Protein or Peptide Antigen?

Transcription

1 Protein or Peptide Antigen? Liyan Pang, Ph.D.

2 Table of Contents Choosing mab or pab Characteristics of a Good Antigen Protein Antigen Peptide Antigen How to Choose Protein or Peptide Advantages of GenScript Ab Services 2

3 Monoclonal vs. Polyclonal Monoclonal Produced by identical clone immune cells Recognizes a single epitope Extremely specific Quantitative Polyclonal Produced by multiple immune cells Recognizes multiple epitopes Less specific 3

4 How are Monoclonal Ab Made? 4

5 Pros and Cons of mab Advantages Highly specific recognition of only one epitope of an antigen Immortal hybridoma cell lines have the ability to produce unlimited quantities of antibodies High consistency among experiments Minimal background noise and cross-reactivity Excellent for affinity purification Disadvantages More expensive Longer production time Possible lower signal More vulnerable to loss of epitope through chemical treatment of the antigen. This can be offset by pooling two or more monoclonal antibodies 5

6 How are Polyclonal Antibodies Made? 6

7 Pros and Cons of pab Advantages Robust target signal, even for low expression protein More tolerant to changes to the antigen (denature, polymorphism, heterogeneity, glycosylation) Short production time Low cost Better results in IP/ChIP, and WB More likely to detect across a range of species Useful for non-characterized antigens Disadvantages Prone to batch to batch variation Possible higher background in certain applications 7

8 Antigen Immunogenicity Definition: The ability of a particular molecule to elicit an immune response determined by whether the immune system can recognize the antigen. Two broad categories impact immunogenicity 1. Antigen characteristics 2. Biological system 8

9 Factors Affect Antigen Immunogenicity Phylogenetic distance - How related the antigen origin species and the host are Molecular size - Antigens with higher molecular weight are more immunogenic. Epitope density - A high epitope density in antigen significantly enhances antigen immunogenicity and antibody affinity. Chemical composition and heterogeneity - Molecules with the high internal molecular complexity and heterogeneity are good immunogens. Degradability (ability to be processed & presented to T cells) 9

10 Options on Choosing an Antigen Carbohydrates Lipids Nucleic acids Small molecules (like neurotransmitters) Protein Polypeptides 10

11 Protein Antigen: Pros and Cons Advantages Disadvantages Cost effective if protein supply is available Higher probability of cross-reactivity High likelihood of binding to native protein Higher background in the assay Resulting antibody has a strong signal Can be challenging to produce or purify enough protein antigen 11

12 Criteria of a Good Protein Antigen Homology: - How related the antigen origin species and the host are Molecular size: - Protein with a higher molecular weight show better immunogenicity. Chemical composition and heterogeneity - High molecular complexity and internal heterogeneity Solubility - Should not be soluble in body fluids Degradability - Needs to be degradable to be processed and presented to the T cells 12

13 Peptide Antigen: Pros and Cons Advantages Disadvantages Lower probability of cross-reactivity Possibly not sufficient to induce an antigenic response More flexibility in antigen selection Peptide antibody may fail to recognize the native protein with folded conformation Only protein sequence is needed Easy to synthesize and cost-effective Faster turnaround time 13

14 Considerations for Peptide Antigen 1. Homology 2. Epitope selection 3. Continuous vs. discontinuous epitopes 4. Targeting the N-terminus or C-terminus 5. Sequence Length 6. Avoidance of specific sequence motifs 7. Peptide purity 8. Peptide solubility 9. Carrier protein coupling 14

15 1. Homology Homology: Choose a unique sequence with low homology Why? A unique sequence with low homology will help to enhance immunogenicity and also ensure specificity to the target protein. 15

16 2. Epitope Selection Hydrophilic, surface-oriented, and flexible: Antibodies by peptide antigens are produced against epitope sequences which are most likely to be hydrophilic, surface-oriented, and flexible. Why? Most naturally occurring proteins in aqueous solutions have their hydrophilic residues on the protein surface and hydrophobic residues buried in the interior. 16

17 3. Continuous vs. Discontinuous Continuous epitope: It is better to select a continuous epitope for antibody binding Why? Anti-peptide antibodies may or may not recognize a discontinuous epitope depending on whether the peptide used has a secondary structure similar to the epitope 17

18 4. N-termimus or C-terminus? Targeting the N-terminus or C-terminus: Either the C- termini or N-termini can be chosen for generating anti-peptide antibodies. However, one could be better than the other on certain cases. Why? The C-termini and N-termini of proteins are often exposed and have a high degree of flexibility making either a good epitope candidate for antibody production. Exceptions: membrane protein & secretory protein 18

19 5. Sequence Length Sequence length: The typical length of the peptide antigen for generating anti-peptide antibodies is in the range of residues. Why? Shorter sequence can offer greater specificity, but at the risk of being less likely to be exposed on the native protein. Longer sequences, by contrast, might be slightly less specific, but offer a higher probability of recognizing the native protein. 19

20 6. Avoidance of Sequence Motifs Sequence motifs to avoid: Arginyl-glycyl-aspartic acid (RGD) motif, helix-loop-helix sequence, GTP binding site, Src homology 2 (SH2) domains, etc. Why? Functional motifs are often subject to conformational change and highly conserved between family members, which made them poor choices as antigen and may cause cross-reactivity when chosen as antigen. 20

21 7. Peptide Purity Purity: 75% is usually sufficient for antibody generation and testing, but 95% is recommended for biological activity studies Why? 75% purity is the minimal requirement for antibody generation but certain applications require higher purity for less background 21

