J Med Sci I l0 (3): l93-216,2005
Upsala
InventionsLeadingto the Developmentof the DiagnosticTest
Kit Industry- From the Modern
PregnancyTestto the SandwichAssays
Leif Wicle
Dtparmrent ol Mclfuul Srienrcs.Clinirul Chcnristry,UpltsalaUniversity,Ilpltsolu urul
the Atalemic Laboratort',Dtparnncnt of Clinicul Chenisn'yuul Pharntucttlogl,Univcrsitl'Il<tspirul,
751 tl5 Uppsala.Su'eden
ABSTRACT
by the governmentnowadaysto stimulateinnovations
The universitiesareencouraged
provide
proper
the
machineryfor assistingtheprotectionandcommercialiandalsoto
A betterundentandingof the innovationprocessmay helpto cresationof innovations.
suitablefor inventionsat the university.Examplescanbe trken trom
atean atmosphere
previously
innovations
madeat theuniversity.
successtul
During the 1960'sI madea seriesof inventions,which ultimatelyled to thedevelopment of the diagnostictest kit industry.The first, which I madea.san undergmduate,
This wasfollowed by the
wasa simpleandreliabletestkit for diagnosisof pregniurcy.
assayfor vitaminB12; next,the dual
solidphaseradioimmunoassay
anda solidpha.se
specificnon-competitive
sandwichassayand the in-vitro testfor diagnosisof allergy,
test).Orgiillonin Holliurdwith the pregnimcytest
called RAST (Radioallergosorbent
becamepioneers
kit, and Pharmaciairr Swedenwith test kits for radioimmunoassay,
Diagnosticslaterbe,came
oneof the
amongthediagnostictestkit industries.Pharmacia
leadingdiagnostictest kit companiesin the world and has continuedto be so in the
field of allergydiagnosis.
Each one of theseinventionsstartedwith a few uniqueobservationsleadingto a
The pregnancytestas well asthe allergytestemergedfrom the
technicaldevelopment.
searchfor
of assaymethodswith uniquequalitieswith the subsequent
development
The foreseeingof a commercialvalueon a futuremarketwas
appropriateapplications.
a very importantstep.This wastbllowedby the searchfor a suitableindustryinterested
to exploit the inventionwith irs new businessopportunityi.e. apply for a patent,proreagents
which in my caseconsistedof the necessary
duce and marketthe prrJducts,
had to be settled
and equipmentsfor p:uticulardiagnostictests.Finally,an agreement
andthe inventors.This reportdescribestheseinventionsand
betweenthe entrepreneur
particularly
somecrucialstepsof theinnovatioltpft)cesses.
discusses
Received9 May 2ffi5
Acceoted20 Mav 2005
193
INTRODUCTION
During the 1960's I made a seriesof inventions which ultimately led to the development of the diagnostic test kit industry. The first was a simple and reliable test kit
for diagnosisof pregnancy.This was fcrllowedby the solid phaseradioimmunoassay
and a solid phasebinding assayfor vitamin Bl2; next, the dual specific non-competitive sandwich assaysand the in-vitro test for diagnosis of allergy. At that time
the university showed no real interestto encourageinnovations leading to commercial products.Actually, I even experiencedcritical commentsfrom several university colleagues.This attitude has completely changeclduring the last 20 years.Today,
the universitiesare encouragedby the governmentto stimulate innovations and also
to provide the proper machinery for assistingthe protection and commercialisation
of innovations. A better understandingabout the innovation process may help to
create an atmospheresuitable for inventions at the university. Examples can be taken from some of those innovations previously createdat the university and having
led to successfulcommercial products.The different stepson the mad to a successful innovation are seldom describedin the scientific literature.
The innovatiln process
There is no universal or single innovation processand it may vary considerably for
different successfulproducts. However, each one of the inventions that I made in
the 1960's started with a few unique observationsleading to a technical development. The pregnancytest as well as the allergy test emergedfrom the development
of assay methods with unique qualities with the subsequentsearch for appropriate
itpplications.The foreseeingof a commercial value on a future market was the next
very important step.This was followed by the searchfor a suitableindustry interested to exploit the invention with the new businessopportunity i.e. apply for a patent,
start a prulucfion and market the products,which in my caseconsistedof the necessary reagentsand equipmentsfor particular diagnostic tests. Finally, an agreement
had to be settled between the entrepreneur and the inventors. This report will
describe these inventions and particularly discusssome crucial steps of the innovation process.
THE PREGNANCYTEST
Tlte Boyden technique
The idea to develop an immunological pregnancy test emerged from some crucial
experiments that I did with Boyden's passive haemagglutinationinhibition methul
aiming to measurehuman growth hormone (hGH) in blood. Boyden had found that
protein antigenscould be adsorbedto the surface of tannic acid treated sheep erythrocytes (l). He then used the Salk pattern technique to detect antibodies to the
protein (2). When the protein coated cells sedimentedin a test tube with a hemit94
sphericalbottomin the presence
of antibodiestheyfirrmeda mat pattem,while otherwisethe patternwas a ring or dot in the centre. He also found that additionof
the antibodiesand inhibitedthemat patternformation.This
tiee antigenneutralized
formedthe basisfor a methodto detectthe antigenin a solution.
A uniqueobsert'ation
As a student,I met Dr Carl Gemzellduringthe coursein ObstetricsandGynaecology at the Karolinskalnstituteand he showedme how he determinedhGH in bluxl
with a bioassayusing hypophysectomized
rats. In the autumn of 1959 I told
Gemzellthat I was interestedto start someresearchin parallelwith my medical
studies.Gemzell,who himselfhadno experience
in immunology,askedme if I was
interestedto learn some immunologyand try to measurehGH in blood with an
immunoassayin his laboratoryat the King GustavV:s Researchlnstituteat the
(3) on immunoassay
KarolinskaHospital.Therewasa recentshortcommunication
of pituitaryhCH usingthe Boydentechniqueand the samemethodhad beenused
previouslyfor the assayof insulinin buft-ersolutions.I developedthis methrd for
hGH but found that the instabilityof the hGH-coatedtannicacid treatecl
sheeperywas a greatproblemin the immunoassay.
throc-ytes
I solvedthis by formalintreatment of the cells prior to the treatmentwith tannicacid and the hormone.Formalin
treatmentof erythrocytes
had beenusedsincethe 1920'sin differentagglutination
reactions,but not for theassayof hormones(reviewedin 4).
