DIY Lab Setup

[Inventoriffic]

Has anyone run their own ELISA's at home? It's been a few years since
I did them at the lab and was wondering if there were any cheap ways
to run them for some experiments I want to run.

I don't mind buying the equipment, but I should be able to build
anything (software + hardware) that is needed, and from a cursory look
at it, it doesn't seem that hard to automate the entire thing (xy
plotter + auto pippette, light source + something like
http://blog.makezine.com/archive/2008/11/safety_spectrometer.html#more).

Any ideas/pointers welcome.

[Aaron Hicks]

I'm tooling up to do this; right now, everything's on fire-sale, deep-discount prices on eBay. Fewer people are buying, but the dealers still need to pay their mortgages so the list prices keep dropping.

-AJ

[Inventoriffic]

What sort of machines are you looking at? Got any links for some good
prices?

On Mar 23, 8:42 am, Aaron Hicks <aaron.hi...@gmail.com> wrote:
> I'm tooling up to do this; right now, everything's on fire-sale,
> deep-discount prices on eBay. Fewer people are buying, but the dealers still
> need to pay their mortgages so the list prices keep dropping.
>
> -AJ
>

[Aaron Hicks]

Me? I just check eBay now and again until I find what I need. Gel box: $9.95 + $15 in shipping. When I got it, it was in near-perfect condition. Same model off the company's website: $500. Now I need a power supply, so I'm just patiently waiting for one to meet my criteria.

Small shaker table for under $100 delivered. Genie vortexer for under $100 delivered. A big box of what may be the last gel immunodiffusion plates available in the free world for under $15 + shipping (the method has largely been supplanted with electrophoresis, but I don't need the sensitivity). I need a decent spectrophotometer, so I'm sitting on that just right now. I have a suite of pipettors and snagged a case of small tips, as I already have the big ones. Refurbished centrifuge for under $100. A whole pile of hematology stuff for under $100.

Back when I was in research, all this would have set me back substantially more. I'd hate to guess how much, but the full lab- including the spectrophotometer and the power supply- would probably be $4000-5000 new, ballpark, depending upon how nice the shaker table was. For an incubator, I use a foam plastic cooler and a thermostatic controller (eBay!) with a 20-watt light bulb. It gets the job done. The ELISA strip reader was free; I hit up a company for a donation, stating I was researching a specific disease, and they offered up a used model that had been a trade-in. So, that'd be another $1200, minimum. The other equipment- pressure cookers, forceps, bead sterilizer, etc.- I all had on hand. Petri plates and various bacto media come from eBay or from the local vendor; they're not cheap, but they sell to individuals.

I've always been a scrounger- I have astonished my employers in the past with the things I can come up with. It's improvise, adapt, and overcome when you're doing bargain-basement research. Still- I'd put my results up against those of a commercial laboratory, even in the bio realm. For chemistry, I've managed all kinds of stuff in previous years- a gas chromatograph (virtually unused), a polarimeter (for pennies on the dollar), osmometers, all kinds of stuff to aid me in my research.

None of this is new. When doing research, I met the wife of one of the lead investigators for whom we were doing contract work. She said when she first met him, he came over to his home- an old mansion- that had a scanning electron microscope in the basement. That would have been... 1998, IIRC. At the time I met him, he was working for a large, acronymic federal agency with no graduate degree. There is now a room named after him at one of the federal research agencies. Quite a guy, and he was a bargain-basement researcher himself.

-AJ

[Inventoriffic]

Hi AJ,

Thats amazing. You must spend a lot of time on ebay! I would pay for
such a service :)
In terms of ELISA's, what would be the cheapest way of running them do
you know?

[Aaron Hicks]

I wouldn't say I spend a *lot* of time; when I need a new piece of instrumentation, I just search to find ballpark prices. I drop a bid, and don't look back. None of this sniping or other stuff; if I win it at the price I want, so be it. If nothing suits me, I come back in another few days.

As for cheap ELISAs- my advice will be skewed as I'm mainly looking at gel electroimmunodiffusions, so I'm not looking for bands or anything right now. Later I'll be looking at quantifying antibodies, but not just right now.

First- buy or make a gel box. I prefer buy, as I figure if I can get one for $30-60, that's worth saving 1-4 hours of my time scooting around town to buy parts and stuff- and then having extra methylene chloride around the lab, slowly volatilizing out of the bottle. Then a power supply- not a DIY sort of thing for my money.

