Lyophilization

protocol with open access reagents

Molecular diagnostic assays owe their high performance and reliability to enzymes that catalyze specific chemical reactions. But most enzymes are highly sensitive to temperature, which is why we usually work with them on ice in the lab. They lose activity over time when stored in a solution, most enzymes are stored in either -20°C freezers (like those most people have at home) or -80°C deep freezers, which are found in some labs. To overcome the need for cold storage of enzymes and to enable their shipment at room temperature, we developed protocols for lyophilization or freeze-drying of the enzymes or reagent mixes. This enables one lab to centrally make the freeze-dried reagents, and to ship them out over long distances with minimal costs.

Here's how it works...

Prepare the mix

Prepare either an enzyme mix or a reagent mix with primers and dNTPS too. Include trehalose, a cryoprotectant.

Freeze dry

Plunge-freeze the aliquotted mix in liquid nitrogen. Place in a freeze-drier to sublimate all the ice from the frozen mix.

Store or ship

Store the dehydrated mixes in an air-tight bag with a moisture-absorbing material. Ship over long distances without cooling.

Reconstitute and use

Reconstitute for use when necessary, adding water, buffer and other LAMP reaction components.

The principle of lyophilization

Freeze-drying works by freezing the object and then placing it into a sealed container which maintains very low pressure and a low temperature. This results in the sublimation of ice from the frozen object, ultimately completely dehydrating it. Enzymes, reagents, bacterial strains, pharmaceuticals and more can be preserved in this way in the lab. Since dehydrating with elevated temperatures (like in an oven or a drier) damages the activity of enzymes, freeze drying is the method of choice for preserving enzymes and keeping their activity intact. You may have stumbled upon freeze-dried fruits at the store, so you can taste for yourself just how effective freeze-drying is in preserving the original characteristics of the food while making it shelf stable for a long period of time!

Enzyme mix

The first option for RT-LAMP freeze drying is to lyophilize the enzymes. A concentrated enzyme mix is prepared, trehalose solution is added as a cryoprotectant and the enzymes are aliquoted and frozen in liquid nitrogen. Then, the frozen enzymes are quickly placed in a freeze-drier, and the process of ice sublimation starts. After 16 hours (depending on the machine being used and its settings), the only thing left in the tubes is a white “cake”, a fluffy porous solid consisting of the enzymes and trehalose after all the ice has evaporated. Tubes containing this cake have to be sealed and stored in a place without excess moisture. We’ve had success with storing them in zip-lock bags with desiccant packets (silica gel). When ready for use, the tubes are opened and reconstituted with water or water-glycerol mixture. From that point, one has to handle them like normal enzymes, e.g. work on ice and store in the freezer. The enzymes are combined with buffer, primers, dNTPs, indicator and other components of LAMP reactions to assemble a complete reaction mix.

Reagent mix

The second option is to prepare a reagent mix consisting of enzymes, primers and dNTPs. The reagent mix is advantageous in that it contains all the crucial reaction components that degrade fast at elevated temperatures. Freeze-drying is the same as for the enzyme mix, but it is reconstituted with just water, buffer and indicator (colorimetric indicator HNB or a fluorescent indicator Syto9). After reconstitution, it’s a finished master mix ready for sample addition and testing! A disadvantage of this approach is that you can’t swap out the primers in the mix. On the other hand, you can also lyophilize the reagent mix in single-reaction aliquots, making it very convenient for reconstitution in the field.

Enzyme mix lyophilization protocol

Here, we present a simple protocol to reconstitute RT-LAMP reactions from a shelf-stable “enzyme mix” format, supplying the enyzmes as a freeze-dried pellet in a tube to be reconstituted with a 10% glycerol solution. The reconstituted enzymes can then be stored at -20°C or used in RT-LAMP reactions immediately. This protocol was developed and bechmarked for SARS-CoV-2 detection but can be adapted to the detection of any DNA/RNA sequence with the proper primers. This protocol was developed at the Research Institute of Molecular Pathology and Institute of Molecular Biotechnology at the Vienna BioCenter.

