Immunology Methodology Course # IMMU7040
ELISA demo Preparations October 12 2011
Note to Immunology Methodology # IMMU7040 Students:
This lecture has a hands-on component you carry out just before the lecture.
Don’t forget to bring your clean lab coats (University Of Manitoba Safety guidelines stipulate that
everyone needs to wear a lab coat while working in the Immunology Lab)
On October 12 2011 come to Immunology lab 455 Apotex Centre (750 McDermot Avenue) HayGlass Lab
area 15 minutes prior to lecture start time. Ensure you have read & understood these instructions in
advance.
Purpose: To determine the precision with which you can pipette and titrate liquid, as needed for
successful ELISA (or PCR or …).
Results are expressed as CV (coefficient of variance). This should be <3% (optimally ~ 1%) for all
readings. At the break you will be presented with your personal results.
HayGlass Lab advance preparations:
1. Expired yellow ELISA solution: diluted to read A405 on ELISA reader ~ 0.9 OD with 50 ul / well
~ 1.8 OD with 100 ul / well
2. In advance, prepare ELISA plates (use second–hand plates that have been pre-washed with tap water
& dried, to reduce waste). Each ELISA plate will be divided into two sections (two students will share
one plate). Just prior to the student’s arrival, wells B3+4-H3+4 plus B9+10-H9+10 will have 50 ul of
dilution buffer added to each plate for the students (good idea to prepare a few extra plates for
student error ie. The student drops the plate onto the floor).
3. Designated work stations/bench areas need to be set up for the students (usually 2 students per
station). Each station will require a full box of 200 ul pipette tips, a large mouth plastic beaker for
waste tips, multichannel & single channel pipetors (usually the number of pipetors available for their
use dictates the number of work stations and the number of students per station, borrow as
necessary), and test tube racks with different tubes of diluted and undiluted yellow solutions.
4. In advance, prepare a Softmax ELISA template within an “undergraduate student folder” (this
template will enable the student to see the calculated CV values based on each of the student’s
pipetting abilities).
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Student activity (Please arrive 15 minutes prior to the start of lecture):
5. As soon as the students enter the lab, they will need to wear their lab coats. They will have to wash
their hands before leaving the lab area.
6. A precaution is that the expired yellow solution contains DEA (diethanolamine) in low quantity;
however as per the MSDS it may be harmful if absorbed through the skin, causes severe eye
irritation, etc. Nitrile gloves (all sizes) should be available in the work area; the used gloves can also
be disposed of in the large mouth plastic waste beaker. Point out to the students the eye wash
station locations.
7. Using the single channel pipette the student will pipette 50ul of the diluted expired yellow solution
into each of wells A1, B1, C1 (and a different student/ same plate A7, B7, C7) plus 100ul into each of
wells A2, B2, and C2 (and a different student/ same plate A8, B8, C8) The students will then pipette
100ul of the undiluted expired yellow solution into wells A3+4 (and a different student / same plate
A9+10).
8. Wells B3+4 to H3+4 (and a different student/ same plate B9+10 to H9+10) contains dilution buffer.
Using the multichannel pipette (with 2 tips in a row) the student will serial dilute 50 ul from A3+4
(different student/same plate A9+10) down the plate B3+4(different student/same plateB9+10 etc.
(careful while mixing up and down 5 times in-between each serial dilution). At the last well series
H3+4 (different student/same plate H9+10) the final 50ul with tips will be discarded into the wide
mouth plastic waste beaker.
9. ELISA Reader: The lab instructor will open the designated folder and read their plates and save the
data (with their name). The data will have to be transferred to a USB key for printing. The lab
instructor will print a hard copy from a different computer (the one on the right side of the telephone
in the KH lab, this one has the Softmax program installed).
8X12 96 well ELISA Plate
Student 2
Student 1
4
100
5
6
2
7
50
50
50
8
100
100
100
9
100
10
100
SERIAL DILUTIONS
50 + 50 (MIX 5x)
3
100
SERIAL DILUTIONS
50 + 50 (MIX 5x)
2
100
100
100
SERIAL DILUTIONS
50 + 50 (MIX 5x)
1
50
50
50
SERIAL DILUTIONS
50 + 50 (MIX 5x)
A
B
C
D
E
F
G
H
11
12