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Characterizing Antibody Biomarkers

of Pre-eclampsia

Jane Lin

Marissa Perry

Athena Mitchell

Mackenzie Gorman

Mentor: Serra Elliott

Advisor: Dr. Patrick Daugherty


Issues of Pre-eclampsia

• It affects 5-8% of pregnancies [1] causing 15-20% of

maternal mortalities [2]

• Unable to diagnose Pre-eclampsia (PE) until

symptoms are present

Symptoms of PE

1. Lain & Roberts JAMA (2002)

2. Sibai et al Lancet (2005)

Hypertension

Proteinuria


Searching for a better biomarker

Possible Biomarkers Auto-antibody

▫ Nonspecific, not sensitive

• Additional antibodies may exist


Characterize affinity of peptide

reagents

Michaelis-Menten Kinetics

Mean Cell



Fluorescence


Flmax

[ Antibody ]

[ Antibody ]

K D

Concentration

•Desired:

•Lower K D

•Higher Fl max


Preparation Process for Fluorescence Analysis

= bacteria

= peptide

= biotin

= antibody

= secondary protein


Preparation Process for Fluorescence Analysis

Incubate with

Primary Antibody

= bacteria

= peptide

= biotin

= antibody

= secondary protein


Preparation Process for Fluorescence Analysis

Incubate with

Primary Antibody

= bacteria

= peptide

= biotin

= antibody

= secondary protein


Preparation Process for Fluorescence Analysis

Incubate with

Primary Antibody

= bacteria

= peptide

= biotin

= antibody

= secondary protein

Incubate with

Secondary Protein


Preparation Process for Fluorescence Analysis

Incubate with

Primary Antibody

= bacteria

= peptide

= biotin

= antibody

= secondary protein

Incubate with

Secondary Protein


Fluorescence Activated Cell

Sorting (FACS) Machine

• Organizes prepared bacteria

cells into single-file line

• Utilizes laser to test for

the fluorescence


Side Scatter

Fluorescent intensity depends

on concentration

MCF: 25730

MCF: 19817

MCF: 12286

MCF: 5595

MCF: 577

Red Fluorescence

Decreasing Antibody Concentration


Mean Cell Fluorescence

Determining affinity of peptide

antibody interaction

30000

25000

20000

Peptide A

Fitted A

K D B = 2.765 nM

15000

Peptide B

Fitted B

10000

K D A = 23.417 nM

5000

0

0.01 0.1 1 10 100 1000

Antibody Concentration (nM)


Mean Cell Fluorescence

High fluorescence shows higher

binding affinity

• Fl max is used to

distinguish between

positive and negative

• If the peptide has a lower

K D then it will have a

higher binding affinity

• Peptide B is better than

Peptide A

30000

25000

20000

15000

10000

5000

Peptide A

Fitted A

Peptide B

Fitted B

0

0.01 0.1 1 10 100 1000

Antibody Concentration (nM)


Acknowledgements

• Serra Elliott

• Dr. Patrick Daugherty

• SIMS

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