TITLE: Luminex Magnetic Bead Assay for Assessment of the Immune Response to Vibrio cholerae
AUTHORS: dmslater, Jason Harris

[DESCRIPTION]
This protocol provides instructions for assessing antibody responses to Vibrio cholerae in human blood plasma samples using a multiplex bead assay (MBA). 

Magnetic microspheres (beads) are conjugated with target antigens according to the manufacturer’s instructions. Individual bead stocks are added to a buffered solution to create a pooled, master bead stock and dispensed into  384-well plates. Plasma samples are diluted and tested in duplicate. A serial dilution of a pooled plasma standard, positive and negative controls are included on each plate.  After an incubation step to allow antibody-antigen binding, magnetic beads are washed, and incubated with fluorescently labeled isotype-specific secondary antibodies. Beads are washed once more and then resuspended and analyzed on a FlexMap3D instrument. 


[STEPS]
SECTION: Definitions/Abbreviations
1. Beads: Luminex® MagPlex® microspheres
BSA: bovine serum albumin
MFI: mean fluorescent intensity
PBS: phosphate buffered saline
PPE: personal protective equipment 
rpm: revolutions per minute
O1 Standard: A high-titer pooled plasma sample prepared from convalescent plasma of patients who have recovered from V. cholerae O1 infection. Serial dilutions are used as a standard curve to normalize MFI data to minimize inter-run variation.
O139 Positive Control: A high-titer pooled plasma sample prepared from convalescent plasma, reactive with the O139 antigen.
Sialidase/TcpA Positive Control: A high-titer pooled plasma sample prepared from convalescent plasma, reactive with the sialidase and tcpA antigens.
Negative Control: A pooled heat-inactivated plasma sample is prepared from North American volunteers without prior exposure to cholera.
SECTION: Procedure - Antigen Conjugation to MagPlex Beads
2. Antigens are conjugated to beads following the manufacturer’s instructions in the Luminex xMAP®Antibody Coupling Kit User Manual, RevE, using a ratio of 5µg of antigen for every 1 x 106 beads. Stocks are quantitated using a cell counter and stored at 4C, wrapped lids in parafilm to prevent evaporation and foil to protect from photobleaching. 

The panel consists of the following antigens and bead regions: 
 
              Antigen        Antigen Source        Bead Region             Bead Catalog #          1        V. cholerae O1 Inaba OSP-BSA         Edward Ryan, MGH        44        MC10042-01          2        V. cholerae holotoxin (CTh)        List Biological, 100B        77        MC10044-01          3        V. cholerae toxin, B subunit (CtxB)        Sigma, C9903-1MG        38        MC10077-01          4        V. cholerae sialidase, N16961 strain        Richelle Charles, MGH        35        MC10038-01          5      V. cholerae toxin-coregulated   pilus (TcpA) N16961 strain      Harris   Lab, MGH      49      MC10035-01        6      V. cholerae O1 Ogawa OSP-BSA       Edward   Ryan, MGH      42      MC10049-01        7      V. cholerae O139 OSP-BSA      Edward   Ryan, MGH      61      MC10061-01        8      V. cholerae cytolysin (VCC)      Richelle   Charles, MGH      25      MC10025-01        9      Influenza A HA antigen   HA1(A/California/07/2009) (H1N1)      Immune Technology, IT-003-SW12p      18      MC10018-01        10      E. coli heat-labile toxin, human   isoform (LTh)      Dennis   Clements, Tulane University      55      MC10055-01        11      E. coli heat-labile toxin, B   subunit (LT-B)      Dennis   Clements, Tulane University      20      MC10020-01   
 
SECTION: Preparation of Standards and Controls
3. Standard and controls should be prepared in 1.5-mL microcentrifuge tubes, to allow for thorough mixing using a vortex mixer. 

SECTION: Preparation of Standards and Controls
4. Standard Dilutions
Using sample dilution buffer as a diluent, prepare an 8-step dilution series of the O1 Standard, beginning with a 1:10 dilution of the stock and then performing 7 x 4-fold additional dilutions. The resulting series will represent the following dilutions of standard: 1:10, 1:40, 1:160, 1:640, 1:2560, 1:10240, 1:40960 and 1:163840. 

