ELISA

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BACK TO MICRO 60

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BioRad quick procedure guide for students - Videos : https://www.youtube.com/playlist?list=PL7\_N-H8d6RiPT4saHEvjfpaH0dgyfWz67

### Methods:

#### **1. Make 10x PBS**

See recipe

**2. Make Wash Buffer**

 90 mL 10x PBS
 4.5 mL 10% Tween 20

**3. Make 1xPBS:**

Go carefully on this one and *if* more is needed *check* bottle for remaining volume and work backwards to the amount of water to add. So 4.3 mL of extra 10xPBS gets = 4.3 mL/0.10 = 43.0 mL. 43.0 – 4.3 = 38.7 mL DI water. Can buy 10x PBS or make from scratch.

#### **3. Rehydrate Freeze-Dried Reagents to 50x Stock Solutions:**

**Antigen, Primary Antibody, Secondary Antibody:**

* Add 500μL (1/2 = 250μL) of 1x PBS to each vial.
* \*\*If using 2 infected students with 2 full classes, rehydrate ANTIGEN with 550uL 1x PBS\*\*\*
* Vortex then tap down liquid into the tip and store in fridge at 4\* C. for up to 2 weeks but within 24 hours is best.

#### **4. Prepare Student Tubes**

**Yellow microtubes**: 1/ Student

* Number tubes from 1 to 24 (or however many students there are)

**5. Dilution for Lab:**

Label one 30 mL bottle for each of the diluted solutions below. Use a fresh pipette to add the contents of the appropriate 50x concentrated stock to the corresponding 30 mL bottle. Close caps and shake to mix.

**Creations**: (24 students=1/2 use) NOTE: *use sterile water if splitting kit between two labs. '**Dilute only as much concentrated antibody and antigen as required for each lab session. '**Store 50x in fridge at 4 o C.*

**1x antigen:**

 **50x antigen** | 75.0µL

**1x serum(1\* antibody):**

 **50x 1\* antibody stock.** | 250µL

**1x secondary antibody:**

 **50x 2\* antibody stock** | 250µL

**6. Aliquot dilutions to tubes**

#### **Blue Tubes: - antigen**

* 1/bench (4 students) = 6-7 tubes
* 1x PBS (negative control
* 300uL in each tube x # of benches = 1.8-2.1mL total

#### **Brown Tubes: HRP enzyme substrate (TMB)**

* 1/bench (4 students) = 6-7 tubes
* HRP substrate
* 1200uL in each tube x # of benches

NOTE it is light sensitive. So it is important to use the dark tubes to store and/or distribute this substrate.

**Yellow microtubes**: 1/ Student

* Number tubes from 1 to 24 (or however many students there are)
* UNINFECTED STUDENTS: 750 uL 1x PBS
* INFECTED STUDENT (Can have 3 if 1 class, but enough reagents for 2 per class if there are 2 classes):
	+ 650 1xPBS
	+ 100uL 50x antigen stock solution (UNDILUTED STOCK - Morning of - See Below))

**White tubes: + antigen - Morning of (see below)**

* 1/bench (4 students) = 6-7 tubes
* 1x antigen dilution
* 600uL in each tube x # of benches = 1.8-2.1mL total

#### **Green Tubes: Primary Antibody(PA) - Morning of (See Below)**

* 1/bench (4 students) = 6-7 tubes
* 1x Primary antibody
* 1200uL in each tube x # of benches

**Orange Tubes: Secondary Antibody(SA) - Morning of (See Below)**

* 1/bench (4 students) = 6-7 tubes
* 1x Secondary antibody
* 1200uL in each tube x # of benches

**7. Morning of lab**

* Add 100uL *concentrated antigen* to infected student tubes
* Add 150uL *concentrated antigen* to diluted antigen bottle
* Add 500uL concentrated 1' antibody to diluted 1' antibody bottle
* Add 500uL concentrated 2' antibody to diluted 2' antibody bottle
* Aliquot 600uL antigen to white (+) microtubes
* Aliquot 1200uL 1' antibody to green (PA) microtubes
* Aliquot 1200uL 2' antibody to orange (SA) microtubes
* Put in holders and bring to class

#### 8. **Setup Classroom: Per Bench**

* 2 x 12-well microplate strips

* 2 P-200 uL micropipet
* 1x used tip beaker

* 2x boxes P200 micropipet tips [at least ½ full, lots of tips used]

* 5 x plastic transfer pipets
* 1 x roll of paper towels
* Bottle of wash buffer
* Microtube holder with set of tubes for students (can store in fridge)

* 1 1000mL plastic waste beaker on cart