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