r--~
2~923~ -
..
3 Method for the simultaneous determination of antigens
and antibodies
~9 ~
The invention concerns a method for th~ simultaneous
determination of at least one antigen of a pathogen and
at least one antibody against the same pathogen based on
the principle o~ a heterogeneous immunoassay by
incubating the sample with the receptors R1 to R4, in - ~
9 which Rl and R3 are capable of specific binding to the --
l antigen to be determined, R2 and R4 are capable of
specific binding to the antibody to be determined, R1
and R2 mediate binding to the solid phase, R3 and R4
carry the same label, separating the solid phase from
the liquid phase and determining the label in one of the
two phases.
', ..
Numerous important clinical parameters are determined
with immunological detection methods. There is a wide
selection of homogeneous and heterogeneous methods for
immunoassays. Often a heterogeneous method based on the
sandwich principle or a variant derived from this is
used. The sample solution is usually incubated with a
receptor which is capable of binding to the substance to
3 be determined and carries a label and with a receptor
capable of specific binding to the substance to be
determined which is bound to the solid phase or mediates
~3 binding to the solid phase. In this process bound
complexes of the substance to be determined and labelled
receptor are formed on the ~olid phase via the receptor
mediating binding to the solid phase. A~ter separating
~, the liquid ~rom the solid phase, the label can be
detected in one o~ the two phases~
~ ...
. .
,,~ . , :, :,
2~123~
2 --
f, In order to detect an infection by a pathogen, either
~ff antibodies which are specifically directed towards this
~ pathogen or certain antigens of this pathogen are
7, usually detected. Separate determinations of the
J antibodies and antigens are carried out in order to
detect an in~ection as reliably as possible. These tests
f have a lower detection limit,. If measured signals are
obtained in the test which are ~elow this detection
limit then this sample is declared to be negative. If
the measured signals are above the detection limit then
the sample is declared to be positive. When the samples
are ambiguous it is often necessary to repeat the
measurement. Despite this, some samples which only
contain small amounts of antigen or antibody are falsely
j classified as negative by this limitation of the
detectability o~ the individual substances to be
determinfPd. This is a particular problem for the early
detection of infection parameters.
The object of the present invention was to develop an
J immunoassay for the determination of antigens of a
pathogen and antibodies against this pathogen which
enables an improved differentiation between negative and
pusitive samples.
;- I
This object is achieved by a method for the simultaneous
determination of at least one antigen of a pathogen and
at least one ankibody against the same pathogen based on
the principle of a heterogeneous immunoassay by
~' incubating the sample with at least one receptor R1
whi~-h i~ capable of specific binding to the antigen to
be determined that is not identical to the antigen of
the pathogen towards which the antibody to be determined
i5 directed and is bound to or can be bound to a solid
phase, with at least one receptor R2 which is capable of
,' . .
-- 2 ~ 3 ~ :
~ - 3 - ~
., .
specific binding to the antibody to be determined and is
bound to or can be bound to a solid phase~ with at least
one receptor R3 which is capable of specific binding to
the antigen to be determined that is not identical to
'!
the antigen of the pathogen towards which the antibody
to be deter~ined is directed and carries a label and
with at least one receptor R4 which is capable of
specific binding to the antibody to be determined and
carries the same label as the receptor R3, separating
the solid from the liquid phase and determining the
label in one of the two phases.
A method for the determination of several substances
i.eO several antibodies or an~igens or antigens o~ a
pathogen in combination with antibodies against another
pathogen in an immunoassay is described in EP-A O 379
216. Several receptors R1 are used which bind
specifically to the substances to be determined. The
receptors R1 mediate binding to the solid phase. In
addition various receptors RZ can be used which are
capable of binding to various substances ta be
determined. The receptor~i R2 either carry different
labels or the same label. If different labels are used,
the method can be carried out in a single phase, i.e.
the receptors R2 are added simultaneously. I~ the same
label is used, the method must be carried out in two
phases i.e. the various receptors R2 are added
r successively and the individual labels are determined
successively in this way. A simultaneous determination
of antigens of a particular pathogen and antibodies
which are directed towards this particular pathogen and
which enables a good di~ferentiation between negative
and positive sample~i is not disclosed in EP-A-O 379 216.
