dilution was repeated 2 times. Lastly, calculate the standard deviation of the mean.) Plot the conc. (ug/ml) of ID6 antibody (starting with the highest undiluted concentraiton we used which was 250 ug/ml and diluted by a factor of 10 (so would be on x axis 250, 25, 2.5, 0.25, 0.025, 0.0025 (will only be able to plot up to that) versus the specific OD reading for these concentrations on the Y axis (interval can be 0.00, 0.05, 0.10 so increments of 0.05).