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Dr Margaret Clotworthy
info@humanfocusedtesting.com

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In vitro generated antibodies & antibody-like molecules

Antibodies are ubiquitous in the lab, where they are prized for their high specificity, high affinity to target & ability to detect & label a variety of targets, whether in fixed samples or living cells.

 

That at least is the idealized rationale for utilising antibodies in a huge range of applications, from purifying targets/ antigens, probing for the presence of particular markers on or in a population of cells, interfering with cellular functions by inhibiting receptor activity or artificially inducing receptor dimerisation & even probing molecular interactions under the microscope by the use of techniques such as FRET.

 

The reality can be rather different (particularly where polyclonal antibodies are concerned):

-        Antibodies are suspended in serum that can contain impurities which may interfere with your results.

-        There is often lot-lot variability as each immunised animal is likely to generate slightly different responses to antigen.

-        Antibodies often bind non-specifically to proteins in your sample, or may bind to epitopes common to relatives of the target you are trying to identify or purify.

-        If your antibody will only bind to denatured targets, that rules out live-cell imaging & adds extra steps to your experiments.

-        If your target is not conveniently located on the cell surface, but is rather a nuclear receptor, for example, then fixing and permeabilising your cells before microscopic localisation is a must, meaning that real-time imaging of translocation and other dynamic events is impossible.

-        Although there are many off-the shelf antibodies available, including monoclonal antibodies and batches of polyclonal antibodies, if you want to generate a custom antibody, this will typically take months

 

If you consider the increasing use of antibodies in the clinic, the potential problems become even more acute, as antibodies need to be humanised lest antibodies with Fc portions specific to other species induce an immune response against the treatment. Furthermore, considerable precautions must be taken to ensure that no traces of animal-derived materials, including also bovine serum, which may contain prions or other disease-inducing agents, remain in the therapy to be administered to the patient.

 

Antibodies are such common reagents that we have simply become accustomed to navigating around these problems to the best of our ability. However, given the many pitfalls associated with antibody use, is it time to consider some of the smart alternative tools now available?

 

Type

Description

Strengths

Limitations

Current applications

Time

Antibodies

Phage display library antibodies

Fv portions displayed at high density on the surface of phage. Typically, phage are washed through columns  containing immobilized antigen, leaving phage displaying high-affinity binding Fv bound to the column for elution

- Suitable for generating

polyclonal mixes

- Suitable for generating monoclonals

- High purity

- High yield

- Multiple rounds of mutation (evolution) can be performed quickly

- Multiple rounds of selection can be performed quickly

- Fc portions from any species can be used in conjunction with the Fv selected (useful for detection amplification)

- libraries of billions of possibilities are already available

-libraries can be generated with Fc portions added to match any species

 

 

Any antibody application

Existing libraries can be screened in weeks

Yeast display library antibodies

Fv portions displayed at high density on the surface of yeast

Similar to Phage display, with the additional advantage that:

- Eukaryotic nature of host may lead to more authentic post-translational modifications than in bacterial/phage hosts

 

 

Any antibody application

Existing libraries can be screened in weeks

pre-B cell library display

Full-length human IgG library expressed in pre-B cells transfected by retrovirus. Selection by FACS using soluble fluorescently labelled antigen or antigen anchored to magnetic beads.

- Immobilized & soluble antigen can be used in the selection process

- B lineage cell line use facilitates biologically accurate glycosylation, disulphide bridge formation, folding

 

 

Clinical antibody generation

 

 

weeks

Antibody-like molecules

Aptamers

Nucleic acids / peptides with affinity to antigen. Those artificially  generated through successive rounds of in vitro selection are modified with sugars to increase their half-life.

- Short half-life can be clinically desirable:

    - treating transient conditions

    - imaging

    - can facilitate higher dosing

- Small size facilitates:

    - greater tissue penetration.

    - frapid chemical synthesis

- Unlike some antibodies, do not tend to generate immune responses

 

 

Stability is greater for DNA than RNA reagents, although  aptamers can be further modified to enhance stability.

- Clinical drugs

- Basic research

- In vitro & clinical diagnosis

In vitro selection rounds can be accomplished in a matter of days

Labelling without antibodies

Fluorescent ligands

Fluorophores linked using novel synthetic chemistries to drug-like compounds & ligands

- excellent photostability

- non-toxic

- small size minimises risk of alteration of biological activity, generating pharmacologically accurate reagents

- ideal for live-cell dynamic imaging

- multiple colours for multiplexing

- variety of chemistries available for linking ligand to fluorophore

Not suitable for every antibody application (e.g. Western blots)

- Fluorescent ligand binding kinetics

(including competitive binding assays, investigation of allosteric modulation)

- Microscopy

- HTS

- HCS

- FACS

- FCS

- Dual readout Binding & Function

FRET (e.g. receptor homo/ hetero-dimerisation)

2-4 months for a custom reagent

 

As you can see, there is a variety of in vitro generated antibodies, as well as probes with enhanced antibody-like properties or functions available. Each of these has particular strengths and some may be more to the application you have in mind than others. All share advantages in terms of purity & reproducibility, whilst retaining at least equivalent specificity, diversity and breadth of possible application between each of the available options. Moreover, these probes & in vitro antibodies are often available at similar costs & in similar (or even reduced) time-frames to conventionally produced antibodies.

 

At Human Focused Testing, we specialise in identifying the right tools for your research, and would be delighted to discuss any of those discussed in this document. We can also put you in touch with trusted providers of these tools who will be delighted to help you to deliver the results you need.

 

Please contact us through our website with details of your enquiry, or email us at This e-mail address is being protected from spambots. You need JavaScript enabled to view it for more information. Alternatively, if you have a technology (or an application for a technology) that you think should be mentioned here, please get in touch in the same way.

 

Margaret Clotworthy PhD, Director

 
 

Abbreviations

Fc: Fragment constant

FCS: Fluorescence Correlation Spectroscopy

FRET: Fluorescence/Fӧrster Resonance Energy Transfer

Fv: Fragment variable

HTS: High Throughput Screening

HCS: High Content Screening

FACS: Fluorescence Activated Cell Sorting

 

Phage: Bacteriophage- viruses that infect bacteria


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