22 8. Peptide Solubility Solubility: Recommended to keep the hydrophobic amino acid content below 50% and include one charged residue for every five amino acids Why? This will ensure that the peptide is soluble and accessible to the immune cells in order to generate antibodies. 22

23 9. Epitope Coupling Strategy Epitope coupling: Linking the peptide to a carrier protein via the C or N terminus What is the purpose? - Peptides alone are usually too small to elicit an immune response sufficient to generate antibodies. Conjugation to a carrier protein stimulates T-helper cells. 23

24 How to Choose Protein or Peptide Antigen? 24

25 Types of Work Where Protein Antigen is Better Applications which need to detect conformational epitopes of natural protein such as IP, ICC/IF, FACs etc. 25

26 Applications Where Peptide Antigen is Better Applications which detect denatured or linear protein epitopes such as Western blots, IHC or ELISA 26

27 Decision Time: Protein or Peptide? 27

28 Advantages of GenScript Custom Abs Sequence to purified antibody service with no need to provide an antigen Optimized immunization using our OptimumAntigen design tool and intelligent Antigen Strategy increasing specificity and affinity of antibodies Guaranteed results: quantity of antibodies or hybridoma, ELISA titer, and WB guarantee (varies with specific package) Fast turnaround time: delivery of purified pab or development of specific hybridoma in 45 days. Certified facility: AAALAC International accreditation and OLAW certification, demonstrating our commitment to responsible animal care and use. 28

29 Advantages of GenScript Custom Abs Sequence to purified antibody service with no need to provide an antigen Optimized immunization using our OptimumAntigen design tool and intelligent Antigen Strategy increasing specificity and affinity of antibodies Guaranteed results: quantity of antibodies or hybridoma, ELISA titer, and WB guarantee (varies with specific package) Fast turnaround time: delivery of purified pab or development of specific hybridoma in 45 days. Certified facility: AAALAC International accreditation and OLAW certification, demonstrating our commitment to responsible animal care and use. 29

30 Advantages of GenScript Custom Abs Sequence to purified antibody service with no need to provide an antigen Optimized immunization using our OptimumAntigen design tool and intelligent Antigen Strategy increasing specificity and affinity of antibodies Guaranteed results: quantity of antibodies or hybridoma, ELISA titer, and WB guarantee (varies with specific package) Fast turnaround time: delivery of purified pab or development of specific hybridoma in 45 days. Certified facility: AAALAC International accreditation and OLAW certification, demonstrating our commitment to responsible animal care and use. 30

31 Advantages of GenScript Custom Abs Sequence to purified antibody service with no need to provide an antigen Optimized immunization using our OptimumAntigen design tool and intelligent Antigen Strategy increasing specificity and affinity of antibodies Guaranteed results: quantity of antibodies or hybridoma, ELISA titer, and WB guarantee (varies with specific package) Fast turnaround time: delivery of purified pab or development of specific hybridoma in 45 days. Certified facility: AAALAC International accreditation and OLAW certification, demonstrating our commitment to responsible animal care and use. 31

32 PolyExpress TM Custom pab 32

33 MonoExpress TM Custom mab 33

34 Variety of GenScript Antibody Services 34

35 Over 500 Publications Citing Our Ab Services Methylation protects micrornas from an AGO1-associated activity that uridylates 5 RNA fragments generated by AGO1 cleavage. Yu B, Chen X, Vinovskis C, etc. PNAS, (Apr 2014) HYPERSENSITIVE TO HIGH LIGHT1 Interacts with LOW QUANTUM YIELD OF PHOTOSYSTEM II1 and Functions in Protection of Photosystem II from Photodamage in Arabidopsis. Wang HB, Wang J, Qi K, etc. Plant Cell, (Mar 2014) Tousled-like kinases phosphorylate Asf1 to promote histone supply during DNA replication. Groth A, Jensen ON, Nielsen ML, etc. Nature Communications, (Mar 2014) Dirigent domain-containing protein is part of the machinery required for formation of the lignin-based Casparian strip in the root. Hosmani PS, Kamiya T, Danku J, etc. PNAS, (August 2013) PfSETvs methylation of histone H3K36 represses virulence genes in Plasmodium falciparum Jiang L, Mu J, Zhang Q, Ni T, etc. Nature, (July 2013) Wheat Mds-1 encodes a heat-shock protein and governs susceptibility towards the Hessian fly gall midge. Liu X, Khajuria C, Li J, Trick HN, Huang L, etc Nature Communications, (Jun 2013) 35

36 About GenScript Gene Cell Line Peptide Discovery Biology GenScript Products & Services Protein Antibody Introduction 36

37 Thank you for your participation We wish you all success in your Research Register for other webinars in the GenScript Webinar June 5, 2014/ 2:00 pm EST Stem cell culture: choosing optimal conditions for expansion and differentiation - Matthew Riolo, Ph.D. June 11, 2014/ 1:00 pm EST Recombinant protein expression & purification: challenges and solutions - Liyan Pang, Ph.D. June 18, 2014/ 2:00 pm EST Can CRISPR/Cas9 off-target genomic editing be avoided? Ways to improve target specificity - Maxine Chen, Ph.D June 25, 2014/ 2:00 pm EST Building a Synthetic Eukaryotic Genome Sc2.0 - Leslie Mitchell, Ph.D., NYU Langone Medical Center Conclusion 37