Reagents

deoxynucleotide triphosphate (dNTP) mix, 25 mM each of dATP, dTTP, dCTP and dGTP sodium salts in water, store at -20C
deoxyuridine triphospate (dUTP) solution, 100 mM sodium salt in water, store at -20C
RT-LAMP primer stocks in 20x concentration (see reagent setup below), store at -20C
D-trehalose, 50% (w/v) solution in nuclease-free water, prepare fresh on day of lyophilization
- [CRITICAL] Storing the trehalose solution might result in microbial growth.
10X Isothermal amplification buffer for lyophilization, prepare from stock solutions as indicated below, store at -20C
- [CRITICAL] It is necessary to omit magnesium in the buffer for freeze-drying. We found that if magnesium is present in the buffer for freeze-drying, the colorimetric readout with HNB is not reliable.
- The final composition of 10x Isothermal amplficiation buffer for lyophilization is 500 mM KCl, 200 mM Tris-HCl (pH 8.8), 100 mM (NH4)2SO4, 1% Tween-20. Sterile-filter the complete buffer, aliquot, and store at -20C for moths.
nuclease-free water (not DEPC treated), store at room temperature
in-house purified Bst LF enyzme in glycerol-free buffer, store at -80C
in-house purified HIV-1 RT enyzme in glycerol-free storage buffer, store at -80C
in-house purified BMTU UDG enyzme in glycerol-free storage buffer, store at -80C

Equipment

set of micropipettes in the 1 – 10, 10 – 100, and 100 – 1 000 ul range and corresponding filter barrier tips
- [CRITICAL] It is necessary to use filter tips to avoid possible contamination from pipetting.
  PCR hood or equivalent template-free space
  bleach for disinfecting surfaces, 10% solution of household bleach in water
- [CRITICAL] Hypochlorite contained in bleach is one of the few truly effective agents for mitigating DNA contamination. Bleach solution can be prepared from household bleach (typically 5% sodium hypochlorite) by diluting it in a 1:10 fashion with water. Do not store bleach solution longer than a few days, as dilute hypochlorite solutions tend to decompose. Apply to surfaces liberally and let it work for 5 minutes before wiping off with paper towels. Decontaminate pipettes and other items by wiping them down with bleach-soaked paper towels. The work surfaces can then be wiped down with deionized water to remove the bleach.
- [CAUTION] Work only with gloves, goggles and while wearing a lab coat. Pay attention to the MSDS and general guidelines for working with bleach, working in a well-ventilated area.
SafeLock RNase-free tubes, 1.5 ml
liquid nitrogen and a container for freezing tubes in liquid nitrogen
safety equipment for working with liquid nitrogen, e.g. goggles, cryogloves, faceshield etc.
styrofoam or metal inlets for freezing down 1.5 ml tubes in liquid nitrogen
Christ Alpha 2–4 LDplus freeze-drier, set to main drying program with chamber temperature at -78°C and pressure at 0.091 mbar
- University labs and reseach institutions usually have freeze-driers used for work with proteins. Those are the best choice, but likely freeze-driers for many different applications can be used in this protocol. Try to optimize the freeze drying time for the specific machine you are using.
sillica gel desiccant packets
secure-sealable plastic bags

PROCEDURE

Thaw all reagents at room temperature. After the reagents have thawn, pipette up and down gently, briefly spin down in a microcentrifuge and keep on ice.
[CRITICAL] Vortex the reagents after thawing. The isothermal amplificaiton buffer can have some precipitate present, this needs to be fully dissolved and in solution.

While thawing the reagents, calculate the volumes of reagents to add according to the mixing table below. Count with a 10% excess to account for pipetting error.

RT-LAMP Enzyme Mix (20X)
- Reagent Reaction conc. Mix conc.
  Bst-LF 20 ng/ul 400 ng/ul
  HIV-RT 7.5 ng/ul 150 ng/ul
  BMTU UDG 0.0025 ng/ul 0.25 ng/ul
  Trehalose 0.5% 10%
  IAB (no Mg) 1X 1X
  water to 20 ul to 1 ul
- Preparing an RT-LAMP Enzyme Mix requires the enzyme concentrations to be at 20X the normal concentrations in the reaction. This way, when resuspended, one microliter can be added to a master mix without enzymes to make a full RT-LAMP master mix.

