Parallel immunization of rabbits using the same antigens yield antibodies with similar, but not identical, epitopes

Bar bara H jelm1, Bjorn Forsstrom1, John Lofblom1, Johan Rockberg1, Mathias Uhlen1,2*

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Content we are going to study:Polyclonal antibodies Epitopes Epitope mapping Peptide bead arrayWestern blotting Immunohistochemistry

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Article review:Generation of polyclonal antibodies is the potential difficulties for obtaining a new resource, due to batch to batch variation when the same antigen is parallel immunized in a 3 rabbits.Investigate this issue through epitopes of antigens & antibodies generates by immunization.They have used recombinant antigen, correspond to 10 human target proteins They were perform epitope mapping by two different techniquesSuspension bead array Bacterial display approach

Binding of antigen with antibody were determine by fluorescent based analysis.

Same antigen is immunized in several rabbits produce polyclonal antibody with similar epitopes, but great difference occur in the relative amount of antibodies to different epitopes, because in some case unique epitope were observed

This suggest that polyclonal antibody generated by repeated immunization dont display an identical epitope pattern, although many epitope are similar Main objectives:

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Introduction

Why we are studying this article?? Antibodies have proven to be an exceptional tool to study the proteins of human biology and disease. An important issue in this regard is the renewability of antibodies.Here, the limited availability of many polyclonal antibodies is a great concern, since there only exist a limited supply from the original immunization.

In the diagnostic arena, this problem has been over-come by immunizing large animals, such as sheep or goat, to generate large quantities of antibodies.

Alternatively, many animals, such as rabbits, are immunized with the same antigen and the sera from many animals are pooled to generate a large supply of antibodies with the same batch number.

Few studies have been performed in the past to estimate the degree of reproducibility when a new batch of polyclonal antibodies have been generated by immunization of a second animal.

In the work of Larsson, They concluded that all immunizations detected the correct band in Western blotting, but they rendered different staining patterns in IHC possibly related to their different epitope patterns.

In the work by Geysen et al, the comparison of seven sera from outbred rabbits immunized with myohemerythrin, showed that no antibody specificity was common in all seven rabbits.

Methods for epitope mapping:Established methods for epitope mapping of antibodies involves chemical synthesis of peptidesor peptide display on phages. Recently, we have described two independent methods for epitope mapping of antibodies

Bacterial surface display on Staphylococcus carnosus

suspension bead arrays with color-coded beads

Bacterial surface display on staphylococcus carnosusGene encoding the target protein is fragmented, cloned into an expression vector and subsequently introduced into S. carnosus host cells. Gene expressed on the surface of the cell. The cells are incubated with the antibody to be mapped labeled with a fluorescent dye and the cells are analyzed in a flow cytometer so that cells expressing fragments bound by the antibody can be collected. These cells are grown.

suspension bead arrays with color-coded beads

In which each has a synthetic peptide bound to its surface.

The bead mixture with overlapping peptides spanning the whole antigen sequence is incubated with the fluorescently labeled antibody and the beads are analyzed on a flow sorter capable of identifying each color-coded bead.

What are the polyclonal antibodies?

Antigen: They were prepared antigen by using ten human targeting protein including the protein from diverse protein families, such as cell surface protein, nuclear protein, and mitochondrial protein.

Materials and Methods:

Techniques:Generation of polyclonal sera Epitope mapping using bacterial display of polyclonal seraEpitope mapping using peptide bead arrays of polyclonal sera Affinity purification of epitope specific fractions Western blotImmunohistochemistry Structural analysis of epitope Data analysis

Generation of polyclonal sera:Antigen designed on a sequence similarity to ten human target protein by using software PRESTIGE .

Designed gene fragment were then amplified from a pool of RNA isolated from human tissues

Antigen were then cloned into vector pAff8c, in fusion with hexa-histidine tag and an albumin binding protein and expressed in Escherichia coli

This purified and recombinant protein fusions were used for immunization supplemented with Freunds complete adjuvants followed by booster immunizations at four, eight and twelve week together with Freunds incomplete adjuvant.

