Note: Descriptions are shown in the official language in which they were submitted.
TITLE OF INVENTION
IMMUNOASSAY USING ANTIBODY-ANTIGEN CONJUGATES
FIELD OF INVENTION
The present lnvention relates to the detection of
materials by the immunoassay method and, in particular~
to a novel procedure for effecting the same.
BACKGROUND TO THE INVENTION
One of the most sensitive methods for detecting
small quantitles of materials, is the immunoassay
method, which is based on the combination of the
material to be detected, usually called an antigen, and
an antibody, raised in an animal, to that antigen.
Either the antigen or the antibody is labelled in some
fashion to allow for detection. From being a research
tool, the method is now used routinely for the screening
of large numbers of samples, especially human plasma,
for pathogens and foreign substances, such as drugs.
The combination of antigen and antibody, which
usually gives a complex of lower solubility than either
of the two starting materials, in a test system, is
detected by a variety of methods. Initially detection
was achieved by radioactively labelling one of the
reactants and determining the amount of radiation,
either in the separated antigen-antibody complex or in
the solution after removal of the complex. More recently
detection has been achieved by labelling one of the
reactants with an enzyme, which can cause a colour
change in the test system, when a substrate is added. A
well known example of such an enzyme is horse radish
peroxidase, and the test system is known as the
enzyme-linked immunosorbent assay (ELISA).
This principle of using an antigen-antibody complex
is employed in a number of assays for the detection of
pathogenic organisms, or their products, which have
invaded a mammalian organism. The method can also be
used for the detection of antibodies whlch the organism
has raised to the pathogen. This latter assay ls used
~ 2 ~ 5
to determine whether the mammalian organism has been
previously exposed to the disease. Examples of these
two assays are the detection of the Hepatitis B surface
antigen (see, for example, U.S. Patents Nos. 3,867,517
and 4,197,361) and antibody to this antigen (U.S.
Patent No. 4,230,683).
A number of different types of immunoassayr using
the antigen-antibody complex principle, have been
developed. One of the most useful immunoassays is the
so-called 'sandwich-type' assay, in which one reactant
is bound to a solid-phase, for example, a plastic tube,
bead or microplate well. This complex is exposed to the
second reactant and this mixture, after washing, again
is exposed to the first reactant, which has been
labelled in some fashion. From this sequence of
operations the quantity of the second reactant in the
sample can be determined by determining the quantity of
labelled material. The second reactant is sandwiched
between two layers of the first. Habermann
2~ [Z.Clin.Chem.Clin.Biochem.,8,51,(1970)] used the method
for the detection of tetanus toxin present in test
samples. The sandwich immunoassay also has been
successfully used for the detection of the five
different types of antibody present in human and animal
sera-
In all the above techniques, the sandwich is builtup sequentially by contacting the individual components
with the underlying solid-phase for a period of time and
then any unbound material is washed off. At no time has
a mixture of antibody and antigen been used, since it is
generally considered that the formation of the
antibody-antigen complex diminishes the immunoreactivity
of both components.
SUMMARY OF INVENTION
We have now surprisingly found that macromolecular
antigens in combination with their antibodies can be
used to form immune conjugates with very little decrease
in the immunoreactivity of the antigen. Essential to
the present invention is the use of macromolecular
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antigens which con~ain multiple copies of epitopes and
the use of a limited amount of antibody, which then
leaves sufficient epitope sites to react with the
solid-phase-bound antibody.
The surprising ability to employ antibody-antigen
conjugates in an immunoassay permits labelling of the
antibody as the indicator rather than the antigen.
Labelling of antigens often can be difficult to achieve
and can be ineffective, since antigenic sites are
destroyed.
Accordingly, the present invention provides a
method of detecting antibodies to antigen, which
comprises (a) contacting a solid-phase substrate with an
antigen to form a solid-phase substrate antigen complex;
(b) contacting the solid-phase complex with a test
sample in which the presence of an antibody to the
antigen is required to be known; (c) contacting the
resulting complex with an antibody-antigen conjugate in
which the antibody is labelled with a detectable moiety
~0 and the antigen has at least one free antibody binding
site in the conjugate; and (d) detecting the presence of
the moiety on the solid phase substrate as a detection
of the antibody to the antigen in the test sample. The
concentration of the moiety on the solid phase substrate
may be determined as a measure of the concentration of
the antibody to the antigen in the test sample.
