Custom Antibodies

Reliable and significant immunological assays require reliable and reputable antibodies and antibody services. metabion provides a one-stop solution to all our customers' antibody needs. Our custom antibody services include recombinant protein expression, peptide design and synthesis, polyclonal and monoclonal antibody production, affinity purification, conjugation, and immunoassay development.

Our service at a glance:

1. We develop and purify the antibody for you. Once an adequate polyclonal response has been reached, the crude serum can be purified by two distinct methods:
   
   • Protein A (or G) affinity chromatography is used for purifying all the IgGs present in the sample. For better results and to enrich specific IgG antisera, we recommend the elimination of antibodies with low affinity and specificity but higher binding capacity, such as IgM by purification of the antiserum. This type of purification is useful for relatively non sensitive systems like Western Blotting.
   • Immunoaffinity purification is recommended for purifying antigen specific antibodies from a pool of polyclonal antibodies, principally when they are to be used in immunocytochemistry or in immunofluorescence, where a high background would interfere with analysis. In this procedure, the pure antigen (free peptide or purified protein) is covalently bound to a solid support: CNBr-Sepharose, then the specific antibodies within the pool bind themselves to the antigen-sepharose, while the unbound antibodies are removed by washing. Finally the specific antibodies are eluted from the column and collected in TRIS buffer; their positivity and titer are reconfirmed by ELISA testing. We normally purify half of the total serum and the purified antibodies are then shipped in solution.
2. Upon request, we can label the antibody. Purified antibodies can be labelled by us with fluorochromes, biotin, or with enzymes, such as horseradish peroxidase or alkaline phosphatase: please choose the best label for your technique.
3. We can set up immunometric assays for you. This service includes the development of specific polyclonal and monoclonal antibodies, the preparation of antigen standards (extractive or recombinant proteins), and the establishment of immunometric assays (i.e. ELISA), with specificity, accuracy and reproducibility.

We provide an efficient service for the production of polyclonal antibodies from rabbits, mice and rats. The service is offered in three different formats, based on the antigen used for immunization:

1. Custom polyclonal antibodies against synthetic peptides
2. Custom polyclonal antibodies against recombinant proteins (his-tag fusion)
3. Custom polyclonal antibodies against other proteins or antigens supplied by the customer

a. Custom polyclonal antibodies against synthetic peptides

The use of synthetic peptides as immunogens is generally applied where either the complete protein is not available in sufficient quantities to carry out an adequate immunization protocol, or to obtain antibodies able to recognize only specific regions of a polypeptide chain. The selection of the amino acid sequence of the peptide to be used as immunogen is a crucial step towards success.

It is impossible to guarantee a priori, that antibodies raised against specific peptide sequences will efficiently recognize the intact protein from which the peptide sequence was originally derived. Nevertheless, it is possible to exploit certain parameters based on hydrophilicity, accessibility, and flexibility criteria, together with considerations of T-cell epitopes to judge the possible immunogenic power of a given peptide, and thus to increase the possibility that anti-peptide antibodies do recognize the complete and often native conformation of the protein chain.

We will assist our customers with the selection of the best candidate peptide sequences to be used as immunogens. Specific sequences will be proposed to our customers and submitted for their approval prior to the start of the chemical synthesis. In addition, in some cases the use of more than one peptide from a given protein chain is recommended. After synthesis the peptides are combined and used as a single antigen towards the preparation of antibodies able to recognize all the peptides. Through such an approach, the resulting antibodies have a higher capability to recognize the intact and native protein chain. Generally, synthetic peptides used as immunogens are synthesized on a small scale (5 - 10 mg) and are made of approximately 15 amino acid residues.

Peptide conjugation
Small molecules, such as peptides, have low immunogenic potential. To increase their immunogenicity, it is necessary to link, by chemical conjugation, the peptide sequence to a larger protein molecule, which serves as a carrier. In general, the few required features for a carrier to be efficient are its molecular weight, which should be higher than 20,000 Daltons, and its immunogenic potential. Most of the immunopeptides that we produce are conjugated with carriers such as OVA, BSA or KLH.

