Contents

4 Advanced Technologies Driving

Cancer Research
Novel tools have helped develop more precise therapies,
and ultimately improved patient outcomes

6 Optical Imaging in Cancer Immunology

A practical guide for in vivo imaging

10 Antibody-drug Conjugates:
Smart Chemotherapeutics
Promising class of anticancer therapeutics demonstrates improved
precision and reduced off-target toxicity

17 DNA Methylation Detection Using

Droplet Digital™ PCR
Novel technique has a number of advantages

26 Trust Your Results with Rigorously Validated

ELISA Kits
Proprietary validation process helps ensure precise and
reproducible results that you can trust

2
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Advanced Technologies Driving
Cancer Research
Novel tools have helped develop more precise therapies,
and ultimately improved patient outcomes

Cutting-edge technologies have been behind significant benefits by enabling researchers to vi-
many of the recent breakthroughs that have ad- sualize tumor cells and their behavior in real-time
vanced our understanding of cancer biology, di- within living organisms. This technology allows for
agnosis, treatment, and prevention. By leveraging the monitoring of tumor dynamics, assessment of
advanced tools and technologies effectively, re- treatment responses, and the identification of key
searchers have uncovered novel insights, devel- biological processes influencing cancer progres-
oped more precise therapies, and ultimately im- sion. Chapter 2 describes the use of optical in vivo
proved patient outcomes. imaging for investigating cancer immunotherapy.
One key area where new tools have made a sig- The field of immunotherapy has seen remarkable
nificant impact is in genomics. The advent of progress with the development of new tools and
next-generation sequencing (NGS) technologies
technologies. Immune checkpoint inhibitors, chi-
has revolutionized our ability to analyze the ge-
meric antigen receptor (CAR)-T cell therapy, and
netic alterations driving cancer development. By
cancer vaccines are just a few examples of innova-
sequencing the entire genome or specific regions
tive immunotherapies that have shown promising
of interest in tumor samples, researchers can iden-
tify mutations, gene fusions, and other genomic results in treating various types of cancer.
aberrations that contribute to cancer progression. Antibody-drug conjugates (ADCs) are another
This information not only helps in understanding promising class of cancer therapeutics that im-
the underlying mechanisms of cancer but also en- prove precision and reduce off-target toxicity in
ables the development of targeted therapies tai- targeting cancer cells. The development of ADCs
lored to individual patients based on their unique
involves careful consideration of target-specific an-
genetic profile.
tibodies, cytotoxic payloads, and linkers. Chapter 3
In addition to genomics, advances in imaging explores the structure, mechanism of action, evo-
technologies have also played a crucial role in can- lution, clinical success stories, challenges, and fu-
cer research. For example, in vivo imaging offers ture directions of ADCs.

4
Advancements in immune profiling techniques, validation of ELISA kits to ensure that they accurate-
such as single-cell sequencing and mass cytome- ly detect the analyte of interest without artifacts
try, have provided valuable insights into the com- or interference and produce a standard curve that
plex interactions between tumors and the immune closely mirrors antibody-antigen binding in the
system, paving the way for more effective immu- test samples.
notherapeutic approaches.
Moreover, the rise of artificial intelligence (AI) and
Recently, methylation status has been deter- machine learning has transformed cancer research
mined to have utility as an indicator of ear- by enabling rapid analysis of vast amounts of data.
ly-stage cancer development as well as a meth- AI algorithms can sift through massive datasets
od for monitoring tumor response to therapy.
to identify patterns, predict treatment responses,
Chapter 4 reviews the use of Droplet Digital
and even discover new drug targets. By leveraging
PCR (ddPCR) to analyze methylation changes in
AI-driven approaches, researchers can accelerate
cell-free DNA (cfDNA). By combining tradition-
drug discovery processes, optimize clinical trial de-
al methylation detection methods with ddP-
signs, and personalize treatment regimens based
CR, researchers can enhance the sensitivity, ro-
bustness, and cost-effectiveness of analyzing on a patient’s unique characteristics.
cfDNA samples. This approach not only aids in From genomics and imaging to novel cancer ther-
early-stage cancer detection but can facilitate apeutics and AI, advanced solutions are reshaping
monitoring tumor response to therapy. our understanding of cancer biology and trans-
ELISAs are widely used in cancer research due forming the way we diagnose, treat, and prevent
to their high sensitivity and specificity in de- this disease. By continuing to innovate and collab-
tecting and quantifying proteins or antigens in- orate across disciplines, we can unlock new possi-
volved in cancer development and progression. bilities in cancer research and ultimately improve
Chapter 5 covers the importance of proper outcomes for patients worldwide.

5
Optical Imaging in
Cancer Immunology
A practical guide for in vivo imaging

Cancer remains a significant global health chal- including immune-related side effects and variable
lenge, prompting extensive research into effec- treatment responses.
tive treatments. Among the innovative approach-
es is cancer immunotherapy, which explores the The role of in vivo experiments
intricate interplay between cancer cells and the
immune system. Animal studies, particularly in Understanding the complex mechanisms underly-
vivo experiments, play a crucial role in advancing ing cancer and immune system interactions neces-
cancer immunotherapy research. This article high- sitates in vivo experiments using animal models.
lights the benefits of optical in vivo imaging for in- These experiments provide insights into cancer
vestigating cancer immunotherapy. pathophysiology, drug mechanisms, and therapeu-
tic efficacy. However, conducting such experiments
In vivo studies in requires specialized equipment tailored to the
cancer immunology unique demands of cancer immunology research.