The method of the present invention may be used to
detect classes and subclasses of antibodies which are
specific to a given antigen. A method, in accordance
with this aspect of the invention, comprises (a)
contacting a solid-phase substrate with an antibody
specific for a class or subclass of antibodies to form a
solid-phase substrate-antibody complex; (b) contacting
the solid-phase complex with a test sample in which the
presence of a class or subclass-specific antibody to a
desired antigen is required to be known; (c) contacting
the resulting complex with an antibody-antigen conjugate
in which the antibody is labelled with a detectable
moiety and the antigen has at least one free antibody
4 ~ 85
binding slte in the conjugate; and (d) detecting the
presence of the moiety on the solid phase substrate as a
detection of a class or subclass of antibodies which are
specific to a given antigen in the test sample. The
concentration of the moiety on the solid phase substrate
may be determined as a measure of the concentration of
the antibody to the antigen in the test sample.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 is a graphical representation of results
obtained for an assay of antibodies to Hepatitis B
surface antigen;
Figure 2 is a graphical representation of results
obtained for an assay of IgM antibodies to Hepatitis B
core antigen; and
Figure 3 is a graphical representation of results
obtained for an assay of IgM antibodies to Hepatitis A
virus.
GENERAL_DESCRIPTION OF INVENTION
The present invention encompasses methods for the
~ detection and determination of antibodies to
macromolecular antigens. The invention can determine
either antibodies of all classes to a given antigen or,
with minor modifications, can determine the specific
sub-class of immunoglobulin to the specified antigen.
For the determination of antibodies to a given
antigen, the antigen, or a significant part of it, first
is bound to a solid support by adsorption or chemical
bonding, to form a complex which will bind antibodies
specific to the antigen. This antibody binding
"reagent" then is exposed to the test sample containing
the antibody to be measured and, after a suitable time,
the sample is removed and the test reagent containing
bound antibody washed free of test sample. These two
steps are the same as those carried out in the
conventional sandwich immunoassay procedure.
In accordance with the present invention, the
resulting complex then is treated with a conjugate of
the macromolecular antigen and a labelled antibody to
the antigen.
The conjugate may be preformed by reacting the
labelled antibody, which is prelabelled, in accordance
with the detection method to be adopted, with the
macromolecular antigen by adding solutions one to the
5 other. Alternatively, the conjugate may be formed in
situ in the presence of the solid phase complex by the
addition of antibody solution and antigen solution in
either order. In order to ensure that the conjugate is
effective in achieving antigen-antlbody binding to the
solid-phase-bound sample, the antigen used in the
conjugate is macromolecular and has multiple numbers of
epitopes and the antibody is used in a ratio which
leaves unbound epitopes for binding to the sample.
Generally, there is an optimum ratio of antigen to
antibody, depending on the antigen, which will give the
best performance in the assay. This optimum is best
determined by comparing the sensitivities of mixtures
with different proportions of antigen and antibody in
the immunoassay. This optimum then is adhered to in
~0 subsequent assays for consistent performance. The
preferred ratio depends on the antigen but is generally
greater than unity.
Following addition of the conjugate of the antigen
and a labelled antibody, unbound conjugate is removed by
washing and the amount of label, associated with the
reacting solid support complex is determined. In our
invention, the use of an enzyme ]abelling system is
preferred as a method of determining the concentration
of the labelled conjugate, but other methods, such as
radioactive labelling, equally may be applied.
In the application of the invention to the
determination of the amount of a class or sub-class of
antibody to a given antigen that is in a test sample,
the first step is the formation of a complex wherein an
antibody capable of binding a specific class, or
sub-class, of immunoglobulin, such as IgA or IgM, is
absorbed onto a solid phase. This specific
antibody-binding reagent is contacted with the test
sample, and after a suitable time is washed to remove
6 ~ 8~
unbound substances. In this way, all antibodies of the
appropriate class or sub-class in the test sample are
captured by the binding reagent. In accordance with the
present invention, the resulting complex then is
contacted with the specific antigen-antibody conjugate,
formed as described above and containing the labelled
antibody, and, after washing, the label amount is
measured. This procedure provides the class or
sub-class of antibody which is associated with the
macromolecular antigen, in the test sample.
The advantages of the present invention are
multifold. Firstly, the antigen need not be labelled,
rather a complementary antibody is labelled. Many
antigens are labile and the purification and labelling
processes can destroy antigenic sites, thus decreasing
the sensitivity of the reaction. Secondly the assay, as
applied to class specific antibodies, can be performed
as a three step process, instead of the four step
procedure in current practice. These features greatly
~ reduce the time and cost for the immunoassay process,
according to the present invention. In addition, the
immunoassay technique of the invention exhibits an
enhanced sensitivity when compared with conventional
immunoassay procedures.
EXAMPLES
Example I: This Example illustrates the provision of an
assay for all antibodies to Hepatitis B surface antigen.
