Quant Tetramer Kit Starter Guide

● Products lineup ● Kit components ● Materials required but not supplied ● Workflow ● Protocol  A. Generation of New Specificity Tetramer    Using Peptide Exchange  B. Quantification of Peptide Exchange Using    Flow Cytometric Sandwich Immunoassay ● Determining Percentages of Peptide Exchange

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Product name	Code No.
	PE-labeled	APC-labeled	BV421-labeled
QuickSwitch™ Quant HLA-A*01:01 Tetramer Kit	TB-7308-K1	TB-7308-K2	TB-7308-K4
QuickSwitch™ Quant HLA-A*02:01 Tetramer Kit	TB-7300-K1	TB-7300-K2	―
QuickSwitch™ Quant HLA-A*03:01 Tetramer Kit	TB-7306-K1	TB-7306-K2	TB-7306-K4
QuickSwitch™ Quant HLA-A*11:01 Tetramer Kit	TB-7304-K1	TB-7304-K2	TB-7304-K4
QuickSwitch™ Quant HLA-A*24:02 Tetramer Kit	TB-7302-K1	―	TB-7302-K4
QuickSwitch™ Quant H-2Kb Tetramer Kit	TB-7400-K1	TB-7400-K2	TB-7400-K4

Component name		Description	Size	Storage
QuickSwitch™ Tetramer		Exiting Peptide loaded MHC tetramer	500 µL	2-8°C
Peptide Exchange Factor		The proprietary peptide exchange factor	13 µL	-20°C
Magnetic Capture Beads		Magnetic beads conjugated with a capture antibody specific for tetramers	500 µL	2-8°C
Exiting Peptide Antibody-FITC (25x)		FITC conjugated antibody reacting against Exiting Peptide	25 µL	2-8°C
Reference Peptide 1 mM		The peptide for a positive control	13 µL	-20°C
Assay Buffer (10x)		Washing/Dissolving buffer	1.7 mL	2-8°C

■ Peptides for new specificity tetramers	■ Flow cytometer
■ Magnetic tray for microplate	■ Vortex
■ Microtubes	■ Round or conical bottom microplates
■ Micropippettes and disposable tips	■ Aluminum foil
■ Ultra pure water	■ DMSO
■ Plate shaker (Labline model 4625 or equivalent)
■ Sonicator (Branson Ultrasonic Cleaner Model #B200 or equivalent)

 WARNINGS AND PRECAUTIONS 

A Generation of New Specificity Tetramer Using Peptide Exchange

B Quantification of Peptide Exchange Using Flow Cytometric Sandwich Immunoassay

A Generation of New Specificity Tetramer Using Peptide Exchange

1 Dissolve each lyophilized peptide to be assayed in DMSO to a 10 mM solution.
(For high affinity peptides, a 1 mM stock solution is a reasonable starting concentration for the assay.)

2 Pipet 50 µL of QuickSwitch™ Tetramer into a microtube.

3 Add 1 µL of peptide and mix gently with pipetting.

4 Add 1 µL of Peptide Exchange Factor and mix gently with pipetting.

5 Repeat steps 1 - 4 for each additional peptide.

6 Prepare Reference Peptide as follows;
　(1) Pipet 50 µL of QuickSwitch™ Tetramer into a microtube.
　(2) Add 1 µL of Reference Peptide (1 mM) and mix gently with pipetting.
　(3) Add 1 µL of Peptide Exchange Factor and mix gently with pipetting.

7 Incubate at least for 4 hours* at room temperature protected from light.
(*Reaction time varies depending on the kit. For details, please refer to the datasheet for each kit.)

8 Refrigerate tetramers at 2-8°C protected from light when not used.

B Quantification of Peptide Exchange Using Flow Cytometric Sandwich Immunoassay

The following assay measures the percentage of original peptide replaced by a competing peptide to help determine whether the resulting tetramer is suitable for antigen-specific CD8⁺ T cell staining. All control samples are required for the assay. The QuickSwitch™ Calculator on the website can be downloaded for determining percentages of peptide exchange.
 WEB  http://www.mblintl.com/quickswitch-peptide-exchange-calculator/

Control #3: Beads that have captured QuickSwitch™ Tetramer. FITC conjugated antibody reacts against Exiting Peptide.
Reference Peptide: This peptide typically undergoes about 100% exchange. FITC conjugated antibody does not react against Reference Peptide.
User’s Peptide: Peptides selected by users. The figure above shows the flow cytometry data for 50% peptide exchange.

Preparation of 1x Assay Buffer

1 Prepare 1x Assay Buffer as follows:
For 1-5 peptide exchanges, prepare 7.5 mL by mixing 750 µL of 10x concentrated Assay Buffer with 6.75 mL of distilled water.
For 6-8 exchanges, double the volumes.

Dispensing Capture Beads

2 Immediately before use, vortex the tetramer capture beads for 30 seconds, followed by a 30-second sonication in a water bath sonicator. If no sonicator is available, vortex an additional 30 seconds.
3 Prepare a round or conical-bottom 96 well microtiter plate.
4 Pipet 20 µL Magnetic Capture Beads into each of wells #1-#7. Pipet the beads into wells for three essential controls, Reference Peptide, and tetramers generated in Step A. The below figure shows the case when three new tetramers are used.

Tetramer Capture

5 Pipet 5 µL 1x Assay Buffer in well #2.

6 Pipet 5 µL QuickSwitch™ Tetramer in wells #1 and #3.

7 In wells #4-#7, pipet 5 µL taken from the difference peptide exchange microtubes as described in Step A.

8 Shake plate for 45 min. at 550 rpm, protected from light with an opaque cover such as a piece of aluminum foil.

Rinse

9 Dispense 150 µL of 1x Assay Buffer in wells #1-#7.

10 Place the plate on a plate magnet for 5 min. to sediment beads.^※

11 Flick the plate held tightly to the magnet and blot on a paper towel to minimize cross-contamination of wells.

12 After returning the plate upright, vortex for 2 seconds to disperse the beads.

※It is possible to pellet by centrifugation using a plate holder at 1,000 x g for 5 min.

Preparation of 1x Exiting Peptide Antibody

13 Dilute 25x Exiting Peptide Antibody to 1x as follows: Determine the number (n) of samples to stain with the antibody, including control #2, control #3, Reference Peptides and tetramers generated in Step A. Add one (+1), to account for pipetting errors. In a microtube, pipet (n+1) x 24 µL of Assay Buffer and then add (n+1) x 1 µL of Exiting Peptide Antibody-FITC. Mix by pipetting.

Beads incubation with Exiting Peptide Antibody

14 Pipet 25 μL of 1x Exiting Peptide Antibody-FITC prepared in step 13 in wells #2-#7.

15 Pipet 25 µL of 1x Assay Buffer in well #1.

16 Shake plate for 45 min. at 550 rpm, protected from light.

Rinse

17 Dispense 150 µL of 1x Assay Buffer in wells #1-#7.

18 Place the plate on a plate magnet for 5 min. to sediment beads.^※

19 Flick the plate held tightly to the magnet and blot on a paper towel to minimize cross-contamination of wells.

20 After returning the plate upright, vortex for 2 seconds to disperse the beads. Then remove the plate from the magnet.

21 Resuspend beads in 200 µL 1x Assay Buffer.

※It is possible to pellet by centrifugation using a plate holder at 1,000 x g for 5 min.

1 Pipet 200 µL 1x Assay Buffer and 5 µL Magnetic Capture Beads into well #X.

Now they are ready to be analyzed by flow cytometer.

For more detailed information, please see the product datasheet.