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CLIN . CHEM.37/10, 1667-1668(1991)
CLINICA LCHEMISTR Y,Vol .37 , No.10 , 1991 1667
Sero log ica l Diagnosis wi th Recombinant Peptides /Prote
insDetection of antibodies directed against specific viral
antigens has been th e traditional m an ner of document-ing
prior infection. The viral antigens available forserological
testing are usually in th e form of a lysateprepared from whole
virions. For those viruses thatassemble and bud from host cell m em
branes, v irionpurification frequently resu lts in cop urifica tion
of h ostcellula r p rotein s. T hese cop urifyin g cellular
proteinscan be recognized by antibodies p re se nt in in div id ua
lsw ho h ave been previously exposed t o fo re ig n a ntig en s(
e.g ., v ia transfusion or transplantation, or prior preg-n an cie
s), re su ltin g in fa lse-p ositiv e r ea ctio ns.The dramatic r
ev olu tio n in s er olo gic al te gtin g w ith inth e past two
decades is directly attributable to theexplosive grow th of the
discipline of m olecular biology.The ability to determ ine rapidly
the entire geneticsequences of viruses and to clone and produce
specificviral antigens in large quantities has perm itted thed ev
elo pm en t o f h om og en eo us antigen preparations. Inturn ,
these homogeneous preparations have perm ittedthe developm ent of
highly reproducible sero logical as-says. T he absence of host
cellu lar proteins in the recom-binantly derived an tig en
preparatio ns h as also dramat-i ca ll y d ec re as ed the rate of
f al se -p os it iv e r ea ct io ns .In addition to tests for Type
1 human immunodefi-ciency v irus (H IV -1 ), recom binantly d er iv
ed a nt ig en shave been used for the developm ent of other
serologicalassays for the detection of b lo odbor ne i nf ec ti ou
s agents.H epatitis C virus is the best example of the power ofmole
cu la r b io lo gy tec hn iq ues, both for identification ofth e
causative agent and subsequent developm ent ofd ia gn os tic re ag
en ts . The huge blood bank m arket hasbeen the im petus f or th e
c omm erc ia l fo cu s on deve lop ings er olo gic al a ss ay s fo
r b lo odbor ne in fe ct io u s ag en ts: all o fthe estim ated 12
m illion units of donor b lo od c ol le ct edper year need to be
tested for the presence o f t ransmi s -sible agents. H owever, now
that the needs of th e bloodbanks have been a de qu ate ly a dd
resse d, o th er orp ha ndiseases (e.g., p ara sitic d is ea se s),
fo r which better diag-nostic reagents are desperately needed and
for which aglobal m arket exists, are finally being addressed.HIV-1
recom binant antigens currently com e in tw oform s: synthetic
peptides and engineered recom binantp ro te in s. S eq ue nc es fo
r sy nthetic peptides are typ icallychosen by identifying areas of
the viral genom e m ostlikely to induce an immune res po nse . T
his identificationis accomplished by entering th e primary n ucleic
acidsequence of the entire viral genome into a com puterprogram,
which then determines th e predicted am inoacid sequence an d
generates a p lo t o f h yd ro ph ob ic an dhydrophilic regions. H
ydrophilic reg ions are more likelyto be antigenic; thu s,
synthetic p ep ti de s c or re sp ond in gto sh ort segments of the
hydrophilic regions (typically10-20 amino a cid s) a re synthesized
and tested for their
a bility to detect antibodies in sera obtained from know nH IV
-1-infected individuals. R ecom binant proteins, incontrast, are
know n as fusion proteins and are con-structed by splicing a
specific viral-pro tein-coding se-quence into an e xp re ss ed b ac
te ri op h ag e g en e. T ra ns la -tion is controlled by the
bacteriophage gene, and theviral protein segm ent is translated as
a part of a largerprotein that includes the rem ainder of the
bacterialgene into which the viral gene w as spliced.In this issue
(C lin C hem 1991;37:1700-7), Pollet et al.
re po rt th eir fa vo ra ble ex pe rie nce w ith a co mm
erciallya va ila ble r ec omb in an t antigen-based assay (LIA
HLV;Im munogenetics, Ghent, Belg ium ) as a confirmatorytest for
evid en ce o f H 1V -1 in fectio n. T his a ss ay c on ta in sb oth
r ec omb in an t a ntig en s (p ro du ce d in E sc he ric hia c
oli)an d syn th etic p ep tid es. C on sisten t w ith p reviou sly
p ub -lish ed a cc ou nts a bo ut sim ila rly constructed H IV -1
co n-firm atory assays, they have also observed that the LIAH1V
reliably detected HIV -1 antibodies and yieldedfewer false-pos it
ive results th an did con vention al imm u-noblot. Pollet et a l.
