APPLICATION NOTE

The ultimate flex: pseudotype specific LV titer withLeprechaun
Introduction
VSVG is the most common envelope protein usedfor pseudotyping lentivirus (LV) due to the broadcell tropism and high transduction efficiency it
provides. While ideal for ex vivo modificationof selected cells, these properties make VSVG
pseudotyped LV poorly suited for in vivo therapies
due to the high chance of off-target delivery.
Modifying the LV pseudotype by replacing VSVG
with an alternative envelope protein with a more
specific cell tropism can increase LV deliveryefficiency and reduce off-target effects1. As aresult there is growing interest in manufacturing
non-VSVG pseudotyped LV, engineered to target
disease specific cell types.

Figure 1: Leprechaun is the only platform that simultaneously
checks your LV has the correct envelope protein, contains acapsid and RNA, and is the right size.

protein is specifically bound by Flex antibodies
spotted on the Luni surface (Figure 2B). Once theLuni has been functionalized with the desired antibodies the sample is added, and immuno-staining
performed to check for the presence of the viralcapsid and RNA (Figure 2C).

Amongst the challenges for anyone modifying theLV pseudotype is the inability to easily check that thecorrect envelope protein is consistently expressed
throughout production and across batches.
Conventional methods for assessing LV quality, e.g.
capsid ELISAs or qRT-PCR, don’t look at the viralenvelope, while alternative approaches such as
western blotting are slow and only semi-quantitative.
Consequently, confirmation of the viral pseudotype
is often indirectly confirmed by functional cell-based
assays which measure the efficiency of transductionof the target cells by the virus.

The number of viral particles with and withoutcapsid or RNA is combined with single particle sizing
data to generate a LV titer for each structural
population – LV w/ capsid & RNA, LV w/ capsid
w/o RNA, LV w/o capsid w/ RNA, LV w/o capsid
w/o RNA and aggregates. As an alternative to theLeprechaun makes it easy to obtain a pseudotype-specific LV titer at any stage of your production
process (Figure 1).

ALeprechaun’s Lentivirus Flex RNA Kit utilizes Abcam’sLightning-Link™ technology, allowing users tocustomize the antibody used for LV capture fromthe comfort of their own labs. All you need to do
is start with an antibody which recognizes theenvelope protein of your choice, incubate it withthe linker protein provided in the kit (Figure 2A),
add the quencher reagent and then incubate thelinker-antibody conjugate on a Flex Luni. The linker

BC

Figure 2: Step by step functionalization of the Lentivirus Flex Luni.
(A) An antibody specific for the desired envelope protein (green) isincubated with the Flex linker (gray) to form an antibody-linker conjugate. (B) The antibody-linker conjugate is then incubated on theFlex Luni and the linker specifically binds to the corresponding Flex
capture antibody, which is pre-spotted on the Luni surface. (C) LV is
captured by the custom antibody via the viral envelope protein.

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THE ULTIMATE FLEX: PSEUDOTYPE SPECIFIC LV TITER WITH LEPRECHAUN

A

B

Figure 3: Available Lentivirus Flex Luni designs. (A) All Flex Lunis feature six Flex 1 spots for LV capture, three Flex 2 spots which can befunctionalized with isotype controls and three anti-p24 spots for quantification of soluble p24. (B) Lentivirus Flex EV Contaminant Lunis
also have an additional set of three anti-tetraspanin spots for analysis of non-viral vesicle contaminants.

Flex RNA Kit (Figure 3A), an EV Contaminant Kit
is available which includes additional Luni spotsfor capture and quantification of non-viral vesicle
contaminants alongside viral titer (Figure 3B).
Each kit contains a pair of linker reagents, each ofwhich recognizes a different set of Flex spots on
the Luni surface. This design allows for conjugationof one antibody to capture the viral envelope,and a second antibody to function as an isotype
control for the first.

fixation and permeabilization by Solution C and D, allLunis were incubated for 1 hour at RT with kit suppliedanti-p24-CF647 (1:250). Lunis were washed, dried
and run on Leprechaun using the Lentivirus RNA
application or Lentivirus EV Contaminant application.

qRT-PCR Analysis
Analysis was performed by SydLabs using theLenti-X qRT-PCR Titration Kit (Takara Bio), according
to the kit protocol. Three dilutions of each sample
were run in duplicate and results averaged toobtain genomic titer.

This app note describes how Leprechaun’s Lentivirus
Flex RNA Kit and Lentivirus Flex EV Contaminant
kit deliver pseudotype specific quantification LV
and non-viral contaminants, providing a one
stop shop for confirming the quality of your LV,
whatever the pseudotype.

Results
Highly Specific
The specificity of the Flex system was confirmed
by incubating Lunis with and without a flex-linked
custom antibody in the presence of LV sample. In
the case where the custom antibody was excluded
the linker was still added to the Flex spots, but withno antibody attached. Signal was only detectedon the fully functionalized Luni (linker and antibody
both present), verifying that the linker alone is
not sufficient for particle capture (Figure 4 – left
pair of bars). A fully functionalized Luni was also
analyzed without sample present and no signal
recorded (right bars), confirming that particle
counts are only detected when sample is bound to
the Flex conjugate. EV contaminants were tracked
across the three experimental designs, with readings
taken from the same Flex Luni as LV titer. As thetetraspanin antibody is spotted directly onto theLuni rather than added via Flex, this acted as apositive control to confirm sample had been added
where appropriate and there were no technicalissues with the reading.

