Merck Immunoassay - External Short

TIPS PEMILIHAN TEKNIK IMMUNOASSAY UNTUK ANALISA BIOMARKER :
“ELISA VS MULTIPLEX IMMUNOASSAY CYTOKINE/CHEMOKINE

USING LUMINEX TECHNOLOGY”
7th MCC, ITD UNAIR 2023
Taufik Indamawan
Merck Life Science ID
ELISA LUMINEX XMAP
(SINGLE ANALYTE) (MULTI ANALYTE)
Antigen Detection
Antibody Detection
IgG Antibody
(Analyte)
Sandwich Assay Procedure :
MERCK: ELISA 1. Bring all reagents and samples to room temperature (18 - 25°C) before
use. It is recommended,all standards and samples be run at least in
duplicate.
2. Add 100 µl of each standard and sample into appropriate wells. Cover
wells and incubate for 2.5 hours at room temperature or overnight at
4°C with gentle shaking.
3. Discard the solution and wash 4 times with 1X Wash Solution. Wash by
filling each well with Wash Buffer (300 µl) using a multi-channel Pipette
or autowasher. After the last wash, remove any remaining Wash Buffer
by aspirating or decanting, invert the plate,blot it against clean paper
towels.
4. Add 100 µl of 1x prepared Detection Antibody to each well. Cover wells

and incubate for 1 hour at room temperature with gentle shaking.
5. Discard the solution. Repeat the wash procedure as in step 3.
6. Add 100 µl of prepared Streptavidin solution to each well. Cover wells

and incubate for 45 minutes at room temperature with gentle shaking.
7. Discard the solution. Repeat the wash as in step 3.
8. Add 100 µl of TMB One-Step Substrate Reagent (Item H) to each well.

Cover wells and incubate for 30 minutes at room temperature in the
dark with gentle shaking.
9. Add 50 µl of Stop Solution (Item I) to each well. Read absorbance at

450 nm immediately.
MERCK: MILLIPLEX (Multiplex)
1 3
7
IMMUNOASSAY PLATFORM
ELISA Vs Multiplex ELISA
ELISA MILLIPLEX® - LUMINEX

Plate Reader Compatibility Biomarker Screening
Single Protein Analysis Multiplex Assay
picogram/ml picogram/ml
50-100 ul ~25 ul
2-3 log dynamic range 3-4 log dynamic range

LUMINEX XMAP
UNIT BASE
Lab Genomic CSC
ITD Unair Lab Hepatitis Lab Sentral

& Lab TB
Lab Virologi Litbangkes &

B2P2VRP
Lab FK Lab Terpadu FK-KMK

Prodia Widyahusada
Kramat
Lab FK
Lab FK Terpadu, Collab Lab FMIPA

Bio UI, Lab IMERI - Oxford
University Clinical Research Unit
MERCK: MILLIPLEX® Multiplex Immunoassay
1 day 5-15 day

Comparison of Immunoassays: Multiplex (Milliplex) vs. ELISA
For detection of 20
biomarkers in 38 samples MILLIPLEX® ELISA
(run in duplicate)
Number of plates required

20
Total time to result 3 hours 10 hours
Results per plate 1,520 76
Total volume required per sample 10-50 µL 0.5-1 mL
Dynamic range 1-10,000 pg/mL 10-2,500 pg/mL
Lower limit of detection ~1 pg/mL ~1 pg/µL

3 Proteins
How to Choose right Immunoassay
Sample size
Limited sample types such as cerebrospinal Speed and high-throughput

fluid, synovial fluid, tears, mouse serum, etc
Many Analytes in Single well!!

Save time, money, and manpower….
Singleplex ELISA similar workflow, multiplex assay results

Less than half the cost of the standard ELISA (less capture
antibody used due to the smaller surface area) Material No. MILLIPLEX ELISA
1 Analyte 16,785,000 9,000,000
2 Analyte 20,617,000 18,000,000
3 Analyte 27,747,000 27,000,000
4 Analyte 33,092,000 36,000,000
5 Analyte 38,433,000 45,000,000
6 Analyte 43,774,000 54,000,000
7 Analyte 49,116,000 63,000,000
8 Analyte 54,457,000 72,000,000
9 Analyte 59,799,000 81,000,000
10 Analyte 65,142,000 90,000,000
MILLIPLEX® MAP
Background
• xMAP® is multi-analyte profiling technology
• Luminex Corporation is the inventor of

xMAP® technology
• Merck partners with Luminex® to develop

MILLIPLEX® MAP assays and kits
Overview
MILLIPLEX® (Multiplexing)
Microsphere-based assay for simultaneous
detection of multiple biomarkers/analytes, in
one single sample
 Microsphere – Higher surface-to-volume ratio
 Low sample volume (25 uL)
 Save time & valuable sample material
Magplex – 6.5 micron