Preparation of Reagents
Hepatitis B surface antigen (HBsAg) was purified
from a pool of human carrier plasma containing the two
major sub-types of virus, by standard methods, and used
to coat microwells according to the procedures of Catt
and Tregear, as published in Science, 158, 1570 (1967).
This same HBsAg was used to immunize chimpanzees as
described by Wilson and Logan in Develop. Biol.
Standard, vol. 30, pp.240 to 243 (1975), from which
specific antibody was obtained. The chimpanzee
antibodies (anti-HBs) were labelled with the enzyme
peroxidase according to the procedure of Nakane as
7 1~ 8~
published in the Journal of Histochemistry and
Cytochemistry, Vol. 22, pp.1084 to 1091 (1974).
200ug of biosynthetic HBsAg ~prepared as described
in Hsiung et al in Journal of Molecular and Applied
5 Genetics 2:497 to 506 (1984) was incubated with 40ug of
the peroxidase-labelled chimpanzee anti-HBs in 2ml of
phosphate buffered saline at pH 7.4 for 2hrs. at 37~C
and then diluted to the required concentration (0.125ug
HBsAg/ml) with phosphate buffered saline containing 50%
fetal bovine serum, 1.5% normal human serum and 0.01~ of
gentamicin sulphate.
HBsAg purified from human carrier plasma also has
been used successfully formed into a labelled
antibody-antigen conjugate and used as a reagent in the
assay.
Assay Procedure
Aliquots (0.2ml) of test and control samples were
added to the HBsAg coated microwells and held overnight
at room temperature. The wells then were aspirated and
20 washed five times with 0.4ml aliquots of deionised
water. Aliquots of the peroxidase-labelled chimpanzee
antiHBs:HBsAg conjugate were added to all the
microwells, which then were incubated at room
temperature for 4 hours. The wells next were aspirated
and washed five times with 0.4ml aliquots of deionised
water. The amount of peroxidase associated with the
solid phase was determined by adding 0.2ml aliquots of
the enzyme substrate (0.2mg/ml of tetramethyl benzidine
in dilute hydrogen peroxide) and leaving -the mixture in
the dark for 30 minutes. It was found convenient to
stop the colour reaction by the addition of 0.05ml of lN
sulphuric acid. The absorption of the solution in each
microwell was measured at 450nm using a microplate
spectrophotometer.
The results obtained were plotted graphically and
are reproduced in Figure 1. As may be seen from Figure
1, the present invention yields an assay with
considerably improved sensitivity over a commercial
assay where the enzyme is bound to the antigen (HBsAg),
Z8~5
using an avidin-biotin linkage. The present invention
also shows a much lower false positive rate than
commercial radioimmunoassays to detect antibodies
against HBsAg.
Similar results are obtained using Hepatitis B core
antigen (HBcAg) in place of the HBsAg.
Example II: This Example illustrates the provision of
an assay for IgM antibodies to Hepatitis B Core Antigen
Preparation of Reagents
Murine monoclonal antibody specific for the heavy
chain of human IgM (M2 antiu) was prepared according to
Kohler and Milstein as described in Nature, 256, 495
(1975) and then was used to coat microwells as described
in Example I. Biosynthetic Hepatitis B Core antigen
(HBcAg) was expressed in E.Coli and purified by standard
recombinant technology methods. The immunoglobulin
fraction from the plasma of an individual with a high
titre of antibodies to HBcAg (defined as anti-HBc) was
digested with pepsin, in the conventional manner, to
remove the Fc portion of the heavy chains and yield the
(Fab)2 anti-HBc. This (Fab)2 anti-HBc antibody fraction
was coupled to peroxidase by the method of Nakane
described in Example I. 1.5ug of the labelled antibody
fragment was incubated with lug of purified,
biosynthetic HBcAg in 2ml of phosphate buffered saline
at pH 7.4 for 2 hours at 37C. The peroxidase-labelled
antibody:antigen conjugate was diluted to the required
concentration (0.006ug HBcAg/ml) by dilution with
phosphate buffered saline containing 50~ of fetal bovine
serum, 10% of normal human serum and 0.01% of gentamycin
sulphate.
