the assay format, the standard should cover at !east
thesequencefortheepitopeused for immunization.
It
is also possible to produce synthetically the
whole sequence of the protein (up to 100 amino
acids). In any case, the synthetic proteins do not
necessarily have exactly the same folding, con–
figuration orposttranslationalmodifications as the
nativeproteinseausingdifferences in theaffinityof
antibodies between native and synthetic proteins.
Forthisreason, theanalyticallevelmustbecorrected
with theaidofthenativeproteinoran assay system
using the native protein as a standard. Benefits of
syntheticproteins includethat theyarecommercially
available at reasonable cost in !arge amounts and
highly purified forms.
ELISA for intact osteocalcin as a model for the
useofsyntheticpeptides inantibodyproduction
and assay standardization
We used the novel technology described above for
productionofantibodiesforan immunometricassay
ofintact humanosteocalcin (Parviainen et all994;
Kuronen et al 1993). The epitope analysis of the
protein sequence of osteocalcin produced a blot
shown in Fig l. Human osteocalcin is composed
of49 amino acids, and it contains severa! potential
antigenie determinants (the Jameson andWolf in–
dex is high), which are surface-oriented in the
nativeprotein. Thesesequencesaregoodcandidates
combination of antibodies gave the standard prof–
ile shown in Fig 2with synthetic osteocalcin as a
A405
0.1
1
10
100
Osteocalcin (JJgll)
Fig 2. Typical standard profile of the Mo-site ELISA
assayforintact osteocalcin in human serum. Eachpoint
is amean ofthree analyses.
standard. The tracer was arranged by coupling the
detection antibody to horse raddish peroxidase
(HRP) via its carbohydrate moieties. This con-
2,0-.----------------,
for successful antibodyproduction.After synthesis
A405
of themost promising peptictes and coupling them
--o--
standard 1-49
--.-- peptide 1-29
to a carrier protein, the immunization was carried
out following thenormal scheme inmice andhens.
Developmentoftheantibody titer in the immunized
animals was followed by an antigen-coupled
enzyme-linked immunosorbentassay (ELISA). As
expected, the antibody titer (the highest dilution
giving a positive reaction in ELISA) in mice was
higherthan l to 12000 and in hens l to l0000. For
assay system, we established mouse hybridomas
producing antibodies against the osteocalcin
sequences using routine techniques for hybridoma
production. We purified polyclonal antibodies
recognizing osteocalcin from hen egg yolks with
the method reported by Akita and Nakai (1992).
The hybridomamedium and hen IgY preparations
werefurtherpurifiedwithantigencoupledSepharose
CL4B. At this stage, we evaluated themonoclonal
and polyclonal antibody preparations for their
suitability for the two-siteELISA format. Thebest
50
1,5
-o-
tryptic digest
1,0
0,5
0,0
+---r-.-'T'"1"T'TT't'r--r'""'r'T"T"f"T1T''-...,--"T"'T.,...,..,.,.,.j
'1
1
o
100
1!9 perl
Fig 3. Effect of tryptic digestion ofosteocalcin protein
and a synthetic sequence 1-29 on assay results. Each
point is amean of three determinations.
KliniskKemi
i
Norden
2,
1995