The methodworkedwell with pituitaryhGH addedto a buffersolution,but not to
blood serumor plasma.As the erythrocyteshad beenstabilizedto particlesafter
formalintreatmentthey wcrenot sensitiveto changesin saltconcentration
and pH
like the intact red blood cells.After the failure to developa methodfor hGH in
blood I tried to measurethe hormonein plain urine.The mostperfectSalk pattems
were achieved,but hGH could be detectedonly after additionof pituitaryhGH to
the urine.The concentration
of endogenous
hGH in normal urine was too low. It
seemsthat no one had evertried to measureany compoundin plain urine with the
Boydentechnique.This trivial experimentwas the first crucialstepin the innovation processof the pregnancytest. I had a pert-ectmethodfor urine assaysarrd
lookedfor a suitableapplication:a hormolreknown to be presentin largeamounts
in urine.
The imnrunologicalprcllnancytest
Urine sampleswere sent to the hospitallaboratoryto test whethera woman was
pregnantor not. Animals were then used in the laboratoryto detecta hormone,
(hCG),excretedin urine in largeamountsearly in pregchorionicgonadotrophin
nancy.Couldthe immunoassay
be usedto measurehCG in plain urine?In February
1960I decicledto abandonthe hGH projectand startedto immunizetwo rabbits
with hCG to developa pregnancytest and an assaymethodfor hCG in urine.
Gemzellwas in USA when I got the idea to switch to hCG and few weeksIater
when he returnedI had,to his surprise,the first resultswith the hCG test.Gemzell
195
immediately organizedthat urine sampleswere obtained from his ward. Only urine
fiom pregnamtwomen gave a positive reactiort in the test. The first immunological
pregnancytest with a high accuracyhad beendeveloped.
Frcc:e-dricd reaRents
I had a strong desire that my dircovery woulcl be widely used and generally replace
the animal tests for pregnancydiagnosis.I realized that only a few larger laboratories could prepare the pregnancy test re:rgents for their own use. To perform the
tests in smaller laboratories,out-patient clinics and phzrmaciesit was necessaryto
buy the reagentsfrom some manufacturer. lt would then be an advantage if the
reagentscould be freeze-dried.Therefore, I made some experiments which were
crucial for the innovation. First, I investigatedthe possibility of freeze-drying the
hCG-coated particles and the antiserum in two different bottles for about 20 tests.
These reagentsgave accurateresults and could be stored at ditTerenttemperatures.
Then, I obtained ampoules with a hemispherical bottom and freeze-dried the two
reagentstogether in these for single tests. The pregnancy test was now so simple
that only urine and buffer had to be addeclto the ampoule. The ampoules were
sealedand could be storedfor future use and seemedsuitableas 'test-kits'for a cloctor's office or a pharmacy.
Test-kirJbr a doctor's tffic'e
The principle of the immunological pregnancy test developed to a 'kit' for a doctor's office is shown in Fig. l. One drop of a woman's urine plus 0.5 ml buffer solution is added to an ampoule containing lieeze-drietl reugents.The freeze-dried
pregDincytest,| 960
lnrnrLrnogical
hCG In a urine smdo
tont a pregnaolwormn
*
Reamts:
Prcedre:
Freezedred
hCG{cEted
parlrcles
+
artibodies
agarrethCG
Add m
*op ofuilne
+ 0.5 ml
brffr{$lutorl
/ltVls
t'
Uone smdefron
a
od-pr4nant wma n
Antmdrcs
"snrn'ir'ccfi[6
...,)rL!
tiJ
tr5;* hCG c€ted f I.-+
*+ +*""'itiX
++
+
4rfx
@,.ilT*'(1-)
l'ru?nnnl
N(q!pregnilil
Figure L A test kit lbr diagnosisof pregnancybasedupon immunorssayof hCC in urine. hCC in urine trrrm a pre-qnantwornanbinds the antibodiesand prohibits the hCG-coatedparticlesto fomr a mat
panent.The hCC-coatetl
paniclesweresbeepredbloodcellstreatedwith formalinand tanniclcid and
finally coatedwith hCG.
t96
reagents consist of antibodies to hCG and hCc-particles. The hCG-particles are
sheep red blood cells pre-treatedwith formalin and tannic acid and finally coated
with hCG. After 90 minutes the test is read by looking in a mirror undemeath the
test tube at the pattem formed by the particles on the hemispherical bottom of the
ampoule. lf the wtman is pregnant her urine contains the hormone hCC which
binds the antibodies,and the hCG-particles slide along the glass wall and settle as a
sharp ring or disc. Urine from a not-pregnant woman contains no detectable hCG
and the antibodiesreact with and cover the hCC-coated particles. When these antibody-hCG-coatedparticles sediment they adhere to the glass wall and form a mar
paftem.
Readv to approach a ntonufacturcr
ln May 1960 I felt that we were ready to approach a possible manufacturer of the
reagentsfbr the pregnancytest.The method was remarkably reliable. Tests on over
300 morning urine sermpleshad not given any incorrect result. The hCG-coatedparticles could be made in batches for 20,000 reactions and they were stable for
months. The antiserum from one of the rabbits could be used for over one million
reirctions. The results from quantitative determinations of hCG in urine of ll0
women in early pregnancy indicated that such determinationswould be of clinical
value. I made a draft of a publication with the title: 'An lmmunological hegnancy
Test' with l-rif Wide and Carl Gemzell as authors (5). My experience with the
freeze-driedreagentswas so exciting, that it seemedto be the right time to contact a
suitable industry, before finishing the manuscript.
Organon, u suilable pharma<'euticalcontpunv
Gemzell had earlier obtained financial support from N.V. Organon in Holland and
he suggestedthat he should write a letter to explore their interest. On l'r of June
1960, Dr Marius Tausk, managing director of N.V. Organon in Holland, received a
letter from Dr Carl Gemzell at che Karolinska Hospital, Stockholm, the first paragraph of which read as follows:
'A young
doctor in my laboratory,L. Wide, has developed a new pregnancy test
which seems to have many advantages.lt is an immunological method, takes only
one to two hours. gives a quantitative answer and can be perfbrmed in every laboratory or even in a d<rctor'soffice. lt is also very inexpensive.A pre-requisitecondition is that some material can be preparedand ,,,fumished pref'erablythrough some
pharmaceuticalcompany.This material, as we have found, can be stored and used at
convenience....'(Thelener publishedby M Tauskin ref 6.)
Pregnastit'on
Dr Tausk found the letter exciting and appearedin Stockholm shortly thereafter.I
demonstratedturd discussedthe pregnancy test with him and two weeks later with
Mr Polderman, who becameresponsiblefor marketing the test by Organon under
the name of Pregnosticon.I also showed that the antiserum and the hCG-particles
t97
in bottlesfor e.g. 20 or 100tests.Thesewere suicould be freeze-dried
separately
table for labomtoriesperforminglargerseriesof pregnancytestsusing test tubes
with a hemispherical
bottom.By testinga serialdilutionof the urine,the concentrain lU/L.
tion of hCG in urinecouldbe estimatedandexpressed
Dr Tauskwasimpressed
andOrganondecidedto bring it up immediately,and an
agreementwas proposedwhich was acceptedby us. Organonthen neededa t'ew
weekstbr patentapplication,
and afterthat we could sendthe manuscriptfor publion this projectand
Organonput in largeresources
cationin Acta Endtrcrinologica.
hopedto launchthefirst kit in 6 morrths.However,thetieeze-dryingof the reagents
that had gonewithoutany problemftrr me in laboratoryscaletumedout to be the
kit wasnot
maintechnicaldifficultyfor Organonto solve,andthe first Pregnosticon
launcheduntil spring1962.
