Antibody Production


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Antibody Production

The Peptide Approach to Protein Antibody Production

The requirement for region-specific antibodies capable of detecting and differentiating members of structurally similar proteins is ever increasing. This is made even more challenging when the demand for increased sensitivity is taken into account. The peptide approach to protein antibody production is ideally suited to achieving this goal.

All too often, however, antiserum production is undertaken by researchers and contract organisations with little thought being given to overall project structure with the consequent widely varying success rates.

Q. - Why choose AFFINITI's custom antibody services?

A. - We listen to you, the user,and discuss your needs, design each project individually based on mutual experience and expertise, and deliver a high quality product.

Some factors to consider:

Choose peptides wisely: Peptide choice may be based on a combination of predictive methods and guesswork but it should at least be inspired! AFFINITI will assist.

Design peptides for the specific purpose intended: As the molecule is to be synthesised why not build in the elements required? For example, if being used for antibody production consider the method of peptide-carrier conjugation to be used prior to synthesis, design accordingly, and optimise the chances of subsequent success. AFFINITI will advise.

Start with a defined peptide: The widely quoted phrase “immunological grade peptides” should be interpreted warily, often describing products of ill-defined integrity. Why start any project with an unknown entity? All peptides, for whatever purpose, should be analysed both chromatographically and mass spectrometrically to assure a high degree of chemical integrity. Detailed analysis is standard for all products from AFFINITI.

Peptide conjugation is chemistry not alchemy: Conjugation of peptides to carrier proteins is necessary in order to obtain a high level of immune response. A well designed and prepared conjugate is more likely to produce a desired result. AFFINITI offers a variety of tried and tested procedures.

Species for immunisation: Rabbits are often first choice for polyclonal antibody production but others should be considered. Sheep have advantages for many users, chickens for others. AFFINITI offers a range of species.

Prepare immunogens correctly: The single most common reason for poor antiserum production lies in incorrect preparation of an antigen emulsion prior to immunisation. AFFINITI does it correctly.

Immunisation programs should not be rushed: Whilst everyone wants the best antibodies in the shortest possible time, the immune response is a natural phenomenon and follows a well recognised time-course. Peptide-reactive antisera are seldom the goal. Protein-reactive IgG antibodies arise and peak later in an immunisation schedule. AFFINITI uses an optimised protocol that routinely provides success.

Screen throughout an immunisation program: By screening in an appropriate system for the desired reactivity, programs can be modified accordingly with consequent benefit. AFFINITI provides test samples throughout a program for Client screening and report.

Guaranteed success: NOBODY can offer this; however, with a little planning and careful execution the chances of a successful outcome are greatly enhanced.

Click here to view detailed schematic showing the
peptide approach to protein-reactive antibody production

Antibody screening

AFFINITI believes that its Clients should be given the opportunity to monitor an immunisation program throughout its course. Consequently, small samples of each test bleed are routinely supplied for Client testing. Projects may then be terminated once the desired response is obtained

Antibody purification

A polyclonal antiserum should only be purified where it proves to be necessary. Criteria for purification include high non-specific staining, multiple bands on Western blots and low signal strength.

AFFINITI applies a number of approaches to antibody purification which have been optimised over time and which have been demonstrated to result in dramatic improvements in signal selectivity.

Precipitation techniques

In the first instance, the polyclonal antiserum is subjected to a caprylic acid precipitation procedure removing the bulk of extraneous serum proteins such as albumins. This is followed by an ammonium sulphate precipitation step to provide an immunoglobulin enriched fraction. At this stage of purification an antibody may prove sufficiently pure for further use with complicating reactivities having been removed.

Affinity chromatography

If further purification proves necessary, the immunoglobulin fraction may be subjected to affinity chromatography using immobilised Proteins A, G or L. When carried out under optimal conditions this may lead to highly purified and concentrated immunoglobulins.

Whilst it is possible to use such an approach in isolation with crude serum, the above two precipitation techniques are recommended, and always employed, by AFFINITI, as a preliminary stage. Their utilisation ensures more efficient subsequent chromatography and a longer column working life.

Peptide/protein immunoaffinity chromatography

AFFINITI has particular expertise in the design and application of peptide (and protein) immunoaffinity chromatography. In such instances, an enriched immunoglobulin fraction obtained by use of the precipitation procedures mentioned above is further purified by immunoadsorbtion on an immobilised peptide-matrix and subsequent removal of contaminating activities.

The efficient preparation of a matrix and its subsequent use is key to successful purification and here Affiniti has much experience in a wide variety of immobilisation chemistries and has developed a number of procedures that facilitate the purification of antibodies whilst retaining high affinity for the desired peptide/protein.

WARNING - Highly avid antibodies may be difficult to detach from affinity matrices without irreversible damage occurring to the antibody. This fact must be taken into account before peptide/protein immunoaffinity chromatography and purification is undertaken.

Antibody analysis

Material is retained after each step of a purification protocol and subjected to analysis for peptide/protein reactivity in appropriate systems. Protein concentration is determined spectrophotometrically and expressed in milligrams of immunoglobulin per millilitre of material supplied.

Opposite is a Western blot following SDS-PAGE of rat whole brain lysate probed with synthetic peptide derived antibody to glutamic acid decarboxylase. Key: (a) crude serum; (b) after caprylic acid precipitation; (c) after caprylic acid and ammonium sulphate precipitation; (d) after precipitation and immunoaffinity chromatography on peptide-agarose column; (e) as per (d) but antibody pre-absorbed with peptide.

a b c d e

Product development

AFFINITI has the capability of assisting with antibody characterisation using a variety of techniques including ELISA, Western blotting, immunoprecipitation and immunohisto-chemistry.

Additionally, AFFINITI offers a full antibody modification and derivatisation service. Whether you require Fab, Fab’, F(ab’)2 fragments, immobilisation of the antibody onto a solid support, or conjugation to an enzyme, chromophore, fluorophore, or a magnetic or electron-dense particle, we shall be happy to advise accordingly.

Our senior staff will be happy to discuss your needs in detail without obligation and in total commercial confidence.

AFFINITI expertise

Senior staff at AFFINITI have been recognised as being amongst the earliest and most practiced exponents of the synthetic peptide approach to antibody production world-wide. We have many years experience of, and proprietary expertise in, project planning, antigen selection and synthesis, immunogen preparation, immunisation and antibody characterisation. This expertise is made available on a custom basis; please contact Customer Service for further details or a quotation.

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