~,
"1
.. ;, ~ :
~ ~.
''`',:~
:',:'~ . . '
2 ~
'' ,1, _
ThP simultaneous determination of antigens of a pathogen
and antibodies which are directed towards this
particular pathogan is made possible since, according to
the present invention, those receptors are used as
receptors R1 and R3 which speci~ically bind the antigen
to be determined but are not directed towards an antigen
against which the antibody to be de~ermined is directed.
If receptors were used which also specifically bind the
antigen that is bound by the antibody to be determined
then a mutual neutr~lization of antigens and antibodies
would occur.
Since -the method according to the present invention
enables the simultaneous detection of antigens of a
pathogen and antibodies that ~re directed towards this pa~ogen
pathogen in samples, there is a summation of khe
measured signals caused by the antigens and antibodies.
Samples with a low content o~ antigens and antibodies
which yield borderline signals when the antigens or
antibodies are determined separately, i.e. signals which
are near to the detection limit and which may be falsely
classified as negative or require a repeated
determination, can be classi~ied as positive using the
method according to the present invention. This
therefore obviates the time-consuming repeated
determination. In additio~ after an infection has
occurred it is possible to detect patients earlier as
being in~ected and thus to start treatment earlier. This
was surprising since it was assumed that at thei
beginning of an infection only antigens can be detected.
When selecting receptors R1 to R4 it is important, in
case the method is intended for an early detection of
in~ections, to use thoss receptors which are directed
towards early antigens i.e. antigens which occur very
:- . :-.
: :
.
~:
,
:".
21~92~9
soon after infection or are directed towards antibodies
~i which occur very soon after infection in high titres in
the samples. If it is only intended to enable an
improved differen~iation between negative and positive
samples using the method according to the present
invention and it does not serve specifically for the
early detection of infectious diseases, but instead for
,~!j,Y~ example the monitoring of an infectious disease, it is
3 also possible to use receptors R1 to R4 which are
. ,,
directed towards antigens or antibodies which do not
occur until later in the course of an infection. The
. ,~,;
selPction of the receptors therefore depends on which
stage of the infectious disease is intended to be
determined. The method according to the present
invention is particularly suitable for the early
detection of infectious diseases. ~ -
,",
The method according to the present invention is carried -~
out according to the principle of a heterogeneous
immunoassay. A solid phase is used on the surface of
which the receptors R1 and R2 are directly ~ound or can
bs bound.
~ .
Known materials such as plastic, glass, paper carriers,
ceramics, latex and magnetic particles can be used ~or
the solid phase. The solid phase can for example be
present in the form of reaction tubes, reagent carrier
strips or spheres. In a preferr~d embodiment the solid
phase is present in the form of a reaction tube the
inner surface of whose walls is or can be at least
partially coated with the receptors Rl and R2. Known
materials are suitable as the material for the reaction
vessel. Polystyrene, copolymers of polystyrene,
polycarbonates, polyacrylates and polymethacrylates are
preferred.
,.
;'~
~1 -
2 3 ~
.,5',~ -- 6 ~
r
- The coating of the solid phase with receptors Rl and R2
,r,.,~, is carried out either directly or indirPctly. Processes
for the direct binding of receptors to a solid phase are
known to a person skilled in the art. Adsorptive binding
is preferred.
:~r t
~' Indirect binding is understood to mean that receptors R1
,i and R2 are bound to the solid phase only shortly before
or during the immunological reaction. In this case a
partner P1 of a specific binding pair Pl/P2 is bound to
~ the solid phase. The other partner P2 of the specific
`~ bindiny pair is coupled in each case to the receptors R1
and R2.