Pregnant'ydiagnosisfiom unc'ientda;"tol960
Thereis no diagnosticproblemthathascreatedmoreideasfor teststhanfinding out
whethera womanis pregnantor not. The oldestrecordedpregnallcytest is found in
Egyptianpapyri about 3,350 yearsago and was basedupon the germinationof
seedsinducedby a factor in the pregnantwoman'surine. Severalhundredsof
methodshavebeendescribedfrom antiquityto the presentday (reviewedin 7, 8.
9,10).Therewas no simpletestfor diagnosisof pregnancyin 1960.They were all
basecluponinjectinganimalswith plain urineor extractsof urineto detecta biological effectin the animalcausedby the possiblepresence
of hCG in urine.Two rabbits werecommonlyusedfor a testancl8 ml of the woman'surinewas injectedinto
theirear veins.Twu dayslatertherabbitswerekilled andtheir ovarieswereinspectTherabbitsoftendiedclueto toxic effectsof the urine
ed for presence
of a bleecling.
and thetesthadto be repeated
with an extractof the urine.Otherlaboratories
useda
mousetest with five mice which were injectedtwice daily for threedaysand then
killed the fbllowingday for inspectionof the ovaries.Toadsandfrogs,whereeither
the laying of eggsor the expulsionof spermatozoa
was observed,were used in
many clinical chemicallaboratories
and by pharmacists.
Theseanimalscoulclbe
usedseveraltimes.
The idea to developan immunologicalpregnancytest was not new in 1960.
Numerousattemptsweremadefrom 1902to the 1950'susingantiserafiom animals
immunizedwith extractsof humanplacentaor pregnancyurine,and around1950,
with purified preparations
of hCG (referencesin 4). It can be noted that all the
infbrmationneededto preparethe reagentsfor this pregnancytest were available
nine yearsbeforeI madethe invention.In l95l the Boydentechnique(l), the for(ll), purified hCG preparations
(12) and specific
malin treatmentof erythrocytes
antiserato hCG (13) had beendescribedin the scientificliterature.A scientistwho
was very closeto developan immunologicalpregnancytest was Helen Strausser
making a PhD thesisin 1958at RutgersUniversity,N.J., USA on somemethodologicalstudieswith a modifiedBoydentechnique(14).However,sheanalysedfor
of hCG only in kaolinconcentrates
of lfi) ml of pregnancyurine and
the presence
198
not in plain urine.lts potentialuseasa pregnancytestis not mentionedin the thesis,
and thestudywasneverpublished.
Frorn bioasscrys
to ltouseholdkirslbr pregnoncydiagnosis
The pregnancytestsmadein rabbitswerereplacedby the immunologicaltestat the
UniversityHospitalin Uppsalain January1961,whenI movedto Uppsala,where
Soonthereatler
Gemzellhad becomethe professorof Obstetricsand Gynaecology.
pregwith
Sweden
started
immunological
severalotherhospitallaboratories
in
the
presented
that I finally
in my PhD
nancytest assistedby the detailedprescriptions
thesisthe following year.I reportedin this thesis,defendedat UppsalaUniversity,
that the pregnancytestwaspositiveabout2l-23 daysafterestimatedday of ovula(4).
ticrnandthatthe testhadan accuracyof 99.8per cent on2,23Ourinespecimens
Pregnosticonwas a successfor Organonand becamethe most sensitiveanclaccurate pregnuncytest during a 20 yearsp€riod.The variantwith both reagenLs
tieezedried in the ampoule(seeFig. l) wa.smarketedas Pregnosticon-all-in.
A 'household
kit' of kegnosticonwas sold to the public underthe narneof hedictor. The women
testsat home.
couldfrom now on makethepregnancy
Pregnancytestspertbrmedon a glassplate,with the resultsgiven after only 2 minutes,were soon on the marketboth by Ortho in USA and by Organonin Holhmd.
lt wasnot until the 1980's
However,thesetestswerelesssensitiveandlessaccurate.
that pregnancytests basedupon the sandwichtechnique(see below), some using
monoclonalantibodies,replacedthepregnancytestsdeveloped20 yearsearlier.
oJ'hCG
CIinical significanceol'quantilatiyeintnrunoassays
(4)
of hCG
In my PhD thesis I describedthe valueof a quantitativeimmunoassays
in urine in variousclinical conditionsbaseclupon more than 10,000assaysof the
in urine.lnformationaboutthe hCG levelin urinewasshownt<l
hCG concentration
abortion,intra-uterine
be of importancetbr diagnosisof ectopicpregneurcy,
threatened
The immunoassays
replaced
foetaldeath,hydatidiformmole and choriocarcinoma.
the bioassays
lbr hCG in urinewhich hadbeenusedfor manyyearsin particularfor
(15).Quantitative
of hCG in urine
of choriocarcinoma
immunoassays
thediagnosis
could be madewith the Pregnosticon
kit fi'omOrganon,which becamethefirst testassayof a hormone.
kit orrthe marketfor a quantitative
SOLID PHASE COMPETITIVE BINDING ASSAY
Att c.rperintentgiving risc to trcn'inventions
Two of the other inventions, the solid phase competitive and the non-competitive
binding assays,emer-eedfrom one particular experiment with the hCG-coatedparticles used in the pregnancy test. The first crucial observation was that the particle
pottern could be transformed from a mat to a ring and then back to a mat and then
again to a ring by adding antiserum. honrrone, arrtiserum and ht>nnone in a sequence. This was true even when I washed the particles between the additions of
t99
Two altematives
Antibody
coated
particle
Anligen
added
s3
\
re "t *33
+
\
83
OO
\
+
%
tf
oo
.o
(,
+
Slidedown and
setle as a sham disc
"&g
\#
ft*
Figure2. Altemativereaclionsafterthe aclditionof excessilmountsof antigen(hCC) to the antibodycoatedpaniclesusedin thepregn:rncy
test.
Figura 3. The el'fec'tofalternately adding polyclonal antibotlies against hCG and rrsl-labelleclhCC to
hCC-coated panicles. Formation of several layers of antibodies and labelled hormone.
200
Antibody-maed
formaliniirdretl
bloodcell
A.
