Note: Descriptions are shown in the official language in which they were submitted.
~62~1
1 BACKGROUND OF THE INVENTION
. .
This invention relates, in general, to a method for
detecting the presence of antigens associated with hepatitis.
In particular, it relates to an immunoassay for antigens
associated with hepatitis involving the use of an enzyme-tagged
antibody which reacts with an antigen to detect the presence of
hepatitis.
Hepatitis, which means "an inflammation of the liver",
is due to an infection or obstruction of the bile channels.
There are thought to be two varieties of viral hepatitis, one hav-
ing a longer incubation period than the other. In the past, when
a patient contracted hepatitis and had a known parenteral
exposure, the hepatitis was termed "serum hepatitis". If the
patient did not have a known parenteral exposure and he con-
tracted hepatitis orally it was called "infectious". However,~
it has been documented that in addition to having overlapping
incubation periods, "infectious hepatitis" can be contracted
parenterally, and, the so-called "serum hepatitis" can be
contracted orally. Thus, although there would appear to be two
forms of hepatitis caused by at least two distinct agents, the
terms "serum hepatitis" and "infectious hepatitis" should not be
used to distinguish them. Accordingly, it has been suggested
that the terms "hepatitis A" be used to designate the form most
closely resembling "infectious hepatitis", and, the term "hepatitis
B" be used to designate the form most closely resembling "serum
hepatitis".
The examples appearing in this specification are
directed to the detection of the antigen or antigens associated
with hepatitis type B. Patients who contract the form most
30 closely resembling serum hepatitis, no matter how contracted,
~ .
~1~62~1
1 often have these antigens in their blood. At this point, it
should be noted that there is no reliable assay for determining
the presence of an antigen associated with hepatitis A or a
hypothesized hepatitis C. Thus, the examples in this specifi-
cation are directed to the detection of the presence of those
antigens associated with hepatitis B. However, there is no
reason why the process of the present invention could not be
used to detect the presence of antigens associated with other
types of hepatitis once their antigens have been identified.
Contraction of "serum hepatitis" or hepatitis B
creates a serious clinical problem that cannot be ignored.
Because of the severity of this problem, a variety of test
methods for the detection of hepatitis have been developed.
These included Micro-Ouchterlony, immunodiffusion, complement
fixation, immunoelectro-osmophoresis, haemagglutination and
haemagglutionation inhibition, electron microscopy, and solid
phase radioimmunoassay. See British Medical Bulletin, 1972,
Vol. 28, No. 2 (Viral Hepatitis) pages 138 - 141 for a brief
description of each.
Immunoelectro-osmophoresis or counterelectrophoresis
(CEP) provides a rapid, simple method for the detection of the
hepatitis antigen and its antibody. However, this technique is
somewhat less sensitive then, for example, complement
fixation. Its principle advantage is that tests can be completed
within two hours. However, because of its low sensitivity level,
CEP is no longer approved by the Food and Drug Administration.
The application of radio-immunoassay (RIA) for
routine diagnostic purposes is believed to be somewhat limited,
not only because of the relatively complex, specialized, and
expensive equipment necessary for conducting the test, but also
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~3LG6281
1 because of the strict precautions required in handling radio-
active isotopes. Isotope tagging presents a serious potential
health hazard, requires monitoring, and Atomic Energy Commission
licensing (for user and manufacturer), and presents waste
disposal problems. Nevertheless, this technique is now rather
well established for immunoassay.
Immunological methods depend, of course, upon a
primary characteristic of all antibodies and antigens, i.e.,
their ability to react with a specific complementary antigen or
10 antibody. Thus, if an antibody is added to a serum containing
its antigen, the antibody and antigen will complex and may
precipitate from the solution. In most of the above-mentioned
test methods, the presence of antigens in human sera is detected
by making use of this simple fact.
Labeled antibodies have been used previously for
identifying various antigens. If an antibody known to be
specific for a particular antigen is isolated from the globulin
portion of serum or plasma of a host animal which has been
stimulated to produce that antibody, it can be labeled or
tagged by known means. By conjugating the antibody with a
labeling agent, e.g., a physically detectable substance such as a
radio isotope, as above-mentioned, or fluorescent chemicals,`
the presence of the anitbody can be detected. Thus, when used
diagnostically, if the counterpart antigen is present in some
prepared test sample, the labeled antibody will attach itself
to that antigen, and the presence of the antigen can be confirmed
through detection of the labeled antibody in the sample.
A labeled antibody, for diagnostic purposes, should be
made sufficiently specific so that it will react only with those
antigens whose detection is desired and without cross-reaction
- 3 -
62~
1 with other closely related antigens which may have quite dis-
similar or insignificant consequences. Thus, it is apparent
that both the source and the manner of preparation of the anti-
body is quite critical in any immunoassay.
