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Recombinant Antibodies Technical Information

1. Technology Summary and FAQ’s >

2. Protocols and Strategies >

3. Example Certificate of Analysis >

1. Technology Summary and FAQs

Our new range of Recombinant Fab Monoclonal Antibodies were derived from mouse and developed using phage display technology, recombinant techniques and the Luminex platform. We now offer matched antibody pairs with exceptional and clinically-relevant performance characteristics to a wide range of biomarkers, with more in development. These Fab antibodies are highly scalable and reproducible, and comprise engineered features including a Cysteine for site-specific conjugation.

Recombinant Fab Monoclonal Antibodies

Q: Why are these Fab antibodies so good?

The antibodies were selected using proprietary phage display selection technology. For each biomarker, typically:

  • Immunisation is performed
  • A library of 109 phage members is screened
  • 1 nanomolar is used as the affinity cut-off, so the antibodies have sub-nanomolar affinities
  • The best 96 antibodies are tested against each other – 96 x 96 – for matched pairing.
  • Final candidate pairings are tested in patient samples to ensure clinical significance
  • We provide an evaluated Range of Detection (ng/ml) for each antibody pair

Plus there are many different features of the antibodies which improve on standard monoclonal antibodies (see below)

Q: How do these Fab antibodies differ from standard antibodies?

The antibodies are recombinant Fab fragments of mouse monoclonal antibodies. Apart from outstanding performance, there are several important advantages of these Recombinant Fab antibodies over full mouse IgG monoclonal antibodies.

  • Improved scale up for batch consistency – Our processes are fast, reliable and consistent because we express in and purify from E.coli.
  • More efficient binding so less antibody required – Fab fragments retain the antibody binding site but with the unwanted regions of the antibody removed. This results in >36% more antibody binding sites per milligram of product.
  • Precise binding meaning fewer conjugation issues – our recombinant Fab antibodies are specifically engineered with a free Cysteine residue at the C-terminus of the heavy chain for site-specific conjugation away from the antigen binding site. When conjugationg to standard monoclonal antibodies, the conjugation process is less precise and can interfere with the antigen binding site of the antibody.
  • No heterogeneity issues and no further processing – Fab fragments prepared from full IgG antibodies via proteolytic processing with Trypsin, are a heterogeneous mixture of Fab, Fab2 and undigested material due to partial processing and the fact that Trypsin can cleave the protein at various lysine residues. The resulting material is difficult to reproduce and often requires further purification with associated loss of material. However, our recombinant Fabs are completely homogeneous as no proteolytic processing is required, and as a consequence of this no further purification is required.
  • Improve results by removing NSB – the Fc region, which can lead to non-specific binding issues within full IgG antibodies, has been removed.
  • Increased flexibility for purification and labeling – a C-terminal His tag on the heavy chain allows for purification or directed secondary labeling strategies.

Q: What data do we provide?

These antibodies typically exhibit subnanomolar affinity. We also state a Range of Detection. This is the tested range of concentrations over which the antibodies have been shown to detect the antibody in a quantitative manner. The selection process has been designed to find antibodies detecting molecules over a clinically relevant range. For example, our Calcitonin pair can detect between 6 and 2000 pg/ml, and “Patients with calcitonin levels >100 pg/mL have a high risk for medullary thyroid carcinoma (~90%–100%)” Toledo et al., (2009). The antibodies are also tested in patient samples. Please note that antibody sensitivity is assay dependent. Our antibodies were optimised for detection of a particular clinical condition, and it is likely that our stated Range of Detection can be extended beyond the information we have published. For example, we have shown our CRP antibody BR228-D4A3 can detect considerably lower CRP levels (as low as 2ng/mL) than the Range of Detection we have stated.

Q: Are there any other technical considerations?