,, ~
The coating of the solid phase with the specific binding
partner P1 can either be carried out directly, via a
carrier material or a spacer. Binding to a soluble
':'.! protein with a molecular weight over 500000 which is
~ then adsorbed to the inner surface of the reaction
'!'~ vessel is for example suitable. Binding via a spacer
,~, which can be covalently or adsorptively bound to the
~!;: surface of the reaction vessel via a functional group is
~t also suitable. Processes and agents for this are known
to a person skilled in the art. In a preferred
!': embodiment a arrier material is used as the solid phase
q':
which has been manufactured according to the process
dascribel in DE-A-3640412.8. In a further preferred
embodiment the solid phase is a reagent carrier that has
, been obtained by activating a fibre fleece made of a
cellulose/synthetic fibre mixture by treatment with
't' periodate and coated with the specific binding partner
Pl which was previously treated with acid. A process for
the manufacture of such reagent carriers is described in
DE-A-3543749.
:~
',,:;
~ ' t
",
Antigen/antibody, hapten/antibody, lectin/carbohydrate,
biotin/anti-biotin antibody, biotin/avidin,
biotin/streptavidin are for example suitable as specific
binding pairs P1/P2. Partners capable of binding to
biotin, in particular strep~avidin or avidin are
preferably immobilized on the solid phase.
The quantity ratio of the receptors R1 and R2 bound
directly or indirectly to the solid phase can be varied
over a wide range. The preferred quantity ratio of the
receptors R1 to R2 is betw~en 10:1 and 1:10~ The optimal
quantity ratio Rl:R2 for each special test can be easily
determined by binding different amounts of R1 and R2 to
the solid phase. If a specific binding pair P1/P2 is
used to bind the receptors R1 and R2 to the solid phase,
the quantity ratio of the bound receptors R1 and R2 ;
corresponds approximately to the mixing ratio of the
receptors R1 and R2 in the solution that is used for the
coating since the binding af~inities of both receptors
are approximately equal. If a direct binding of the
receptors R1 and R2 is carrield out, the quantity ratio
of the directly bound receptors R1 and R2 can differ
from the mixing ratio of receptors R1 : R2 in the
solution that is used for the coatin~ since the
receptors often have di~ferent affinities to the solid
phase. I~ this is desired, the exact quantity ratio of
the receptors R1 and R2 bound to the solid phase can be
subsequently determined according to methods known to a
per~son skilled in the art, for example by determining
the binding capacity for the antigens and antibodies to
be determined. The quantity ratio R3:R4 is also selected
in accordance with the quantity ratio selected for the
receptors Rl:R2. The quantity ratio of the receptors
R3:R4 is also between 10:1 to 1:10. The quantity ratio
R3 :R4 can in this case be the same or similar to the
, .,
~'` ii
,,, :. .
v,l :
3 ~
-- 8 -
quantity ratio Rl:R2, deviations can, however, also
occur. The optimal ~uantity ratio R3 :R4 can easily be
determined for each individual -test by mixing different
amounts of R3 and R4.
Receptors are used as receptor Rl which have binding
sites for the antigen to be determined. The antigen to
be determined must be different from the antigen which
is recognized and bound by the antibody to be
determined. The receptor R1 must specifically recognize
a different antigenic determinant or a different epitope
of the pathogen than is recogni~ed by the an-tibod~7 to be dete ~ ned. It
is necessary that receptor R1 and the antibody to be
determined do not strongly cross-react with the same
binding site. The antigenic determinants are known for
known pathogens such as for example hepatitis viruses or
HIV. For example in order to detect a hepatitis B virus
infection, a receptor against the hepatitis B surface
antigen (HBsAg~ can be used ac; receptor R1 and
simultaneously anti-hepatitis B core antibodies
(<HBc>Ab) are detected as the antibodies to be
determined. In order to detect a HIV infection, a
receptor R1 can for example be used which binds the p24
protein while anti-gp32, gp41 or gpl20 antibodies are
determined. If the antigenic determinants are not known
for a pathogen then suitable receptors R1 that do not
compete with the antibody to be determined for the same
binding site can be selected by a person skilled in the
art based on known preliminary experiments for ~-
determining cross-rsactivity.
, .