One manner of detecting hepatitis associated antigen,
as earlier mentioned, involves solid phase radio-immunoassay.
Such a precedure is disclosed in United States Patent No. 3,867,517.
As disclosed therein, in the performance of the assay, a
tube well, or insert for use therewith, of molded polystyrene
10 is first coated with antibody. This is accomplished by
incubating the member to be coated with an antibody solution.
Afterwards, the unknown sample is incubated with the coated well
or insert to react the antibody with antigen present in the
sample. The well is then washed and incubated with an antibody
labeled with the radioactive isotope I-125. It is then again
washed to remove any unbonded labeled antibody. Thus, in the event
any antigen is present in the test sample, a sandwich is formed
from the polystyrene well (or insert), the antibody, the antigen,
and the I-125 tagged antibody. The radiation emitted from the
I-125 tagged antibody is then counted and compared against a
control.
It has also been disclosed that a disc of poly-
tetrafluoroethylene onto which is grafted a substituted poly-
styrene, e.g., isothiocyanostyrene, might be useful in performing
radioimmunoassay. This polystyrene is an insoluble material
having specifically designed surfaces of protein-reactive
groups which may be used to covalently bond proteins to provide
a reagent useful in bioassay procedures.
Recently, an important alternative to labeling anti-
bodies with radio-isotopes or fluorescent chemicals has been
11~;36~
1 developed. This involves labeling or tagging an antibody with
an enzyme. Such a procedure is described in U.S. Patents
3,654,090 and 3,791,932. In Clinica Chemica Acta, 48 (1973)
15-18, an enzyme-linked immunoassay for alphafetoprotein by either
the competitive or the sandwich procedure is disclosed.
Compared to radio-immunoassay, enzyme labeling offers
several important advantages. For example, every enzyme tagged
molecule in the final mixture participates in the readout. On
the other hand, only a very small proportion of isotopic atoms
10 in the final mixture undergo decay during readout to participate
in the assay. In enzyme tagging, every tag repeatedly parti-
cipates in readout, by attacking many substrate molecules to
form a detectable end product, e.g., up to 100,000 times per
minute, hence greatly enhancing sensitivity. An isotope tagged
atom decays only once during readout, after which it is lost
from participation. An enzyme tagged reagent has a long shelf
life; whereas, the isotope tagged reagent is constantly
decaying, and presents serious shelf life problems. While there
is minimal health hazards associated with using an enzyme
20 tagged reagent, serious potential health hazards are encountered
with isotope tagging. Lastly, an immunoassay involving enzyme
tagging can use simple, relatively inexpensive equipment for
readout. The success of isotope immunoassay, in contrast, is
dependent on the efficiency of detecting decay, and hence on the
quality of very expensive detection equipment.
Although the finding of an antigen associated with
hepatitis in one's blood may not be the equivalent of obtaining
a clinical history of hepatitis, investigations have revealed
a high incidence of hepatitis infection when a patient has
received blood which tests positively for antigens associated with
~281
1 hepatitis. Since decisions on whether to use particular blood
units available from a blood center must often be made in a
realtively short time period, a sensitive,rapid, easy to
perform screening test ~or hepatitis, without need for expensive
equipment, is of extreme importance. Although the various
tests used in the past for the detection of the antigens
associated with hepatitis have been satisfactory to a degree,
they are all attendant with one or more disadvantages.
Each year hepatitis causes thousands of deaths and
hospitalizations. It has long been thought that the key to
bringing the disease under control would be a technique for
screening blood which could be made available world-wide and
could be conducted simply and routinely. While, as indicated
above, there are now a number of techniques available, because
they are relatively insensitive, require a lengthy and detailed
procedure, require the use of sophisticated equipment not
readily available in most hemotology labs, or require the use
of reagents which are highly unstable and thus cannot be main-
tained at-hand, they have not satisfied the demands of the ideal
test.
SUMMARY OF THE INVENTION
The invention broadly involves a direct immunoassay
for antigens associated with hepatitis involving the "sandwich"
principle. In the performance of the assay, antigen to be
detected is sandwiched between antibody layers which react
with it. One antibody layer is labeled with an enzyme. The
other layer is covalently bonded to an insoluble member. The
enzyme is exposed to a chemical substrate which will undergo a
chemical change to produce a reaction product in the presence
of the enzyme catalyst. The presence of hepatitis is determined
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.
~1~362~1
1 by determining the presence of the reaction product.
