Note: Descriptions are shown in the official language in which they were submitted.
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BACKGROUND OF THE INVENTION
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This invention discloses an improvement in solid
phase immunoassay methods for the detection and determina-
tion of antigens and antibodies (markers) relating to hepa~
S titis.
There are at least two distinct types of viral
hepatitis. Hepatitis A is believed to be caused by the hepa-
titis A antigen (HAVAg) and is generally characterized by an
incubation period of two to six weeks, mild prodromata and a
10 relatively mild clinical illness. The disease is generally
transmitted by contaminated food or liquid, but has also
been shown to be transmitted by systemic inoculation. Hepa-
titis A is frequently called "infectious hepatitis", and in
the United States the number of reported cases is over 50~000
15 annually.
The hepatitis B virus is believed to be the most
probabl~ etiologic agent for "serum hepatitis". Hepatitis B
infection is generally transmitted by blood products or con-
taminated instruments such as needles, but it may also be
20 transmitted by other means. Previously, a hepatitis B in-
fection was associated with an incubation period ranging from
six weeks to six months. However, incubation periods as
short as two weeks have been documented. The illness may be
mild or asymptomatic, but if symptomatic, manifestations may
25 be especially severe. Prodromata may include arthralgias,
arthritis, rash, fever, anorexia, fatigue and pruritis with
or without jaundice.
At least three distinct antigen-antibody systems
can be associated with hepatitis virus B: the surface
(HBsAg anti-HBs), the "core"(HBcAg: anti-HBc), and e-anti-
gen(HBe~g: anti-HBe). The hepatitis B surface antigen(HBsAg)
is found in the blood as 22nm spheres and as elongated tu-
bules which are 22nm in diameter and variable in length and
is believed to represent the protein coat of the hepatitis B
35 virus.
A 42nm particle containing DNA and a D~A polymerase
is considered to represent the infectious virus (Dane parti-
cle). The surface of the Dane particle is similar or identi-
cal to HBsAg. In detergents, the Dane particle is degraded
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to a 27nm electron dense core, HBcAg. The latter is seen in
nuclei of hepatocytes of patlents with serum hepatitis during
the acute infection stage.
Thus, patients with viral hepatitis type B might
5 be expected to produce antibodies to the surface antigen
(anti-HBs), and also to the protein core (anti-HBc). HBsAg in
serum has been a consistent marker for the presence of the
hepatitis B virus, and anti-HB usually appears during early
viremia, often accompanying antigenemia (HB Ag), at the height
10 of liver disfunction and long before the appearance of anti-HBs.
Anti-HBc is generally associated with prolonged circulation of
HBsAg suggesting that anti-HBc is produced in response to the
active replication of the virus.
In 1972, the hepatitis e antigen (HBeAg) was de-
15 tected and characterized. The e marker has been found in HBsAg-
positive serum and is thought to occur more commonly in serum
of chronic HBsAg carriers with active liver disease than in
healthy carriers. In patients, during the incubation period
of hepatitis B, the e antigen was shown to appear just after
20 the appearance of HBsAg and before clinically apparent liver
injury. Logically, its presence in such sera would be indica-
tive of a poor prognosis and on-going liver damage. Converse-
ly, the presence of e antibody (anti-HBe) would be indicative
of a good prognosis. These correlations are not absolute, but
25 may be useful clinical guides.
Since the serolo~ic markers for hepatitis appear
in consistent, sequential order during the course of infection,
acute disease and recovery, an analysis for two or more markers
would be valuable to assess the time-course of the disease.
An assay for more than one marker can also provide
an important double-check for HBsAg in blood doners or patients
in an effort to reduce the incidence of type-B hepatitis. Such
a need has been demonstrated by Goldfield et al; Am. J. Med.
Sci., 270: 335-342 (1975), who in careful follow-ups of recip-
35 ients of blood negative for HBsAg, found evidence of exposure
to the hepatitis antigen in 7 of 465 patients. Clearly, a
method of detecting more than one marker to the hepatitis B
virus should decrease or prevent the occurrence of false nega-
tives. Similarly, in many instances a
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positive response to two tests minimizes the possibility of
a false positive occurring in one test.
While the reagents and methods described and claim-
ed herein are similar to known commercial products and proced-
5 ures for detecting the various markers to viral hepatitis,heretofore there has been no disclosure or suggestion describ-
ing any techniques for detecting said markers simultaneously.
SU~MARY OF THE INVENTION
Accordingly, this specification describes in de-
10 tail reagents and a method useful for detecting simultaneously
in a sample at least two different markers evidencing exposure
to hepatitis virus.