Magnetic microspheres
Overview
MILLIPLEX® xMAP® Technology
Infrared Red
dye dye
Microsphere beads Precise concentration Distinctly-coloured

of fluorescent dyes bead sets
Materials Required :
Luminex 200
Instrument
Milliplex Kit
DI H2O 0.1 M NaOH

Luminex 200 10% bleach 70% alcohol
Calibration & Verification
14
Materials Required :
Reagent reservoir
Microtube
1.5 mL
500-800 rpm
Spin Down 4 to 65°C range temp.
Duran Schoot 500 mL
Handheld
Magnetic &
Additional Black Mylar
Plate
Seal Plate 96 Well

Single & Vortex
15 Multichannel
Pippete
Luminex Instrument Positioning
500 Plex
500 Plex 20 minutes
20 minutes
Multiplexing
100 Plex
45 minutes
FM3D
Intelliflex RUO
LX 200
Price and Throughput

Overview What is xMAP® Technology?
Type of Sample :
Serum
Plasma
Cell Culture Supernatant
*Cell Lysate
*Urine
* Spesific Anayte only
https://www.sigmaaldrich.com/life-science/immunoassay-platform-solutions/milliplex.html
Multiplex Capabilities
Bead sets are

combined into a single
well
Antibody/Antigen-conjugated magnetic beads

PE fluorescence is
quantifiable by Luminex®
instrument
Analytes/Antigen
reporter molecules
Capture
with fluorescent dye
Antibody
(Streptavidin - PE)
Biotinylated Detection
Antibody
Protocol
Detection System
Green laser Quantify assay

(525 nm) reporter dyes
FLOW CYTOMETRY
Luminex® 200
& Red laser Identify bead
(635 nm) colour for analytes
FlexMAP 3D® identification
Chicken
Immunoassay Portfolio: Target Breakdown • Cytokine/Chemokine
Human
• Cytokine/Chemokine • Liver proteins Primate
• T Lymphocytes • Neurodegenerative • Cytokine/Chemokine
536 71
• Cytokine Receptors • Neuropeptide • Metabolic Hormone
Human Primate
• Complement • Adipokine/Adipocyte • Pituitary
• Immunoglobulins • Metabolic hormone Bovine
• Cardiovascular • Myokine Multiplex • Cytokine/Chemokine
Rat
• Cancer and I/O • Pituitary MILLIPLEX® • Cytokine/Chemokine
• Metabolic Hormone
• Thyroid
• MMPs
• Bone Metabolism 193 Kits and growing • Adipokine/Adipocyte
• TIMPs • Metabolic hormone
• Skin
• IGFBPs
• Kidney injury
1,130 Soluble Analytes • Pituitary
• Angiogenesis 162 • Stress hormone
• Liver injury 128 Cell Signaling Analytes
• Metastasis
Single End Point Rat • Thyroid
• Bone
1800+ ELISA Assays • Neuropeptide
SMCxPRO™ 30+ Assays Ovine
• Cardiac injury • Cytokine/Chemokine
Signaling • Kidney toxicity
• Y/S/T Phosphorylation
Intra-
• Liver injury
• AKT, MAPK, STAT, SFK, SRC cellular
• Vascular injury
• NF-κB, TGFβ, RTK, Multi-Pathway, TCR 236
• Apoptosis, Protein Translation Mouse
• DNA Damage/Genotoxicity
80
Porcine, Mouse
Feline, 45 • Cytokine/Chemokine • Pituitary
Equine Canine • T lymphocytes • Thyroid

• Immunoglobulins • Bone
• MMPs • Cardiovascular
Equine
Cytokine/Chemokine • Adipokine/Adipocyte • Angiogenesis
• Metabolic hormone • Neuropeptide
Feline Canine • Kidney toxicity
• Cytokine/Chemokine • Cytokine/Chemokine
• Metabolic hormone Porcine • Metabolic hormone
Virus • Cytokine/Chemokine • Pituitary
SARS Cov 2 Antigen • Kidney toxicity
Data Analysis – Human COVID Cytokine A
Data Analysis – Human Cytokine B
Data Analysis – Human Cancer
Data Analysis – Chicken Cytokine
Data Analysis - Bovine
Data Analysis - Mouse
Data Analysis - Dog
Publication
MILLIPLEX® Assay Kits (Over 12000 publications)
Publication List MILLIPLEX®