Unlabelledrutfen
Labelledmtigor
go
-)..{-
\
.?1"r'
;ii
j{x
?;,
I
t
,o 'J.
t'
B.
Moreof
unlabelhdantigen
I-abellu.liurtigur
ooo%o
ooo
l.!;-.
jii
Ti
re
{
\-
f
\
*
,{
I
A
rlc
1(,' ".i:
oa
Figurc 1. Unlabelledartigen inhibits the binding of labelledantigento the antikxly<oated panicles.
Effectsof n smaller(A) anda larger(B) amountof unlubelledantigen.
antisemm and hormone. There were two possible explanationsto this phenomenon
as shown in Fig. 2. I got a chanceto explore this with rzsl-labelledhCG in 1965 at
the Department of Clinical Chemistry, University Hospital, Uppsala. I found, as is
shown in Fig. 3, that antibodiesand hormone could be added in several layers on
the hCC coated particles. Next crucial observation was that the amount of labelled
hCC that bounclto the particles was directly proportional to the amount of antibody
added in the previous step. This observation led to a new assayprinciple: the non'sandwich
competitive
technique' (see below). Another observation was that unlabelled hCG competitively inhibited the binding of labelled hCG to the antibtxly
coated particles.The effect of two different amounts of unlabelled hCG is shown in
Fig. 4. The particles were separatedfrom the solution by centrifugation and then
carefully washed severaltimes. This was followed by detennination of the radioactivity bound to the panicles which decreasedin relation to the amount of unlabelled
hCG. This competitive binding assay, using antibody coated fonnalinized sheep
erythrocytesand lrsl-labelledhormone, was evaluatedin our departmentin 1965 for
determination of hCG, luteinizing hormone (LH), follicle-stimulating hormone
(FSH) and insulin in human serum. The highly purified human pituitary
gonadotrophin preparationsused in the assay were isolated by Dr Paul Roos, Departnlent of Biochemistry, UppsalaUniversity.
201
Theprinciple ot'the (ompetitivebinding 4.r.r4,1,s
The principleof the competitivebindingassaywith a gradedresponse,shown in
Fig. 5, was first developedby Arquilla anclstavitskyin 1956for immunoassay
of
humaninsulin(16). In theirmethodthe signalwasthe antibodytriggeredreleaseof
haemoglobinfrom intacterythrocytes
attachedto insulin.A considerable
improvement of the competitivebindingassaywas madea few yearslaterby RogerEkins
(17) in London,uK, and RosalynYalow and salomonBerson(ls. 19) in New
Xrrk' USA. They independently
developedconsiderably
moresensirivecompetitive
binding assaysby introducingradioactivelylabelledcompetinganalytesusedfor
the assayofthyroxin andinsulin,respectively.
Analyte
(ligand)
Labelled
ligand
Binding
reaoent
t
;rG
,
N
3r
Figurc5. Theprinciple
ofctxnpetitive
bindirrg
assays
usinga lubelletl
(t6-19).
ligirnd.
dcscribcd
19.56-m
Theanalyteanda tixedarnount
of labelled
ligandcompete
lbr a limitedamountof bindingreagent.
The
anrount
of labelled
lignndbound
is invene(l he amounl
oianalvte.
F rom.lbrmulized ervthrocvtes to Sephade-rpartit les
The lack of infolmation about the levels of the protein hormones in blood iurd urine of
the patientshad beena frustrating experiencefor me when I worked as a gynaecglogist
at the Uppsala Univenity Hospital. In spite of its great potentials to measurethesehorrnones,the radioimmunoassaydescribed by Yalow and Berson had not become used in
the clinical routine in the hospital chemical laboratories.I assumedthat this was most
likely due to the cumbenome and laborious chnomattrclectrophoreticmethod they used
to separatefree and bound labelled hormone (19). The techniqueI had developed using
antibody coatedparticles facilitated this separationprocedureto a simple washing step.
However. even if the performanceof the radioimmunoassaynow w.rs simple, very tbw
hospital laboratories would have the facilities to prepare the reagentsneeded for the
assay.I saw a large potential market for test kits containing the necessarymaterials for
radioimmunoassays.It then seemeddesirable to replace the formalized sheeperythrocytes, usal as a solid matrix, with some insoluble polymer like cellulose or cK)ssI inkecldextr:rn(Sephaclex).
202
had developeda
Dr JerkerPurarh,ar the Departmentof Biochemisnyin uppsala,
(20)' I conmethql to coupleenzynesto Sephadexusingan isothiocyanate-derivative
against
antibcxlies
tacteclhim and obtainedactivat;d sephadexto which we coupled
non-specific
low
:rnd
hormones.These:urtibotlycoatedparticleshad a high specific
wasso ptxx thatthe fint
tinAing of the radioactiveiylabelledantigenbut the precision
Eventuallyl -solvedthis problem
pr"p*rrkol* coul6 not be usedin the immunoassay.
Rolf Ax6n' working in
Ly ultrasonicdisintegratio*of the Sephadexparticles.Dr
porarh,s group, suggestedthat we siroul<tinvestigatecNBr-activated Sephadex.
I reportto him' that
Already oit., itr" firs experimentwittr coupledantibodiescould
:
ffi;ffi,
+*
Laberbd \\
antigen
\\
,4
ry +H
...1..
soJpn"*
ffir,,Iil,
usingsolid phasecoupledantibody
A competitiveimnrulroassay
Fi1lurt,6.Soliclphaseradioimnrunoassay.
pnr-eduretrf antitxxlyboundand
separalitnr
the
phase
t'acilitares
*fi.f
nin
u..
The
antigen.
labelled
ancla
step'
wirshing
freelabelledurtigento a simple
The CNBr-methuj
this immunosrlrbent was perfect fbr use in the immunoassay.
(21)
into my.own laboratory'I
was simpleto performuul I could immetliatelyintroduceit
sizesfor usein the
starte6to inueitigateSephadexand celluloseparticlesof diftbrent
(RIST)tt^ssay'
raclioimmunosorbent
iclea
ancla neu'husiness
ToPharnnciau'ithint'entions
the inventionand conln March 1966,I contactedPhannaciain uppsalato present
kits ttl hospitallabreagent
vincethemt0 enterinto a new businessarea:marketing
in blood'
compounds
other
of honnonesand many
oratoriesf.r radioimmunoassay
that
suggested
I
time.
that
There were no radioimmunourruykits on the market at
radioimphase
competitive
rhey shoul4startwith a patentaipticarionl'or the solid
particles'I represented
Sephadex
and includetire useof CNBr-activated
munoassay
Directortlf Pharmacia.