Everything else is pretty routine- hot water for melting the agarose (which can be bought off the web), pipettors off eBay for loading the gels. I need to make antigen, and that's a matter for the shaker table, some Erlenmeyers, the media, the autoclave, and so forth. I don't have a supplier yet for the reagents, but I'm not quite there yet; I'm still working on generating the antigen. That's what the gel immunodiffusions are all about; they don't make those components anymore, so to make the wells you can either make a die for cookie-cutting the holes with a suction apparatus, or use a positive mold (from brass or aluminum) when you pour the plates.

Instead, I made a positive mold out of glass tubes, and then cast a negative mold out of silicone (again, from eBay); from this negative mold, I can cast as many positive molds as I want to out of silicone so I can pour as many immunodiffusion (Ouchterlony, or "ouch") plates as I want.

Quick, cheap, easy- Ouch plates are all three. Different Ouch plates can even be used to quantify antibody response without any need for additional reagents. The whole spectrum of tools afforded by gel immunodiffusion- and gel electroimmunodiffusion- has largely been passed by, but there's no reason why they should be ignored. It's a lot of bang for the buck.

-AJ

[Jake]

Speaking of bang for the buck... I've had great success using food
grade agar for petris. The quality seems to vary a bit as far as the
color of the agar, but I've never had a problem with it gelling, even
making "soft" agar plates.

I haven't tried doing electrophoresis with food grade agar yet, but I
imagine that even if the quality and gel strength varies a bit it
should at least be consistent enough across the gel slab.

Since food grade agar is SO much cheaper and usually locally available
at food co-ops or asian markets it makes sense to use it where ever
possible.

-Jake

[Aaron Hicks]

Some of the older texts (1960s and 1970s) discuss purification of agar to increase clarity. It's a pretty involved process, but if anybody would like the references so they can go read about it themselves, I can look them up.

The plant tissue culture grade stuff is a notch up from food grade; not as readily available, of course, but certainly less expensive than highly purified agarose.

-AJ

[Meredith L. Patterson]

I would be interested in these. Clearer agar is easier to look through
from above when incubating plates upside down.

Cheers,
--mlp

[Tom]

here is a good washed agar protocol, cleans up contaminants. It
involves acetone, easy to find at home depot, but dont be smoking.
Also the washed agar will gel at a lower concentration, 1% instead of
1.5% or 2%. As store bought stuff is less pure than Difco, it would
certainly benefit more from cleaning. I have only done the second
protocol.

http://www.fgsc.net/neurosporaprotocols/How%20to%20wash%20agar.pdf

Tom

On Mar 23, 7:57 pm, "Meredith L. Patterson" <clonea...@gmail.com>
wrote:

> I would be interested in these. Clearer agar is easier to look through
> from above when incubating plates upside down.
>
> Cheers,
> --mlp
>

> On Mon, Mar 23, 2009 at 6:35 PM, Aaron Hicks <aaron.hi...@gmail.com> wrote:
>
> > Some of the older texts (1960s and 1970s) discuss purification of agar to
> > increase clarity. It's a pretty involved process, but if anybody would like
> > the references so they can go read about it themselves, I can look them up.
>
> > The plant tissue culture grade stuff is a notch up from food grade; not as
> > readily available, of course, but certainly less expensive than highly
> > purified agarose.
>
> > -AJ
>

[Bryan Bishop]

On Mon, Mar 23, 2009 at 6:57 PM, Meredith L. Patterson
<clon...@gmail.com> wrote:
> I would be interested in these. Clearer agar is easier to look through
> from above when incubating plates upside down.

I don't mean to hijack the thread, but it's been a while since I've
had a chance to mention a neat table that I came across the other day
that lists alternatives to agar- like wheat flour, laundry starch,
potato powder, rice powder, etc. IIRC, historically potato was used
first for culturing little critters, but now I don't know whether or
not that would be useful if you're looking for clear agar ;-). You
could probably take thin slices of potato and then shine a light
through them, but does that count?

http://heybryan.org/books/papers/graph1_alternate_agar_formula.png

- Bryan
http://heybryan.org/
1 512 203 0507

[Tom Knight]

Silica gel is another possible solidifying agent. Likely totally
inert, and stable at high temperatures for thermophiles.