Cancer arises from various factors, including ge- Advantages of optical

netic mutations and environmental influences, in vivo imaging
leading to the transformation of normal cells into
malignant ones. Tumors can proliferate and spread Optical in vivo imaging technology offers sever-
to other organs, complicating treatment strategies al advantages for studying cancer immunology.
and diminishing patient prognosis. Traditional can- Unlike invasive methods, optical imaging enables
cer treatments, such as surgery, radiation therapy, non-invasive monitoring of disease progression
and chemotherapy, have evolved over time. The and therapeutic responses in real-time. It allows
advent of immunotherapy represents a paradigm researchers to track drug distribution, pharmaco-
shift, harnessing the body’s immune response to kinetics, and efficacy within living organisms accu-
combat cancer cells. While immunotherapies offer rately. Longitudinal imaging studies enhance data
promising outcomes, they also present challenges, reliability by minimizing biological variability and

6
reducing the number of animals needed for exper- confirm whether activated T cells can be effectively
iments, aligning with ethical guidelines. observed even in in vivo conditions (Figure 1).1 This
study can be applied to research on immune cell
Optical imaging modalities therapy such as CAR-T.

Among imaging modalities, optical imaging stands

out for its user-friendly interface, wide availability
of imaging agents, and cost-effectiveness. Unlike
radioactive or ionizing radiation-based techniques,
optical imaging poses minimal risks to animals and
researchers. Luminescence and fluorescence imag-
ing techniques enable precise targeting of cancer
cells and immune components, facilitating com-
prehensive studies in cancer immunology.

Application examples
There are various targets in cancer immunology
research.
Figure 2. Biodistribution of Antibody. The mice were imaged
at 4 and 24 h after injection of antibody alone and anti-
body-nanoparticle complex (a). White circles indicate the
sites of tumors. Antibody-nanoparticle complexes accumu-
lated more in tumor than antibody (b) and other tissues (c)
at 4 h after injection.2

In another paper, a therapeutic antibody delivery

platform was developed by combining antibodies
and nanoparticles and confirmed in vivo. In this pa-
Figure 1. Biodistribution of nanoprobe in the body. The
fluorescence images (c) were taken after subcutaneous in-
per, it was confirmed whether the antibody of PD-
jection of the T cells into the backs of mice before (0 min) L1 using the platform goes to the tumor site in vivo
and at 5 min post-injection, and the relative intensity were and has anticancer effect (Figure 2).2 This could be
quantitated (d). The nanoprobe can detect the site of acti- applied to other immune checkpoint inhibitors.
vated T cells in vivo through fluorescence imaging. The red
circles indicate the injection sites.1 Jiang’s research showed how nanoparticles that
reduce the secretion of lactic acid from tumors,
which can inhibit T-cell activity in TME, were de-
One study illustrates how a fluorescence-labeled veloped and tested in vivo. It was confirmed that
nanoparticle probe for monitoring and tracking T the therapeutic effect of PD-1, an immune check-
cell was developed and then injected into mice to point inhibitor, was increased up to three times

7
 accessible to individual laboratories. These systems
facilitate high-quality imaging and data analysis,
empowering researchers to conduct sophisticated
in vivo experiments effectively.

Conclusion
Optical in vivo imaging represents a valuable tool
for advancing cancer immunotherapy research. By
providing real-time insights into disease dynamics
and treatment responses, optical imaging contrib-
Figure 3. Tumor targetability and distribution behavior utes to the development of novel therapeutic strat-
of nanoparticles in vivo fluorescence imaging of the tu- egies. As the field of cancer immunology continues
mor-bearing mice were taken at 2, 4, 6, 8 and 24 h after to evolve, optical imaging systems offer a practical
intravenous injection (D). White circles indicate the sites of
solution for researchers seeking to explore new
tumors. ex vivo fluorescence images were taken with the tu-
mor and normal tissues extracted from the mice at 24 h (E)
frontiers in cancer treatment.
and peak time (G) and analyzed quantitatively (F). From in
vivo and ex vivo imaging experiments, it is identified that References
DiR/MCA group accumulate mostly in tumor.3
1. Chen, M. et al. Biomacromolecules (2020) Apr
compared to the control (Figure 3).3 This presents 13;21(4):1587-1595.
another possibility to increase the response rate of
immune checkpoint inhibitors. 2. Lim, J. et al. Journal of Controlled Release (2021) Feb
10;330:1168-1177.
The versatility of optical 3. Jiang, Z. et al. Advanced Healthcare Materials (2020)
imaging systems Jun;9(12):e2000075.

Optical in vivo imaging systems, such as the VIS- 4. 4. BIO, QLS Advisors, Informa UK Ltd. (2021) Clinical
QUE InVivo Smart-LF System, offer compact de- Development Success Rates and Contributing Factors
signs, ease of use, and affordability, making them 2011-2020, Feb.

Additional Resource
In Vivo Systems

8
Antibody-drug Conjugates:
Smart Chemotherapeutics
Promising class of anticancer therapeutics demonstrates improved
precision and reduced off-target toxicity

Antibody-drug conjugates (ADCs) have emerged ventional chemotherapy agents. The mechanism
as a promising class of anticancer therapeutics, involves the monoclonal antibody binding to an
demonstrating improved precision and reduced overexpressed target antigen on cancer cells, lead-
off-target toxicity in targeting cancer cells.1 The ing to internalization and formation of endosomes.
fundamental mechanism involves monoclonal an- Lysosomal fusion triggers detachment of cytotox-
tibodies binding to overexpressed target antigens ic payloads, which specifically target subcellular
on cancer cells, leading to internalization, lyso- structures such as DNA or microtubules leading to
somal fusion, and subsequent release of cytotoxic cell death. Cytotoxic payloads can also be released
payloads, inducing tumor cell death.2 Additional at the cell membrane and induce a bystander ef-
mechanisms like antibody-dependent cellular cy- fect, targeting neighboring cells and contributing
totoxicity (ADCC), complement-dependent cyto- to overall tumor cell destruction.4 Additionally,
toxicity (CDC), and antibody-dependent cellular mechanisms like ADCC, CDC, and ADCP may en-
phagocytosis (ADCP) also contribute to the over- hance the ADCs anticancer effect. The Fab segment
all anticancer effect.3 The development of ADCs binds to the antigen, while the Fc segment engag-
involves careful consideration of various compo- es immune cells and components of complement
nents, including target-specific antibodies, cyto- system for direct cell death.5 Moreover, monoclo-
toxic payloads, and linkers.4 This article explores nal antibodies can directly inhibit downstream
the structure, mechanism of action, evolution of signal transduction, exemplified by trastuzumab
three generations, clinical success stories, chal- blocking HER2-related pathways for apoptosis.4,5
lenges faced, and future directions of ADCs.
ADC building blocks
Mechanism of action
An ADC consists of a target-specific mAb, a cyto-
ADCs target cancer cells with improved precision toxic payload, and a chemically synthesized linker
and reduced off-target toxicity compared to con- that covalently links the toxin and the antibody.4,6,7

