Læknaneminn - 01.10.1996, Page 113

Rannsóknarverkefni 4. árs læknanema, útdrættir
annað sem innihélt genóm KV1772kv72/67 frá basa nr.
7769 að 3’ enda. Þessu plasmíði var skotið inn í synovial
frumur og félekst þá fullgerð ný veira sem nefnd var VOl.
Gerðar voru samanburðartilraunir með VOl og móðurveir-
unum tveimur, LVl-lKSl og KV1772kv72/67, í plexus-
frumum og einkjarna átfrumum. A sýnum sem tekin voru 2
- 4 sinnum á sólarhring var gert reverse transcriptasa próf til
að meta magn veiru í ræktinni.
Niðurstöður: PCR og raðgreining leiddu í ljós að VOl
innihélt bútinn sem fluttur var úr LVl-lKSl. I plexus-
frumurækt virðist klónninn VOl ekki valda jafn mikilli sýk-
ingu og KV1772kv72/67 eða LVl-lKSl. í einkjarna át-
frumum hefur VOl sýkingarkúrfu sem líkist kúrfu
KV1772kv72/67. Sá eiginleiki LVl-lKSl að vaxa illa í ein-
kjarna átfrumum, ásamt sérstakri smásjármynd með stærri
risafrumum, fluttist ekki í VOl með w/geni.
Efnisskil: Álcveðið var að flytja bút úr wj/'geni vegna þess
að amínósýrubreytingarnar 3 voru sértækar fyrir LVl-lKSl
og ekki að finna í systurveiru hennar. Fleiri sértækar stökk-
breytingar er að finna í LVl-lKSl og líklegt er að þessi eig-
inleiki sé bundinn einhverri þeirra. Einnig er mögulegt að
þessi eiginleiki veirunnar flytjist ekki með einu geni heldur
fleiri. Rökrétt framhald af þessari rannsókn er að flytja stökk-
breytingar frá öðrum stöðum í genóminu milli veiranna.
Mannan-binding protein:
Evaluation of three different assays and
measurement in breast milk and serum of
allergic versus non-allergic women
Rósa Þórunn Aðalsteinsdóttir1.
Steffen Thiel2.
'LHI, 2Institut for medicinsk Mikrobiology
og Immunology, Árhus Universitet.
Introduction: mannan-binding protein: Mannan-bind-
ing protein (MBP) is found in serum and a few other body
fluids. It is produced in the liver and the serum levels of the
normal population varies highly, the mean value being
around 1.7pg/ml. The MBP gene is on chromosome 10 and
several mutations in that gene have been found. Heterozy-
gotes and specially homozygotes for the mutant genes have
low concentrations of MBP in serum. MBP has affinity for
mannan (a carbohydrate rich constitutent of yeast cell walls)
and a few other sugars. It has thus the ability to bind to the
surface of pathogen microbes. When bound to the surface of
microroganisms it can activate the complement system.
MBP is part of the first defence against infection by acting in
two ways; directly opsonizing microbes leading to
phagocytosis or direct lysis of the microorganism. Low levels
ofMBP have been found to mediate increased susceptibility
to certain infections.
Assays for MBP: Different methods for MBP-measurem-
ent are used by different groups. In this project I have com-
pared the methods used in Aarhus, Iceland and in Kilpat-
rick's group in Scotland. They will be called TRIFMA (time-
resolved immunofluorometric assay). Sandwich ELISA
(enzyme linked immunosorbent assay) and ELLA (enzyme
linked lectin assay) respectively in this paper. They are all
immunoassays, i.e. they use an anti-MBP antibody to detect
MBP bound in microtiter wells.
Atopy and MBP: Allergic reactions (type I hypersensiti-
vity) are sometimes referred to as atopy. A connection has
been found between atopy in infants and low MBP levels. It
is known that atopy is inherited more from the maternal than
paternal side but the reason for that remains to be explained.
In order to get an idea of whether it is possible that low MBP
concentrations in breast milk can be an allowing factor for
atopy to develop, I measured MBP in breast milk and serum
of women who had recently given birth.
Materials and methods: MBP assays: TRIFMA: The
plastic surface of the wells was coated with anti-MBP anti-
bodies and samples added. Eu*1 labelled anti-MBP antibod-
ies were used to detect the MBP from the samples and an en-
hancement solution to chelate the fluorescent Eu*’ ions from
the antibody complex. The Eu*1 and thus MBP amount
could then be measured in a time resolved fluorometer.
Sandwich ELISA: Anti-MBP was used for coating and
thus used to catch the MBP from the samples. Biotin labelled
anti-MBP detected the MBP bound to the surface, and alka-
line phosphatase (AP) conjugated avidin was added followed
by substrate (para-nitro phenylphosphate, PNPP). The add-
ed PNPP solution turned yellow in the presence of AP and a
microplate reader detected the color. ELLA: Poly-lysine and
glutaraldehyde were used to bind mannan to the plastic sur-
face. Following sample-addition, anti-MBP detected the
bound MBP and then AP conjugated anti mouse IgG detect-
ed the anti-MBP bound. The intensity of the yellow color
was then detected by a microplate reader. Each method was
performed at least three times. The sensitivity, dynamic
range, the influence of pH and speed of assay were examined.
MBP and IgA in breast milk and serum: Voluntary women
in the labor- and delivery- department of the National
Hospital in Iceland filled out a list concerning allergy and
gave breast milk and blood samples with informed consent.
12 had allergy (7 diagnosed by a physician), 13 had no
known allergy and 4 were uncertain. The samples were kept
frozen until measured. They were assayed by TRIFMA and
ELLA.
Results and discussion: MBP assays: The TRIFMA had
a sensitivity of 0.1 ng/ml and a dynamic range up to 1150
ng/ml. The Sandwich ELISA had a sensitivity of 5 ng/ml
and a dynamic range up to 100 ng/ml. The ELLA had a
sensitivity of 10 ng/ml and a dynamic range up to 360
ng/ml. MBP and IgA in breast milk and serum: There was
no clear correlation between MBP in breast milk and serum.
LÆKNANEMINN
103 2. tbl. 1996, 49. árg.