In accordance with another aspect of the invention,
a hepatitis detection kit or set is provided. The three main
reagents of the test set are the insoluble polymeric solid having
antibodies reactive with antigens associated with hepatitis
bonded thereto, enzyme tagged hepatitis antibody reagent, and an
enzyme substrate capable of being chemically changed under the
catalytic influence of the enzyme to form a detectable end
product. In the preferred embodiment, the enzyme of the con-
jugate is an alkaline phosphatase and the enzyme substrate isp-nitrophenylphosphate. The test set may also contain control
sera including sera negative, weakly positive, and strongly
positive for hepatitis antigens, a solution of a horse globulin
additive for the test sample for minimizing the frequency of
non-specific reactions between the test sample and the insoluble
polymeric solid, a buffer designed to maintain the pH of the
enzyme substrate solution in the optimum range for reaction,
and a plurality of vials of a size calculated to promote
contact between the insoluble solid and the small quantities of
reagents used in the incubations. When alkaline phosphatase
and p-nitrophenylphosphate are used as the enzyme-enzyme
substrate system, the buffer is an aqueous solution 0.02 8M in
Na2C03 and 0.001 M in Mg
In another aspect of the invention, an immunologically
active purified hepatitis antibody conjugated, i.e., chemically
linked, with a functional en2yme such as alkaline phosphatase
is provided for use in the immunoassay.
In still another aspect of the invention, a disc-like
insoluble member or matrix is provided for use in the immunoassay.
The matrix has a plurality of groups reactive with proteins
grafted uniformly to its surface layer. Purified hepatitis
il~6281
1 antibody is covalently bonded to the reactive groups to provide
an exterior layer of hepatitis antibody.
In the immunoassay, the insoluble member is placed
in a flat-bottomed vial and just covered with a liquid test sample.
For the test to be reproducible, it is important that the
antigens in the test sample be exposed to the entire surface of
the antibody coated discs. It has been discovered that use of
a flat or planar surfaced disc resulted in loss of sensitivity.
~ Accordingly, the two opposed surfaces of the disc of the
invention are distorted to an irregular configuration to reduce
the disc surface-vial bottom contact. In one preferred embodi-
ment, the opposed surfaces are rendered waffle-like by passing
the disc through a press prior to attachment of the antibody.
Accordingly, it is a primary object of the invention to
provide a hepatitis detection kit and a procedure for its use
such that the presence of antigens associated with hepatitis~
may be rapidly, simply, accurately, and routinely determined
in body fluids.
Another object of the invention is to provide a
unitized test set which is adapted for the performance of a
sensitive, reproducible immunoassay of antigen& associated with
hepatitis on a routine basis by relatively unskilled persons.
Another object of the invention is to provide such a
test set which is designed to minimize procedural errors in the
performance of the immunoassay and which contains all the
necessary reagents, reaction containers, etc. in a form designed
to optimize the accuracy and sensitivity of the test.
Another object of the invention is to provide a
test set containing laboratory equipment designed to standardize
the various steps performed during the assay.
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1~6%81
1 Still another object of the invention is to provide a
test set containing standard control samples, negative, weakly
positive, and strongly positive for hepatitis associated antigen,
with which the test samples may be compared.
Another object of the invention is to provide a
hepatitis antibody-enzyme conjugate for use with the immunoassay
wherein both the antibody and the enzyme retain their respective
desired chemical characteristics.
A further object is to provide such a conjugate which
can be stored for extended periods without losing its
immunological or catalytic characteristics and which is highly
reactive with antigens associated with hepatitis.
Still another object of the invention is to provide
hepatitis antibody conjugated with an enzyme which is capable of
catalyzing a reaction to produce an end product at a high
reaction rate, the concentration of the end product being
precisely detectable using a photometric detector.
Another object of the invention is to provide a
conjugate as disclosed above which is highly purified, and thus
contributes heavily to promoting sensitive and accurate immuno-
assays of antigens associated with hepatitis.
Another object of the invention is to provide an
insoluble matrix useful as described in the above-mentioned test
which has a high concentration of purified, hepatitis associated
antibody distributed uniformly over its entire surface.
A further object is to attach hepatitis antibody to a
solid such that it will not be dislodged by mechanical or
chemical forces to which it may be subjected during use. In
this regard, it is imperative to effect the attachment such that
the antibody remains functional, i.e., capable of participating
in its immunological reaction and immunochemically unaltered
_ g _
1~62t31
by its attachment.
A further object of the invention is to provide an
improved shape for an insoluble antibody coated solid which
is easy to handle during immunoassay and which is designed to
expose a constant and a greater portion of its coated surface
to the solutions used during incubations.