10
 development requires consideration of the target
antigens, antibodies, cytotoxic payload, and linker
(Figure 2).

Antibodies
Target antigens for ADCs should be non-secreto-
ry, mainly on tumor cell surfaces, and internalize
post-binding for effective cytotoxic payload re-
lease into tumor cells.8 Popular ADC targets in-
clude HER2, trop2, nectin4, EGFR, CD19, CD22,
CD33, CD30, BCMA, and CD79b.1,3,4,7,9 The evolving
landscape includes emerging targets like EpCAM,
Figure 1. Mechanisms of Antibody Drug Conjugates (ADCs) to CD70, CD25, CD166, showing promising results.10
selectively kill cancer cells.
Immunoglobulins (IgGs), particularly IgG1, are
commonly employed in clinical studies for ADCs.1
IgG1, abundant in serum, induces strong effector
The mAb binds to specific antigens on the surface
functions, including ADCC, ADCP, and CDC, due to
of tumor cells, and ADCs are internalized into tu-
high Fc receptor binding affinity.1,3 Ideal antibodies
mor cells during the formation of antibody-antigen for ADCs require high affinity, efficient internaliza-
complexes.1,3 ADCs are typically transported from tion, low immunogenicity, and a long plasma half-
the endosome to the lysosome, where the linker is life.3,7 Current ADCs predominantly use fully hu-
cleaved and the small molecule cytotoxins are re- manized antibodies, minimizing immunogenicity
leased, leading to tumor cell death.1,4,7 Overall, ADC compared to earlier mouse antibodies.1,3,9

Figure 2. Basic components of the ADC include antibody, linker, and cytotoxic drug. Antibody targets binding, payload destroys cancer cells,
and linker controls drug release.

11
Figure 3. Featured Target Proteins from Sino Biological

Novel approaches in payload design for ADCs in-

Cytotoxic payload
clude the exploration of non-toxic payloads like
Less than 2% of administered ADC reaches target- RNA inhibitors, immuno-agonists (such as Toll-
like receptors and STING agonists),1,4 and apopto-
ed tumors.5 Therefore, high potency (nano and
sis-promoting Bcl-xL inhibitors,1 aiming to enhance
picomolar IC50) is essential for ADC payload com-
anti-tumor immune responses, broaden treatment
pounds.5 Current cytotoxic payloads include tubu-
windows, and improve safety and efficacy. Recent
lin inhibitors and DNA damaging agents.1,7,11 Ideal developments include PROTAC (Proteolysis-target-
cytotoxins need high toxicity, low immunogenicity, ing chimeras) molecules that aims to combine cat-
and stability, with modifiable functional groups for alytic properties to target undruggable proteins;5
mAb linkage.1 The drug-antibody ratio (DAR) is cru- and the development of hybrid payloads that com-
cial; low DAR may reduce efficacy, while high DAR bine different mechanisms to achieve synergistic
may cause instability and off-target toxicity in ADC effects, targeting multiple refractory cancers with
development.1,4 heterogeneity and drug resistance.5

12
 with mouse-derived antibodies, facing potency
Linker
and immunogenicity concerns. Further improve-
Cleavable and non-cleavable linkers are used to ments, seen in gemtuzumab ozogamicin, incorpo-
control cytotoxic payload release.3,4 Cleavable rated humanized antibodies and potent cytotoxic
linkers leverage environmental differences between agents but retained challenges like uncontrollable
circulation and tumor cells, utilizing chemical cleav- payload release.1,3,5 The second generation, fea-
turing brentuximab vedotin and ado-trastuzum-
age (hydrazone and disulfide bonds)4,9 or enzyme
ab emtansine, optimized mAb isotypes, cytotoxic
cleavage (glucuronide and peptide bonds).1,9 Hydra-
payloads, and linkers, enhancing clinical efficacy
zone-linked ADCs remain stable in circulation and
and safety.1 Despite progress, issues like off-target
release cytotoxic payloads in lysosomes (pH 4.8) and
toxicity and high drug-antibody ratios persisted.1,7
endosomes (pH 5.5–6.2) upon internalization into
The third generation, represented by polatuzumab
cancer cells.1 Non-cleavable linkers depend on lyso-
vedotin, enfortumab vedotin, and fam-trastuzum-
somal degradation of the entire structure, retaining
ab deruxtecan, focuses on overcoming limitations,
charged amino acids in the payload.3,4,9 The linker’s
employing specific conjugation technologies for
role is critical in ensuring effective release within tar-
homogenous ADCs with consistent drug-antibody
get tumor cells, preventing premature release in the
ratios, resulting in improved anticancer efficacy,
plasma that could harm normal tissues. lower toxicity, and increased stability.1

