1. ELISA for mAb detection and Quantification
miniBIOMAN 2017

2. Properties of IL-8 IL-8 is a small 72 amino acid peptide
Molecular weight 7.8 kDa

3. CHO DP-12 derived anti-Human IL-8 Ab Properties
Monoclonal
Mouse
IgG

4. ELISA Enzyme Linked Immunosorbent Assay
Use antibody reagents and a microtitre plate reader to determine the concentration and/or the activity of a protein of interest.
Used for identity testing – if drug is a Mab could also be used for activity, i.e binding to ligand
In the mAb production experiment we will use it to quantify mAb (API) levels in medium by assaying against a standard curve

5. ELISA ELISAs are read on a microtitre plate reader
several types of ELISAs
direct
indirect
sandwich
ELISAs are read on a microtitre plate reader
a mini-spectrophotometer that determines the absorption or transmission of a beam of light of a particular wave length passing through a solution of the protein of interest.
Using standards to generate a standard curve, one can determine the concentration of the protein of interest in a sample.
Indirect
Indirect ELISA detects the presence of a certain antibody in a sample (serum)
antigen is coated on the plate, then sample is put on (antibody will bind if present), then add enzyme-conjugated antibody to detect

6. ELISA Animation The animation may be found at:
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7. Indirect ELISA
The indirect, two-step method uses a labeled secondary antibody for detection.
1. Wells of plate coated with antigen
2. primary antibody is incubated with the antigen –mAb API
3. incubated with a labeled or enzyme conjugated secondary antibody that recognizes the primary antibody
4. Substrate of enzyme conjugated to secondary Ab added which results in color change
5. Measured in plate reader

8. Microtiter plate

9. ELISAs ELISAs are read on a microtitre plate reader
Secondary antibody conjugated to enzyme Horseradish peroxidase HRP
In presence of H2O2 HRP catalyzes chromogenic substrate TMB (3,3’,5,5’ – tetramethylbenzidene) to form a blue product
standards used to generate a standard curve, one can determine the concentration of the protein of interest in a sample

10. ELISA Results
Concentration ng/ml OD
0.071
6.25
0.169 25
0.426
100
0.951
400
1.156
Sample 1
1.320
Sample 2
1.290
Sample 3

11. Standard curve To accurately determine the mAb
content of our conditioned
medium samples, it is important that
when we assay them we get a
reading that falls with the linear
portion of the standard curve
This may mean diluting the samples first
Remember ELISAs are extremely sensitive assays

12. Commercial anti IL 8 mAb
Anti-IL8 Mouse mAb

13. Summer Internship 2016 Robin M. Zuck
ELISA Development for CHO DP12 derived Mouse anti-Human IL-8 monoclonal Antibody
Summer Internship 2016
Robin M. Zuck

14. Plate and Coating Buffer Selection
Plates screened:
Nunc Maxi Sorp
Corning high Protein binding
Polystyrene
Coating Buffers tried:
1X PBS
Sodium Bicarbonate (NaHCO3 ) pH 9.57

15. IL-8 concentrations screened for coating
1000 ng/well
500 ng/well
250 ng/well
125 ng/well
62.5 ng/well
31.25 ng/well
15.6 ng/well
0 ng/well
Each plate and coating buffer combination was used to coat wells at each of the concentrations listed.

16. Plate and Buffer Combinations tried
1X PBS
Na HCO3
NUNC Maxi Sorp –
Mixed hydrophilic/hydrophobic domains,
Corning High Protein Binding –
Hydrophobic and ionic (negatively charged) molecules greater than 10 kDa
Polystyrene -
(hydrophobic, large molecules >20kDa)
Produced the best linear binding range.

17. CHO DP-12 derived anti-human IL-8 mAb dilutions screened
IL-8 coating
Each of the following dilutions of Protein A purified antibody was screened for a linear response range using each of the coating concentrations listed
800ng per well was used moving forward
Ab dilution
1:2,000
1:1,600
1:1,000
1:800
1:500
1:400
1:200
1:50

20. Calculation of the concentration of anti-IL-8mAb dilutions
Anti IL-8 mAb eluted in a sharp well defined peak. The height of the peak in Absorbance units was
Sylvia Donnelly Summer Internship Notebook2 page 11.

21. Washes and blocking buffers
Coating Buffer used: 1X PBS
Washes: 1X PBS
Blocking Buffer: a 3% (w/v) solution of BSA in 1X PBS was used to block any non-specific binding to the ELISA plate.
Volume of blocking buffer used is at least twice the volume used to coat the plate.
Antibody diluent: a 3% (w/v) solution of BSA in 1X PBS, Blocking Buffer.

22. Summary of Method
1. Coat plate overnight at 4°C. (or possibly 2 hr at room temperature, not tested).
Wash the plate twice.
Block plate, 2hours at room temperature. (blocked plates can be sealed and stored at 4°C for 1 week after removing the blocking buffer)
Wash the plate three times.
Add CHO DP-12 anti-human IL-8 antibody standard and sample dilutions. Incubate for 1 hour at room temperature.
Wash plate three times.
Apply Rabbit anti-Mouse IgG peroxidase conjugated antibody.
Add TMB substrate solution, monitor color development.
Add Stop solution
Read plate, Absorbance at 450nm.
Plot the standard curve. Calculate the concentration of unknown solutions

23. References Antibodies a Laboratory Manual
Ed Harlow, David Lane, Cold Spring Laboratory, 1988
ELISA Handbook
Improved antibody production in Chinese hamster ovary cells by ATF4 overexpression
Ahmad M. Hardy, Kohsuke Honda, Akitoshi Ohya, Hisao Ohtake, Takeshi Omasa
Cytotechnology (2013) 65: DOI /s
(description of ELISA method for determining the concentration of CHO DP12 derived antibody)