It is a further object of the present invention to
provide a process for determining the presence of antigens as-
sociated with hepatitis in a sample comprising the following
steps:
(a) incubating the sample with an antibody immobilized on
an insoluble member, the antibody being reactive with the ant-
igens associated with hepatitis, the incubation enabling a bond
to be formed between the antibody and an antigen present in the
sample to produce an insoluble member having an antigen bonded
thereto in the event antigens associated with hepatitis are
present in the sample;
(b) separating the insoluble member from any unbonded
substances;
(c) incubating the insoluble member with a solution con-
taining tagged antibody reactive with an antigen associated
with hepatitis, the tagged antibody being tagged with an enzyme
capable of effecting a reaction of a substrate to produce a
detectable reaction product, the incubation being conducted to
enable the tagged antibody to bond to any antigen bonded in
step (a) to the antibody on the insoluble member;
(d) separating the insoluble member from the enzyme
tagged antibody solution to remove any unbonded tagged anti-
body therefrom;
(e) exposing the insoluble member to a substrate solution
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11~362~31
which the enzyme of the tagged antibody reacts upon to enable
a chemical change in the substrate to take place and to produce
a detectable reaction product; and
(f) detecting any reaction product present in the sol-
ution to determine the presence of antigens associated with
hepatitis.
These and other objects and features of the invention
will be apparent to those skilled in the art from the following
description of a preferred embodiment.
BRIEF DESCRIPTION OF THE DRAWING
Fig. 1 is a flow chart summarizing the steps of the
process of the present invention;
Fig. 2 is a perspective view of the preferred insol-
uble member used in the test set and the process of the present
invention;
Figs. 3, 4 and 5 depict three incubation stages of
the immunoassay of the present invention; and
Fig. 6 is a schematic representation showing the
"sandwich" structure developed during positive immunoassay of
a test sample tested in accordance with the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENT
At the outset the invention is described in its
broadest overall aspects with a more detailed description fol- -
lowing. The method of detecting hepatitis according to the
present invention is carried out in four distinct stages as is
illustrated in Fig. l.
The first stage involves reacting the immobilized anti-
body on the insoluble member l0 (see Fig. 2) with an antigen
present in the test serum as is shown in Fig. 3. This reaction
- 10a -
11~6281
immobilizes antigen so that on reaction in the next stage with
an enzyme tagged antibody, as is shown in Fig. 4, the tagged
antibody is also immobilized. As is shown in Fig. 5, the enzyme
is exposed to a suitable substrate which is reacted upon by
the enzyme to produce changes in colour. The colour change acts
as an indication of the presence of antigens associated with
hepatitis. The last stage involves reading the degree of colour
and comparing the value obtained with a standard of control.
The hepatitis detection test set of the instant
invention which contains all materials needed in the test out-
lined above is designed for 100 tests. Obviously, larger or
smaller sets may be manufactured by proportionally increasing
or decreasing the quantities of reagents, etc. disclosed herein-
after. The kit is distributed, in, e.g., a box, containing
reagents and controls to be stored at 2-8C until used. In
addition, vials sufficient for conducting 100 tests are provided.
One hundred to one hundred and five polymeric discs,
having waffle-like surfaces, as shown in Fig. 2, and having a
layer of antibody reactive with hepatitis antigens bonded to
their exterior and lyophilized are supplied. The exact nature
of these insoluble solid discs and their method of manufacture
are disclosed hereinafter.
The second reagent in the set comprises a sample of
hepatitis associated antibody-alkaline phosphatase conjugate.
The exact nature of this reagent, the method for its preparation
and alternative useful reagents are disclosed hereinafter.
This reagent is supplied as a solution ready for use.
A third reagent supplied in the test set is 400 mg
of p-nitrophenylphosphate enzyme substrate. This compound is
stable in its powdered form, but, when dissolved in buffer to
-- 11 --
11~62~31
1 form a 1 mg/ml solution, becomes relatively unstable. Con-
sequently, the solution must be prepared just prior to use.
The other materials used duxing the procedure include
a quantity of horse globulin, sample diluent, various buffer
solutions, and various wash solutions. The exact nature and
method of preparation of these reagents and materials are
indicated below.
A. Preparation of Antibody
In order to practice the present invention, it is
necessary to obtain antibody that will react with an antigen
or antigens associated with hepatitis. It should be noted that
such an antibody exists, and thus, the present invention is not
intended to be limited to the use of any particular antibody.
A reactive antibody may be prepared by purifying
bleeding from a host animal which has been injected with a known
sample of antigen. An antibody which is reactive with an antigen
associated with hepatitis may, in general, be prepared by the
process disclosed in British Patent No. 1,387,625.