Conjugation methods Clinical application of ADCs

Two types of conjugation techniques are com- As of February 2024, the FDA has approved 12
monly used; stochastic and site-specific.1 Stochas- ADCs for various cancers, encompassing lym-
tic conjugations by amide coupling in ADCs like phomas, leukemia, myeloma, and solid tumors
gemtuzumab ozogamicin, T-DM1, and inotuzum- like HER2+ breast cancer, urothelial cancer, met-
ab ozogamicin, leads to variable drug-antibody astatic triple-negative breast cancer, and gastric
ratios (DAR 0–8) due to randomness among nu- cancer. Examples include trastuzumab emtansine
merous lysine residues.1 This may cause instability, (T-DM1) for HER2-positive breast cancer and ino-
premature payload release, off-target toxicity, and tuzumab ozogamicin for acute lymphoblastic leu-
challenges in achieving consistency.3,7 In contrast, kemia. Additionally, there are over 100 ADC candi-
site-specific conjugation relies on cysteine-based dates in clinical trials, with many of them already
coupling, exposing disulfide bonds for a controlled demonstrating remarkable success in advanced
approach, reducing heterogeneity.1,5 However, it phases.12 These success stories underscore the
may compromise antibody integrity.1 potential of ADCs to transform the landscape of
cancer treatment.12
Evolution of ADCs
Challenges and future directions
ADC drug development has evolved through three
generations. The initial generation, exemplified by ADCs encounter several challenges like complex
BR96-doxorubicin, utilized non-cleavable linkers pharmacokinetics, side effects, limited tumor tar-

13
Table 1. FDA Approved ADCs1,9

geting, and drug resistance.4,7,9 In addition, tumor Sino Biological are at the forefront of advancing
penetration is hindered by ADCs’ large molecular ADC research with a comprehensive suite of prod-
weight.1,4,5 To overcome these challenges, next-gen- ucts and services. Our portfolio boasts a diverse
eration ADCs focus on optimizing monoclonal an- range of ADC target proteins for precise cancer cell
tibodies, linkers, and payloads, utilizing bispecific drug delivery, complemented by MMPs, Cathep-
antibody technology, and employing dual-payload sins, and uPA enzymes for optimal linker cleav-
ADCs.1,4,5 Smaller molecular weight ADCs, like PEN- age. High-quality FACS antibodies and reliable Fc
221, aim to enhance penetration efficiency, partic- receptor proteins aid immune cell analysis and
ularly in challenging tumors.1 Finally, the use of two therapeutic development. Specialized anti-pay-
cytotoxic agents as payloads to reduce ADC resis- load antibodies assess efficacy. Beyond products,
tance is also promising.8 our services encompass rigorous in vitro bioactiv-

14
ity validation, internalization assay studies, bind- 7. Shastry, M. et al. Rise of Antibody-Drug Conjugates:
ing activity assays, neutralizing/blocking assays, The Present and Future. American Society of Clinical
anti-idiotypic antibodies development for PK/ADA Oncology Educational Book (2023) doi:10.1200/
assays, and control antibody production, ensuring EDBK_390094.

a holistic approach to ADC research. 8. Abuhelwa, Z., Alloghbi, A. & Nagasaka, M. A

comprehensive review on antibody-drug conjugates
References (ADCs) in the treatment landscape of non-small cell
lung cancer (NSCLC). Cancer Treatment Reviews
1. Fu, Z., Li, S., Han, S., Shi, C. & Zhang, Y. Antibody drug vol. 106 Preprint at https://doi.org/10.1016/j.
conjugate: the “biological missile” for targeted cancer ctrv.2022.102393 (2022).
therapy. Signal Transduction and Targeted Therapy
9. Nguyen, T. D., Bordeau, B. M. & Balthasar, J. P.
vol. 7 Preprint at https://doi.org/10.1038/s41392-022-
Mechanisms of ADC Toxicity and Strategies to
00947-7 (2022).
Increase ADC Tolerability. Cancers vol. 15 Preprint at
2. Díaz-Rodríguez, E., Gandullo-Sánchez, L., Ocaña, A. & https://doi.org/10.3390/cancers15030713 (2023).

Pandiella, A. Novel adcs and strategies to overcome 10. Menon, S., Parakh, S., Scott, A. M. & Gan, H. K.
resistance to anti-her2 adcs. Cancers vol. 14 Preprint Antibody-drug conjugates: beyond current approvals
at https://doi.org/10.3390/cancers14010154 (2022). and potential future strategies. Exploration of
Targeted Anti-tumor Therapy vol. 3 252–277 Preprint
3. Gogia, P., Ashraf, H., Bhasin, S. & Xu, Y. Antibody–Drug
at https://doi.org/10.37349/etat.2022.00082 (2022).
Conjugates: A Review of Approved Drugs and Their
Clinical Level of Evidence. Cancers vol. 15 Preprint at 11. Koster, K.-L., Huober, J. & Joerger, M. New antibody-
https://doi.org/10.3390/cancers15153886 (2023). drug conjugates (ADCs) in breast cancer—an
overview of ADCs recently approved and in later
4. Samantasinghar, A. et al. A comprehensive review stages of development. Explor Target Antitumor Ther
of key factors affecting the efficacy of antibody 27–36 (2022) doi:10.37349/etat.2022.00069.
drug conjugate. Biomedicine and Pharmacotherapy
12. Kesireddy, M., Kothapalli, S. R., Gundepalli, S. G. & Asif,
vol. 161 Preprint at https://doi.org/10.1016/j.
S. A Review of the Current FDA-Approved Antibody-
biopha.2023.114408 (2023).
Drug Conjugates: Landmark Clinical Trials and
5. Wang, Z., Li, H., Gou, L., Li, W. & Wang, Y. Antibody– Indications. Pharmaceut Med 38, 39–54 (2023).
drug conjugates: Recent advances in payloads. Acta
Pharmaceutica Sinica B vol. 13 4025–4059 Preprint at
https://doi.org/10.1016/j.apsb.2023.06.015 (2023).

