Great to have you all here. I thought that sharing the kits and reagents that we use in the lab might help our field be more consistent and unified. I’ll start with some of the kits and techniques that we have found works:
HBsAg and HBeAg ELISAs - We have purchased ELISA kits from Wantai Biopharm kits and they seem to work quite well (though some dilution required to get into the linear range for measuring supernatant from infected cells). The kits are cheap too!
cccDNA quantification - We use cinqPCR: https://en.bio-protocol.org/en/bpdetail?id=3986&type=0, and A novel method to precisely quantify hepatitis B virus covalently closed circular (ccc)DNA formation and maintenance - ScienceDirect
HBc IF - With DAKO discontinued, this has been an issue with us. But Cell Marque has been working well for us: Hepatitis B Virus Core Antigen Rabbit Polyclonal Antibody
Anyone got any other kits, antibodies, etc. they like to use? I’m looking forward to your hints, tips, and tricks for your research!
On our side in the Matthews group we use:
HBsAg and HBeAg ELISA: Ig Biotechnology ones HBsAg CLIA Kit – Ig Biotechnology
They are re-sellers of the Autobio ones so going directly to Autobio can be cheaper, however from experience i had difficulties in making contact with their sells team + their shipment are often stoped at border control (last one our manager had to argue on a daily basis for 2months with border control to get them in the country…). so for ease we use the IgBiotech ones. And they are keen to decrease the prices significantly when buying bulk (20 kits) so we get them at 150-170£ if i remember correctly.
HBV DNA quantification: we use this Taqman kit Product Details
cccDNA quantification: we unfortunately do not use your method, but only because i was used to the pK extraction + T5 one (with recent update here https://gut.bmj.com/content/72/5/972) and if i’m correct you found similar results than with the cinqPCR?
Sequencing for serum samples: we are currently still trying to get a better sensitivity for lower VL (<3log) and there should be a last iteration of the protocol soon out, however the last clean one with our pangenotypic capture probes is here: https://www.biorxiv.org/content/10.1101/2023.02.20.529276v1
We use the Kappa ProbeForce Universal qPCR mixes for amplifying partially purified HBV DNAs during our drug screening studies. it is the most contaminant-tolerant qPCR mix we’ve tried. We also use the Qiagen Protease as a reliably inactivatable protease (70C for 10 min).
To continue from John’s post the Tavis Lab also uses the following:
HBV particle assay and HBcAg Western Blot - We have found that the 2AHC24 (Hyb- T2221) antibody from Tokyo Future Style/ Institute of Immunology works well at 1:1000. But this antibody has not worked in our hands for IF.
Yep, we did! We just find it a bit cleaner to do the normalisation as a duplex reaction, rather than separating out into exo+ and exo- reactions.
Cool! What DNA extraction technique are you using? Something cheaper that QIAGEN column-based kits would be great for those times that we’re not doing something that requires high yield/sensitivity/purity.
Thanks @Daniel.bradley. Yep, I talked to a few colleagues about the troubles we were having in HBc WBs at the international meeting and this antibody came up. We ordered it before I got back from Japan and are expecting it soon. It also detects HBeAg and other core-related antigens, right?
Please keep sharing, this is great!
And I’m not necessarily limiting this thread to wet lab. For example, whenever I need to know if something (DNA, protein, etc.) will be highly conserved over the different HBV genotypes, my first point of call is hbvdb (HBVdb::Home - Hepatitis B Virus Database; citation: HBVdb: a knowledge database for Hepatitis B Virus | Nucleic Acids Research | Oxford Academic), then downloading all sequences to do an alignment in Jalview. Super useful!
And please feel free to post requests. I’ll start: I’m looking for a good and simple core-protection assay for detecting intra-capsid RNA/DNA. Any suggestions?
Our 96 well qPCR assay for HBV DNA is detailed in Li, Q., Edwards, T.C., Ponzar, N.L., and Tavis, J.E. (2021). A mid-throughput HBV replication inhibition assay capable of detecting ribonuclease H inhibitors. J. Virol. Methods **292:**114127. Note that this only partially purifies the DNA, and for reasons we don’t understand, the plates need to be used for qPCR right after the prep is done (freezing or extended incubation ad 4C causes poor data).
Does anyone do non-radioactive Southern blots for HBV? We’ve got a series of them to do, and we’re not getting adequate sensitivity from the DIG probe labeling approach. Any tips would be appreciated.
You might want to ask @LenaAllweiss - she’s the senior author of this: A sensitive and rapid Southern blot assay based on branched DNA technology for the detection of HBV DNA in cell culture and liver tissue samples – ICE-HBV
Thomas is right, we use branched DNA probes for our Southern blots. We found that they´re much more sensitive (and easier to handle) than the DIG probes. The protocol should be pretty detailed, but if you have more questions don´t hesitate to write to me.
Let me know if it works!