The preparation of hepatitis antibody depends first of
all on obtaining blood which is known to be positive for antigens
associated with hepatitis. Consequently, blood units obtained
from various sources must first be evaluated to determine their
suitability for preparing the immunospecific purified antibody
of the invention.
A blood bag segment, containing blood which is believed
to be positive for hepatitis antigens, is held at 2-8C in an
upright position to allow the blood cells to settle to the lower
half. The plasma is separated from the cells and a titer is
run on the undiluted plasma and a sample diluted 1:16 in normal
saline against a standard antibody by the well-known technique
~62~31
1 of counterlectrophoresis (CEP). When both the undiluted plasma
sample and the 1:16 diluted sample are positive, the blood unit
is considered acceptable for use in the preparation of the
purified antibody of the invention as described hereinafter.
Portion of antigens isolated from blood units are used
for either stimulating antibody production in a host animal or
purifying the antibody produced by that animal. The antigens
must be subjected to a preliminary isolation process prior to
either of the above uses.
The plasma is transferred to a sterile vacuum container
and clotted by adding a 5M solution of a CaC12 on the basis of
0.75 ml CaC12 per 200 ml of plasma. This solution is then in-
cubated at 37C in a water bath for one hour or until a clot
forms. After a firm clot forms, the plasma is frozen at -20 C
and allowed to thaw at 2-8C to allow clot retraction. The
serum is separated from the clot and filtered if necessary,
then is ready for preparation of a hepatitis antigen pellet
for immunization or for use in lmmunoabsorbent columns.
(1) Preparation of Antigen Pellet for Immun ation
Hepatitis positive sera which have been subjected to
the above process are centrifuged at 10,000 rpm for 30 minutes
at 4C. The supernatant of this centrifugation is distributed
into ultra centrifuge tubes and centrifuged in, for example,
a Beckman L2-65B ultra-centrifuge, at 40,000 to 50,000 rpm for
4 - 20 hours at 4C. The supernatant in each tube is removed
and discarded; the pellet, which contains antigens, is given
- a preliminary rinse with normal (.15M) saline.
A small volume of normal saline is then added to each
centrifuge tube and the contents are subjected to sonication
to break up the pellet. The suspensions in the sonicator tubes
- 13 -
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1 are then pooled and redistributed equally into clean tubes which
are filled with normal saline. This solution is again centrifuged
in the Beckman L2-65B centrifuge at 40,000 to 50,000 rpm for
4 - 20 hours at 4C, as mentioned above.
The procedure in the preceeding paragraph may be
repeated 5 or more times.
The pellet material, after removing the supernatant
from each centrifuge tube, is pooled in the minimal volume of
normal saline. A sample is assayed against a standard hepatitis
antibody. If the pellet titers at 1:25 or higher by CEP, it can
be used for immunization. The pooled antigen pellet may be
divided into 3 ml aliquots and frozen at -20C for future use.
(2) Production and Preliminary Purification of Hepatitis Antibody
A sample of the antigen pellet, prepared as described
above, is added to an equal volume of FREUND'S complete adjuvant
the morning of the immunization and an emulsion is prepared in
accordance with procedures well known to those skilled in the
art. The antigen is then injected into a host animal, e.g.,
a horse, in accordance with techniques known per se, to produce
hepatitis antibody. The immunized horses are bled or subjected
to plasmaphoresis according to conventional techniques. Alter-
natively, or in addition, preparations without adjuvant can be
used with other routes of immunization.
These bleedings must be treated to isolate the
hepatitis antibody in anticipation of the final immunoabsorption
purification step. Broadly, this preliminary purification is
accomplished in three steps. First, plas~a from the host animal
is recalcified. Second, the serum is mixed with a sufficient
amount of normal human plasma (NHP) to precipitate antibodies
other than those associated with hepatitis by inducing insoluble
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1~36~
1 antigen-antibody complex formatibn. The absorbed antiserum is
assayed for hepatitis antibody using CEP. Third, -the antibody
reactive with antigens associated with hepatitis is precipitated
with ammonium sulfate. This material can he frozen until used.
(3) Pre~ tion of Charcoal Immunoabsorbent Column
The preparation of the purified antibody which is used
to produce the reagents of this in~ention is accomplished by
subjecting the henatitis antibody produced as disclosed
above to an immunospecific extraction process. For a general
discussion of this procedure, reference should be made to sritish
Patent No. 1,387,625 to Bradish et al., March 19, 1975, entitled
"Immunospecific Separation of Antigens and Antibodies" ~
In general, this purification process as utilized in
the present invention, takes advantage of the ability of anti-
bodies reactive with hepatitis antigens to complex with these
antigens to the exclusion of other extraneous antibodies and
proteins which are inevitably present in the antibody sample
extracted from the bleedings.