are also to be commended f or t he irrigorous docum entation of th
e lot-to-lot variability ofbo th qua li ta t ive an d quantitative
resu lts ob se rv ed withc onv en ti ona l immunob lo t c on firm a
to ry a ss ay s.A comm on misperception is that, if a s er ol og ic
al assayis based on recom bin an t an tig en s, th en r es ults o
bta in edby that assay mu st, b y d efin itio n, b e c omp letely a
cc ura te .In fact, how ever, certain types of sera (e.g., those ob
-tained fro m individuals with connective tissue d ise ase o rlipem
ic or hemolyzed sara) known to generate false-positive results i n
c onv en ti ona l serological assays canstill give f al se -p os it
iv e r ea ct io n s in recom binant antigen-based assays. To the
credit of the designers of the LIAHIV assay, no false-positive
reactions were observed in aset of these k nown p ro blema tic se
ra . S ec on dly , if re com-binant antigens are produced as fusion
pro teins andcontain additional bacterial epitopes, sam ples from
in di-viduals who have antibacterial antibodies may reactwith th e
c ar rie r p ortio n o f t he fu sion protein and not withth e v ir
al epitopes. In this study, no reactivity patternsthat m ight be
attribu table to interference of humananti-E. coli antibodies were
observed . Thus, the twom ost likely sources of n onspecific
reactivity w ere testeda nd d id n ot in te rfe re with the LIA
H1V-with th e reser-vation that the number of specim ens tested in
thesecategories was a de qu ate fo r th e study but was
relativelysmall in c om pariso n w ith th e v olum e o f su ch p ro
blema ticsera seen daily in a c lin ic al la bo ra to ry .It should
also be apparent, however, t ha t f a ls e -po si ti vereactions
can occur with recombinant antigen-based as-says f or r ea son s
unique to recombinant technology. Forexample, immunoreactive
epitopes may rely on eitherprunary a min o ac id se qu en ce or
conform ational shape fo ra ntig en icity . T herefo re , n on sp
ecific reactivity may be
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1668 CLINICALCHEMISTRY,Vol .37, N o. 10. 1991
observed if sim ilar ep itopes exist on different viruses.
Infact, Pollet and colleag ues cite tw o a rtic le s re po rtin
gepitopes common to H 1V -1 p2 4 an d th e picornav iral V P2coat
protein or the core protein of h um an T -cell leukem iavirus-V
(HTLV-V ). A lthough only a single isolate ofHTLV-V h as been
described th us fa r, p ic or na viru se s a rewidespread and are u
su ally re sp on sib le fo r yearly epi-d em ics of
gastrointestinal and respiratory flu. It is easyto speculate that
th e w id esp re ad ex po su re to picornavi-ruses would result in
a sm all su bset o f inf ect ed individ-uals w ho w ould m ake
antibodies against th e ep ito peshared with H1V-1 p24. T his could
ex plain th e observa-tion that H IV -1 p24 is the H IV -1 protein
m ost prone to fa ls e- po sit iv e r ea ct io ns (i.e., r ea cts w
ith antibodies inthe sara of indiv iduals not infected w ith H IV
-1). W ew ou ld h av e to infer, however, that this subset of
picorna-v ir us -i nf ec te d i nd iv id ua ls who m ake antibo
dies reactiv ewith H1V-1 p24 must be small, b ec au se th e s en
sitiv it yan d sp ecificity of a ll H 1V -1 assa ys in u se
(recombinant- orn on -r ec omb in an t-b as ed ) a pp ro ac h 9
9.9% .Another cons idera t ion unique to r ec omb in an t t ec hn
ol-ogy is that v ir al p r ot ei ns produced a s f us io n proteins
donot typically r ep re sen t th e en tir e v ira l p rotein as
encoded,tr an sla te d, a nd processed acco rding to th e
virus-specifiedlifecycle. T he viral com pon en t of fu sion
proteins is typi-cally the segm ent of the viral genom e that
coincidentallyis lo ca te d b etw ee n tw o sites recognized by the
restrictionenzymes in use. In this manner, new epitopes may becrea
ted b y engineered v ir al p ro te in segm ents th at nowcontain
junctions betw een tw o proteins that ordinarilyw ou ld b e c lea
ved during the v irus ma tu ra ti on process orby truncation of the
com plete viral protein , generatingnew epitopes at either the N -
or C -term inus. Som e recom -b ina nt-b ased assays h av e avoid
ed this potential problemby engineering au th en tic H IV -1 v ira
l p ro te in s th ro ug hm anipulation of the expression vector to
include the vi-ra lly en co de d p ro te ase a nd en su re fa ith
fu l p ro te in p ro ce ss-in g a fte r translation within th e
host bacterium.A third consideration is unique to synthetic
peptideantigens. Synthetic peptides are only short segm ents
ofamino a cid s, co rrespo ndin g to highly im munogenic re-gions
of viral proteins. The shortness of these peptidesmay preclude
recognition of epitopes, which may bed ep en den t on con fo rm
ation al sh ap e an d in vo lv e lin kin gof distant sites via
disulfide bonding or hydrophilic/h yd ro ph ob ic in te ra ctio ns.
