Screening and Immunotyping Monoclonal Antibody using the...
Transcript of Screening and Immunotyping Monoclonal Antibody using the...
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Screening and Immunotyping
Monoclonal Antibody using the
V8 Nexus
Ankara – July 2019
Tony Aitchison – Helena Biosciences
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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
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Serum Protein Screening using Helena
Helena platform can address
entire serum protein screening
requirements:
• High throughput CZE screening
• Sensitive immunotyping
• Powerful integration
• Comprehensive training
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V8 Nexus
• Capillary electrophoresis
• 8 Channel
• Full walk away automation
• Multi test analyser
• Automated maintenance
• Gel integration
• Track and network integration
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SAS Gel Electrophoresis
• Semi automated
• SAS-3 Electrophoresis
• SAS-4 Stainer
• Automated sample prep
using V8 Nexus
• Common database for Gel
and CZE results
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Serum Protein Screening
• CZE separation on the V8
• Split beta separation
• High throughput
• Accurate monoclonal measurement
• Easily compare to historic results
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Capillary Electrophoresis
• Sample cups loaded at anode
• Vacuum applied to load capillary
• Current applied to facilitate
separation
• Absorbance measured as
protein passes the detector
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Electro-Osmotic Flow (EOF)
• Silica capillaries have
negative charge
• Run buffer coats capillary
neutralising charge
• Remaining buffer flows
through capillary
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Sample Stacking
• Differences in protein mobility
between diluent and buffer
stack protein prior to separation
• Diluent – fast mobility
• Run buffer – slow mobility
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Serum Protein Electropherogram
Pre-Albumin
Albumin
Alpha-1-acid Glycoprotein
Alpha-1-antitrypsin
Haptoglobin
Alpha-2-macroglobulin
Haemopexin
Transferrin
Complement
CRP
Immunoglobulin
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Six Band Serum Protein Trace
Albumin
Alpha-1
Alpha-2
Beta-1
Beta-2
GammaGamma
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Additional Peak
100 200
(sec)
0
50
100
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Additional Peak
100 200
(sec)
0
50
100
Gamma M-spike 1
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Relative Peak Percentages
0 20 40
(sec)
0
50
100
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Relative Peak Percentages
0 20 40
(sec)
0
50
100
Beta-2 M-spike 1
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Immunoglobulin Migration – Heavy Chains
IgMIgGIgA
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Immunoglobulin Migration – Light Chains
Kappa
Lambda
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Monoclonal Migration – Relative Incidence
HighLow
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Polyclonal Response
IgG Polyclonal Response
Normal
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Polyclonal Response – Clone Specific
IgMIgGIgA
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Polyclonal Response – IgG and IgA Response
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Immunotyping Monoclonal Antibody
• Confirm the presence of monoclonal
• Immunotype monoclonal
• Two methodologies
• CZE immunodisplacement
• Gel immunofixation
• Both have pros and cons
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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
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Immunodisplacement
• Antisera added to sample
• Antisera will bind all
immunoglobulin of a specific
immunotype
– Monoclonal
– Polyclonal
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Reduced Mobility
• Antisera bound
immunoglobulin migrates
significantly slower than
unbound immunoglobulin
• Immunoglobulin removed
from trace
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Immunodisplacement – IgM Monoclonal
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Immunodisplacement – IgM Monoclonal
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Immunodisplacement
Immunodisplacement Serum Protein Screen
• Five antisera required
– IgG, IgA, IgM
– Kappa & Lambda
• Five capillaries
• Linked to original
screening trace
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Immunodisplacement
Immunodisplacement
Serum Protein Screen
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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
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IgM Kappa Monoclonal
IgG IgA IgM
Kappa Lambda
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Polyclonal Removal
IgG IgA IgM
Kappa Lambda
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Example Case Study
100 200
(sec)
0.00
0.01
0.02
0.03
• Serum protein
screening trace
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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
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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
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Example Case Study - Immunodisplacement
• Two monoclonal
peaks
• IgA Kappa x2
Kappa Lambda
IgM
IgG
IgA
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Immunotyping
How to choose between methods
• Immunodisplacement
• Medium & large peaks
• Suspected co migrating peaks
• Fast / automated
• Immunofixation
• Small peaks
• Manual
• Batches
Immunodisplacement
Immunofixation
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Immunofixation
• Customise for purpose
• Screening versus / typing
• Suggested dilution schemes
• Screening
• 52233
• Immunotyping
•Monoclonal specific
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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
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IFE Dilutions
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IFE Dilutions
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Any Questions
Ankara – July 2019
Tony Aitchison – Helena Biosciences