Screening and quality control
Titer and quality of the polyclonal antibodies are verified by ELISA using free peptide. Results of the test are supplied with the antibodies. Normally, dilutions of the serum ranging from 1:1,000 to 1:10,000, can be used in most immunological applications.

b. Custom polyclonal antibodies against recombinant proteins

We offer a very competitive service based on the production of recombinant proteins following gene cloning or synthesis. Starting from the nucleotide gene sequence or from a gene ID number/name related to public genomic databases, we will proceed to the isolation of the cDNA clone. In case of human genes, we have an almost complete and constantly updated gene library from which the desired gene targets can be isolated. When the gene target is from unusual organisms or species and a reliable RNA source is not available, the customer may be requested to supply the gene. Depending on the protein size and features, our team will attempt to clone and express the full length gene. Alternatively, a selected domain of 100-300 amino acids will be chosen for expression. For instance, in case of very hydrophobic or transmembrane proteins, the expression and purification of full-length versions is not the preferred choice. In such cases, our scientists will select domains of 100-300 amino acids in the native protein for antibody recognition. Recombinant proteins or protein domains will be produced as His-Tag fusions from E. coli cells and purified in sufficient quantities to be used in the immunization protocol and for ELISA or Western Blot testing.

In the unluckily event that we fail to express any of the suggested domains, the price charged will include the immunization with a mix of peptides.

At the end of the immunization protocol, you will also receive an amount of recombinant protein (maximum 500 µg) and a small amount of plasmid DNA (about 1 µg).

c. Custom polyclonal antibodies against custom delivered proteins

If you have a purified soluble protein, a partially purified soluble protein, inclusion bodies with the insoluble protein, a protein in a polyacrylamide gel slice, or any other type of antigen, you can send your sample to us. For the development of polyclonal antibodies, the total quantity of antigen required for immunization of two rabbits is 1.5 mg.

The antigen can be supplied either lyophilized or in solution at a concentration not less than 0.5 mg/ml. In this case, we test the serum in ELISA after the third boosting and we can guarantee a final serum titer of 1:1,000-1:10,000 against the antigen you have supplied. For an extra charge, there is the possibility for the customer to test the serum simultaneously.

We have a very efficient production pipeline of monoclonal antibody using mice immunized with antigens either provided by the customer or produced by us (recombinant proteins - synthetic peptides). On average, 1-2 mg of protein are necessary for immunization and analysis. This service includes the generation of monoclonal antibodies through somatic fusion and the possibility to choose either 2 or 4 hybridoma clones after the first screening. We can provide both, the cell lines and the purified monoclonal ABs. Upon request, a cell banking service is available for the storage of the desired clones.

The development and production of monoclonal antibodies goes through different phases:

• Production of recombinant protein (if requested)
  Gene cloning
  Protein expression
  Protein purificaton
• Synthesis of peptide and conjugation (if requested)
  Synthesis of about 10 mg of peptide, purity > 70%
  Conjugation of 3-4 mg of peptide to a carrier protein (as antigen)
  Conjugation of 1-2 mg of peptide to a different carrier protein for ELISA screening
• Immunization Phase (1.5 months*)
  Immunization of 4 BALB/c mice with the antigen
  Setup of a procedure for quantitative titration of specific immunoglobulins in the sera of immunized animals. ELISA testing of the immune response and selection of the animals for subsequent spleen cell fusion.
  *does not include time for antigen preparation
• MAb Development Phase (3-3.5 months)
  Splenectomy and fusion of splenocytes with myeloma cells
  Selection of hybridomas by ELISA: freezing of the more positive mother clones
  Delivered products: supernatants of mother clones (about 5 ml each)
  
  Cloning and subcloning of positive clones (2 or 4) by limiting dilution
  Freezing of the hybridomas (2 aliquots/clone)
  Viability, productivity and stability assays after thawing of one aliquot.
  Delivered products: frozen hybridomas (3 cryo-vials of each selected sub-clone + 2 cryo-vials of each mother clone)
• Production and purification
  of 0.5-2 mg of each monoclonal antibody in cell culture

Delivery time of a monoclonal antibody should be estimated at 7-10 months depending on the complexity of requested services. Close interaction with the customer at any step of production is of the essence!

Please submit your inquiry to info@metabion.com