I Mixtures of different receptors can also be used as ;
I rèceptors Rl which are capable of specific binding to
diff~rent antigens of the pathoyen. In a test for HIV
infection, receptors for the antigenic determinants p24
.
2 ~
and gp32 and/or gp41 can for example be used. In this
case an anti-gpl20 antibody is for ex~mple selected as
the antibody to be det2rmined. In the case of a test for
a hepatitis B virus infection, a mixture of two
receptors for HBsAg and HBcAg can for example be used as
receptor R1 while the antibody to be determined is
directed towards another epitope of HBcAg or against
HBeAG.
Complet~ antibodies of all subclasses which are capable
of binding to the antigen to be determined can be used
as receptors Rl. 0~ course their fragments such as Fab, ~ -
Fab' or F(ab')2 fragments can also be used instead of
the complete antibodies. The antibodies can be
polyclonal or monoclonal. Since there should be no
cross-reactivity with the antibodies to be determined it
is advantageous to use highly purified specific
polyclonal antibodies. The use of monoclonal antibodies
is most suitable.
In a preferred embodiment of the method according to the
present invention a partner P2 of the specific binding
paix P1/P2 is couplad to the rleceptor R1. Immohilization ~-
on the solid phase is effected via the partner P2 which
binds to the partner P1. A hapten such as FITC, p-
nitrophenol, saponin, digoxin and particularly
preferab~y biotin is preferably used as the partner P1.
Methods for coupling partner P2 to the receptor R2 are
known to a person skilled in the art.
The antigens are utilized as receptor R2 to which the
antibody or antibodies to be determined speci~ically ~ -
bind. Complete or purified parts of pathogens can be
used as the antigen such as antigens, protozoal antigens
or viral antigens. The antigens can be comprised of
- 10 -
native material, recombinant material or of chemically
synthesized or modified proteins or carbohydrates. Thus
for example in a test for HIV antibodies different
antigenic determinants such as p24, gp41, gp32 or gpl20
are used as receptor R2. In a test for hepatitis
diseases, the viral antigens HBcAg, HBsAg, HBeAg, HAV
etc can be used. As described above the selection of the
receptor R2 depends on the receptor Rl used.
: .: '
As described ~or receptor Rl, a mixture of different
receptors can also likewise be used for receptor R2
which are capable of specific binding to dif~erent
antibodies which are directed towards different
antigenic determinants of a pathogen. For example a
mixture o~ the viral antigens gp32, gp41 and gpl20 can
be used in a test for a HIV infection. In this case
receptor R1 would for example be directed towards p24.
Receptor R2 is either directly or indirectly bound to
the solid phase. In the pre~erred embodiment of the
method according to the presen~: invention a partner P2
o~ the specific binding pair P1/P2 is coupled to the
receptor R2. In a particular test the same substances
should be coupled as partners P2 to the receptor R2 as
to the receptor R1. The selection of the partner P2 from
various substances and the coupling is carried out in an
analogous manner to that described for receptor Rl.
The receptors which are used ~or receptor R3 are
analogous to receptor R1 those which have binding sites
or the antigen to be determined but are not directed
toward~ the antigen which is specifically bound by the
antibody to be determined. The same antibodies can be
used as receptor R3 which are also used for receptor R1.
In a particular test the antibodies used for receptor R3
' ~:
can be identical to the antibodies used for receptor R1
or different from this depending on the determination to
be carried out i.e. the infection to be detected. For
example a complete antibody can be used as receptor R1
and an antibody fragment as receptor R3 which both
specifically bind to the same antigen. Receptors R1 and
R3 must specifically bind the same antigen. However, in
doing so they can bind to different epitopes of the same
antigen. It is preferred that receptors R1 and R3 do not
compete for the same epitope of the same antigen. This
condition can particularly easily be fulfilled when
monoclonal antibodies are used as R1 and R3. As for
receptor Rl, a mixture of different receptors can also
be used for receptor R3.
