TITLE: Split Luciferase Binding Assay (SLBA) Protocol
AUTHORS: Elzerackaityte, joe

[DESCRIPTION]
HiBit is an 11 amino acid protein tag that binds with high affinity to a larger subunit called LgBit. The bound complex has luciferase activity and will release luminescent signal in the presence of added furimazine substrate. HiBit is highly quantitative, extremely sensitive, and much faster than conventional immunoassays. When added to C-terminus of protein/peptide, the luminescence from tag ensures entire construct has been translated. The tagged protein/peptide is incubated with patient sera and antibody/antigen complexes are captured with protein A/G beads. Luminescence of captured complexes is measured. 


[BEFORE_START]
Make SLBA buffer
Prepare sepharose beads


[STEPS]
SECTION: Protocol overview
1. Step 1: Design insert within Hibit construct, DNA synthesis
Step 2: PCR amplify constructs
Step 3: In vitro transcription-translation 
Step 4: Quantify protein RLU, normalize protein
Step 5: First run an experiment for the positive control titrations and some other negative controls to determine if the protein of interest have any issues of sticking to the beads or the PVDF membrane. Run a titration for the serum.
·Relevant controls are: 
oProtein only 
oProtein + beads 
oProtein + negative serum or isotype control 
Step 6: Run SLBA: experimental samples + positive controls (commercial antibody or known positive serum) + negative controls (healthy control sera) + blank wells.

SECTION: I. Design insert with Hibit Construct
2. ·Design insert in frame with the construct below

 

This construct contains 5’ CMV promoter, T7 promoter, Kozak. In 3’ contains GS linkers, Hibit (underlined), stop.
SECTION: I. Design insert with Hibit Construct
3. ·Order from oligo synthesis company (e.g. IDT, Twist)
SECTION: II. PCR amplify construct
4. Resuspend lyophilized DNA to 1uM (can do less, but test PCR first)
SECTION: II. PCR amplify construct
5. Prepare 100uL PCR reaction per construct as below:

 
          1x            Water         72            5x PhusionHF Buffer         20            10nM dNTP         2            10uM Ultra Hibit FW         2            10uM Ultra Hibit REV         2            Phusion HF         1      

PCR primers: 


 

 
Add 1uL 1uM lyophilized DNA into 99uL of master mix (can use a lot less, test depending on your construct)
SECTION: II. PCR amplify construct
6. Run PCR program in thermocycler:

 
 
       Temp         Time         No.   Cycles            98         2 min         1            98         30s         25            68         30s            72         30s            72         5 min         1            10         inf         1      


SECTION: II. PCR amplify construct
7. Optional: Purify PCR with Ampure XP SPRI beads, using 1x volume beads : PCR ratio following manufacturer’s instructions. Elute in 50uL water.
Optional: Using Qubit HS DNA quantification kit, quantify DNA following manufacturer’s instructions. 
Optional: run gel to confirm product is correct size (recommended if first time)

 

SECTION: III. In vitro transcription translation (TNT)
8. Optional: Normalize purified amplicon DNA to 0.125ug/uL

 

SECTION: III. In vitro transcription translation (TNT)
9. Using rabbit reticulocyte lysate (Promega quick couple Kit) prepare master mix. One TNT master mix is good for 4 rxn + 1 neg control (no DNA) 



 
 
 
       TNT rabbit   reticulocyte master mix         40                PCR enhancer         1                Methionine (non-radiolabeled)         1                   42         ul MM /well                8         DNA (0.125 ug/uL)      

 

SECTION: III. In vitro transcription translation (TNT)
10. Transfer to the PCR tubes in the thermocycler to proceed with transcription/translation. Incubate for 3h at 30C
 

SECTION: IV. Normalize protein RLU
11. Dilute protein 1:100 in SLBA buffer, perform each RLU measurement in triplicate

SECTION: IV. Normalize protein RLU
12. Add 50uL diluted protein/well to 96-well white flat bottom plate
SECTION: IV. Normalize protein RLU
13. Add 50uL luciferase reagent/well. Prepare master mix (shown here for 1 reaction): 
 
       Nano Glo  Hibit Lytic Reagent         50            Hibit substrate         1            Lg Bit  protein         0.5      

SECTION: IV. Normalize protein RLU
14. Incubate 30 min RT dark, measure counts using luminometer
SECTION: IV. Normalize protein RLU
15. Dilute protein with SLBA buffer to add 2e6-2e8 RLU per well 
 

SECTION: V. SLBA Day 1
16. Calculate the amount needed for 2e6-2e8 RLU protein per well and RLBA buffer in total volume of 50uL per well. Dispense into PCR plate.
SECTION: V. SLBA Day 1
17. Add 1-5ul of serum to antigen+buffer 

 

SECTION: V. SLBA Day 1
18. Orbital shake 15 min 500rpm at 4C, incubate overnight 4C.
SECTION: V. SLBA Day 1
19. Block the 0.2um PVDFFilter 96-well Plates PVDF plate with 200ul of RLBA buffer overnight at RT
SECTION: VI. SLBA Day 2
20. Remove wash buffer from blocked PVDF plates by placing on vacuum manifold, seal bottom of plate. 
SECTION: VI. SLBA Day 2
21. Using wide bore P200 tips, add 25ul of beads/well (1:1 protein A:G; SLBA buffer to beads should be 1:1 ratio) to filter plate
SECTION: VI. SLBA Day 2
22. Add 55ul of serum + Ag mix to each well that contains the beads
SECTION: VI. SLBA Day 2
23. Seal the plate with foil cover and orbital shake (500rpm) in the cold room for 45mins.
SECTION: VI. SLBA Day 2
24. Place PVDF plate on vacuum fold with gentle vacuum to remove unbound antigen.
SECTION: VI. SLBA Day 2
25. Add 200ul of SLBA buffer to each well, repeat for a total of 3 washes

SECTION: VI. SLBA Day 2
26. Add 150uL SLBA buffer. Seal the bottom plate with foil cover. 

SECTION: VI. SLBA Day 2
27. Orbital shake (500rpm) for 5 mins at 4C. 

SECTION: VI. SLBA Day 2
28. Connect the vacuum seal and wash it with 200ul of SLBA buffer, repeat for a total of 3 washes

SECTION: VI. SLBA Day 2
29. Release the pressure after wash and put the paper towel on the vacuum seal and applied pressure to remove excess fluid. Do it until almost little buffer are seen on the paper towel from the vacuum pressure. 

SECTION: VI. SLBA Day 2
30. Gently place a white seal on bottom of plate

SECTION: VI. SLBA Day 2
31. Resuspend beads in 50uL SLBA buffer

SECTION: VI. SLBA Day 2
32. Add 50uL luciferase reagent (prepared as above) to each well, mix

SECTION: VI. SLBA Day 2
33. Incubate 1h room temperature in the dark

SECTION: VI. SLBA Day 2
34. Read counts using luminometer