Multiplex Assays Using Luminex®
https://www.sigmaaldrich.com/life-science/immunoassay-plat
form-solutions/milliplex/publications.html
ELISA Parameter Multiplex – Luminex
<3 Total Analyte ≥3
Limited Volume sample Unlimited
Low Cost <3 Analytes = High

≥ 3 Analytes = Low
+ Save Time +++
+ Standard Curve Range +++
+ Screening Capabilities +++
++ Invesment +
+ Custom Kit ++
MILLIPLEX® Panel List :
Transplantation of ACE2-
Mesenchymal Stem Cells Improves
the Outcome of Patients with COVID-
19 Pneumonia
Figure. The profile of serum cytokine/

chemokine/growth factors. The ratio of serum cytokines
IL-10 (A), growth factor VEGF (B), the chemokine IP-10
(C) and TNF-α (D) before and after MSCs treatment
were detected in severe patients compared with the
control group without MSCs using HCYTA-60K (Merck)
Luminex® 200
• Luminex® based multiplexing
• Multiple bead types
• Runs MILLIPLEX® MAP kits
• 100-plex potential
• 96-well
BioTek® 50™ TS • Laser-based acquisition
FLEXMAP® 3D &
Microplate Washer INTELLIFLEX®
• Simple dispense and aspirate
routines typical of many ELISA –
• Luminex® based multiplexing
8 Strips • Multiple bead types
• Color touchscreen makes • Runs MILLIPLEX® MAP kits
programming quick and easy • 500-plex potential
• Automated switching of up to 3 • 96- & 384-well capable
buffers for even greater • Laser-based acquisition
automation
Supported Immunoassay
Detection Platforms
Solutions
for your
samples.
SMCxPRO™
• Single Molecule Counting (SMC™)
BioTek® 800 TS
• BioTek® Absorbance Reader
• 384-well capable
• Covers the majority of our ELISAs
• Femtogram/mL level detection
• NIST certification plate
• Laser-based
• CFR 21 Part 11
• Bench-top system
• Gen5 Secure Software
• CFR 21 Part 11 Compliance Fit for your
purpose.
Thank You!

Merck Immunoassay - External Short

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TIPS PEMILIHAN TEKNIK IMMUNOASSAY UNTUK ANALISA BIOMARKER :

“ELISA VS MULTIPLEX IMMUNOASSAY CYTOKINE/CHEMOKINE

7th MCC, ITD UNAIR 2023

4. Add 100 µl of 1x prepared Detection Antibody to each well. Cover wells

5. Discard the solution. Repeat the wash procedure as in step 3.

6. Add 100 µl of prepared Streptavidin solution to each well. Cover wells

7. Discard the solution. Repeat the wash as in step 3.

8. Add 100 µl of TMB One-Step Substrate Reagent (Item H) to each well.

9. Add 50 µl of Stop Solution (Item I) to each well. Read absorbance at

ELISA MILLIPLEX® - LUMINEX

Single Protein Analysis Multiplex Assay

2-3 log dynamic range 3-4 log dynamic range

Lab Genomic CSC

ITD Unair Lab Hepatitis Lab Sentral

Lab Virologi Litbangkes &

Lab FK Lab Terpadu FK-KMK

Lab FK Terpadu, Collab Lab FMIPA

1 day 5-15 day

Number of plates required

Total time to result 3 hours 10 hours

Results per plate 1,520 76

Total volume required per sample 10-50 µL 0.5-1 mL

Dynamic range 1-10,000 pg/mL 10-2,500 pg/mL

Lower limit of detection ~1 pg/mL ~1 pg/µL

Limited sample types such as cerebrospinal Speed and high-throughput

Many Analytes in Single well!!

Save time, money, and manpower….

Singleplex ELISA similar workflow, multiplex assay results

• Luminex Corporation is the inventor of

• Merck partners with Luminex® to develop

Magplex – 6.5 micron

MILLIPLEX® xMAP® Technology

Microsphere beads Precise concentration Distinctly-coloured

DI H2O 0.1 M NaOH

Seal Plate 96 Well

Price and Throughput

* Spesific Anayte only

MILLIPLEX® xMAP® Technology

Bead sets are

MILLIPLEX® xMAP® Technology

Green laser Quantify assay

Equine Canine • T lymphocytes • Thyroid

MILLIPLEX® Assay Kits (Over 12000 publications)

Publication List MILLIPLEX®

ELISA Parameter Multiplex – Luminex

<3 Total Analyte ≥3

Limited Volume sample Unlimited

Low Cost <3 Analytes = High

+ Standard Curve Range +++

+ Screening Capabilities +++

Figure. The profile of serum cytokine/

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