  • Site-Specific Conjugation – the antibodies contain an engineered Cysteine residue for site-specific conjugation that won’t interfere with the antigen binding site. View our conjugation summary table >
  • Purification – the antibodies contain an engineered His Tag for simple purification.
  • Reduction to Monomers – during standard fermentation and purification, a recombinant Fab antibody can have a portion of the molecules as the disulfide-linked homodimer. If you wish to use the Cysteine for conjugation it is therefore advisable to first reduce to monomers. The monomers can be covalently “capped” with a thiol-directed alkylating reagent such as N-ethylmaleimide.
  • Use as you would for normal Fab fragments – other than the above improvements introduced to the reagents, and inherent homogeneity of the reagent due to avoidance of proteolytic processing, there are no other differences between using these reagents and other Fab fragments.

Q: What if I want antibodies to a biomarker that are not within your current offering?

We may have antibodies to other biomarkers available. Please contact us to find out . Also, follow this link to see if your biomarker of interest is on our “in-development” list

If there is enough interest we may be able to speed up a given project, so do contact us.

Q: What if I have another question?

These Fab antibodies have unique features not available on ordinary IgG’s. We understand that some of our users will not have experience of using these features or working with Fab fragments. If you have any technical questions please do contact us as we can share our experience in using these reagents to help you in your work.

2. Protocols and Strategies

We offer Custom Conjugation Services and Protocols to help you handle these reagents. Please contact us at antibodies@bbisolutions.com for more information.

Conjugation Summary Table

Antibody ApplicationRecommended Conjugation Strategy
ELISA Conjugate to enzyme or Biotin through engineered Cysteine
Lateral Flow (i) Conjugate Fab to BSA. BSA-Fabs can be adhered to nitrocellulose or colloidal gold (ii) Conjugate Fab to Biotin. Biotin-Fabs can be adhered to anti-biotin colloidal gold conjugate (iii) BBI GoldLink Kit (iv) Conjugate Fab to Chicken IgY, then conjugate gold to this complex
Free (Monomeric) Antibodies Reduce and cap the free Cysteine residue with thiol-directed alkylating reagent (i.e. NEM or NHEM)
Western blots Conjugate to reporter enzymes or to Biotin through engineered Cysteine

Please remember to remove azide prior to conjugation.

Protocols for Reducing Homodimers and Capping Monomers

Recombinant Fab antibodies may have a portion of the molecules as the disulfide-linked homodimer. If you wish to use the engineered Cysteine residue for conjugation (recommended) it is therefore advisable to first reduce to monomers. The monomers can be covalently “capped” with a thiol-directed alkylating reagent such as N-ethylmaleimide.

Antibody reduction

  1. Prepare 1 ml of antibody at 5 mg/ml (use PBS if antibody stock needs
    to be diluted)
  2. Add 100 mM TCEP to antibody solution to final concentration of 1 mM
    and incubate for 30 minutes.
  3. Purify antibody on 10DG column (Biorad Cat#732-2010) equilibrated with PBS:

Add 1 ml of sample collect 1 ml waste,

Add 2 ml PBS wash and collect 2 ml waste,

Add 1.3 ml PBS collect and save

  1. Determine A 280nm (dilute antibody for measurement 1:10 in PBS) and use extinction coefficient of 1.6.
  2. Add 1 ml of final antibody solution to beads, mix and cover with foil and place on rocker overnight at RT.

Capping and Storage

Please note that sodium azide will interfere with this procedure and thus it is best to reduce and purify the antibodies prior to performing it.

  1. Add 100 mM BME to final concentration of 2 mM and incubate 30 minutes on rocker.
  2. Add 500 mM NHEM (or NEM) to final concentration of 6 mM and incubate for 30 minutes on rocker.
  3. Remove excess BME and NHEM using 10DG columns (same as in Antibody Reduction section) and store.

BIOTIN-ANTIBODY Conjugation

  1. Use the same Antibody reduction procedure as above.
  2. Dilute Biotin-maleimide (Life Technologies M-1602) to 48 mM in DMSO.
  3. Add final 1 mM to 2mg/ml of Antibody (1 mL) and mix
  4. Let sit 1 hour and purify of 10DG columns same as in Antibody Reduction section 3).

Determine A280nm of Antibody for final Biotinylated-Antibody concentration.

3. Example Certificate of Analysis

Example Certificate of Analysis Recombinant Antibodies

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