RT-LAMP Reagent Mix (5X)

Reagent	Reaction conc.	Mix conc.
Bst-LF	20 ng/ul	400 ng/ul
HIV-RT	7.5 ng/ul	150 ng/ul
BMTU UDG	0.0025 ng/ul	0.25 ng/ul
dNTPs	1.4 mM	7 mM
dUTP	0.7 mM	3.5 mM
F3/B3 primers	0.2 uM	1 uM
LB/LF primers	0.4 uM	2 uM
FIP/BIP primers	1.6 uM	8 uM
Trehalose	2%	10%
IAB (no Mg)	1X	0.8X
water	to 20 ul	to 4 ul

Preparing an RT-LAMP reagent mix requires the reagents, in this case enzymes, dNTPs and primers, to be at a 5X concentration compared to the normal final concentration in LAMP reactions. Thus, one reaction’s worth of RT-LAMP reagent mix is 4 ul, for a 20 ul final reaction.

Assemble the master mix for lyophilization in a sterile, RNase-free plastic microtube. Calculate the appropriate amount of enzymes and reagents to add according to the concentrations given below. Add the reagents in the given order from top to bottom, adding the enyzmes last. Work on ice.
After assembling the master mix for lyophilization, make sure the entire mixture is homogeneous, mix it by pipetting up and down or by gently vortexing and spinning down.
Prepare the tubes you want to lyophilize the mix in. For 1.5 ml eppendorf tubes, prepare sterile, RNase-free tubes with separate pierced lids. If you want to prepare single-reaction lyophilisates, prepare your 8-well or 12-well sterile and RNase-free PCR strips. Place the tubes on ice.
Aliquot the lyophilization mix into the 1.5 ml tubes or PCR strips for lyophilization. Cap 1.5 ml tubes with pierced lids. Leave PCR strips uncovered.
Transfer the tubes to a freezing rack.
[CRITICAL] Once the tubes are out of liquid nitrogen, it is necessary to proceed very quickly. Set up your work area carefully so that no time is lost from this point until the tubes are on the freeze-drier.
While wearing appropriate safety equipment for working with liquid nitrogen, submerge the bottom of the tubes into the liquid nitrogen.
[CRITICAL] It is essential that the contents of the tubes are fully frozen and have reached the temperatrue of the liquid nitrogen. Keep the bottoms of the tubes submerged until the liquid nitrogen stops violently boiling around them, usually a few minutes.
[CRITICAL] Make sure the liquid nitrogen doesn’t get into the tubes themselves, as that could contaminate the reagents and compromise the results of the assay when the enzymes are reconstituted.
Once the contents are fully frozen, transfer the tubes quickly into the suction vessel used for freeze-drying.
[CRITICAL] It is necessary to move very quickly so the tube contents don’t thaw.
Once the tubes are in the vessels for freeze-drying, place the vessels onto the suction valve of the freeze-drier. Carefully and slowly start twisting the vacuum knob on the freeze-drier until the suction vessel is fitting snugly on the suction valve of the freeze-drier.
[CRITICAL] Turn the knob slowly, otherwise you risk that the change in pressure inside the suction vessel will result in your tubes moving around and spilling the tube contents.
Check the temperature and pressure gauges on the freeze-drier for the correct values and let the freeze-drying process take place for 16 hours.
[PAUSE POINT] Freeze-drying is typically performed overnight.
After 16 hours, remove the suction vessel from the freeze-drier by carefully turning the knob of the vacuum valve down until normal pressure is reached within the freeze-drier. Remove the suction vessel.
[CRITICAL] Work quickly from this point on. Prolonged exposure to air and the moisture contained in atmospheric air could decrease the shelf-life of the lyophilized enzymes or reagents..
Carefully cap the tubes or strips with RNase-free caps.
[CRITICAL] It is necessary the caps fit perfectly, as that protects the freeze-dried product from atmospheric moisture.
Place the finished lyophilized capped tubes or strips in sealable plastic bags with sillica desiccant packets for long-term storage. Stable at room temperature for over one month.