[Aaron Hicks]

Crowe's "Immunodiffusion," 2nd ed., 1973:

Soak agar shreds or granules in "several changes" of distilled water (DI). Make a 4% gel, slice, dialyze or electrodialyze it, then use that to make a more dilute gel (no specified dilution) through reheating. Alternatively- dry and dissolve flakes later as needed.

Dissolve agar "in the solvent to be employed," and hot-filter through several layers of "lintless gauze, coarse filter paper, shredded paper or diatomaceous earth, or centrifuge at high speed (eg, 5000 g) for 10 mins in a rotor pre-heated to 80 C" (small volume technique: pull into 10 mL pipette with loose cotton plug. Remove plug and deliver to plate or slide).

Make agar gel from this, then chil, freeze, and thaw it to disrupt gel and "express the water and dissolved impurities." The reader is referred to Crowle 1961 (first edition of Immunodiffusion) for more detailed explanations.

Clausen in "Laboratory Techniques in Biochemistry and Molecular Biology" (volume 1, part 3, edited by Burdon and Knippenberg) describe preparation of agarose with cetylpyridinium chloride "or other tertiary ammonium compounds." Too technical to describe here.

There was another text- I cannot find it right now- with excellent descriptions; it mostly consisted of making slabs of gel that are then allowed to sit in distilled water, which is changed every day for a week or more. It *might* be in "Handbook of Immunoprecipitation-in-Gel Techniques," edited by Axelson (1983). In fact, that book is a very good read for anyone who wants to work with antibodies and antigens; the techniques are surprisingly powerful given the materials required.

-AJ

[Daniel C.]

On Tue, Mar 24, 2009 at 11:44 AM, Aaron Hicks <aaron...@gmail.com> wrote:
> Soak agar shreds or granules in "several changes" of distilled water (DI).

Several of the procedures involve soaking agar in water and changing
it a few times. Doesn't agar dissolve into (or create a colloid with)
water? Or do you have to heat it up for that to happen?

Dan

[Aaron Hicks]

Once it's gelled, water can diffuse in and out, but not a whole lot. This is actually a problem in microbiology; poured plates get condensation on their surface, which makes for problems when it comes to creating clean colonies. I've seen plant tissue cultures with significant condensation that accumulates on the surface of the gelled substrate- and even though the medium must have a substantially higher osmotic concentration than that of the condensate (which is pretty much distilled water), the liquid stays on the surface, often for weeks or months if the vessel is sealed and it can not evaporate.

So- once you have an agar slab, it can be dunked in water without fear of it dissolving. Bump up the heat, and- yes, it can dissolve.

-AJ

[Tom]

Unless you heat it, the agar will just precipitate to the bottom over
time, pour off water, repeat. At least the bacto agar type I have
used, I have never tried the asian store stuff, sounds like I should
at least try it based on cost.

On Mar 24, 1:53 pm, "Daniel C." <dcrooks...@gmail.com> wrote:

[Tomasz Suchan]

Aaron,
could you point me to some simple protocols for generating the
antigen, please?
And for Ouch plates... do you mould them out of some special kind of
silicone or the regular transparent sanitary one?

Cheers, Tom

[Jake]

Maybe I should clarify... When I mentioned the color varying with food
grade agar I was referring to the solid powder not the final gelled
product. I bought agar from a local food co-op several times and
stored it in the same containers. I noticed that two of the purchases
were a slightly different color and one was a bit layered with
slightly different colors.

When made into petris it has always seemed plenty clear. The PD and
ME always added a lot more color than the agar.

If I were going to purify the agar, and I probably will just as a
comparison to see if it's needed for electrophoresis, I'd probably
just add a 3X volume of dH2O and let it sit for a day or two, then
filter to recover the agar. A simple water wash like that should
probably be enough. Then again they probably already do that during
manufacturing, so it might not help any.

-Jake

[Aaron Hicks]

Antigen production is a huge topic, and- in the case of bacteria- is far too broad to discuss here. In most cases, you will need to culture the bacterium, break it in some fashion, and purify it- unless crude extract is OK. That second step is the picky one, and that varies depending upon what you're working with. Best bet: go to the literature and see what others have done, and emulate their work.