the inventorsand negotiatedwith GijstaVirding,Managing
at Pharmaciaan agreementwas settled
After about six weeis of considerations
Porath.The patentapplicabetweenPharmaciaand the inventors:wide, Ax6n and
t i o n g o t a p r i o r i t y c l a t e o f 2 o f J u n e l g 6 6 , a f t e r w h i c h w e c o u l d p u b l i s Aushourresults
prioritydate,Kevin Cattet al in
(22,23).ln the samemonth,afterPharmacia's
a similar solid phase
tralia submitteda manuscriptfor publicationdescribing
(24).The generalprincipleis shownin Fig' 6'
radioimmunoassay
203
During the negotiations
with PharmaciaI sressedthat the soliclphasecompeti.
tive assaywasnot restrictedto immunoassays
and I wantedthe agreement
to cover
non-imnrunological
systemsas well. They askedme to confirm this with an example' A few monthslaterI presentecl
Pharmaciawith a nrethodfor the assayof vitamin Bl2 in bluxl- lt was a soliclphasecompetitivebindingassay
using CosTlabelledvitaminBt2 as a tracerand intrinsicfactorcouplei to
cNg._rctivated
A) No'-competitive assay,ofantibodiesusinglabelledantigen
k
bY
F + *-->
T
AntibuJy
+
#-,
*f
$*r
..$
Figurt 7' A) solid phasecoupledantigenin excessbinds rhe
;xrlyckrnalanrikxlies.Labelle4anrigenin
excessbinds to the antikrdies.unbourid.labelled
antigenis remoi,ecluy *r-r,i"g -[p. B) Soliclphase
coupledJxllyclonalantibodiesin excessbind the anrigJn.Labellettpolycional
" anritfflie,
in exces.s
bintl to
the anrigen.Unb,ound
labelledantibodiesarerenroved'bya washing'srep.
Sephadexas a binding reagent (25). An agreemenrwas settletl
between the invenkrrs and the company, and in october 1966 phamracia appried
for a patent on the
solid phasevitamin B l2-assav.
FROM SANDWICH ASSAY TO ALLERCY TEST
A neu,ussayprinciple - a non-conryetitirte,sandu,ic.h
techniclue,
As mentioned above, the experiment with hCG-coated formalinized
erythrocytes,
anti-hCG and r2sl-labelledhcG gave rise to the solid phase
competitive binding
assays as well as to the non-competitive sandwich asiays.
The iron_competitive
methul was first used as a very simple method to detect
antibodiesusin-{ a latettea
antigen (Fig 7a). I then examined a non-competitive assay
of artige.s using the
2M
combinationof solidphasecoupledantibodiesand labelledantibodies(Fig 7b).The
antigenhad to haveat leasttwo bindingsites.ln this experirnent
the antiserumwas
polyvalentwith antibodies
to differentepitopeson the hCG molecule.The IgG fraction of the antiserumwaspurified and usedboth for couplingto the solid phaseand
for labelling.The differenceof the closeresponsecurvesof competitiveand n<lnusinga labelledantigenanda labelledantibcxly,
respeccompetitiveimmunoassays,
Compotilivobbding ac*y
Bumd
P*ccrt
r.rirly
hihton
Non-oorqotlivo ($ndwioh) asseY
Buvrd
rcildU
10000
1000000
10 0 0 0 0
10 0 0 0
1000
1
r0
'10)
Analyteconcenfation( Log scale )
j10x0{Df,co
AnaMecon@nfatbn ( Log scale )
Figurc 8. Conrparisonof doseresponsecuryesof competitiveiurd non-cornpetitive(sandwich)binding
assays.The txruntl fructionof labelledligand and labelledbindingreagent,respectively.is shownon the
Y-axis.
tively, is shown in Fig 8. ln both casesthe bound fraction of the labelled reagent is
indicated on tlte Y-axis,
The clesigrtof a dual specific:alle rgy test
The non-competitive assayusing labelled antibodies had not been described in the
literature. I had observedthat the methfil had a higher sensitivity and a better precision than the competitive immunoassay.lt also had a shorter reaction time, as the
reagentswere added in excess.If the labelled and the solid phasecoupled binding
reagents were directed to different sites on the analyte, a dual specific assay was
obtained.Excited over the wide potential of this assaymethod with its unique qualities, I was looking for an application with a novelty and clinical significance simil:u
to the successfulpregnancytest.
I got the idea to develop an allergy test early in the autumn 1966 while listening
to a dennatologist,Lennart Juhlin, at a rneetingof the Society of Experimental Biol-
20s
i
by Pharmacia
andorganizedby the Departogy in Uppsala.This society,sponsored
was
a forum to increaseinterdisci\ mentof MedicalChemistry,UppsalaUniversity,
plinary collaborationand exchangeof ideasand knowledgein Uppsala.Juhlin had
just returnedfrom a yearin Philadelphia,
USA andgavea lectureon urticaria,hista\ mine and reaginicactivity. I askedhim during the lecturewhetherit had been
1 shownthat the reaginicactivityin allergywasassociated
with an antik)dy.He told
us that a groupin Denver(leadby K tshizaka)had recentlyshownthat the reaginic
j
@u€
Eagenl
Soll
phase
L*dsJ
brdrg
r€gent
I
A'1I
-
Appliedfor thediagnosisof allergy:
*3
{
e,
TheRudioi ll e tgr.rSrrlx,ltTcst (R.4S7)
Allcryencouplcdto sohdphasc
Reaginic(lgE)antibcxlyspcific fbr tlreallcrycn
Labellcdantibodyspcciliclbr r.-aginic(lgE )antibodies
Figurc 9. Generalprincipleofthe dual specificnon+onrpetitivesandwichassay.The analytebindsto the
capturcreagentaswell asto the labelledbindingreagent.The capturereagentandthelabelledbindingreagent areaddedin excess.Unboundlabelledbindingreagentis rentovedby a w::r.shing
step.A wirshingstep
nrily alsobe essentialafter the anirlyteis boundlo the capturereagent.The applicationtbr diagnosisof
allergyis de*-ribedin the text.
activity
was associated with antibodies. These antibtdies
did not belong to the
known immunoglobulinclassesyG, yM, 1A or yD-globulin(lgA, IgM, lgA or lgD)
but to a fifth unique immunoglobulin class called rE-globulin (26-28).
With this infonnation, I designedduring the lecture a dual specific sandwich test
for reaginic antibodies,as shown in Fig 9. The reaginic antibcxlyin a serum sample
was t'irst bound to the specific allergen attachedto a solid phase.The allergen specific reaginic antibody was then detected with a labelled antibody specific against
the yE-immunoglobulin (lgE) class.A prerequisitefor the test was that the reaginic
antibody could be bound simultaneously bolh to the allergen and to the labelled
antibody.A signal (e.g. radioactivity,fluorescence)from the label bound to the solid
phase indicated that the patient had reaginic (IgE) antibodies and was allergic
ag:rinstthe particular allergen.