Adjust the volume for dilutions based on the number of plates and/or antibody isotypes to be tested. 5 µL of each dilution will be used per replicate (384-well plate).
SECTION: Preparation of Standards and Controls
5. Controls
Prepare Positive and Negative Controls by diluting 1:100 in sample dilution buffer. Factor in any previous dilutions of the plasma stock. 5 µL of each dilution will be used per replicate (384-well plate).

Prepare an additional aliquot of sample dilution buffer in a 1.5 mL centrifuge tube to use as a blank (plate background measurement). 
SECTION: Sample Preparation
6. Sample dilutions are prepared in low-protein binding round-bottom 96-well plates to allow for use of multi-channel pipet when transferring samples to the 384 well plate.

Prepare plasma samples by diluting 1:100 in sample dilution buffer, factoring in any previous dilutions of the plasma sample. 

Using a multichannel pipet, add 148.5 µL of sample dilution buffer to 96-well plate. 

Mix samples briefly by pulsing on a vortex mixer and then using a P2 pipet, transfer 1.5 µL of plasma into buffer to make the 1:100 dilution.

Seal plate with a foil plate seal and place on a microplate shaker at room temperature to mix at 550 rpm while preparing bead stocks (approximately 30 minutes). 


SECTION: Preparation of Pooled Bead Stock
7. A 10x concentrated bead stock is prepared and mixed well before diluting to the final 1x stock concentration dispensed into the 384 well plate. The final 1x stock contains 810 beads for each bead region (antigen type) in a 45 µL volume. An overage of 20% is included to account for loss during pipetting. 

The table below provides an example calculation. 

     Column Letter (For Formula Reference)      B      C      C        Row Number (For Formula Reference)      Calculation/Raw Number      Example      Formula        30      number of samples      24      24        31      number of controls      12      12        32      replicate number      2      2        33      number of secondary Abs      1      1        34      total number of wells      72      =(C30+C31)*C32*C33        35      number of 384-well plates      0.2      =C34/384        36      number of beads/region/well      810      810        37      total number of beads + 20%      69984      =C34*C36*1.2        38      volume beads per well (µL)      45      45        39      total volume of 1x bead stock + 20% (mL)      3.888      =C34*C38*1.2/1000        40      total volume of 10x bead stock    (mL)      0.389      =C39/10   
 
SECTION: Preparation of Pooled Bead Stock
8. Prepare bead pool in a 50-mL centrifuge tube. Protect tube from light by wrapping in foil. 

Vortex bead stocks for 1 minute to resuspend beads and then sonicate for 2 minutes in an ultrasonic water bath at a frequency of 46 KHz. 

Add the predetermined volume of Bead Stock Buffer to the 50-mL tube for the 10x pooled bead stock, then add beads for each of the antigens in the panel. Vortex each bead stock for 5-10 seconds to mix immediately before making the transfer. 

Vortex 10x pooled bead stock for 1 minute, then add the remaining Bead Stock buffer to dilute bead stock to final 1x concentration. Vortex 1x pooled bead stock again for 1 minute to mix.


SECTION: Incubation Steps
9. Transfer half of the 1x pooled bead stock to a reagent reservoir and use a multichannel pipet to transfer 45 µL to the wells of the 384-well plate. Ensure that the bead pool in the reservoir remains mixed by pipetting up and down 3-5 times before each transfer. Mix the 50-mL tube for 1-minute before transferring the remaining portion of 1x pooled bead stock to the reservoir. 

SECTION: Incubation Steps
10. Using a 12-channel pipettor, transfer 5 µL of diluted sample from the 96-well plates to the 384-well plate. Cover plate with an opaque foil seal and incubate for 2 hours at room temperature on a microplate shaker set to 850 rpm. 
SECTION: Incubation Steps
12. After the 2-hour incubation, wash beads on a magnetic plate washer (e.g. Tecan HydroSpeed) using instrument dispense and aspiration rates suitable for magnetic beads. Use 6 cycles of 50 µL of Wash Buffer. 