Methods
Leprechaun Analysis
LV samples were acquired from Vectorbuilder andLeprechaun analysis performed using the Lentivirus
Flex RNA Kit or Lentivirus Flex EV contaminate Kit(Unchained Labs). Flex Lunis were functionalized, asper kit instructions, with SARS-CoV-2 spike protein
RBD antibody (R&D Systems). Crude LV was diluted
1:5 and purified LV 1:30 in manufacturer supplied
Incubation Solution with added 1% FBS. For RNA
detection kit supplied SYTO14 was diluted to 10 µM
in Incubation Solution and added to the diluted LV
sample at a dye to sample ratio of 1:10. The dye-sample mixture was incubated for 16 hours at 37 °C. Postdye incubation, 50 µL SYTO14 labeled sample wasincubated for 1 hour at RT on Lentivirus Flex RNA
Lunis. For contaminant analysis diluted sample wasincubated directly on the Luni for 1 hour at RT. After2

THE ULTIMATE FLEX: PSEUDOTYPE SPECIFIC LV TITER WITH LEPRECHAUN

Pseudotype Specific LV Analytics
As soon as your Flex Lunis are functionalized andready to go you can start digging into LV titer,
RNA content, aggregation and contamination,using less than 25 µL of sample. Analysis of crude
and PEG purified samples of SARS-CoV-2 spike
protein pseudotyped LV reveals that purification
successfully concentrates LV particles containing
both capsid and RNA (Figure 6A). The titer ofRNA-containing LV increased 100-fold in thepurified sample, in line with the change in genomic
titer as measured by qRT-PCR. LV containing botha capsid and RNA was enriched from 14% to 35%.

Figure 4: Validation of signal specificity from Lentivirus Flex Lunis.
All components (linker, custom antibody and sample) must bepresent for LV titer to be recorded. n = 3 for each condition andoutput, error bars are SD.

Contaminant analysis, however, shows that there
are still a high titer of non-viral EV contaminants inthe final product (Figure 6B). Standard approaches to LV purification tend to co-purify EVs due totheir similar size and density. As a consequence,
there is growing focus on the need to measure
and characterize the EV content of LV related
drug products in order to meet regulatory requirements2. Interestingly, the proportion of particles
which are non-viral EVs rises from 58% in crude,
to 72% in the purified sample, indicating that PEG
purification is completely ineffective at removing
these contaminants.

Linear and Precise
LV engineered to express the SARS-CoV-2 spike
protein was used to test the linearity and precisionof Leprechaun analysis of non-VSVG pseudotypedLV. An antibody specific to the spike protein wasimmobilized on the Flex RNA Luni via the Flex
conjugation system. Total LV titer and LV w/ capsid& RNA titer are linear down to 1x107 particles/mL
in both crude and purified samples (Figure 5),
matching the performance of the VSVG Luni andapplication. Assay precision can be altered by thecustom antibody clone used for capture, but forthe spike protein antibody tested % CV was typically around 12%. Equivalent results were obtainedfor the Flex EV Contaminant Luni.

Figure 5: Linearity of LV titer for SARS-CoV-2 spike protein pseudotyped LV as measured byLeprechaun. Error bars are SD.

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THE ULTIMATE FLEX: PSEUDOTYPE SPECIFIC LV TITER WITH LEPRECHAUNA

BFigure 6: Leprechaun analysis of crude and purified SARS-CoV-2 spike protein pseudotyped LV. (A) Data from the Lentivirus Flex
RNA Kit provides information on the structural composition and RNA content of LV particles. (B) The Lentivirus Flex EV Contaminant Kit
delivers LV titer alongside contaminant analysis. n = 3 for each condition and output, error bars are SD.

ConclusionTake your process and analytical development to
the next level with the help of Leprechaun, the only
platform to measure pseudotype specific LV titer,
contamination and aggregation in both crude andpure samples. Whatever the envelope protein, nomatter how complex the sample matrix, monitor yourLV quality from harvest to final product.

Modification of LV pseudotype is a useful tool forincreasing the specificity and efficiency of target
cell transduction. The majority of LV analyticaltechniques focus on either the protein capsid orRNA payload and provide no information on theviral envelope protein. Leprechaun confirms LV
pseudotype while simultaneously checking for theviral capsid and RNA, meaning you can be confident
you’re measuring the right stuff.

References
1 Pseudotyping lentiviral vectors: when the clothes
make the virus. Duverge A. and M. Negroni. Viruses,
2020. 12, 1311

Flex Lunis are easily customized with any antibody
and come in two flavors. If you want to know thetiter of structurally complete RNA-containing LVthen the Flex RNA Kit can provide it. Alternatively,to monitor LV purity and contaminant removal theFlex EV Contaminant Kit steps in.

2 Critical assessment of purification and analyticaltechnologies for enveloped viral vector and vaccine
processing and their current limitations in resolving
co-expressed extracellular vesicles. Vaccines, 2021.
9(8): 823

Unchained Labs
4747 Willow Rd
Pleasanton, CA 94588
Phone: 1.925.587.9800
Toll-free: 1.800.815.6384
Email: info@unchainedlabs.com© 2023 Unchained Labs. All rights reserved. Leprechaun
is a trademark and Unchained Labs is a registeredtrademark of Unchained Labs. All other brands or productnames mentioned are trademarks owned by their respective organizations.

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