6. Ferraro, E., Drago, J. Z. & Modi, S. Implementing

antibody-drug conjugates (ADCs) in HER2-positive Additional Resource
breast cancer: state of the art and future directions. Emerging ADC Target Antigens in
Breast Cancer Research vol. 23 Preprint at https://doi. Solid Tumors
org/10.1186/s13058-021-01459-y (2021).

16
DNA Methylation Detection
Using Droplet Digital™ PCR
Novel technique has a number of advantages
Hamutal Bonen, Ph.D., Jeremiah McDole, Ph.D., and Eddy van Collenburg

status as an indicator of early-stage cancer devel-

Abstract
opment and as a method for monitoring tumor re-
Thirteen years have passed since the advent of Drop- sponse to therapy. Detecting methylation changes
let Digital PCR (ddPCR™) technology. In that time, in cell-free DNA (cfDNA) is especially attractive giv-
ddPCR platforms have revolutionized the absolute en the noninvasive nature of blood draws.
quantification of nucleic acids. With a host of tech- Unfortunately, sensitive, robust, and cost-effec-
nical advances and enhanced automation in recent tive methods to perform methylation analysis on
years, ddPCR is moving from a specialized research cfDNA samples remain an unmet need. Here we
technique to a robust tool in clinical and translation demonstrate how common methylation detection
research applications. This white paper reviews the methods combine with ddPCR to enhance analysis
use of ddPCR for detection of DNA methylation. We of these precious samples.
review ddPCR technology, compare different work-
flow strategies, and explain critical parameters for op- DNA Digestion Using
timal assay design to get unprecedented sensitivity,
linearity, and robustness when detecting DNA meth-
Methylation-Sensitive
ylation by ddPCR. Restriction Enzymes (MSRE)
By far, the fastest and easiest method of DNA
Introduction
methylation quantification incorporates MSRE di-
DNA methylation plays a critical role in many nor- gestion. Further, this method does not have the
mal developmental and sustaining physiological drawbacks of bisulfite conversion, such as exten-
processes. However, aberrant methylation can sive DNA degradation and high starting material
lead to a variety of disease states, including several input requirements.
types of cancer. For instance, global hypomethyla- As shown in the schematic below, methylation-sen-
tion can contribute to the activation of oncogenes sitive restriction enzymes do not cleave restriction
while hypermethylation within tumor suppressor sites in the presence of a methylated cytosine, lead-
genes can result in silencing. Mounting evidence ing to PCR amplification (Figure 1), while cleavage
demonstrates the utility of analyzing methylation of an unmethylated site inhibits amplification.

17
 To detect the methylation status at a specific locus,
the sample is digested by a methylation-sensitive
restriction enzyme and analyzed with Droplet Dig-
ital PCR (Figure 2). When using probe- based de-
tection methods, multiplexing several methylation
sites or targets is possible. For best results, a du-
Figure 1. Schematic overview of methylation-sensitive plex reaction with a reference target (no restriction
restriction enzyme digestion. M, methyl; MSRE, sites) is advised (e.g., RPP30).
methylation-sensitive restriction enzyme.
The reference assay is used to normalize and cor-
rect for small input differences caused by pipetting
There are a variety of commercially available MSREs
or samples with unstable copy number.
available (Table 1). Specific primers can be designed
to amplify a specific sequence of interest that con- Methylation percentages are calculated with high
tains an MSRE. See the Primer Design section for precision via Droplet Digital PCR by measuring
recommendations on how to design primers. DNA concentrations in samples with (MSRE+) and
Table 1. Overview of MSREs and
without (MSRE–) enzyme. The percentage of meth-
restriction site sequences. ylation, or methylation fraction, is calculated using
Restriction Enzyme (all 4-base cutters) Restriction Site the precise ddPCR concentrations measured for
CGCG samples either digested (MSRE+) or undigested
AccII (BstUI, Bsh1236I) (MSRE–), as follows:
GCGC
CCGC
AciI (SsiI) MSRE+ ratio =
GGCG target concentration/reference concentration
CCGG
HpaII (HapII)
GGCC MSRE– ratio =
GCGC target concentration/reference concentration
HhaI (CfoI)
CGCG % methylation = MSRE+ ratio/MSRE– ratio

Figure 2. Typical ddPCR MSRE workflow. Sample is split. One aliquot is digested with a restriction enzyme and the other without restriction
enzyme. Both aliquots are run as individual samples using a standard ddPCR setup. MSRE, methylation-sensitive restriction enzyme.

18
Note that copy number results are available from Droplet reading can be performed on a QX200™ or
the undigested (MSRE–) samples and can be used QX600™ Droplet Reader. All the data shown in this
to detect differences in samples (assuming the use white paper were generated using a QX200 Drop-
of a stable reference gene). let Reader.

Samples are prepared as follows (incubations can

be done in a thermal cycler such as the T100 Ther-
mal Cycler):

• MSRE+ samples — 50–100 ng of DNA is

incubated for 1 hr at 60°C with 2–10 U
of BstUI and 0.5 µl 10x CutSmart Buffer
(New England Biolabs, Inc., discontinued;
rCutSmart Buffer [#B6004S] is a suitable
alternative) in a total volume of 5.0 μl.

• MSRE– samples — 50–100 ng of DNA is

incubated for 1 hr at 60°C with 0.5 μl 10x
CutSmart Buffer in a total volume of 5.0 μl.

In a 22 µl experiment, 1–100 ng of incubated DNA

samples can be analyzed using 11 µl ddPCR Super-
mix for Probes (No dUTP)

(Bio-Rad Laboratories, Inc., catalog #1863024) and

primers and probes in a final concentration of 900
and 250 nM, respectively.