An example wash protocol for a Tecan HydroSpeed Instrument is provided below: 

Grenier 384-well plate
Aspiration Rate: 1

Stage 1
Cycles: 1
Soak 
Intensity: Off Shake
Time: 90 seconds
Stage 2
Cycles: 6
Aspirate
Z-position: Bottom, Normal
Set: 8.0 [mm]
Time: 1 second
Head speed: 5 [mm/s]
Dispense
Z-position: Bottom
Set: 8.0 [mm]
Dispense rate: 50 [µl/s]
Channel: 2
Volume: 50 [µl]
Soak
Intensity: Medium Shake
Time: 15 seconds
Soak
Intensity: Off Shake
Time: 60 seconds
Cycles: 1
Aspirate
Z-position: Bottom, Normal
Set: 8.0 [mm]
Time: 1 second
Head speed: 5 [mm/s]
SECTION: Incubation Steps
11. Prepare a 1:150 dilution of each secondary antibody in sample dilution buffer at the tail end of the incubation step and protect from light. Prepare sufficient volume to cover addition of 40 µL per well plus 15-20% overage for loss during pipetting and vortex gently for 10 seconds to mix.  
SECTION: Incubation Steps
13. Using a reagent reservoir and a multichannel pipet, add 40 µL of secondary antibody to each well. Cover plate with a foil seal and incubate at room temperature for 1 hour on a microplate shaker set at 850 rpm.   
SECTION: Incubation Steps
14. After the 1-hour incubation, wash beads on a magnetic plate washer (e.g. Tecan HydroSpeed) using instrument dispense and aspiration rates suitable for magnetic beads. Using 6 cycles of 50 µL of Luminex Sheath Buffer and a final resuspension step with 60 µL of Luminex Sheath Buffer. 

An example wash protocol for a Tecan HydroSpeed Instrument is provided below: 

Grenier 384-well plate
Aspiration Rate: 1

Stage 1
Cycles: 1
Soak 
Intensity: Off Shake
Time: 90 seconds
Stage 2
Cycles: 6
Aspirate
Z-position: Bottom, Normal
Set: 8.0 [mm]
Time: 1 seconds
Head speed: 5 [mm/s]
Dispense
Z-position: Bottom
Set: 8.0 [mm]
Dispense rate: 50 [µl/s]
Channel: 1
Volume: 50 [µl]
Soak
Intensity: Medium Shake
Time: 15 seconds
Soak
Intensity: Off Shake
Time: 60 seconds
Cycles: 1
Aspirate
Z-position: Bottom, Normal
Set: 8.0 [mm]
Time: 1 second
Head speed: 5 [mm/s]
Stage 3
Dispense
Z-position: Overflow
Set: 8.0 [mm]
Dispense rate: 50 [µl/s]
Channel: 1
Volume: 60 [µl]

SECTION: Incubation Steps
15. Cover plate with a foil seal and sonicate in an ultrasonic water bath for 1 minute to disperse beads well before removing foil seal and loading on the Luminex instrument. 
SECTION: Luminex Run Protocol
16. Luminex acquisition settings:
Volume: 50 microliters
Timeout: 200 seconds 
Bead Type: MagPlex 
Plate Name: Greiner Luminex 
DD Gating: 7500 to 15000 
Reporter Gain: Enhanced PMT (High) 

Assign the antigens to their corresponding beads regions (see table in step 2). Set the target bead count to a minimum of 50 for each antigen.

We also typically add 4-8 alcohol flush steps at regular intervals throughout the 384-well plate, depending on how many samples are being tested. 
SECTION: Luminex Run Protocol
17. Run QC:

Retest plates if over half of the dilutions of standards had 5 or more antigens with a coefficient of variation greater than 20% (calculated from triplicate MFI measurements) 
Exclude any measurements with a bead count less than 30