A lter na tiv ely , a sh or t p ep tid emay in fact represent an
epitope found commonly innature an d m ay not show a reaction
specific fo r th e v iru sunder considera ti on .A no th er p
otential p rob lem exists fo r a ll se ro lo gic alassays,
regardless of source of antigen. The first anti-bodies produced in
resp onse to an tigen ic challenge aretypically broadly
cross-reactive and are produced byCD5+ B cells. Continued antigen
ex po su re re su lts inrecruitment of a different subset of B
cells (CD5-),w hich are destined to differentiate into m ature
plasm acells that secrete antibodies with high affinity andavidity
for the inciting antigen. For those medicalconditions associated
with an increased num ber of cir-
culating CD 5+ B cells (e.g., r he um a to lo gic al d is ord er
s),th e no nspecific im mun oglobu lin s produced m ight alsoreact
with antigens, but in a n on pre dic tab le , n on un i-form
fashion. F or re comb in an t a ntig en -b ase d assays,this could
result in a pattern of reactiv ity sufficient foran in determ in
ate or ev en p ositive in terp reta tion .T he idiotype netw ork
theory of antibody regulationpredicts a potential problem in
individuals who haveb een tre ate d w ith r ec omb in an t CD4
peptides/proteins.CD 4 binding of H1V-1 gpl2O is the p rim ary m
ech an is mby which HIV-1 binds a nd in fec ts CD4+ T cells.
Con-tinued exposure to recom binant CD4 would perm it thehost im
mune system to generate anti-C D4 antibodies,some of which w ill be
directed against the CD4 sitedirectly involved w ith H IV -1 gpl2O
binding. A nti-id io-type antibodies could then be generated
against theseanti-C D4 antibodies. Som e of these a nti- id io ty
pic a nti-bodies w ould undoubtedly react in any H IV -1
serologi-ca l assay in a m an ner in distin guish ab le from an
au-th en tic a nti-g pl2O a ntib od y.The issue of which criteria
to use for LIA interpreta-tio n so as to av oid fa lse-p ositiv e
resu lts is w ell a ddressedb y P olle t an d colleagues. A lthough
the opinion is fre-quently voiced that reactivity against a single
HIV -1envelope protein (gp4l) should be sufficient for confir-m
ation of HIV -1 infection, m ost laboratorians wouldtake the m ore
conservative approach and dem and to seee vid en ce o f r ea ctiv
it y against p ro te in s d er iv ed from atleast tw o different H
IV -1 genes. This conservatismarises from the fact that
false-positive reactions havebeen observed w ith every single H IV
-1 protein , recom -binant or authentic-an im portant
consideration, giventhe ram ifications of a diagnosis of H IV -1
infection.L astly , o ne must consider the cost of performing
theserecom binant antigen-based assays. C urrently they arequite
expensive, w ith reagent costs for HIV-1 assayspriced to be com
parable w ith conventional im munoblot(..$20-.3O per strip), and
assays of hepatitis C virus arep ric ed a t $7-8 p er sin gle m ic
ro tite r w ell. (In c ompa riso n,r ea ge nt c osts fo r b io ch
em ica l en zyme a ssa ys a re ty pic ally