PCR mixtures are partitioned into ~20,000 droplets

using the Automated Droplet Generator (Bio-Rad,
#1864101). Subsequent PCR is performed in a ther-
mal cycler using the the protocol outlined in Table 2.

Table 2. Thermal cycling protocol. Ramp rate set to Figure 3. PTGER4 promotor (FAM) and RPP30 (HEX). One-
2°C/sec for all steps. dimensional plots show the positive and negative droplet
Cycling Step Temperature, Time Number clusters from two targets run in duplex, PTGER4 (FAM, AssayID
°C of Cycles dHsaEXD82842910) and RPP30 (HEX, AssayID dHsaCP2500350).
Enzyme activation 95 10 min 1 Two-dimensional plot shows the four clusters from the same
Denaturation 95 30 sec reaction. HpaII is used for digestion. Concentrations (copies/
μl) are shown in the bottom graph and used for calculating the
Annealing/extension 55 1 min 40
percentage of methylation from each sample (see calculations).
Enzyme deactivation 98 10 min MSRE, methylation-sensitive restriction enzyme.
Hold 12 <48 hr 1

19
 Figure 4. SEPT9 CGI3 promotor (FAM) and RPP30 (HEX). One-
Figure 5. Probe Mix Triplex WIF1 (FAM), NPY (FAM), and RPP30
dimensional plots show the positive and negative droplet clusters
(0.4x FAM, 0.25x HEX). One-dimensional plots show the
from two targets run in duplex, SEPT9 CGI3 (FAM, AssayID
positive and negative droplet clusters from three targets run
dHsaEXD18126781) and RPP30 (HEX, AssayID dHsaCP2500350).
in triplex. WIF1 (FAM, AssayID dHsaEXD31159278), NPY (FAM,
Two-dimensional plot shows the four clusters from the same
AssayID dHsaEXD18238370), and RPP30 (0.4x FAM, AssayID
reaction. BstUI is used for digestion. Concentrations (copies/
dHsaCP2500313, 0.25x HEX, AssayID dHsaCP2500350). Two-
μl) are shown in the bottom graph and used for calculating the
dimensional plot shows up to eight clusters from the same
percentage of methylation from each sample (see calculations).
reaction. A mixture of AciI and HhaI was used for digestion.
MSRE, methylation-sensitive restriction enzyme.
Two replicate reactions of an unmethylated control sample
were used to compare the digestion in sample (buffer: digestion
overnight) with the digestion in master mix (sample: 45 min
at room temperature). Concentrations (copies/μl) are shown
Examples of Assay Performance in the bottom graph and used for calculating the percentage
of methylation from each sample (see calculations). MSRE,
In this section, we highlight assay performance us- methylation-sensitive restriction enzyme.
ing a few examples (Figures 3–5).

20
 Table 3. PTGER4 promoter methylation calculations. • Look for restriction enzymes that have 2 or
Methylated Unmethylated more restriction sites close together
MSRE+ Ratio 175/185 = 0.946 2.39/249 = 0.0096
• Design primers outside these restriction
MSRE– Ratio 179/183 = 0.978 272/252 = 1.079 sites to produce an amplicon with 2 or more
Methylation, % 0.946/0.978 = 97% 0.0096/1.079 = 0.9% restriction sites

Table 4. SEPT9 promoter methylation calculations. • A restriction site can be included in the
Methylated Unmethylated primers but only in the last 4 nucleotides of
MSRE+ Ratio 335/208 = 1.611 2.33/266 = 0.0088 the 3’ end of each of the primers. Restriction
MSRE– Ratio 350/217 = 1.613 271/272 = 0.996 sites outside the primers are preferred
Methylation, % 1.611/1.613 = 100% 0.0088/0.996 = 0.9% • Short amplicons (<100 nucleotides)
enable fragmented sample analysis (e.g.,
PTGER4 Promoter (FAM) and RPP30 (HEX) formalin-fixed paraffin-embedded [FFPE] or
circulating tumor DNA [ctDNA])
The percentage of PTGER4 promoter methylation
was calculated as shown in Table 3. • Combining different restriction enzymes
is possible and used to perform double
The percentage of SEPT9 promoter methylation digestions in the same reaction
was calculated as shown in Table 4.
Example Assay Design
ddPCR Assay Design Figure 6 shows an example assay design for the tran-
scription factor one cut homeobox 2 (ONECUT2).
Since analysis is based on restriction enzyme cleav-
age, the amplicon sequence must include cleavage
sites. This approach provides high specificity, how-
ever only specific restriction sites can be analyzed,
which can be a limitation. To reliably measure DNA
Figure 6. Example assay design for the transcription factor
methylation, we recommend including at least two
ONECUT2 (BstUI restriction sites). Bio-Rad PrimePCR AssayID:
but not more than four restriction sites in the am- dHsaEXD31605634. BstUI sites are bolded, primer sites are
plicon. If a single MSRE cannot cut at least two sites underlined, and the probe site is highlighted yellow. Chr,
in an amplicon, combining multiple MSREs that chromosome; hg, human genome.
cut within the amplicon is a good alternative. For
an extended overview on how to design specific
ddPCR assays, please refer to the Assay Design for
Cytosine Deamination
Droplet Digital PCR section in bulletin 6407.

Consider the following when designing primers: While MSRE digestion protocols are rapid and
reliable, sodium bisulfite conversion is the cur-
• Check for restriction sites within the area of rent gold standard of methylation analysis. In
your target sequence this method, DNA is denatured and treated with

21
sodium bisulfite. This process deaminates un- dium bisulfite protocols typically require between
methylated cytosine in DNA to uracil and leaves 0.1 and 2 μg of DNA (depending on the manufac-
methylated cytosines unchanged. Amplifying the turer) while the enzymatic approach can be per-
treated DNA with PCR converts uracils further to formed with much less.
thymines while the methylated cytosines remain
Since both methods rely on the conversion of un-
cytosines. An example of cytosine deamination is
methylated DNA to thymine, primers and probe
shown in Figure 7.
designs are the same for both.