The method comprises contacting ~he sample with a
solid support which has been coated with at least two differ-
15 ent, non-complimentary immunoreactants which are complimentary
to the unknown markers; incubating the sample with the coated
solid support for a period up to 24 hours;
separating the coated solid support from the sample;
washing the coated solid support;
contacting the washed solid support with a liquid
reagent comprising at least two different hepatitis markers
or immunoreactants, each selected to either react or compete
with one of the unknown hepatitis markers and each labeled
with detectably distinct tags;
separating the solid phase from the liquid reagent;
and determining the presence of labeled markers on
the solid support by detecting each distinct tag.
DESCRIPTION OF THE PREFERRED EMBODI~NTS
The following examples will demonstrate the prepa-
30 ration and use of representative reagents within the scope of
the claimed invention. The ~irst example describes the method
of preparing a solid support coated with two different non-
complimentary immunoreactants which are complimentary to
markers evidencing exposure to hepatitis.
More specifically, a solid support (bead) has been
coated with two different, noncomplimentary immunoreactants
in such a manner that both retain their reactivity and are
able to combine with complimentary markers in an unknown
8~5~
ds ~
specimen.
In the first example a polystyrene bead has been
coated with an antibody to HBsAg and with the core antigen,
HBcAg. The antibody retains its avidity and will react with
5 any HBsAg in the unknown specimen and the affixed core anti-
gen will retain its antigenicity and react with any antibody
to the core (anti-HBc) in the unknown specimen. The crux of
this invention is that both immunoreactions will occur
simultaneously with a single solid phase reagent.
EXAMPLE I
PREPAR~TION OF COATED SUPPORT
A formulation of HBcAg was made by exposing Dane
particles to a solution of 2% 2-mercaptoethanol (2-ME) and
5~ Tween~80 in Tris-EDTA-saline (TSE) buffers at 37C for 2
15 hours. That solution was diluted 10-fold in 5% 2~-TSE and
allowed to stand overnight before diluting to final concen-
tration of 1:8000 in TSE. Separately, polystyrene beads
(1/4") were coated with a 1:2000 dilution of anti-HBs serum
~guinea pig) in phosphate buffered saline (PBS) by soaking
20 for two hours. The beads were removed, washed and dried.
The core preparation was then poured over the coated beads
and the core antigen was allowed to adhere to the bead for 2
days at room temperature. The beads were removed from the
buffer solukion, washed, coated with a 10~ sucrose solution
25 to stabilize the adherents and air dried.
Although polystyrene beads are preferred because
they are easily coated and manipulated, any solid support of
either macro or micro dimensions, fashioned from a variety of
plastics, metal and glass could just as easily be coated and
30 used to demonstrate the claimed invention.
While it is possible that the beads may be coated
first with core antigen and subsequently coated with anti-
HBS, it has been observed that coating the heads first withthe guinea pig anti-HBsserum significantly augments the ad-
35 herence of core antigen.
It is also important to note that other combina-
tions of non-complimentary immunoreactants relating to hepa-
titis virus may also be employed. The immunoreactants must,
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of course, be non-complimentary because a complimentary at-
traction would diminish the antigenicity and avidity of the
reagent.
The following example will demonstrate the prepara-
5 tion of a liquid phase rea~ent containing an immunoreactant,anti-HBs , tagged with a radio label (125I) and a marker, anti-
HBC , tagged with a reactive enzyme label (horseradish
peroxidase).
While the terms "marker" and "immunoreactant"
10 might be used interchangably, it is preferable if the term
"marker" is used to designate the antigen or antibody, and
their equivalents, to be detected in the unknown sample. The
term "immunoreactant" is used to define either the antigen
or antibody complimentary to the markers to be detected.
Accordingly, in the following examples, the anti-
gen and antibody employed on the solid support are always
complementary to one of the unknown markers, and, therefore,
are referred to as immunoreactants.
In the liquid phase reagent, the labeled surface
~0 antibody is complimentary to the unknown marker (HBsAg) and
is, therefore, referred to as the labeled immunoreactant. The
labeled core antibody, however, is identical to the marker to
be detected and is, therefore, referred tc as a labeled marker.
EXAMPLE II
TAGGED ANTIBODY REAGENT
The iodination (125I) of antibody to HBsAg (anti-HBs)
entailed adding approximately 0.1 ml of a 0.5M phosphate
buffer at pH7.5, a small volume of 5-6 mci of Nal25I, and 100
mg. of purified anti-HBs to a one dram vial, adjusting the pH
to 7.5-8.0 and mixing all ingredients. To this mixture was
added 50 microliters of a freshly prepared solution of
chloramine T (35mg in 10 ml. 0.05M phosphate buffer, pH 7.5).
