Scientific Forum

Claudin in human colon

Hi BiCell,

It’s especially nice because we just finished making a polyclonal in rabbit against claudin-2 that works really well in both mouse and human on FFPE. As you may be aware, the available anti-claudin-2 antibodies (Invitrogen, Abcam, etc.) work poorly on FFPE. The Invitrogen monoclonal is fair by Western blot. Strangely, our claudin-2 polyclonal does not seem to be able to recognize mouse claudin-2 on Western blot. However, we have claudin-2 knockout mice and have validated that the staining is specific.
If you’d like, we’d be happy to test the serum on a TMA we have made that includes claudin-2 knockout mouse intestine, control mouse intestine, intestine from colitic mice (where claudin-2 is upregulated), and intestine from healthy and colitic humans. We’d also be happy to run your polyclonal rabbit anti-claudin-2 on the same TMA and provide you with pictures. These might be useful to you in promoting the product. From what I see on the page, it’s far better than anything else commercially available and I will publicly tell people that. Unfortunately, I will not be able to share my claudin-2 polyclonal with people because the yield was just too low (we only did two rabbits).
All these things are pretty easy for us, so it might be perfect collaboratively. It looks like the stained you did were on frozen tissue. We really want things to work on FFPE.
We could also test all these on Western blot if it would be helpful. We would use Caco-2 for human and isolated mouse intestinal epithelial cells.
Please let me know what you think, and congratulations on progress so far as well as your polyclonal rabbit anti-claudin-2 antibody.

Best wishes,
Jerry

Comments

  • June 15, 2022

    bicell-admin

    Hi Dr Turner,

    High-affinity binders for Cldn2-hum epitope are still elusive. We have found that the epitope peptide is highly hydrophobic, which might have rendered the enrichment step less effective or even inactive. We are beta-testing a new technology for this type of antigens. Since this epitope worked superbly for IF/IHC, we would like to stick to the epitope but find new ways to identify high-affinity binders. We will update you soon on progress of Cldn2-hum project.

    BiCell Team

  • June 15, 2022

    bicell-admin

    Hi Dr. Turner,

    Please review the attached progress report for project Cld2 (project 20210821 phase 3 progress report). Since Cld2 peptide is very hydrophobic, we have tested a new enrichment protocol and it worked out very well. We have found high affinity binders that will likely work for you applications.

    BiCell Team

  • June 15, 2022

    bicell-admin

    Hi BiCell,

    Li and I have carefully reviewed all the clones we have tested from claudin-2 projects. First, I want to congratulate you. Please be sure to echo this to the people in the labs at BiCell. For each of these targets I tried twice with Pro Mab. They been successful in making excellent ZO-1 and occludin antibodies with me, but failed twice each with MLCK 1 and claudin-2.
    You have been successful! I’m sure that part of this reflects your engine design approach and plasma cell enrichment method, but I really think that your overall skill is another critical difference.

    For the claudin-2 project, there are several that seem to work well on cultured cells and FFPE, even as initial supernatants. However, I think would be best if we did additional testing of purified/concentrated antibodies from anti-claudin-2 clones G2, G3, G4, G5, G7, E4, E5, E6, F4, B7, A8, and F7. I know this is 12 clones, and not 10. I have listed them in order of priority included a few that are strongly positive on your plate but were negative by immunofluorescence on cultured cells and FFPE.

    Thank you again, I am certain that these will be outstanding reagents for the entire scientific community once we validate them.

    Jerry

  • June 15, 2022

    bicell-admin

    Hi Dr Turner,

    We are very glad that your projects worked out nicely! Indeed, our approach is becoming attractive to customers who want high end antibodies such as FFPE or primary cilia or neuronal receptors. We think that our short-epitope technique combined with mAb selection can in the long term provide a precision toolbox for investigators to accurately read protein biology.

    BiCell Team

  • June 15, 2022

    bicell-admin

    Hi BiCell,

    We’ve now screened the claudin-2 antibodies. Several of them are outstanding.
    On tissue (FFPE), E5 is clearly the best on mouse and human intestine (bicell mAb cld2 E5 clone.pdf). Others (including G2 and G3) stain, but the background is high, especially on human tissue. On Western blots, G2 is much brighter than E5. Interestingly, it does seem to also pick up some non-claudin-2 bands while E5 is cleaner. Nevertheless, the improved sensitivity of G2 on Western blot makes it something that would be useful.
    So, for the mouse anti-claudin-2 monoclonals, I think we would like both clones G2 and E5.
    I looked back and found the attached is the most recent quote in my emails. Please correct me if there is a more recent quote I should be seeing. I have a question about a second hybridoma. The quote says that additional clones can be purchased for $750. Does that include purified antibody, as for the first clone that costs $4,462.50, or is there an extra charge for the second antibody? Please let me know.
    Once we have purified antibodies, we will do some more characterization. If you would like, we will provide the documentation to you for the Bi-Cell website. We will also begin using these antibodies in our publications immediately, which should help publicize them. I don’t know how widely people realize this, that certainly within the tight junction community it is well-known that available commercial anti-claudin-2 antibodies are really of poor quality.
    Also, as a separate point, I am really impressed with your approach. As I’ve said before, we tried multiple times previously to create monoclonal and polyclonal antibodies to claudin-2 and MLCK1. We have had limited success generating polyclonals and no success generating monoclonals. You have generated outstanding monoclonals to both (we are still trying to sort out which MLCK1 antibodies we want, but it does appear that there are some great ones in the group). I’d be happy to write a testimonial for your website if it would help and I look forward to continuing to work with you.

    All the best,
    Jerry

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"I am really impressed with your approach. We tried multiple times previously to create monoclonal and polyclonal antibodies to claudin-2 and MLCK1. We have had limited success generating polyclonals and no success generating monoclonals. You have generated outstanding monoclonals to both. I look forward to continuing to work with you."

Jerrold R. Turner, M.D., Ph.D.

Brigham and Women’s Hospital | Harvard Medical School

"The polyclonal antibody you generated for KIAA0408 is stunning! KIAA0408 is a novel cilium molecule that has never been studied. So, clearly there will be a lot of demand for it as we have discovered a very interesting finding and the story will be published in a high impact journal. I am strongly inclined to generate monoclonal antibody for this protein too and we should think about patenting it."

Univ.-Prof. Jay Gopalakrishnan PhD

Heinrich-Heine-Universität | Universitätsklinikum Düsseldorf

"Your ARL13B antibody works beautifully!!! We’re so happy to have a cilia-specific antibody made in rat! I can send you high resolution images to be posted on your website."

Julie Craft Van De Weghe, PhD

School of Medicine | University of Washington

"The assay is a homophilic interaction mediated cell adhesion on purified protein (in this case, immobilized purified Pcdhga9 to Pcdhga9 expressed on cell surface). Compared to control, cell adhesion is reduced in the presence of Pcdhga9 monoclonal antibody supernatants!"

Divyesh Joshi, PhD

School of Medicine | Yale University

"The rabbit hybridoma supernatants of anti-APOBEC3 project are tested positive by ELISA, and we are very happy about it! We previously tried a company, Abclone. Their Project "A" has immune response that is <10,000 titer in antiserum, which would explain why there is no positive mAb after fusion. Their project "B" didn't have any immune response in rabbit."

Harshita B Gupta, PhD.

School of Medicine | UT Health San Antonio

"We have tested anti mouse T cell antiserum samples from both rabbits you sent to us.

They worked very well! Thank you!"

Victoria Gorbacheva, PhD.

School of Medicine | Cleveland Clinic

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