Conjugation of anti-ORF1 Dynabeads and attempt to reduce IgG leakage
Date: 09/08/21
Dynabeads M-270 Epoxy (Invitrogen Dynal, Cat. No. 143.02D); Beads were from NL (lot #00886548, expiration date 2021-11-30)
Desalted antibody: Combine the rest of two batches of Abmart (5310-1-4M1234 4H1-140313, received on 9/1/2021, 0.7mg/ml, 100 mM Glycine, 50 mM Tris, with no Proclin 300)
Concentrate the antibody first by reducing volume in speed vac and then desalt into 0.1M NaPhosphate, pH7.4, using Zeba 7k columns (0.5ml). Did two batches of 700ul each. Combined the desalted antibody, total volume 410ul, final concentration 1.86mg/ml (762ug total). Will use this to make 25mg of beads @ 15ug or 10ug antibody per mg of beads.
Antibody recovered by AmSO4 precipitation
1 tube of anti-ORF1 antibody recovered from old antibody mix; 2.8mg/ml, 400ul in 0.1M NaPhosphate (1120ug total, enough to make 75mg of beads @ 15ug/mg of beads)
1 tube of anti-ORF1 antibody recovered from by precipitating new ORF1 antibody by increasing AmSO4 concentration to >3M and then resuspended in 0.1M NaPhosphate. 1.2mg/ml, 250ul in 0.1M NaPhosphate (total 300ug; Take 250ug of antibody to make 25mg of beads @ 10ug per mg of beads).
Make antibody mix separated, take 2ul of each antibody mix before adding it to beads
1) Recovered old antibody_15 (ug Ab per mg of beads)_pre
2) AmSO4 precipitated new antibody_10 (ug Ab per mg of beads)_pre
3) Desalted new antibody_15 (ug Ab per mg of beads)_pre
4) Desalted new antibody_10 (ug Ab per mg of beads)_pre
All beads are equilibrating with 0.1M NaPO4, pH7.4
Make 20 or 25mg aliquots based on amounts of beads to make
Take 2ul of antibody mix before coupling for gel analysis
1) Recovered old antibody_15 post
2) AmSO4 precipitated new antibody_10 post
3) Desalted new antibody_15 post
4) Desalted new antibody_10 post
Add antibody mix to beads pre-equilibrated with 0.1M NaPO4, pH7.4; mix well
Leave the tube on a Thermomixer at 37°C (1100rpm), O.N. (18 hr)
Take 2ul of antibody mix after coupling for gel analysis
Keep the rest of the antibody mix @ 4°C
Wash the beads 1x 1ml 100mM Glycine-HCl, pH 2.5
Wash the beads 1x 1ml 10mM Tris, pH 8.8
Wash the beads 1x 1ml fresh 100mM Triethylamine
Wash the beads 4x 1ml 1x PBS (5’ each)
Wash the beads 1x 1ml 1x PBS + 0.5% Triton X-100, 5’
Wash the beads 1x 1ml 1x PBS + 0.5% Triton X-100, 15’
Resuspend beads in 50% Glycerol/1xPBS/0.5mg/ml BSA for long-term storage at -20°C
Testing new anti-ORF1 beads
Date: 09/08/21
Cell line: MT302 Dox 24-hour induction (8/26/21)
Extraction: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors)
Scale: 50mg per IP reaction with 5ul of anti-ORF1 beads
Weigh out 2x 125mg of MT302 powder, add 500ul of extraction buffer to each tube (1:4 w/v), vortex to mix
Sonicate 5 x 2 sec at 2 Amp;
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Split clarified lysate into 5 equal parts and set up 5x 50mg IP reactions with 5ul anti-ORF1 beads
IP @ 4°C for 30’
wash 3x 1ml extraction buffer
Switch beads to fresh tubes at 2nd wash step
Elute the beads with 10ul of 1.1x LDS, 70°C for 5’ with mixing
Save 1ul aside (E, in case for Western)
Add DTT to 50mM to all samples
Heat the samples @70°C for 10’
Run 4-12% Bis-Tris gel to analyze antibody mix pre- and post-coupling and anti-ORF1 IP elution
1) Marker
2) Recovered old antibody_15 pre
3) Recovered old antibody_15 post
4) AmSO4 precipitated new antibody_10_pre
5) AmSO4 precipitated new antibody_10_post
6) Desalted new antibody_15 pre
7) Desalted new antibody_15 post
8) Desalted new antibody_10 pre
9) Desalted new antibody_10 post
10) E_anti-ORF1_Old_052721
11) E_Recovered old antibody_15
12) E_AmSO4 precipitated new antibody _10
13) E_Desalted new antibody_15
14) E_Desalted new antibody_10
15) BSA_150ng
All new anti-ORF1 beads have IgG leakage problem. From the gel above, beads made with AmSO4 precipitated new antibody @ 10ug per mg of beads and desalted new antibody @ 15ug per mg of beads have worse IgG leakage. Combine those tubes and repeat all wash steps in conjugation protocol.