After additional mixing, the reaction was allowed to proceed
at room temperature for 60 seconds. Fifty microliters of a
freshly prepared solution of sodium metasulfite (35 mg in 10
ml of 0.05M phosphate buffer at pH 7.5) were added to the
reaction to reduce the chloramine T and thereby stop the re-
action.
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The reaction efficiency was checked by placing 5
microliters of the reaction mlxture on a strip of Whatman
No. 1 paper strip and chromatographing the mixture in 70%
methanol. The iodinated protein remained at the origin while
5 the free 125I migrated with the solvent. The ~ of 125I in
protein is~ccns de~ed t~ be th~ %l o~ re~c~i~o~ efic~ncy.
The crude iodinated antibody was purified using
a Sephadex~G-50 gel column with 0.1 molar Tris buffer con-
taining 0.9% sodium chloride at pH 7.8. The column was pre-
10 treated with a small volume of a 30~ aqueous solution ofbovine serum albumin followed by an equal additional volume
of 0.1 molar Tris buffer solution containing 0.9% sodium
chloride at pH 7.8. The iodinated antibody was added to the
top of the column and washed through using the saline
15 buffered Tris solution. The labeled antibody was the first
eluate collected from the column.
The conjugation of antibody to hepatitis B core
to horseradish peroxidase (HRP) entailed activating peroxi-
dase by using sodium meta periodate. The excess periodate
20 and by-products were seParated ~rom the active peroxidase by
~ e~
gel filtration (4~u~x~ G-25) column. The activated peroxi-
dase was then reacted with the antibody (anti-HBc). The re-
action occurred spontaneously. Sodium borohydride was then
added to stabilize the bond formed between peroxidase and
25 the antibody, and acetone was added to destroy the remaining
sodium borohydride.
~hen employed according to the teaching of this
invention, the two tagged antibodies are diluted into a
diluent containing:
~0 50% fetal calf serum
15~ normal human serum
.005 M EDT~
0.1% Tween-20
0.01% ~ in PBS
It should be apparent that a variety of detect-
able tags may be employed. The only requirement, naturally,
is that they be detectably distinct. Any of a variety of
isotopes could be utilized just as easily as 125I. It is
not necessary that one marker or immunoreactant be radio-
5~
labeled and t~e other enzymatically or florescently labeled
since different isotopes are, themselves, detectably distinct.
Similarly, all labeled components of the reagent might just
as easily employ different enzymes requiring different sub-
strates yielding detectably distinct reaction products.
It should be noted that any of the antibodies and
antigens of hepatitis A and B could be labeled to perform
according to the disclosed invention. The only immunochemical
requirements are that the labeled components not be compli-
lO mentary with each other or to the markers to be determined.
EXAMPLE III
SIMULT~EOUS DETECTION OF HB Ag AND ~TI-HBC
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Serum samples containing the "unknown" hepatitis
markers were added to the individual wells of a reaction tray
and a polystyrene bead coated in accordance with Example I was
added to each specimen sample and allowed to incubate for 2
hours at 45C. The beads were removed from the reaction well,
washed with water to remove any excess reagent, and added to
0.2 ml of the liquid phase reagent prepared in accordance
20 with Example II. The solid and liquid phases were incubated
for a period of one hour at 45C. The solid phase was then
separated from the antibody reagent and washed four times with
water to remove excess reagent. The washed beads were placed
in test tubes containing o-phenylenediamine(30 mg in 10 ml of
0.1 M citrate buffer, pH 5.5) and allowed to incubate for 30
minutes. Following that period the enzyme reaction was stopped
by the addition of 1 ml of 1 M HCl and the tubes were visually
examined for the presence or absence of color resulting from
the reaction of the enzyme tag and the substrate. The ab-
sence or low incidence of color indicated that anti-HBc was
present in the unknown sample and competed with the labeled
marker for reaction sites on the coated support. Next, radio-
activity on the beads was counted in a gamma counter and the
CPM's were recorded. Radioactivity on the bead indicated that
there was HBsAg in the unknown sample which adhered to the
affixed antibody and provided a binding site for the radio-
labeled antibody.
The invention may now be practiced in the various
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ways suggested by the foregoing elaboration and all such
ways are now defined by the terms of the following claims.