Receptor R3 in addition carries a label. There are many
possibilities known for labelling which are suitable in
the method according to the present invention. For
example radioactive isotopes such as 125I, 121I, 51Cr,
35S and 3H, enzymes such as peroxidase, B-galactosidase
or alkaline phosphatase, fluorescent or chemiluminescent
substances or substances which can be detected in
another manner can be used. Apart from a direct label,
receptor R3 can also carry an indirect label by binding
a labelled component capable of binding receptor R3 to
thi~ receptor during the course of the test procedure~ A
labelled antibody which binds the Fc part of receptor R3
can ~or example be usad. Coupling via a hapten antibody
binding is preferred. In this case a hapten such as
digoxin is coupled to the receptor. A labelled anti-
hapten antibody such as an anti-digoxin antibody
peroxidase conjugate binds to the hapten.
A receptor is used as receptor R4 which is capable of
specific binding to the antibody to be determined and
- 21~923~
- 12 -
which carries the same label as receptor R3. The same
detectable substances as defined for R3 are to be used
as the labelO The same antigens which are also used for
receptor R2 can be used as receptor R4. A mixture of
different receptors can be used for receptor R4 as in
the case of receptor R2.
In a further variant of the method an antibody or a
fragment thereof which specifically recognizes the
antibody to be determined can be used for the receptor.
There are two possibilities for this variant of the ~
method. The first possibility is that an antibody is ~-
used as receptor R4 which only specifically recognizes
the antibody to be determined and does not bind to
receptors R1 and R3. This can for example be achieved by
using an antibody as receptor R4 which specifically
recognizes human antibodies and using antibodies solely :
as receptors Rl and R3 which are not of human origin,
such as mouse antibodies. ~ :~
In the second variant of khe method an antibody is used
as receptor R4 which specifica:Lly recognizes the
antibody to be determined as well as receptor R3 but
does not recognize receptor Rl. An antibody, preferably
a Fab or Fab' fragment, can ~or example be used as
receptor R4 which specifically binds the Fc part of an ~ :
antibQdy. In this case an antibody fragment is used as
receptor R1 which no longar contains an Fc part. A ~ ~-
complete antibody must in this case be used as receptor
R3. Receptor R3 thus does not carry a label which is :
directly coupled to 1t but instead receives the label
via binding to receptor R4 which carries a label.
Receptor R3 thus carries an indirect label.
''~ ~. ''
, .. . ...
2 ~
- 13 -
In order that receptor R3 does not become cross-linked
during the incubation by binding of receptor R4, a
monovalent antibody fragment, for example a Fab or Fab'
fragment, is pre~erably used as receptor R4. Th~ sample
is preferably firstly incubated with the receptors R1,
R2 and R3. After the complexes bound to the solid phase
have been formed, it is incubated with receptor R4 after
prior removal of excess receptors and subsequently the
la~el is determined.
~: -
The sample solution can be incubated simultaneously withall receptors. Simultaneous incubation is preferred when
R3 and R4 are directly labelled and receptors R1 and R2
are directly bound to the solid phase. If the receptors
R1 and R2 are indirectly bound to the solid phase, these
two receptors can firstly be preincubated with the solid
phase. The sample is subsequently incubated together
with receptors R3 and R4. Further variants of the method
are possible and known to a person skilled in the art.
The simple procedure enables the determination to be
carried out on automated analysers. The method is also
suitable for a rapid diagnosis. The simultaneous
detection o~ antigens and antibodies of an infection
parameter enables blood samples which contain this risk
parameter to be immediately eliminated without the
necessity for a more exact diagnosis. On the other hand
in examinations a rapid test can be used to check
beforehand whether risk factors are present and if this
is the case then this can be followed by a more exact
diagnosis for example by a DNA test. Thus according to
the present invention it is possible to very rapidly
determine whether particular indications for a disease
are present.
21~239
- 14 -
Using the method according to the present invention, ik
is possible to simultaneously detect at least one
antigen of a pathogen and at least one antibody which is
capable of binding to the same pathogen. In this
connection all pathogens come into consideration as
pathogens or infection parameters in which an antigen
and antibody titre can be detected in blood, especially
viruses such as hepatitis viruses, HIV, CMV, EBV and
other human viruses, prokaryotes as well as eukaryotic
pathogens and protozoa.