The immunizations were performed by Agrisera AB (Sweden), Harlan laboratories (USA), and Beijing Proteome Research Center (China)

Epitope mapping using bacterial display of polyclonal seraTen different peptide libraries were generated as follows.

Gene fragments of each of the target proteins were amplified by PCR separately.

Each product pool (4.8 ml) was sonicated for 75 min

The library was then electroporated intoS. carnosusTM300 as described previously.

Epitope mapping using peptide bead arrays of polyclonal seraSix different peptide. N-terminally biotinylated. neutravidin coupled beads Reaction in PBS-B (PBS supplemented with 1% Bovine Serum Albumin) 60 min at room temperature in dark before washing, pooling of each beadmix and storage in storage buffer Approximately 1000 beads per ID were incubated with antibody in dark under agitation. After washing, a secondary incubation with anti-rabbit IgG-PE was performed in dark under agitation. A final washing was ended by adding 100 l PBS-B and analyzed on a Luminex FlexMap 3D system.

Western blotThe antibodies were analyzed on western blot membranes

The proteins were separated according to size on SDS-PAGE gradient gels under reducing conditions.

The proteins were subsequently transferred to PVDF membranes using Blotting Sandwiches

After 1 h incubation the membranes were washed.

Secondary HRP-conjugated swine anti-rabbit antibody was diluted 1/3000 in blocking buffer and the membranes were incubated 1 h.

Unbound antibodies were removed with another round of washing before addition of HRP-substrate and chemiluminescence detection was carried out using a Chemidoc CCD-camera system

Band intensities of epitope-specific fractions on tissue lysate were analyzed using ImageJ (http://imagej.nih.gov/ij/,

ImmunohistochemistryAutomated immunohistochemistry was performed on TMAs with breast cancer tissue from paraffin embedded specimens.Glass slides were deparaffinized, hydrated and blocked before antigen retrieval by boiling for 4 min in Target Retrieval Solution, TRS, pH 6.0 in a decloaking chamber. Slides were immunostained in an automated staining instrument, Autostainer (Dako). Diaminbenzidine was used as chromogen and Harris hematoxylin for counter-staining before scanning.

Structural analysis of epitopes:The software MacPyMol was used to highlight epitope sequences in structures 1N8Z.psd (ERBB2), 2ENO.pdb (SYNJ2BP) 1D4B.pdb (CIDEB), 2CFS.pdb (PDXP) and 2JOF.pdb (TYMP).

Data analysis:Propensity scores for the selected antigens were calculatedPropensity tables for Hopp and Woodsas well as Rockberg and Uhlnwere used to calculate average propensity values on a sliding window of 9 amino acids throughout the antigen sequence. Scores were normalized between 0 & 1 and plotted along corresponding amino acid coordinates.

RESULTS:

Protein target used in the study:Antigen prepared by using 10 human target protein

PrEST was expressed in E.coli as a fusion protein to a N-terminal hexa histidine tag followed by ABP (antigen binding protein)

size of the antigen was 120 amino acid approx., excluding the fusion tag.

Recombinant protein was purified by using IMAC & the size of fusion protein was confirmed by mass spectrometry.

This is the protein epitope signature tag.In our experiment the Qtag is antigen binding protein.

Select the target protein

Expressed in E.coli

Purify the protein using IMAC

Size of the fusion protein confirmed by mass spectrometry

What is IMAC?Immobilized metal-affinity chromatography (IMAC) to purify recombinant proteins containing a short affinity tag consisting of polyhistidine residues. IMAC is based on the interactions between a transition metal ion (Co2+, Ni2+, Cu2+, Zn2+) immobilized on a matrix and specific amino acid side chains. Histidine is the amino acid that exhibits the strongest interaction with immobilized metal ion matrices

Immunization of antigen:Antigen were used to immunized separate New Zealand White rabbits.

Sera were collected and the polyclonal antibodies were purified.

