Useful Reminders/Links

Requirements:

1. Antibody amino acid (or DNA) sequence is available.
2. Antigen (for activating system) is commercially available at a reasonable cost.
3. Antigens should not significantly affect our mammalian cells other than their involvement in activating our system (for example, avoid EGFR or TNF-alpha as antigens).  Additionally, antigens shouldn't present significant health dangers to us (for example, anthrax spores).
4. Antigen-antibody interaction should be well-characterized.

Leader sequences

Summary Table

PLEASE READ:  After a long search, there are two antibodies against two different antigens that I can recommend for testing our system.  For future reference, when searching for antibodies here are some tips:

• Most antibody databases are woefully incomplete so they actually aren't a great place to look for antibodies.  They do give you a good idea of what kind of antigens people are raising antibodies against, though.
• If you have an antigen in mind, searching PDB can get you to some antibody names.  Then use those antibody names to do a deeper literature search to see if you can find the sequence or binding information you need.
• Cross-check peptide sequences from companies that sell your antigens to make sure the sequence matches that of the antibody's binding site.  Try to do this early on or you might be sad.

My rationale for recommending the alternative antibodies HyHEL-10 (anti-hen egg lysozyme) and VRC01 (anti-HIV1 gp120):

• Chicken lysozyme and HIV1 gp120 as antigens:
  • There are many antibodies against both of these antigens, a number of which are monoclonal, sequenced, and have been studied by NMR or crystallography, so there are multiple options to choose from.
  • They are commercially available for not too expensive (chicken lysozyme is a cheap enzyme and gp120 is about the price of an antibody).
  • They can probably be handled at BSL1 or at least at BSL2 (gp120 can probably be handled at BSL1 since active HIV can be handled at BSL2 and gp120 shouldn't infect cells).
  • They should have minimal effects on HEK293 cells (hen egg lysozyme works on bacterial cell walls and gp120 binds CD4, which is an immune receptor so probably not expressed at high levels in HEK293).
  • Chicken lysozyme has been studied extensively in antigen-antibody interactions (so it's a good model system) and detecting HIV1 gp120 could be useful in gene/cell therapy against HIV.
• HyHEL-10 as an antibody:
  • Sequence available
  • Binding site information available (and corresponds to sequence of commercially available hen egg white lysozyme)
  • Potential problem:  antibody originally generated in mouse
• VRC01 as an antibody:
  • Sequence available
  • Broadly neutralizing - According to the discovery study, this antibody neutralized the sera of about 90% of HIV patients tested, which means that even though commercially available HIV gp120 peptides come with a variety of sequences this antibody will probably bind them all.
  • Binding kinetics are available for various strains of HIV1 (good for modeling!)
  • Some binding site information available (alanine scanning)
  • Potential problem:  some small discrepancies between sequences of commercially available gp120 peptides and the proteins used in the study

I still have 0.5beta on this page as a backup antibody for gp120.  VRC01 is better though because it can recognize gp120 from many different strains.  Also, 0.5beta has a recognition sequence that isn't in exactly in commercially-available peptides.  There's a one amino acid substitution which hasn't been studied in the literature.

CLONING STRATEGY:

Cloning steps for each heavy and light chain are in Geneious.  Amino acid sequences were back-translated using IDT and BsaI cut sites were manually eliminated.  Since we had issues with mutations when we ordered the gantenerumab heavy chain as one gBlock and because ordering the entire heavy chain costs ~$

BUDGET:

Plus oligos for PCR of IgM heavy region

Hen Egg Lysozyme

Chicken lysozyme derives from chicken egg white.  Breaks down bacterial cell walls.

mAb name:  HyHEL-10 (from mouse)

Sequence:  this paper

Binding interactions: this paper (interacts with discontinuous parts of peptide)

Peptide sequence: UniProt

Cross-checked peptide sequence with antibody binding interaction sites.  None of those sites have known variants/mutations.  However, peptide numbering is different on UniProt and in binding interactions paper (18aa difference).

Peptide suppliers:

Sigma-Aldrich:  1g/$49.50

Merck-Millipore:  1g/$39

HIV1 gp120 (HIV envelope glycoprotein)

gp120 is a glycoprotein derived from gp160.  gp160 is processed into gp120 and gp41, which together form a complex that binds to CD4, permitting the fusion of viral and cell membranes.

gp120 antibodies review

Peptide suppliers:

Full-length gp120 peptides are not readily available.  Check the manufacturing details to ensure that the epitope is included in the peptide.  Also check that peptide comes glycosylated.

HXB2 strain:

Abcam:  10ug/$228

MyBioSource:  50ug/$365

YU2 strain:

MyBioSource

JRCSF strain:

Abnova:  50ug/$770

MyBioSource:  50ug/$365

Eenzyme:  50ug/$259

0.5beta

VRC01

Really cool paper about gp120 single-chain antibodies in E. coli

Everything you ever wanted to know about this antibody:

Antibody identification paper (and supplementary materials, which includes sequences) - isolated from human

• Supplementary materials includes VRC01 variable regions' amino acid sequences
• Supplementary materials includes HXB2 strain gp120 core reference sequences that were used for screening antibodies (Fig. S1B).  They consisted of the core sequences separated by GAG linkers.  
   There is a mutation from serine at position 334 in commercial peptides (from env HXB2 sequences) to alanine in this paper, but:
  • Many of the gp120 core mutants they designed and analyzed computationally had a threonine at this position (334), including the one they eventually chose for in vitro screening (RSC3).  Based on this information, it seems that the amino acid present at this position isn't critical to binding, and that binding can still happen even when alanine is replaced by a hydroxyl-containing amino acid like threonine (so by extension maybe serine is ok too).
  • The reference sequence for YU2 strain gp120 also has a serine at the position corresponding to 334 in HXB2 (contained within a conserved amino acid sequence), and VRC01 is shown to have bound YU2 gp120 (wild-type) well (Table S1).
  • The position in JRCSF strain gp120 that corresponds to 334 in HXB2 is not identified as a binding site in the binding information paper (see binding information paper, Fig 1 - in fact it's not even included in the table).
• Has numbers about binding interactions (including Kd!)
  • SPR
  • ITC
  • Competition ELISA

Binding information

• Includes alanine scanning table

Structural analysis

• Most natural resistance a result of variation in V5 region