20150220 – Set up biotinilation of purified IgG in MN

Objectives

Set up 2 new kits with MN sera.

• NAb™ Spin Kits, 0.2mL for Antibody Purification
• EZ-Link™ NHS-PEO4-Biotin, No-Weight™ Format

*All centrifugation at 5000G 1min RT

Methods

Purification of IgG:

1. Equilibrate column and buffers to room temperature. Set microcentrifuge to 5000 × g.
2. Snap off bottom closure and loosen cap on spin column. Place column in a 2mL collection tube, centrifuge for 1 minute and discard the flow-through.
3. Equilibrate column by adding 400µL of Binding Buffer to the column/collection tube assembly and mix briefly. Centrifuge the column and discard the flow-through. Repeat this step once.
4. Cap bottom of spin column with the included rubber cap, add 100ul of sera and 400ul of binding buffer and cap column.
5. Incubate column at room temperature with end-over-end mixing for 10 minutes, when volumes allow mixing to occur.
6. Loosen cap and remove bottom cap. Place spin column in new collection tube and centrifuge for 1 minute. Note: This first collection tube contains the nonbound sample components and can be analyzed to assess binding efficiency and capacity.
7. Transfer the column to a new collection tube. Wash column by adding 400µL of Binding Buffer. Mix briefly to suspend the resin and centrifuge for 1 minute. Repeat wash two additional times for a total of three washes.
8. Add 40µL of Neutralization Buffer to three collection tubes and place the spin column into one of the tubes.
9. Add 400µL of IgG Elution Buffer to the spin column, mix gently and centrifuge for 1 minute. Transfer the spin column to another collection tube that contains Neutralization Buffer, saving the collected solution as the first elution fraction. Repeat this step two times to obtain three fractions.
10. Determine which fraction(s) contain the purified antibody by measuring the relative absorbance of each fraction at 280nm.

To regenerate the used column for storage or re-use, add 400µL of Elution Buffer and centrifuge for 1 minute. Repeat three times. Wash resin several times with Storage Solution and store column at 4°C. Do not allow the resin to become dry. Typically, the immobilized protein column may be used up to 10 times without significant loss in binding capacity, although the actual number of effective usages may vary.

Quantification of purified IgG:

1. Nanodrop at 280nm
2. Comassie at 595nm

Biotinilation of purified IgG:

All centrifugation at 700G 2 minutes

1. Remove the bottom tab from the HisPur Ni-NTA Spin Column
2. Centrifudge and discard
3. Wash with 400ul PBS and centrifudge 2 times
4. Place the bottom plug in the column
5. Add the purified IgG and incubate 10 minutes
6. Lavar con 400ul PBS y centrifugar x2
7. Centrifudge and discard
8. Wash with 500ul PBS, invert and centrifudge 3 times
9. Apply the bottom plug to column
10. Puncture seal of one No-Weigh NHS-PEG4-Biotin Microtube with a pipette tip and dissolve tube contents by adding 200µL of PBS. Gently pipette up and down
11. Add 190ul PBS and 10ul of the NHS-PEG-biotin prepared solution
12. Disolver un microtubo de No-weight NHS (guardado a -20ºC) con 200ul de PBS
13. Cap top of column with a screw cap. Mix by gently flicking
14. Incubate 30 minutes
15. Centrifudge and discard
16. Wash with 400ul PBS and centrifudge 3 times and discard
17. Prepare elution buffer (50ul imidazole 4M +  950ul PBS)
18. Add 200ul elution buffer to the column and incubate it 10 minutes
19. Centrifudge and store the biotinilated IgG.

Results

There are no IgG biotinilated in elution 2, but there are in elution 1 tested by ICC (Fina).

Conclusions

After several tests performed by Fina, the conclusion is to perform another type of test to select the patients. One explanation is that the biotinilation kit only works for IgG(1-3) and not for IgG4.