Primer Design
There are two main design strategies available to
specifically amplify converted (unmethylated) and
unconverted (methylated) sequences with Drop-
Figure 7. Example of cytosine deamination on template DNA let Digital PCR: methylation-specific primers and
strand. Underline denotes methylated cytosine. Note that
methylation-independent primers (Figure 8).
sense and antisense strands are no longer complementary
after deamination.

Although bisulfite treatment is relatively fast and

straightforward, it requires elevated temperatures
and high pH that can damage DNA, resulting in
fragmentation and depyrimidination. Unmethylat-
ed cytosines are especially sensitive to degradation
with this method, resulting in challenges amplify-
ing GC-rich areas.

A less damaging method for cytosine deamina-

tion was recently developed by New England Bi-
olabs, Inc. (NEB). In this method, APOBEC enzyme
is used to convert cytosine to uracil, resulting in
far less DNA degradation. However, APOBEC can
also deaminate 5-methylcytosine (5mC) and 5-hy- Figure 8. Comparison of methylation-specific and methylation-
droxymethylcytosine (5hmC). To protect the meth- independent design strategies. CpG, 5’-cytosine-phosphate-
ylated cytosine from deamination, NEB uses TET2 guanine-3’.
enzyme and Oxidation Enhancer (#E7129) to mod-
ify 5mC and 5hmC to forms that are not substrates Primer design considerations for methylation-spe-
for APOBEC. cific primers:
When deciding which method to utilize, the re- • Include as many CpG sites as possible,
quired starting material should be considered. So- especially at the 3’ end of the primer

22
 • Consider the same CpG sites in the
primer sequence for methylated DNA and
unmethylated DNA

Maximize specificity by using a detection probe in-

cluding CpG sites Primer design considerations for
methylation-independent primers:
• No CpG sites within the primer sequence
• Use an adequate number of C (no CpG)
• Spanning a maximum number of CpG sites
in the amplicon
• Maximize specificity by using a detection
probe including CpG sites
Example Performance (Rat SNRPN )
Figure 9 shows examples of the performance of
methylation-specific and methylation-indepen-
dent primers.

Summary
Methylation-sensitive primers have the advantage
of detecting methylation levels at the primer-bind-
ing region but have the disadvantage of needing
two different primer sets.

Methylation-insensitive primers have the advan-

tage of a single primer set, which should compen- Figure 9. Two-dimensional plot showing the duplex reaction
sate for PCR bias and can be combined with high for rat SNRPN promotor from a mixture of methylated and
unmethylated control samples. A, methylation-specific primers
resolution melt analysis, resulting in methylation
(target: chr1:111592360-111593159 [reverse complement]); B,
information at the regional level (amplicon). methylation-independent primers (target rn7| chr1:111123593-
111123731). Chr, chromosome.
Neither method gives information on individual
CpG resolution levels.
folio of applications highlights the continuously
Conclusions growing utility of Droplet Digital PCR.

Droplet Digital PCR has been utilized in many new While many conventional methods are available
genetic analysis applications. Adding methyla- for cytosine deamination–based methylation anal-
tion-based detection tools to its already large port- ysis (methylation-specific PCR, high resolution

23
melt analysis, pyrosequencing, etc.), several pub- 4. Larsen LK et al. (2018). Noninvasive detection of high grade
prostate cancer by DNA methylation analysis of urine cells captured
lications have demonstrated the added value that
by microfiltration. J Urol 200, 749–757.
Droplet Digital PCR provides (van Wesenbeeck et
al. 2018, Cho et al. 2020, Hagelans et al. 2017, Leh- 5. Lehmann-Werman R et al. (2018). Monitoring liver damage using
mann-Werman et al. 2018, Larsen et al. 2018, Jen- hepatocyte-specific methylation markers in cell-free circulating
sen et al. 2019). DNA. JCI Insight 3, e120687.

Methylation detection using MSRE-based diges- 6. Nell RJ et al. (2020). Quantification of DNA methylation
independent of sodium bisulfite conversion using methylation-
tion in combination with Droplet Digital PCR
sensitive restriction enzymes and digital PCR. Human Mutat 41,
(MSRE ddPCR) is a relatively new technique (Nell et 2,205–2,216.
al. 2020, Wang et al. 2021, van Zogchel et al. 2021,
van de Leemkolk et al. 2022). Advantages for this 7. van de Leemkolk FEM et al. (2022). Quantification of unmethylated
method include negligible DNA degradation and insulin DNA using methylation sensitive restriction enzyme digital
polymerase chain reaction. Transpl Int 35, 10167.
a minimum amount of required starting material.
These parameters make this method well suited 8. Van Wesenbeeck L et al. (2018). Droplet digital PCR is an accurate
for applications that require liquid biopsy samples. method to assess methylation status on FFPE samples. Epigenetics
Additionally, because the DNA is preserved, other 13, 207–213.
genetic alterations such as copy number variants
9. van Zogchel LMJ et al. (2021). Novel circulating hypermethylated
and single nucleotide variants can be performed RASSF1A ddPCR for liquid biopsies in patients with pediatric solid
on the same sample. tumors. JCO Precis Oncol 5, PO.21.00130.

References 10. Wang D et al. (2021). Development of a liquid biopsy based purely
quantitative digital droplet PCR assay for detection of MLH1
promoter methylation in colorectal cancer patients. BMC Cancer
1. Cho NY et al. (2020). Blood-based detection of colorectal cancer using
cancer- specific DNA methylation markers. Diagnostics (Basel) 11, 51. 21, 797.