Reconstituting the freeze dried reagents

Enzymes are reconstituted with water or a water-glycerol mixture to make a 20X enzyme stock solution. Reagent mix is reconstituted with a reconstitution buffer to yield a complete master mix ready for sample addition and incubation.

Reagent mix reconstitution

Assemble the reconstitution buffer by pipetting the reagents together in an RNase-free Eppendorf tube. Start by adding the water and buffer, and then add other reagents.

Reagent	Stock concentration	To add per 1 reaction (20µl size)
Isothermal amplification buffer (no magnesium)	10X	2 μl
MgSO4	100 mM	1.6 μl
Betaine	5M	1.6 μl
HNB colorimetric dye	3 mM	0.8 μl
Syto9 fluorescent dye (optional)	10X	100 μl

nuclease-free water		to 16 μl
total		16 ul

After assembling the reconstitution buffer, vortex it briefly to mix thoroughly and spin down in a microcentrifuge to get all the buffer from the tube cap and sides of the tube. Place the reconstitution buffer on ice if you plan to reconstitute reactions right away.
[PAUSE POINT] You can store finished recosntitution buffer at -20C for months. You can store it at room temperature for a maximum of 24 hours, the HNB dye undergoes temperature-dependent decomposition in the buffer, diminishing the color and compromising the colorimetric readout.
Prepare the tubes with lyophilized RT-LAMP reagent mix. Spin the tubes down briefly to make sure all of the dried pellet is collected at the bottom of the tubes.
Add the correct volume of reconstitution buffer to the freeze-dried RT-LAMP reagent mix. The volume of reconstitution buffer to add equals to the volume of the reactions minus the sample volume (i.e. 16 ul for 20 ul reactions) multiplied by number of reactions in the mix (i.e. 10, 20, 50 rxns). Mix by pipetting up and down to dissolve the lyophilized pellet. Vortex gently and spin down in a microcentrifuge. [CRITICAL] Check to confirm that all the pellet has been dissolved, if not, vortex and/or mix by pipetting. If the pellet doesn’t dissolve quickly, this points to a problem in the freeze-drying process.
Place the reconstituted RT-LAMP reagent mix on ice from now on. This is now a complete master mix that only requires the addtion of sample and incubation at 63C for 35 minutes. Use within one hour for RT-LAMP testing.

Enzyme mix reconstitution

Thaw all reagents at room temperature. Vortex thawn reagents for about 10 seconds, briefly spin down in a microcentrifuge and keep on ice.
Prepare the tubes with lyophilized RT-LAMP enzyme mix. Spin the tubes down briefly to make sure all of the dried pellet is collected at the bottom of the tubes.
Using a micropipette with a sterile tip, add the correct volume of 10% glycerol to the freeze-dried RT-LAMP enzyme mix. The volume of 10% glycerol solution to add equals to the number of reactions in the lyophilized pellet (i.e. 10, 20, 50 rxns = 10, 20, 50 ul). Mix by pipetting up and down to dissolve the lyophilized pellet. Vortex gently and spin down in a microcentrifuge.
[CRITICAL] Check to confirm that all the pellet has been dissolved, if not, vortex and/or mix by pipetting
[PAUSE POINT] You can aliquot the reconstituted enzyme mix and freeze it at -20C for weeks or months and use when needed. Limit the number of freeze-thaw cycles to preserve enzyme activity.
To immediately use the reconstituted RT-LAMP enzymes,prepare a master mix as per the “Open access RT-LAMP protocol” on this website, omitting the addition of individual enzymes and instead adding the reconstituted enzyme mix in a 20X manner to the reaction master mix. For example, 1 ul of the reconstituted enzyme mix per 20 ul reaction volume.
Unused enzyme mix may be stored in the freezer like normal enzymes.

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