Immune response monitoring

Why make an ELISA screening?

ELISA screening allows to follow the antibody titer evolution in an immunized host, and to determine if an antibody program may be terminated or should be prolonged. To proceed, enough antigen should be available.

To monitor an immune response evolution, we test in parallel dilutions from the pre-immune serum (collected just before starting the immunization) and the large bleed (collected 2 months post-immunization for classical programs, 21 days post immunization for speedy programs). Each sample is tested against the antigen, and possibly positive and negative controls.

If the antigen is a peptide, we will test separately the peptide and the protein carrier

In Double X programs, where hosts are each immunized against two peptides, the serum of each host is tested separately against the carrier and the two peptides to compare the antibody signals from each peptide individually.

In PTM programs, the serum of each host is tested against the carrier, the non-modified peptide, and the modified peptide.

Each ELISA service is accompanied by a test report which including the experimental settings, the signal intensity values and the graphical plot of signal intensity over sample dilution.

It is sent to the customer together with the remaining free peptide (if applicable) and all the tested antisera.

This allows directly checking the serum samples in the final application.

Causes of a lack of titer

A lack of titer evolution may be due to :

Low antigen immunogenicity

Antigens with molecular masses higher than 100 kDa (large proteins, cell extracts, inactivated viruses…) are usually excellent immunogens, while low molecular mass antigens (< 10 kDa), usually referred to as haptens, may escape to the host’s immune system. Should a hapten be used as the antigen, it must be conjugated to a carrier to increase the size.

The most used carriers are:




(Key hole limpet hemocyanin)

  • Not used in ELISA or Western blotting
  • Suggested for vertebrate
  • High number of peptide conjugation sites
  • Might cause problems if invertebrate tissues, or certain plant tissues should be examined with crude serum

(Bovine serum albumin)

  • Better suited for monoclonal production than KLH
  • Can be used as alternative carrier to KLH in chicken
  • Reduced number of peptide coupling sites compared to THY or KLH
  • Lower number of peptide conjugation sites


  • Can be used as alternative carrier to KLH in mammalian hosts
  • Suggested for invertebrates
  • Lower number of peptide conjugation sites


  • Not used in ELISA or Western blotting
  • Can be used in chicken and mammals instead of KLH
  • High number of peptide conjugation sites
  • Prolonged immunisation might cause auto immunity to THY of the mammalian host (induction of thyroiditis)

Légende : Protein carriers: high molecular weight; good antigenicity

Note : * KHL is a respiratory heme protein from the sea snail Megathura crenulata; no homology to vertebrate proteins, and no homologies to proteins that are used in the most common lab applications for blocking, like BSA


MAP carriers

Too low antigen quantities

The antigen quantities vary with the host and the antigen type.

Min. antigen quantity / injection


10 < MW < 18 kDa

Antigen MW
> 18 kDa


40 µg

15 µg

Guinea pig

50 µg

30 µg


50 µg

30 µg


200 µg

100 µg


200 µg

100 µg


400 µg

200 µg


400 µg

200 µg


400 µg in max 5 ml

200 µg


400 µg

200 µg

The dose of an antigen, if too high or too low, may or may not elicit an immune response.

Homology between the antigen and the host

The lower the homology between the antigen and the host, the higher the immune response. Accordingly, highly conserved mammalian proteins may be poorly antigenic if injected in mammals, and the selection of a chicken host may be a good alternative.

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