A column is prepared by packing prewashed, sorbent
carbon into a glass or plastic tube by using conventional
techniques. A pool of antigen is prepared from at least six
individual serum specimens to obtain a diverse mixture of
hepatitis antigens. The pool is then adjusted to a protein
concentration of between 1 and 2 mg protein per ml solvent,
based on UV absorption.
To attach the antigen onto the carbon, this diluted
solution is introduced at a flow rate within the range of 300 to
1,000 ml per hour. Seventy five mg protein should be added per
gram of charcoal in the column. The effluent from the column is
- 15 -
~1
2~
1 collected in 500 ~1 aliquots, each of which are checked for
protein content. The column is considered saturated with the
antigen when the effluent has a proteln content approximately
equal to that of the starting material. The bed is washed
by flowing phosphate buffered saline (PBS) through the column
until the effluent shows no appreciable detectable absorption
at 280 nm.
To elute any loosely attached protein, the charcoal
bed is flushed with freshly prepared 5M sodium iodide solution
containing 200 mg per liter of sodium thiosulfate. Afterwards,
the sodium iodide solution is flushed from the column by running
a sufficient volume of PBS therethrough. After a final washing
of the bed with PBS containing 1 mg per ml sodium azide
(preservative), the column can be stored at 2-8C until ready
for use.
(4) Preparation of Antibody for Use in Enzyme Conjugate and
Insoluble Solid
The antibody, purified as described above, is freed
of ammonium sulfate and diluted with PBS on the basis of 1 part
antibody to 2 parts buffer. The column is set up and situated
so that fractions can be collected. The antibody solution is
added continuously to the column with a flow rate of about 200
ml/hr. Effluent is collected and tested for protein and
hepatitis antibody content to determine when the column is
saturated with antibody. After saturation is achieved, the ~ -
column bed is washed with PBS to remove loosely absorbed protein.
At this point, antigens immobilized on the charcoal
column have formed a bond with the antibodies reactive with them. `~
Other extraneous proteins and antibodies, nonspecific to the
absorbed antigens having passed through the coLumn and have
been separated from the antibody.
- 16 -
28~ (
To break this antibody-antigen bond, and to elute the
purified antibody, a 5M solution of NaI prepared immediately
prior to use is introduced into the column. The volume of NaI
solution used should be sufficient to remove all antibody bound
to the column. With the flow rate of the column set at least
200 ml per hour, the eluate is collected in fractions of appro-
priate volumes. The total amount collected should be at least
equal to the volume of sodium iodide solution added.
As each fraction of the purified antibody is collected,
1~ it is subjected to a dual filtration; first, through a 0.45 p
membrane, and second, throuyh a 0.22 ~u membrane. The filtrate
is diluted 1:3 using distilled water at 2-8C, e.g., 200 ml of
filtrate is added to 400 ml of distilled water. These diluted
antibody fractions are then added to, for example, an AMICON*
concentrator equipped with an XM-50*membrane, and the fractions
are concentrated.
As a last purification step, the concentrated, purified,
antibody is dialyzed. Following dialysis, the antibody is
removed and centrifuged. The supernatant is dialyzed for at
least 24 hours against 0.01M sodium phosphate solution, as com-
pared to against PBS in the first dialysis. After completion of
this final dialysis, protein concentration of the antibody
is measured.
The antibody is then assayed for activity against the
standard antigen according to the CEP technique to determine
antibody content and, if found acceptable, is lyophilized and
stored until used.
B. Preparation of_Anti ody-Enzyme Conjugate -~
Calf intestinal alkaline phosphatase is mixed with a
solution of the reconstituted antibody in a ratio of 3:1, enzyme
* Trade Marks - 17 -
1~62t31
1 t:o an-tibody, -to a final concentr--tion c;reater than 10 mg of to-tal
protein per ml o~ solution in PBS (pEI 7.4). Th~ solution is
dialyzed thoroughly to remove NE~4 ions.
The dialyzed antibody enzyme mixture is then centri-
~uged to remove any insoluble material. The pro-tein content
of the supernatant is adjusted to 10 mg/ml by adding the
PBS-Mg solution. To this solution, 8% glutaraldehyde is
added on the basis of 1 ml gluteraldehyde solution per 10 ml
antibody-enzyme solution. After stirring slowly for 3.5 to 20
~ minutes, during which time the antibody and enzyme are chemically
linked by the glutaraldehyde, the solution of conjugate is
dialyzed against PBS containing O.OOlM Mg to remove the
glutaraldehyde.
