Synthetic Peptides inAntibody Productian
and Standardization ofClinicalAssays
l)
I.
Kuronen, l) M. Parviainen, 2) H. Kokko, l) K. Savolainen and l)
I.
Mononen Departments of
l) Clinical Chemistry and
2)
Biochemistry and Biotechnology, University ofKuopio, Finland
Abstract
Purification of native proteins to homogeneity in
!argequantities is usually laborious andexpensive.
For themost immunization schemes at least200
1-!g
of the homogeneous antigen is needed. Evenmore
high!ypurifiedprotein isneededfor standardization
of assays. Development of the methods for
synthesizing !arge peptictes has offered feasible
apportunities to produce highly specific antigens
for antibody productian and assay standardization.
Atmoderateexpenses
l
arge amounts
(~l
00mg) of
homogeneous peptides are available from many
commercial sources. In this article, we discuss the
problems and advances of this technology in
constructionof immunometric systems for clinical
u
se.
Problems with native antigens
For diagnostic immunometric assay systems,
milligrams ofhigh!ypurified proteins are required
for antibodyproductian and assay standardization.
In many cases, especially with antigens of human
origin, purification of native proteins at required
extent is difficult, tedious or even impossible, and
the antigen of interest is present in nature in very
small amounts. Purified proteins may also be
vulnerable to enzymatic degradation leading to
their limited usefulness as antigens.
Synthetic peptides as antigens and standard
Synthetic proteins and small peptictes to detect the
native protein of interest can be used for antibody
productian and standardizationofan assay system.
The optimal length of the synthetic protein is
dependent on the epitope localization and
accessibitity of the generated antibodies to the
epitopeofthenativeproteins. Therefore, the length
and the position of the synthetic antigen in the
KliniskKemi
i
Norden
2,
1995
native sequencemust be carefullydesigned before
the synthesis.According to someauthors, themini–
mum length of a peptide to be used as an immun–
ogen in rabbits andmice is aroundeightaminoacid
residues (Stem 1991; Jameson and Wolf 1988;
Hopp andWoods 1981). Themaximum lengthofa
linear synthetic protein at present is around 100
amino acids. The purity of the commercially
available peptide preparations varies usually from
50%
to l
00%
depending on the purification steps
requiredby thecustomer. Inmanycases,purification
with HPLC using Cl4 or Cl8 columns produces
almost homogenous peptides.
Antigenicity of the synthetic peptides
Small antigens like haptens and short amino acid
sequences elicit weak or no response in animals.
The reason for the "missing epitope" is mostly a
consequenceoflackingTsuppressorcell activityof
the antigen in the immune system eausing in–
appropriate antigen presentation to the B-cells.
This can be overcome by givinga T-cell stimulus
by coupting the peptide to a suitablecarrier protein
like bovine serum albumin, key hole limpets
haemocyanin or ovalbumin. Applicable coupling
proeecturesareavailable inmostofimmunochemical
handbooks. As a general rule, one should bare in
mind that if antibodies against N-terminus are
needed, the coupting must be done through the
Cterminus of the peptide in order to leave the N–
terminus free, and vice versa.
A complex protein folding may result in forma–
tion of hidden epitopes that are located inside the
threedimensional structureoftheproteinandcause
a sterical hindrance for binding of the antibody to
its epitope. Thismay result in poor cross reactivity
of the antibodies raised against the synthetic
sequences and the native protein. Therefore, the
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