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PLEASE READ: My recommendation for alternative antibodies is to use 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).
- HyHEL-10 as an antibody:
- Sequence available
- Binding site information available
Budget:
| Item | Cost |
|---|---|
| HyHEL-10 kappa chain gBlock (784 nt) | $149 |
| HyHEL-10 VH gBlock (472 nt) | $89 |
| Hen egg lysozyme (1g) - Merck-Millipore | $39 |
| VRC01 kappa chain gBlock (772 nt) | $149 |
| VRC01 VH gBlock (496 nt) | $89 |
| HIV1 gp120 JRCSF (50ug) - Eenzyme | $259 |
| TOTAL | $774 |
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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)
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mAb database: HIV Molecular Immunology 2002 (same as this)
mAb name: 0.5beta - from mouse
Sequence: IMGT
Epitope (recognition sequence):
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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
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