Wash the beads 1x 1ml 100mM Glycine-HCl, pH 2.5
Wash the beads 1x 1ml 10mM Tris, pH 8.8
Wash the beads 1x 1ml fresh 100mM Triethylamine
Wash the beads 4x 1ml 1x PBS (5’ each)
Wash the beads 1x 1ml 1x PBS + 0.5% Triton X-100, 5’
Wash the beads 1x 1ml 1x PBS + 0.5% Triton X-100, 15’
Resuspend beads in 50% Glycerol/1xPBS/0.5mg/ml BSA for long-term storage at -20°C
Testing beads again after attempt to reduce IgG leakage
9/13/21 (same protocol as 9/8/21 above)
Cell line: MT302 Dox 24-hour induction (8/26/21)
Extraction: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors)
Scale: 50mg per IP reaction with 5ul of anti-ORF1 beads
Anti-ORF1 beads tested:
1) Old beads_040921
2) New beads_081621_with extra wash
3) New beads_090921_recovered old antibody with extra wash
4) New beads_090921_new antibody with extra wash
4-12% Bis-Tris gel to check elution
1) Marker
2) Old beads_040921
3) New beads_081621_with extra wash
4) New beads_090921_recovered old antibody with extra wash
5) New beads_090921_new antibody with extra wash
6) BSA_50ng
7) BSA_150ng
It looks like that extra wash steps help reduce IgG leakage. Will do extra wash to the rest of the beads made on 8/16/21 and 9/9/21.
Also, beads made with new antibody have more IgG leakage even after extra wash. Will check beads made with new antibody @ 10ug antibody per mg of beads. Those beads have less IgG leakage comparing to beads made with new antibody @ 15ug antibody per mg of beads.
9/14/21 Making more anti-ORF1 beads
Made more beads with new anti-ORF1 antibody recovered from antibody mix from previous round of conjugation (9/9/21). This time use antibody @ 10ug per mg of beads, 37°C, overnight (18 hours) on Thermomixer @ 1100rpm.
9/15/21
Wash beads coupled on 9/14/21. At the same time, repeat the wash steps with remaining beads made on 8/16/21 and 9/9/21 without adding extra washes previously.
Test beads again using MT302, same protocol as before.
Scale: 50mg per IP reaction with 5ul of anti-ORF1 beads
Anti-ORF1 beads tested:
1) Old beads_040921 (control)
2) New beads_081621_with extra wash
3) New beads_090921_desalted new antibody (10ug antibody per mg of beads)_ with extra wash
4) New beads_091521_recovered new antibody (10ug antibody per mg of beads)
4-12% Bis-Tris gel to check elution
1) Marker
2) Old beads_040921 (send 400ul to NL)
3) New beads_081621_with extra wash
4) New beads_090921_recovered old antibody with extra wash (10ug antibody per mg of beads)
5) New beads_091521_recovered new antibody (10ug antibody per mg of beads) (send 400ul to NL)
6) BSA_50ng
7) BSA_150ng
8) Marker
9) Antibody mix pre-coupling_091421
10) Antibody mix pre-coupling_091521
Will send 400ul of old anti-ORF1 beads (04/09/21) and 400ul of new anti-ORF1 beads (09/15/21) to NL.
Conclusion:
1) New batch of anti-ORF1 is fine for making beads
2) New anti_ORF1 (4H1) received on 9/1/21 is in 100 mM Glycine, 50 mM Tris, with no Proclin 300. The concentration is 0.7mg/ml same as Abmart claimed. It needs to be concentrated and desalted before using it to make beads. You can put the antibody in the speed vac to increase its concentration first and then using Zeba desalting column to buffer exchange it into 0.1M Sodium Phosphate, pH7.4. You can also use ammonium sulfate to let antibody precipitate out, then resuspend the pellet in a smaller volume in order concentrate the antibody. But the recovery is lower then concentrating followed by desalting.
3) New side by side comparison showed using 10ug antibody per mg of beads is sufficient for making anti-ORF1 beads.
4) Save the antibody mix after beads conjugation and recover antibody by increasing ammonium sulfate concentration. You can safely reduce the volume of antibody mix to 1/3 (final ammonium sulfate concentration will be 3M). The recovered antibody is as good as, if not better than freshly desalted antibody for making beads.
5) IgG leakage can be reduced by adding extra wash steps to already conjugated beads (repeat all wash steps in conjugation protocol). After subject to extra wash steps, beads need to be tested to make sure the binding efficiency to target protein is comparable before and after extra wash steps.