The merits of the dual specific sandwich technique for this application are illustrated in Fig. 10. It could be expectedthat the serum sample might contain antibodies of other immunoglobulin classesdirected to the specific allergen. If the solid
phase allergen was in a large excess, these antibodies of other immunoglobulin
2M
classeswould hardlyinterfere.They would not be detectedby the labelledantibody.
Also the labelledantibodyshouldbe addedin excess,and the reaginic(lgE) antibodiesdirectedto otherallergensshouldthen not interfere.However,as a precaution, the reaginicantibodiesagainstotherallergenscould be removedin a washing
stepbeforetheadditionof the labelledantibuly.
,C
.a
{
Sdi(l phisc
cruplcd dlcrgt n
lgc-iltibod]
thc allcrgcn
C,^t
+
e*r
<].r
latx'lbd artitx)dy
alFjnst the lgE dlss
againsl
IgE-antibody agalnst
thc nnr:rgcrr
lgE-antibodv qainst
ancth.t alllrlFn
Figurc 10.Selectivedetectionof lgE-antikxliesagainsta panicularallergenin the presenceof lgG-antikxlies ur the sune allergenrmdlgE antikxliestu anotherallergen.
Penic'illirt allergy-
On tlre following day, I met Dr Jrrhlin and showed him my schematic drawings of
possible methods to detect the reaginic antibodies.Juhlin had a particular interest in
penicillin allergy and suggestedthe use of penicilloyl as allergen in the test. We
agreed upon collaboration on penicillin allergy, which eventually resulted in several
joint publications (29, 30). During the autumn 1966 Juhlin sent me blood samples
from penicillin hypersensitivepatients, and I obtained the penicilloyl which was
supposedto be used in the assay.
A remurkahlc coittcidcncc - a RIST assayof an atvpical ntyeloma proteitt t1'us
tlerclopcd
A remarkable coincidencethen occurred.An atypical myeloma protein that did not
belong to the known immunoglobulins A, D, C, and M had been detectedand puritied by two colleaguesin Uppsala:Gunnar Johanssonand Hans Bennich (31). They
had been unable to find a normal counterpartto the myeloma protein (called myeloma-lgND) when investigating300 seratiom blood donors and patients with a single
radial immunodiffusion (SRD) test.
When I heard about their efforts which were presentedin a seminar, I informed
that radioimmunoassayshad a much higher sensitivity than the SRD test. The chromatoelectrophoresis,used as a separationstep in the radioimmunoassayof Yalow
and Berson (19), may not be applicable for the gammagkrbulins.However, I had
invented,but not yet published,a new principle which I tenned radioimmunosorbent
20'7
(RIST) assay,which may be suitablein this case.I invited them to a collaborationin
which they shouldgive rne the purified myeloma proteinand a specificantiserum,and
I should label the protein and couple the antibodies to Sephadexand develop a RIST
assaytbr the myeloma protein.
With this RIST methcxl,a protein was detectedin normal seraand this pnxein had a
position on electrophoresis,gelfiltration and DEAE<hromatography corresponding to
the atypical myeloma protein. lt was concluded that myeloma-lgND representeda new
classof human immunoglobulin.The study was publishedin lmmunology by Johansson, Bennich and Wide (32) with the title: A New Classof lmmunoglobulin in Human
Serum. ln this study we found that one of 62 blood donors had an lgND level 15 times
higher than the average.
The critic:al rcugent.for the allergv t(st :r''qsin my'laboratory
This bloo<tdonor showedclinical signs of extrinsic asthma(32). It was a fair chamce
that the myeloma-IgND belonged to the immunoglobulin class yE (lgE) shown to be
associatedwith the reaginicactivity (26-28,33, 34). When I, late February 1967,heard
about the asthma diagnosis, I realized that the critical reagent for the allergy test may
have been in my laboratory fbr several months. Within a few days I had obtained sera
of allergic patients from the Allergy Outpatient Clinic in the hospital and common
allergens from the pharmacy for coupling to solid phase:animal d:rndruff from hone,
cat and dogl pollen from birch and grass; and extracts of shellfish and house dust. lt
wa.s an immediate successfor the design of the dual specific allergy test. The antiIgND was used a.san anti-IgE in the a^ssay
to detect allergen specific reaginic antibodies, and the resultsconelatedexccllentlywith thoseof pruvo,cationtestswith the same
allergens. This was a strong support for a similarity between the IgND and the 7Eimmunoglobulin(lgE).
The following month specific antiserato myeloma-lgND and yE-immunoglobulin
(lgE) were exchanged between the laboratories in Uppsala and Denver (K Ishiz:rka).
The two proteins shared major imtigenic determinants,and eventually it w;rs reported
that the myeloma-IgNDand the IgND in normal serumwere of the tgE-immunoglobulin class(35, 36).
kt Phqrnrucictv'ith the raclioallerg,osorbent(RAST) test
I had kept the non-competitive sandwich assays,including the design of the allergy
test, as a secretas I regarded it as a patentableinvention. By the time I tbund out that
the allergy test functioned with the anti-IgND, I disclosed the methql for Hans Bennich and Gunnar Johansson.I suggestedthat Bennich, who had the purified lgND protein, should make a larger batch of immunosorbent purified antiJgND. Then, after I
had shown that this functioned well in the test, we could offer the allergy test to Pharmacia to apply for a patent and to procluceand market a diagnostic test kit. An agreement had to be made betweenthe three of us and Pharmaciaiurd this should include. in
addition to the invention, also the supply to Pharmacia of the necessaryamounts of
rnyelorna-lgND and iutti-lgND to start the production. I suggestedthe name RadioAller-
208
gosorbent Test or RAST in an:rlogy with RIST (see above). My negotiation with Gtista
Virding startedin JLure1967 but was not finalized urtil the beginning of September.
A c'ittrtionc:lassic'
As soon as the patent application was sent, I startedto make a drati of a manuscript by
L Wide, H Bennich anclSGO Johanssonwith the title 'Diagnosis of Allergy by an Invitro Test for Allergen Antibtxlies'. This was published in The Lancet in November
1967 (37\ and became,in 1985,a Citation Classic in Current Contents(38) as it for the
first time described the allergy test as well as the dual specific non-competitive sandwich trssayusing labelled antibdies.
Further upplicatiotts td the santlu'ic'htet'hnique
The use of a catching antibody and a labelled antihxly to measurean antigen was pubr
lished in 1969 and presentedby me at a Knrolinska Symposium on Immunoassay of
Gonadotrophins (39) : 'The gonadotrophin in the unknown serum is first bound to
Dual-specific, non-comp
etit ive sandwichimmunoassay.
; \ r r t i r : c rr v i t l t
ff'**"'**'*
ti\.