For Ouch plates- casting anything that's supposed to be clear is very difficult in that the technology to make it "water clear" and without waves or warping is very tough. For glass, it's done with flat surfaces (to make Petri plates), or a large vat of molten tin ("float" glass). For plastic, it takes a lot of machining and polishing. So- it's just cheaper to buy. I could have gotten away with plastic or glass Petri plates, but the immunodiffusion plates on eBay intrigued me. I still haven't opened the case to see what sets them apart from other labware.

-AJ

[Jake]

> I don't mean to hijack the thread, but it's been a while since I've
> had a chance to mention a neat table that I came across the other day
> that lists alternatives to agar- like wheat flour, laundry starch,
> potato powder, rice powder, etc.

None of those work worth a damn. Agar is clear and indigestable by
bacteria, none of those replacements have those properties.

Guar gum is the only substitue worth trying, but from what I
understand it's a LOT harder to work with. For the price of food
grade agar it just isn't worth trying to save money with a crappy
substitute. Food grade works great for everything I've tried and you
can get it pretty dang cheap online.

-Jake

[Jeswin John]

What's an Ouch plate? Nothing on Google

[Tomasz Suchan]

Try Ouchterlony plates or Ouchterlony immuno diffusion.
http://en.wikipedia.org/wiki/Ouchterlony_double_immuno_diffusion

Tom

[Aaron Hicks]

And Google doesn't even scratch the surface; while a double diffusion plate looks fast and easy and limiting, there are variations- like single radial diffusion where the gel is loaded with the antibody (or antigen), and the well is loaded with the other component; the diameter of the ring can be used to quantify the reaction.

Then there's electrophoresis added on top of the immunodiffusion, allowing another dimension in the whole process- affording protein separation on the cheap. Rocket immunoelectrophoresis (RIEP) is inexpensive, and adds a lot to the whole equation. You go back through these old books where these techniques predominated years ago, and they're just amazing. Now you're lucky to find the apparatus to do it. Fortunately, it's easy enough to cobble it together, or adapt other components to the task.

-AJ

[Patrik D'haeseleer]

On Monday, March 23, 2009 10:35:28 AM UTC-7, Aaron Hicks wrote:

Some of the older texts (1960s and 1970s) discuss purification of agar to increase clarity. It's a pretty involved process, but if anybody would like the references so they can go read about it themselves, I can look them up.

If I remember correctly, Cathal gave that a try. Don't remember if he ever gave a thorough summary of his experience with this, other than that it was very messy...

Patrik

[Patrik D'haeseleer]

On Tuesday, March 24, 2009 6:55:26 AM UTC-7, Bryan Bishop wrote:

I don't mean to hijack the thread, but it's been a while since I've
had a chance to mention a neat table that I came across the other day
that lists alternatives to agar- like wheat flour, laundry starch,
potato powder, rice powder, etc. IIRC, historically potato was used
first for culturing little critters, but now I don't know whether or
not that would be useful if you're looking for clear agar ;-). You
could probably take thin slices of potato and then shine a light
through them, but does that count?

http://heybryan.org/books/papers/graph1_alternate_agar_formula.png

That link doesn't seem to be working.

[Bryan Bishop]

On Fri, Mar 22, 2013 at 6:11 PM, Patrik D'haeseleer <pat...@gmail.com> wrote:
>> http://heybryan.org/books/papers/graph1_alternate_agar_formula.png
>
> That link doesn't seem to be working.

http://diyhpl.us/~bryan/papers2/graph1_alternate_agar_formula.png

[Cathal Garvey (Phone)]

I really should add that to my biohacking protocols repo..
Suffice to say it's doable, and straightforward, but very messy and not cost effective unless you can fractionally distil an alcohol *and* propylene glycol (boils 200+C).

Am pretty sure I shared the protocol on this list previously..

Patrik D'haeseleer <pat...@gmail.com> wrote:

--
Sent from my Android phone with K-9 Mail. Please excuse my brevity.

[jarlemag]

According to a recent article (http://www.ncbi.nlm.nih.gov/pubmed/22809873), food-grade agar can be substituted for bacteriological agar with no apparent effects except colour of the medium.

-J

[Cathal Garvey (Phone)]

Oh purification for micro is pretty pointless, I was talking about agarose purification for gels.