All antigens were immunized in three separate animals.

Functional studies of the antibodies obtained from repeated immunizations:After western blotting result were summarized.

Bands of correct size were observed for all proteins targets except the antibodies generated against the HER2 receptor of (ERBB2)

Some additional bands were also observed for some of the antibodies e.g. PDXP and FBXO28

This suggest that functional polyclonal antibodies are obtained by repeated immunizations in all case, but some of the antibody also show the off-target reactivity in western blot.

Another possibility of cross reactive background could be the use of His- tagged recombinant antigen, which may not fold as native protein and lead to epitope exposure changes during immunization.

What is epitope mapping ?

Epitope mapping of antibodies using bacterial surface displayWhat is bacterial surface display??

Bacterial display(orbacteria displayorbacterial surface display) is a protein engineering technique used forin vitroprotein evolution. Libraries ofpolypeptidesdisplayed on the surface ofbacteriacan be screened usingflow cytometryor iterative selection procedures (bio panning). This protein engineering technique allows us to link the function of a protein with the gene that encodes it. Bacterial display can be used to find target proteins with desired properties and can be used to make affinityligandswhich are cell-specific. This system can be used in many applications including the creation of novel vaccines, the identification ofenzyme substratesand finding the affinity of a ligand for its target protein.

Bacterial surface display libraries were separately generated for all ten antigensThese libraries were used for the epitope mapping of the antibody obtained from the separate immunization3 antibodies from 3 separate immunization were analyzed for each antigen.

Epitope mapping of antibody using suspension bead array:What is suspension bead arrays?In the beadbased arrays (suspension or liquid arrays), capture molecules are immobilized to a microsphere and captured analytes are detected mostly using the flow cytometry principle. Utilization of microspheres as the solid support is not new. The application potential of differently sized beads coated with antigens has already been described.

Overview Of Suspension Bead Array

Antigenic determinants of the antibodies against six of the targets with an independent assay15 amino acid long synthetic peptides, covering the entire antigen sequence in an overlapping manner, were immobilized to separate color-coded beads

The bead mixture for a particular protein antigen was incubatedseparately with the antigen-specific antibodies and analyzed ona Luminex FlexMap 3D instrument

The mean fluorescence intensity (MFI)reflecting the binding interaction was Determined and plotted for each peptide.

Overall results:In summary, the epitope mapping of the antibodies generated towards the six protein targets revealed similar, but not identical epitopes,when the same antigen was immunized into separate rabbits. In all cases, most of the epitopes are similar between the different immunizations, but clear differences can also be observed, i.e. for the antibodies towards TYMP, for which antibody 1 has two unique epitopes

Fractionation of polyclonal antibodies using peptide-specific affinity captureThe three polyclonal antibodies from the separate immunizations of the recombinant fragment of TYMP were used for affinity capture of epitope-specific antibodiesThe selected peptides are highlighted in colors in the binding profile of the antibodies to the peptide arrayThe selected peptides are highlighted in colors in the binding profile of the antibodies to the peptide arrayThe results show that most of the epitopes are common for the three immunizations, but a few are unique for a particular immunization, i.e. peptide 12 (pink) that is unique for immunization 1. The Affinity capture pie charts show the relative amounts of antibodies in the different affinity purified fractions. The results reveal a dramatic difference for the relative amounts of antibodies from the different immunizations despite the fact that essentially the same epitopes are observed in the three immunizations

In-depth analysis of polyclonal antibodies towards TYMP

Analysis with epitope prediction methods

Three-dimensional structural analysis of the epitopes:

The three-dimensional structures.For the targets HNRNPH2 (A), SYNJ2BP (B), PDXP (C) and ERBB2 (D), the epitopes are shown in blue with the epitope silent parts of the antigen shown in white.The epitopes of the antibodies towards TYMP (E) and (F) are also located on the surface.

Summary:

polyclonal antibody generated by repeated immunization dont display an identical epitope pattern, although many epitope are similar