II]"
-q"
\
\
#*,,,:,.,,
Solid phrrsc
Catchingantibody
<-
Solid phasc
Labcllctlantibody
Figure I l. The principle ofthe dual specific sandwich assayusing a catching antibody and a labelled antitxxly clirectedagainst different sites on the arrtigen.
209
polymer-coupledantib<xlies.
After washingthe solid phase,labelledantibodiesare
added.Theybind to thegonadotrophin
on the solid phaseandthe uptakeof radioactivity is in relationto the amountof hormonepresentin the originzrlserum.'[n the same
publication,I introducedthe term 'sandwichtechnique'asa generalterm for the noncompetitiveassays.
The principleof thedualspecificsandwicha"ssay
usinga catching
antibodyanda labelledantibodydirectedagainstdifferentsiteson theantigenis shown
in Fig.I l.
The following yearI clescribed
threedifferentvariantsof the sandwichtechniqueat
a EunrpeanWorkshopin Edinburghon Radioimmunoassay
Meth<xls.
The sensitivityof
the IgE assaywasreportedto be at leastl0 timeshigherwith the sandwichtechnique
thanwith thecompetitiveradioimmunoassay
usingthe sameantiserum(40).
Other namesthan the sandwichassayswere proposed,such as 'Verkniipfungs
Test'(4l),'two-sitei*say'(42) and 'immunoradiometric
assay',the latternameoriginally usedfbr a competitiveimmunoassaywith labelledantikxlies (43). Today the
term sandwichassayis commonlyused,andit coversalsothe non-immunological
systemsusingthesamebasicprinciple,e.g.for DNA analyses.
GENERAL REMARKS
Thc inventors' c'hoiceoJ a suitable entrepreneur
A very important step in the innovation process is the choice of entrepreneur. It was
Gemzell who chooseOrganon for the immunological pregnancytest, and I had no
objection to that, or suggestedany altemative.The amount of resources,both number of people and laboratory spacethat they immediately put into the project, was
impressive.Organonhad an excellent quality control and their product Pregnosticon
was highly reliable. Organon was a possible choice also in 1966 for rhe new invention, the solid phase radioimmunoassay.However, I had lost the scientific contact
with Organon and their reply was unfavourablewhen I askedfor a researchgrant in
r965.
Pharmacia in Uppsala became an alternative.This company, on the other hand,
was much smaller and without any experience within the diagnostic area rtr
immunology. Pharmaciahad the advantageof being close to us, had a tradition of
good contacts with scientists at Uppsala University, and their knowledge about
insoluble polysaccharidescould be essential. lt was also attractive to favour a
Swedish industry, and a maximal profit of the invention was never my objective.
My co-inventors,Ax6n and Porath,had no objection to let Pharmaciahave the first
option. It tumed out to be a successfulchoice.
The innovations Ji'ont the entrepreneurs' vieu,
The entrepreneurs,in this case Organon and Pharmacia,had to estimate the potential market for the product, how the project could fit into their organization, their
possibility to Inanutacturethe product and the chancesto obtain a patent protection.
2lo
The potentialmarketof the pregnancytestmusthavebeenobviousfor Organonin
view of the well documented
desirethroughoutagesof developinga simple and
reliabletest.whichhithertohadfailed.
It was much more difficult for Pharmaciato evaluatethe potentialmarketof test
kits for radioimmunoassay.
Over six years had elapsedsince the first radioimmunoassay
methodsweredescribed,and they werenot usedat the hospitalsin the
imedical service.Pharmaciahad limited experiencein the field of immunology.
Conventionalmarketresearches
seemedmeaningless
to do, astherewereno similar
productson the m:rrket,and only a few peoplecould seethe futuredemand.A pioneerresearchanddevelopment
in a new areahad to be made.I hadseverallong discussionswith GcistaVirding,in which I convincedhim abouta largefruitful future
market for theseproducts,and his never failing supportfor the 'Wide-project'
becameimportant.
There were periods during the clevelopmentof the projects, both at Organon and
at Pharmacia, when strong opinions expressedthat the projects should be abandoned, as resourceshad to be taken from other areas within the companies. lt was
then of vital importance tbr the projects that they were supporteclby the managing
directors, Mauris Tausk and Gdsta Virding, respectively.
The agreementshetu,eenthe inr)entorsand the entrepreneurs
It is a very unequal situation when a young inventor from the university negotiates
with professional businessmenand lawyers. The agreementwith Organon in 1960
was too strictly limited to the particular technical performanceof the rnethod as we
had describedit. Whcn in 1964, Organon heard of Strausser'sunpublishedPhD thesis from 1958 ( l4) (see above) they claimed that this anticipatedthe novelty of the
invention and that they therefore reduced the royalty to half and limited the period
for payment to ten years.On the other hand, they were willing to extend the agreement to include also developmentsand improvementsof the test made by Organon
as long as the basic principle of the original test was applied. We had of course taken the latter for grantedwhen the agreementwas settledin 1960.
The discussions with Organon were useful experienceswhen I later negotiated
with Pharmacia.Pharmaciagot the rights to decide the contentsof the patent applications. ln the agreementwith the inventors they defined the invention as it was
describeclin their patent application. I had objections against this way of limiting
the definition of the invention to Pharmacia's patent application. I could fbresee
many products based upon the basic principle of the invention and not covered by
the patent applications. One example, which I discussedwhen the first agreement
was signed, was the use of a solid phase coupled second antibody, later called
DASP (Double Antibody Solid Phase).Different variants of the sandwich technique
were other important examples, as Pharmacia covered the technique with patents
only in the allergy field. Inl968, I made numerous attemptsto persuadePharmacia
to apply for patentson the other different applications of the sandwich technique.
When tlrcy lrcsitatedto do this, I succeededto get an infonnal agreemerlton the use
2ll
'S:rndwich
basic principle of the non-competitive
rechnique'. I realized that it
1 ofwastheimportant
for the future to describe and define in text antl clrawings the basic
lJ
principles of all our inventions. ln this work I had valuable supporr from Bj0m
lngelman, a successfulinventor at Pharmacia.
During the negotiationsfor the RAST methcxl I wanted to change a paragraph
against which I had objectionsin the previous agreements.[t was about the rights tg
sell patents,patent applicationsand licensesto a third pan. After several meetings
with Virding we decided that Pharmaciawas not allowed to do this with any of the
inventions without the permissionof the inventors.
An independent inventor from the university has his particular view on the
exploitation process.An industrial partner may look at the processanclat the agreement in a somewhatdifferent way. This difference may not have been clarifiecl and
penetrated before the agreementsare signecl.we experienced this with a shock
when the first royalties were paid from Pharmacia.We inventors lived with the view
and took it for granted that the royalties were baseclupon the satesto inclepenclent
external customers. Pharmacia regarded their own foreign sales organizations,
which were subsidiaries,as customers.The royalties were calculateclon Pharmacia's internal price betweenthesesubsidiariesanclthe part of Pharmaciawith whom
we had signed the agreement.This considerably reduced the royalties that we had
expected to get. It is advisablefor b,oth parties to take the time necessaryro agree
on all important principles in order to reach a mutual understanclingan4 creaie a
win-win-situation. A lawyer with experienceand a special interest in these particular problems may help to avoid future surprises.
