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Screening and Immunotyping
Monoclonal Antibody using the
V8 Nexus
Ankara – July 2019
Tony Aitchison – Helena Biosciences
Multiple Myeloma
• Cancerous plasma cell tumour mass
• Clonal expansion of Plasma & B Cells
• Monoclonal antibody production
• Serum monoclonal antibody level
indirectly proportional to tumour mass
• Serum Protein Electrophoresis
Serum Protein Screening using Helena
Helena platform can address
entire serum protein screening
requirements:
• High throughput CZE screening
• Sensitive immunotyping
• Powerful integration
• Comprehensive training
V8 Nexus
• Capillary electrophoresis
• 8 Channel
• Full walk away automation
• Multi test analyser
• Automated maintenance
• Gel integration
• Track and network integration
SAS Gel Electrophoresis
• Semi automated
• SAS-3 Electrophoresis
• SAS-4 Stainer
• Automated sample prep
using V8 Nexus
• Common database for Gel
and CZE results
Serum Protein Screening
• CZE separation on the V8
• Split beta separation
• High throughput
• Accurate monoclonal measurement
• Easily compare to historic results
Capillary Electrophoresis
• Sample cups loaded at anode
• Vacuum applied to load capillary
• Current applied to facilitate
separation
• Absorbance measured as
protein passes the detector
Electro-Osmotic Flow (EOF)
• Silica capillaries have
negative charge
• Run buffer coats capillary
neutralising charge
• Remaining buffer flows
through capillary
Sample Stacking
• Differences in protein mobility
between diluent and buffer
stack protein prior to separation
• Diluent – fast mobility
• Run buffer – slow mobility
Serum Protein Electropherogram
Pre-Albumin
Albumin
Alpha-1-acid Glycoprotein
Alpha-1-antitrypsin
Haptoglobin
Alpha-2-macroglobulin
Haemopexin
Transferrin
Complement
CRP
Immunoglobulin
Six Band Serum Protein Trace
Albumin
Alpha-1
Alpha-2
Beta-1
Beta-2
GammaGamma
Additional Peak
100 200
(sec)
0
50
100
Additional Peak
100 200
(sec)
0
50
100
Gamma M-spike 1
Relative Peak Percentages
0 20 40
(sec)
0
50
100
Relative Peak Percentages
0 20 40
(sec)
0
50
100
Beta-2 M-spike 1
Immunoglobulin Migration – Heavy Chains
IgMIgGIgA
Immunoglobulin Migration – Light Chains
Kappa
Lambda
Monoclonal Migration – Relative Incidence
HighLow
Polyclonal Response
IgG Polyclonal Response
Normal
Polyclonal Response – Clone Specific
IgMIgGIgA
Polyclonal Response – IgG and IgA Response
Immunotyping Monoclonal Antibody
• Confirm the presence of monoclonal
• Immunotype monoclonal
• Two methodologies
• CZE immunodisplacement
• Gel immunofixation
• Both have pros and cons
Immunodisplacement Antisera
-
• One antisera for each major chain
• IgG, IgA, IgM, Kappa & Lambda
• Chemically treated to add strong
negative charge to antibody
• Reduces electrophoretic mobility
Immunodisplacement
• Antisera added to sample
• Antisera will bind all
immunoglobulin of a specific
immunotype
– Monoclonal
– Polyclonal
Reduced Mobility
• Antisera bound
immunoglobulin migrates
significantly slower than
unbound immunoglobulin
• Immunoglobulin removed
from trace
Immunodisplacement – IgM Monoclonal
Immunodisplacement – IgM Monoclonal
Immunodisplacement
Immunodisplacement Serum Protein Screen
• Five antisera required
– IgG, IgA, IgM
– Kappa & Lambda
• Five capillaries
• Linked to original
screening trace
Immunodisplacement
Immunodisplacement
Serum Protein Screen
Immunodisplacement Overlay
IgG IgA IgM
Kappa Lambda
IgG IgA IgMIgG IgA
Kappa
IgMIgG IgA
LambdaKappa
IgMIgG IgA
LambdaKappa
IgM
LambdaKappa
IgA IgM
LambdaKappa
IgG IgA IgM
LambdaKappa
IgM Kappa Monoclonal
IgG IgA IgM
Kappa Lambda
Polyclonal Removal
IgG IgA IgM
Kappa Lambda
Example Case Study
100 200
(sec)
0.00
0.01
0.02
0.03
• Serum protein
screening trace
Example Case Study
100 200
(sec)
0.00
0.01
0.02
0.03
Beta-2 M-spike 1
Gamma M-spike 1
• Two monoclonal
peaks
• Beta-2 monoclonal
• Gamma Monoclonal
Example Case Study - Immunodisplacement
• Two monoclonal
peaks
• Beta-2 monoclonal
• Gamma Monoclonal
IgG
IgA IgM
LambdaKappa
IgG
IgA IgM
IgG
IgA
Lambda
IgM
IgG
IgA
Kappa Lambda
IgM
IgG
IgA
Example Case Study - Immunodisplacement
• Two monoclonal
peaks
• IgA Kappa x2
Kappa Lambda
IgM
IgG
IgA
Immunotyping
How to choose between methods
• Immunodisplacement
• Medium & large peaks
• Suspected co migrating peaks
• Fast / automated
• Immunofixation
• Small peaks
• Manual
• Batches
Immunodisplacement
Immunofixation
Immunofixation
• Customise for purpose
• Screening versus / typing
• Suggested dilution schemes
• Screening
• 52233
• Immunotyping
•Monoclonal specific
IFE Dilution Scheme
Total Immunoglobulin Chain Vs Monoclonal Sensitivity
0
10
20
30
40
50
60
70
0.06 0.12 0.18 0.24 0.3 0.36 0.42 0.48 0.54 0.6 0.66 0.72 0.78 0.84 0.9 0.96 1.02 1.08 1.14 1.2
Monoclonal sensitivity (g/L)
To
tal im
mu
no
glo
bu
lin
ch
ain
(g
/L)
Prozoning
Resolution
IFE Dilutions
IFE Dilutions
Any Questions
Ankara – July 2019
Tony Aitchison – Helena Biosciences
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