[Nathan McCorkle]

I have access to a small soxhlet extractor. If I could clean up agar, I'd sell it to people here for enough to cover the basic costs. Where is that protocol? What's the reality of the cost in time, reagent loss cost (ethanol evaporating, etc), and electricity?

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[Cathal Garvey]

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Well, doing some back-of-envelope math without accounting for solvent
loss or energy input (just lack of ability to perfectly recover
solvents..), I found that cheap agarose is already pretty close to the
floor as far as price is concerned. So, unless you live somewhere
where currency exchanges make even market-floor prices unattainable,
it's simply not worth it.

Having said that, a good protocol is never unwelcome, and there *are*
people on this list and out there who could use an alternative to
currency exchanges to get their agarose, so I'll get the protocol
online in pretty form this week.

Also, it's worth noting that when doing the procedure myself and when
calculating the outcomes, I did *not* have access to a median/large
volume centrifuge, which would have significantly reduced waste and
the need for extra washing, so if you have a centrifuge, you can
probably do this more easily and more cheaply than I did.

Attached is the sketchy protocol I used, which lacks lots of nuance
but is essentially the process in full. If you have access to
pectinase (commonly available in brewshops), you can use this to
pre-treat raw agar to dissolve some of the agaropectin prior to glycol
separation, which will optimise the protocol a lot, but I couldn't
find a protocol for pectinase treatment of agar; the patent referenced
seems to be no longer online/available.

Please note that in the original protocols I pieced this from (found
in ugly patents), it was generally suggested to use ethylene glycol
rather than propylene glycol.. don't do that. Ethylene glycol is
pretty toxic, even without considering fractional distillation! Like
methanol, it gets metabolised to formaldehyde, and it's the reason
that propylene glycol is referred to as "non-toxic" by comparison.

Also, when choosing the alcohol/solvent to dilute the cooled liquor
with or to wash your agar, consider how explosive the results could be
if you get impatient and do something like trying to heat the wet agar
to remove solvent. Acetone is pretty happy to find an ignition source
and blow up, and IPA can form surface vapours that can self-ignite
from a little distance, too. Methylated spirits would be a good bet,
if you can get them locally without nauseating additions, or "biofuel"
ethanol for alcohol stoves, which I've found to be very pure and a
good stand-in for technical grade ethanol.

On 03/23/2013 04:29 PM, Nathan McCorkle wrote:
> I have access to a small soxhlet extractor. If I could clean up
> agar, I'd sell it to people here for enough to cover the basic
> costs. Where is that protocol? What's the reality of the cost in
> time, reagent loss cost (ethanol evaporating, etc), and
> electricity?
>
> On Mar 23, 2013 9:22 AM, "Cathal Garvey (Phone)"
> <cathal...@cathalgarvey.me

> <mailto:cathal...@cathalgarvey.me>> wrote:
>
> Oh purification for micro is pretty pointless, I was talking about
> agarose purification for gels.
>

> jarlemag <jarle...@gmail.com <mailto:jarle...@gmail.com>>

> wrote:
>
> According to a recent article
> (http://www.ncbi.nlm.nih.gov/pubmed/22809873), food-grade agar can
> be substituted for bacteriological agar with no apparent effects
> except colour of the medium.
>
> -J
>

> kl. 10:01:51 UTC+1 l�rdag 23. mars 2013 skrev Cathal Garvey (Phone)
> f�lgende:

>
> I really should add that to my biohacking protocols repo.. Suffice
> to say it's doable, and straightforward, but very messy and not
> cost effective unless you can fractionally distil an alcohol *and*
> propylene glycol (boils 200+C).
>
> Am pretty sure I shared the protocol on this list previously..
>
> Patrik D'haeseleer <pat...@gmail.com> wrote:
>
> On Monday, March 23, 2009 10:35:28 AM UTC-7, Aaron Hicks wrote:
>
>
> Some of the older texts (1960s and 1970s) discuss purification of
> agar to increase clarity. It's a pretty involved process, but if
> anybody would like the references so they can go read about it
> themselves, I can look them up.
>
>
> If I remember correctly, Cathal gave that a try. Don't remember if
> he ever gave a thorough summary of his experience with this, other
> than that it was very messy...
>
> Patrik
>
>
> -- Sent from my Android phone with K-9 Mail. Please excuse my
> brevity.
>
>
> -- Sent from my Android phone with K-9 Mail. Please excuse my
> brevity.
>
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