The invention also concerns a reagent for the
determination of an antigen of a pathogen and at least
one antibody against the same pathogen which contains a
solid phase on the surface of which the receptors Rl and
R2 are bound or can be bound by interaction of the
specific binding pair P1/P2, in each case at least one
receptor Rl, R2, R3 and R4 as defined above and, if
desired, further common auxiliary agents for carrying
out a heterogeneous immunoassay such as buffers, ~ -
detergents, substances for eliminating interferences,
stabilizers as well as, if desired, agents for
determining the label such as enzyme-substrates like
ABT5~.
The invention is elucidated by the following examples.
Exa~p~e 1
HIV-Aa/anti-HIV test
HIV antigens (p24 antigen) and anti-HIV antibodies
(anti-gp32, anti-gp41 and anti-gpl20) are determined in
a pseudo 2-step immunoassay.
2 3 ~
- 15 -
Reagent 1:
- 40 mmol/l phosphate bu~f~r, pH 7.0
- l % by weight Nonide ~ P40
Reaaent 2:
- 40 mmol/l phosphate bu~fer, pH 7.0
- 0.9 % by weight NaCl
- 0.2 % by weight bovine serum albumin
- 100 ng/ml each of one or severial biotinylated HIV ~ ~-
antigens (gp32, gp41 or gpl20)
50 ng/ml each of one or several digoxin-labelled ~:
HIV antigens (gp32, gp41 or gpl20)
- 150 ngtml biotinylated anti-HIV p24 antibody
- 75 ng/ml digoxin-labelled anti-HIV p24 antibody
- 150 mU/ml anti-digoxin antibody-pe~roxidase
conjugate
The following antigens were used in this process:
- chemically synthesized peptides
- HIV I gpl20 ~V3 loop~ (EP-A 0 311 219)
- HIV II gp 41 (Gnann, J.W. et al., Science 237,
1987, 1346)
- HIV III gp32 (EP-A 0 379 216)
Purifi~d human polyclonal anti-p24 antibodies
(Innogenetics Co.) were used as the antibodies.
~he antigens and antibodies were labelled with biotin or
digoxin as described by Leary et al., PNAS, 80 (1983),
4045.
3 9
- 16 -
The test was carried out in a streptavidin thermo-sSA
polystyrene tube as described in EP-A 0 379 216.
100 ~l human serum or human plasma was incubated with
200 ~1 reagent l for 60 minu-tes at 37& ;.n the
streptavidin thermo BSA polystyrene tube. Subsequently
700 ~l reagent 2 was added and incubated for 120 minutes
at 37C. Afterwards it was washed three times with tap
water and 1 ml ABTS~ substrate solution was added for
the test reaction. After a 60 minute incubation at 37C
the absorbance wa~ measured photometrically at 422 nm. ~ -~
The anti~HIV test was carried out using the individual
HIV antigens and a combination of all HIV antigens. In
doing so it turned out that the test procedure with
streptavidin thermo-BSA conjugate polystyrene tubes is
suitable for the simultaneous determination of several
individual antibodies or an antibody population and ~ ~;
antigens regardless of whether the antibodies are
directed towards the same virus or several viruses or
their antigens and of whether the detected antigens ;~
belong to one or several viral strains (Table 1). ;
-` 2~23~ ~
Table
Human serum sampl~s -:
HIV antigens Negative HIV I¦ HIV I¦ HIV I ¦ HIV I¦ HIV II
and control Ab+Ag¦ Ab+~g¦ Ab+Ag ¦ Ab~Ag¦ Ab+Ag
antibodies (absorbances in mA)
: _
gp 120 (pep) 52 21615611 3861 94 63 :-
gp 41 (Pep) 48 25555735 2~72 89 72
gp 32 (Pep) 44 54 247 64 41 1389
.
<p24> Ab 56367 427 76 512 1597
peptides-~ 624123 68955678 643 2763
<p24>Ab
,
~: "' .