Assay Procedure
Aliquots (0.2ml) of test and control samples were
added to the M2~antiu coated microwells, which were held
overnight at room temperature. The wells were aspirated
and washed 5 times with 0.4m] aliquots of deionised
water. The wells were incubated with 0.2ml aliquots of
the labelled antibody:antigen conjugate for 2 hours at
37C, then aspirated and washed 5 times with 0.4ml
9 ~ 2~ .5
aliquots of deionised water. Aliquots (0.2ml~ of
freshly prepared tetramethyl benzidine (0.2mg/ml) in
dilute hydrogen peroxide, were added to all microwells,
which then were allowed to stand in the dark for 30
5 minutes. After the addition of 0.05ml of lN sulphuric
acid to each well, the absorption of the solution was
measured at 450nm in a spectrophotometerO
The results obtained were plotted graphically and
are reproduced in Figure 2. As may be seen from Figure
2, the assay of the present invention, involving three
incubation steps, is equivalent in performance to a
current commercial assay which requires four incubations
(as described in IJ.S. Patent No. 4,273,756).
Example III: This Example illustrates the provision of
an assay for IgM antibodies to Hepatitis A virus
Preparation of Reagents
Murine monoclonal antibody, specific for the heavy
chain of human IgM (M2 anti-u) was prepared according to
the method of Kohler and Milstein described in Example
II and was used to coat microwells as previously
described.
Antigens from the Hepatitis A virus were obtained
by the following method. Human diploid cells infected
with the virus in tissue culture, were lysed and the
cell supernatant, after centrifugation, was treated with
formaldehyde at a dilution of 1:4000 for 3 days at 37C
to render the mixture non-infective. The inactivated
virus suspension was diluted with phosphate buffered
saline at pH 7.3 to 25 times the original volume of
tissue culture fluid. This solution was designated as
Reagent A. The immunoglobulin fraction from the plasma
of an individual with a high titre of antibodies to
Hepatitis A virus (anti-HAV) was digested with pepsin in
the conventional manner to yield the antibody binding
fragment (Fab)2 anti-HAV. This fraction was coupled to
peroxidase as detailed in Example I, and the resulting
labelled antibody fragment was diluted 1:6000 in
phosphate buffered saline at pH 7.3 containing 10% fetal
bovine serum and 4% normal human serum with 0.01%
8.~
gentamicin sulphate as a preservative. This solution
was designated as Reagent B.
Inactivated Hepatatis A virus (Reagent A), diluted
1:2.5 in phosphate buffered saline at pH 7.3 and
containing 0.01~ gentamicin sulphate, was incubated for
2 hours at 37C with the peroxidase labelled antibody
fragment, diluted 1:600 in phosphate/saline at pH 7.3
containing 50~ fetal bovine serum and 0.01~ gentamycin
sulphate. This mixture was diluted 1:10 with the same
diluent to give Reagent C.
Assay_Procedure
Aliquots (0.2ml) of test and control samples were
added to the microwells coated with the M2 anti-u
monoclonal antibody. The microwells were held overnight
at room temperature and then washed 5 times with 0.4ml
aliquots of deionised water. The microwells then were
treated in three different ways with the preformed
reagents to assay the IgM antibodies in the sample:
(a) Reagent A (O.lml) (known antigen) was added to
the wells, followed immediately by Reagent B
(O.lml) (labelled antibody);
(b) Reagent B (O.lml) was added to the wells,
followed immediately by Reagent A IO.lml); and
(c~ Reagent C (0.2ml) (preformed conjugate of
Hepatitis A virus antigens and peroxidase labelled
human (Fab)2 anti-HAV) was added to the microwells.
After the addition of the above reagents, the microwells
were incubated for 2 hours at 37C, and then washed 5
times with 0.4ml aliquots of deionised water. Aliquots
(0.2ml) of a freshly prepared solution (0.2mg/ml) of
tetramethyl benzidine in dilute hydrogen peroxide were
added to all the microwells and the mixtures allowed to
stand in the dark for 30 minutes. After the addition of
aliquots (0.05ml) of lN sulphuric acid, the absorption
at 450nm of the solution in each microwell was measured
in a spectrophotometer.
The results obtained were plotted graphically and
are reproduced in Figure 3. It can be seen from the
results depicted in Figure 3 that the assay of the
present invention involving three incubation steps,
gives a performance superior to a commercial assay using
current methodology requiring four incubations (as
described in U.S. Patent No. 4,273,756). It is further
evident that all three methods of forming the labelled
antibody:antigen conjugate are satisfactory, although
the preferred embodiment of the invention is to add the
antigen followed by labelled antibody to the microwells
(Option [a]), so that the immune conjugate forms at the
same time as the solid phase bound IgM antibodies
capture the added antigen.
SUMMARY OF DISCLOSURE
In summary of this disclosure, the present
invention provides a novel immunoassay procedure using a
labelled antibody-antigen conjugate which is beneficial
in enabling more sensitive assays to be performed
satisfactorily. Modifications are possible within the
scope of this invention.
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