It was not until the 80's, thus after 20 years of experiencewith negotiationsthat I
felt that I coulcl negotiateon more equal terms. All agreementswere rewritten ancl
became valid between the inventors and the pharmacia group, including affiliated
companiesas Wallac OY and Electro-NucleonicsInc. Phiumaciaobtained the rights
to grant patent licenses.We also agreed upon the definitions of the innovations and
signed ftrrmal agreementson the DASP-principle and the'sandwich technique'.
Sonte reflections
Even if the university showed no active interest in commercial innovatipns in the
1960's, I experienced there a stimulating atmospherefor creative research.The
institutions at which I worked possessedsome free researchmoney that coulcl be
used for new ideas.The institutions could supply with technical support in the form
of technicians as well as instruments, and they were equipped with a workshop
where an instrument maker was employed. I experienceda strong support from the
staff both at King Gustav v:s Researchlnstitute in Stmkholm and ar the departments in Uppsala.All this facilitated a creative researchwork. Examples have been
given above on the value of seminarsand meetings in local researchsocieties for
the exchangeof information and start of collaborations.
Innovations are seldom createdon order.The pregnancytest as well as the allergy
test started with the development of methods with uni<;uequalities, and then ttrl-
2t2
lowedby the searchtbr appropriate
applications.
This sequence
of 'havingfound a
solutionand then look for its problem'is not unusualduringan iunovationprocedure. It can, as exemplifiedabove,be rathertrivial observations
that can leadto a
deviationfrom the original researchinto new areas.
It can be difficult to formulatethe claimsin a patentapplicationfor an invention
within a completelynew area.The diagnostictestkit areawasnew for Pharmacia's
patentgroup.They werechemistsandcameto focuson thechemistryin theirpatent
applications.In 1968,I presentedat Pharmaciaseveraldrafts for patentprotections
of the generalprincipleof the sandwichtechnique,
coveringfields outsideallergy.I
stressedthe high sensitivity.precisionand specificity,and furthermorethe short
reactiontime and the possibilityto use other solid matrixesthan panicles,when
We discussed
comparedwith thecompetitiveimmunoassays.
it with a patentbureau
preliminary
were
patent
group
and
drafts
made.However,the
at Phrumaciafearecl
that suchan applicationcouldinterferewith theirpreviouspatentfor the allergytest
andtheydecidedto stoptheseattempts.
As a consequence,
Pharmacia
hadno general patentprotectionwhen,in 1976,they launchedthe sandwichassayof hGH using
a solidphasecatchingantibodytogetherwith a labelledantibody.
EPILOGUE _ CLOBAL GROWTHOF DIAGNOSTICTEST KIT INDUSTRY
Front a pojct't group n Phu'ntuticrDiagnostit's
The so called 'Wide-project'at Phamraciadevelopedslowly during the first year,
but after that in an increasingpace.Un{er the guccessful
leadershipof Carl-Erik
SJgErg it grew to a diagmlsticdivisionwithin Pharmaciaancllaterto the company
PharmaciaDiagnostics.Pharmaciabecameone of the pioneersin the field of
for radioimmunoassay
in 1971,whenmarketinga RIST-kitfilr insulin.
reagent-kits
The following yeartheywerefirst on themarketwith teststbr total lgE andthe solid phaseassayfor vitaminBl2.
The useof paperdiscs insteadof Sephadexparticleswas an improvementof the
for Pharmaciain 1968(44). This eliminated
sandwichtechniquesthat I presented
stepusedin the originalversion.Pharmacia
becamea pioneeron
the centrifugation
sandwichtechnique.
the marketwith kits for assaysbasecl
uponthe non-competitive
RAST)
ln 1974the companylaunchedthe testfor allergenspecificIgE (Phadebas
and two yearslater they introducedthe labelledantibodymethodsfor the assaysof
hGH and total lgE. They were all basedupon the useof paperdiscsas the solid
phase.Pharmaciareachecl'breakeven'for the diagnosticproductsten yearsafter
the startof the project.About ten yearslaterthe numberof employeeshad increased
Pharmato 1,500.Whenthe profitsfrom the diagnosticproductsrapidlyincreased,
cia showed its gratitudeto UppsalaUniversity with a generousdonation for
Diagnosticswasone of the leadresearch.During a long periodof time Pharmacia
ing diagnostictestkit companiesin the world andhascontinuedto be so in the field
of allergydiagnosis.
2r3
The <liagnostit'test kit industry gntu'sJ'aston a global multi thousandnillion dollar
nrurket
The non-competitive sandwich technique combines an ultra-high sensitivity,a high
specificity and a shoft reaction time, which are featuresof crucial importance particularly in many clinical applications.tn 1975 Ktihler and Milstein (45) describeda
method for in vitro synthesisof monoclonal antibodies.Such monoclonal antibodies
are particularly useful in sanclwichmethods and offer advantagesfor the kit manufacturers by the means by which these antibodiesare isolated and produced.Large
amounts of antibodiescan be synthesizedagainsta single antigenicsite.
A few immunoassay-kitmanufacturersrealized the advantagesof the sandwich
technique in the 70's, but during the following decadethe number of such companies increased rapidly. Several companies applied for patentson the use of sandwich techniquesin the 80's causingmany bitter patentdisputes(46). The diagnostic
test kit market grew globally and was a multi thousandmillion dollar market in the
90's. The sandwich technique is now used in most of the immunoassaysand in other fields such as the array-basedDNA-analysis. The pregnancy tests in use today
are also basedupon the sandwich technique.
The radioisotopesthat successfullyhad been used as labels in the binding assays
were to a large extent replaced by enzyme, fluorescence.and chcmiluminescent
labels. Competitive binding techniquesare still applied for assayof small size analytes, and the vast majority usesthe solid phaseseparationsystem.
ACKNOWLEDGEMENTS
I thunk Chritct Betrgtst;on
fir nwkittl;theproton,p(sof tlt( I'igtu'esin L'orcl L)ruu,.
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2r5
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38. CitationClassic.( 1985)CurrentContent28: 19.
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45. KrihlerC, Milstein C ( | 97,5)Continuouscultureof focusedcellssecretingspecificnntihrdyol
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Correspondint!author': ProfessorLeif Wide
Deparrmentof Clinical Chemistry
Univenity Hospital
SE-7-51
85 Uppsala,Swetlen
k it.Wide(a)
medssi.uu.se
216