2. Hagelgans A et al. (2017). Identification of CpG sites of SERPINA5

promoter with opposite methylation patterns in benign and About the Authors
malignant prostate cells. Anticancer Res 37, 6,609–6,618.

3. Jensen SØ et al. (2019). Novel DNA methylation biomarkers show Hamutal Bonen, Ph.D., Jeremiah McDole, Ph.D., and
high sensitivity and specificity for blood-based detection of
colorectal cancer—a clinical biomarker discovery and validation
Eddy van Collenburg are with Bio-Rad Laboratories,
study. Clin Epigenetics 11, 158. Digital Biology Center

Additional Resource
QX600 Droplet Digital™ PCR
(ddPCR™) System

24
Six appeal
QX600 Droplet Digital PCR System
with more colors for greater multiplexing capabilities
Trust Your Results with
Rigorously Validated ELISA Kits
Proprietary validation process helps ensure precise and reproducible
results that you can trust

Enzyme-linked immunosorbent assay (ELISA) is con- detects the analyte of interest without artifacts
sidered the gold standard of immunoassays for the or interference and produces a standard curve
high specificity and sensitivity it provides. Although that closely mirrors antibody-antigen binding
researchers may choose to develop an ELISA in- in the test samples. To ensure confidence in its
house, ready-to-use ELISA kits can save time and
ready-to-use ELISA kits, HUABIO bases its valida-
reduce the risk of errors—provided they have been
properly validated. By basing its ELISA validation tion process on four core performance indica-
process on four core performance indicators, HUA- tors, as described below:
BIO ensures that its ready-to-use ELISA kits deliver
• Sensitivity
precise and reproducible results that you can trust.
Sensitivity is typically reported as the minimum de-
ELISA principles tectable dose (MDD), which is calculated by adding
two standard deviations (SD) to the mean value ob-
ELISA is a microplate-based method for detecting tained from replicates of the zero standard. A com-
and quantifying analytes in solution. The most wide-
parison between HUABIO’s ready-to-use ELISA kits
ly used format, the sandwich ELISA, involves coating
the microplate wells with a capture antibody before for human IL-6, TNF-α, IFN-γ, IL-17A, and IL-4 and
blocking and then incubating with the test samples those of a leading competitor indicates the HUABIO
and a reference standard (added across multiple products to have consistently higher sensitivity, as
wells to produce a concentration curve). After wash- shown in Table 1, spanning a 2.5-fold improvement
ing to remove any unbound material, a detection for IL-6 to an increase of over 300-fold for IL-4.
antibody is introduced and the resultant signal is
measured with a plate reader. The analyte concen- Table 1. Sensitivity comparison for HUABIO and a leading competitor
tration in the test samples is then calculated. Target Competitor 1 MDD HUABIO MDD
(pg/mL) (pg/mL)
Rigorous validation ensures Human IL-6 0.7 0.26

reliability and consistency Human TNF-α 6.23 0.21

Human IFN-γ 5.69 0.36

Before an ELISA can be used for sample testing, Human IL-17A 15 0.26

it must be validated to confirm that it accurately Human IL-4 10 0.03

26
• Stability a pass, while a value of 80–120% indicates excel-
lence. Testing of the five HUABIO ELISA kits men-
Stability is defined as the ability to maintain con-
tioned previously demonstrates excellence across
stant product performance, which is critical for gen-
the panel, as shown in Table 3.
erating consistent results over time. Testing shows
HUABIO’s ready-to-use ELISA kits to have a shelf-life
Table 3. Accuracy testing for a panel of HUABIO
of ≥ 18 months at 4 °, and that storage at 37 ° for one ready-to-use ELISA kits.
day is equivalent to storing at 4° for 48 days, demon- Target Recovery (%)
strating the high stability of these products. Human IL-6 84–115

• Precision Human TNF-α 95–105

Human IFN-γ 89–113
Precision is the degree of dispersion of different Human IL-17A 80–112
measurement values in the same sample. It is de- Human IL-4 88–105
termined by testing samples of known concentra-
tion multiple times on the same plate and calcu-
lating the coefficient of variation with the formula A complete ready-to-use solution
% CV = (SD/average) x 100. An assay is generally
given a pass if the % CV is ≤ 15, while % CV values Besides its rigorous validation process, HUABIO
of ≤ 10, ≤ 8 and ≤ 5 are respectively associated with safeguards researchers’ results by incorporating
good, outstanding, and excellent precision. Repre- several helpful design features into its ready-to-
sentative data for HUABIO’s IL-6 ready-to-use ELI- use ELISA kits too. These include color-coded kit
SA kit are shown in Table 2 and exhibit the highest components, to avoid confusion, and provision of
precision levels. ready-to-use working solutions, to save time and
minimize the risk of dilution errors. Other nota-
Table 2. Precision testing for the Human IL-6 ELISA kit (EH0001), ble features of HUABIO’s ready-to-use ELISA kits
n=22 for each concentration tested.
Parameter S1 S2 S3
are their concise protocols, short run times (just
Average (pg/mL) 24 115.4 263.2
120 minutes from start to finish), and miniaturized
SD 1.1 4.5 9.3
packaging that facilitates storage and transporta-
% CV 5.6 3.9 3.6 tion. In addition, the use of laser-engraving instead
of traditional printed labels simplifies product rec-
ognition by resisting wear.
• Accuracy
To learn more about HUABIO’s ready-to-use ELISA
Accuracy refers to the consistency between mea-
kits and how they can benefit your research, visit
sured values and true values, which is best deter-
huabio.com/elisa-kits
mined by running a spiking experiment. This in-
volves introducing a known concentration of the
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ery coefficient with the formula ((measured val-
Additional Resource
ue–sample background value)/spiked theoretical HUABIO ELISA Kits
value) × 100%. A value of 50–150% is considered

28