The dialyzed material is then centrifuged and the
- supernatant is diluted by addition of an aqueous solution of
0.05M in tris(hydroxymethyl) aminomethane (pH 8) buffer, 1%
normal human albumin (crystalline), .02% NaN3, and .OOlM in
MgC12.
In addition to alkaline phosphatase (EC 3.1.3.1),
other enzymes are useable in the procedure of the present in-
vention. Indeed, there is an almost limitless list of enzymes
which can be covalently bonded to the antibody. Of the various ;~
enzymes, the following table indicates enzymes of particular
interest.
1. alcohol dehydrogenase EC 1.1.1.1
2. glycerol dehydrogenase ~ EC 1.1.1.6
3. glyoxylate reductase ~ EC 1.1,1.26
4. L-lactate dehydrogenase - EC 1.1.2. 3
5. malate dehydrogenase ~ EC 1.1,1. 37
6. glucose 6-phosphate dehydrogenase ~ EC 1,1.1.49
7. mannitol l-phosphate dehydrogenase ~ EC l~lel~17
8. glucose oxidase - EC 1.1.3.4
9. galactose oxidase - EC 1.1. 3 ~ 9
10. L-amino acid oxidase ~ EC 1.4.3.2
- ~8 -
,,,., ~
5''' ~7 ~ '
2~31
1 11. D-amino acid oxidase - EC 1.4.3.3
12. polyphenol oxidase - EC 1.14.18.1
13. ascorbate oxidase - EC 1.10.3.3
14. catalase - EC 1.11.1. 6
15. peroxidase - EC 1.11.1. 7
16. cholinesterase- EC 3.1.1.7
17. phospholipase C - EC 3.1.4.3
18. ~-amylase - EC 3.2.1.1
19. lysozyme - EC 3.2.1.17
20. ~-galactosidase - EC 3.2.1.23
21. amyloglucosidase - EC 3.2.1.3
22. ~-glucuronidase - EC 3.2.1.31
23. carboxypeptidase A - EC 3.4.12.2
24. urease - EC 3.5.1.5
25. inorganic pyrophosphatase - EC 3.6.1.1
26. aldolase - EC 4.1.2.13
27. carbonic anhydrase - EC 4.2.1.1
28. histidase - EC 4.3.1.3
The enzyme that is used for tagging the antibody is
selected with several considerations in mind. These consi-
derations include the stability of the enzyme, the ease of
assay of the enzyme, the ability of the enzyme to withstand the
conditions of the covalent bonding of the antibody, the availabili-
ty of the enzyme, and the cost of the enzyme.
C. Preparation of Antibody Coated Insoluble Member
~ . :
The next step in the preparation of the reagents isto covalently bond a portion of the purified antibody to an
insoluble member. To effect this bonding, the insoluble member
used must be provided with reactive groups or sites capable of
reacting with the specific antibody used in the bioassay.
U.S. Patent No. 3,700,609 entitled "Graft Copolvmers",
to G.W. Tregear et al., discloses an insoluble continuous polymeric
substanae eompris7 ng a ~olymeric bac~bone onto which side chains
-- 19 --
Z~l
1 of another polymer or copolymer are grafted. sy suitable choice
of the grafted polymer, it is possible to chemically link biolog-
ical substances to the insoluble matrix. A product which is
disclosed in the above patent is commercially available in a
disc form under the tradename PROTAPOL DI/l from Imperial Chemical
Industries of Australia and New Zealand (ICIANZ).
The PROTAPOL DI/l comprises a polytetrafluoroethylene
backbone having isothiocyanopolystyrene groups grafted uniformly
over its surface and is designed for use in radioimmunoassay.
The discs, as presently available, are approximately .01 inches
thick and 0.5 inch in diameter.
In accordance with one important embodiment of the
matrix of the present invention, as shown in Fig. 2, each disc 10
is provided with a waffle-like pair of surfaces 11 comprising
a first series of linear ridges 12 and a second series of linear
ridges 14 which form grids. Ridges 12 and 14 are preferably
perpendicular to each other and hence define a plurality of
square depressions 15. The sides of each ridge 12 and 14
taper upwardly from adjacent pairs of depressions 16 to form a
line defining the top of the ridge. It should be noted that
in order to facilitate the description of the invention, the
ridges 12 and 14 are greatly exaggerated in the drawing.
The desired configuration of the disc is achieved by
passing the disc through rollers having projections on the surface
of the rollers designed to impart the desired configuration on
the disc. As is obvious, the rollers are designed to provide a
sufficient amount of pressure to disfigure the polymerlc material
in the disc without actually puncturing the disc. This is
important because the disc has a reactive layer on its surface.
Thus, penetration of the disc would expose interior portions
- 20 -
~36Z81
1 to which no antibody can be bonded. Exposure of the poly-
tetrafluoroethylene layer would actually result in a disc
which would have a lowex bonding capaci-ty.
The main consideration is to provide a disc matrix
with surfaces which, when placed in a flat bottomed vial, will
be substantially in complete contact with the test sample, i.e.,
there should be a minimum of surface-to-surface contact between
the matrix and the bottom of the vial. In another important
embodiment of the disc of the invention, the disc's surfaces
are configured to have a field of high and low points.
The antibody, produced as disclosed above in the
lyophilized form, is reconstituted by adding 100 ml of O.lM
NaHC03 (pH 9.6) for each 5.0 mg of antibody. In general,
the procedure for attachmen-t involves contacting the waffled
discs with the dilute solution at 2-8C for 8 to 16 hours,
with agitation. Afterwards, the antibody solution is discarded
and the discs are washed twice with successive volumes of O.lM
NaHC03, pH 9.6, phosphate buffered saline, and cold (2-8C)
0.3% bovine serum albumin in phosphate buffered saline with
0.5% TWEEN*20. After an additional washing with crystalline
bovine serum albumin, and freezing over dry ice, lyophilization
is carried out and the discs are stored at 2-8C until ready
for use.
Although the description in this specification relates
to the preparation of discs having hepatitis antibody bonded
thereto, it should be apparent to those skilled in the art that
the disc of the present invention is useful to immobilize an
almost limitless number of proteins. For e~ample, the increased -
contact between the test sample and the disc enables the disc to
be used in tests which involve the bonding of the following
* Trade Mark
- 21 -
~,J
6Z~
1 proteins thereto: antibodies to drugs such as digoxin, opiates,
and steroids; antibodies to natural products, for example
insulin and other hormones; and specific enzymes to metabolities
found in blood and other body fluids.
Exam~
The following procedure was used to prepare 8,000 discs,
each of which were first treated with the press to produce the
desired configuration as described. A batch of 8,000 discs
requires 40 mg of hepatitis antibody, i.e., 5 mg per 1,000 discs.
The protein content of the reconstituted hepatitis antibody is
adiusted to .05 mg/ml in a final volume of 800 ml in O.lM
NaHCO3 (pH 9.6). The entire 800 ml of buffered antibody is then
added to a 1,000 ml screw-cap bottle provided with a leak
proof liner containing the 8,000 discs, and the bottle is
rotated for 16 hours, e.g., overnight, at 2-8C to slowly
tumble the discs through each rotation cycle. Afterwards, the
liquid is poured from the bottle and discarded and the discs
are transferred to a wide-mouth 2 liter flask.
The discs are washed twice with successive 1 liter
volumes of cold t2-8C) O.lM NaHCO3, pH 9.6, following which the
buffer is removed. The-discs are then washed again, this time
using two successive 1 liter volumes of cold buffer (O.OlM
sodium phosphate, 0.15M NaCl, pH 7.4). After removing residual
buffer, the discs are washed for a third time, using two
successive one liter volumes of cold bovine serum albumin solution
tO.3%).
The discs are finally washed with two successive 1
liter volumes of a solution of cold crystalline bovine serum
albumin (pH 8) at a concentration of 2 mg/ml. This step is
performed to provide a protein environment for the protein on
- 22 -
11~36281
1 the disc. The cliscs, aEter removing the residual wash, are
then transferred to dishes or trays (9 x 9"), each of which is
lined with a sheet of filter paper and each of which contains
200 ml of -the crystalline bovine serum albumin solution. When
the transfer is complete, a sheet of filter paper is used to
cover them. suffer is thoroughly removed. The discs are then
quick frozen for 30 minutes on dry ice.
The contents of the tray are then lyophilized and the
dry discs are removed and stored in stoppered containers.
0 (1) Preparation of Control Serum
In order to obtain meaningful data from the immuno-
assay, it is necessary to provide negative and positive control
sera for proper comparison with a given test sample. The
preparation of these controls is disclosed in detail below.
The negative control is made from human plasma which
has been tested and found negative for hepatitis associated
antigen, by, for example, the radioimmunoassay technique.
To each unit that is clearly negative for hepatitis antigen,
5M CaC12 is added to induce clotting, on the basis of 0.75 ml
of CaC12 solution per 200 ml of plasma. This plasma is then
incubated at 37C in a water bath until a clot forms. The
clotted plasma units are then frozen at -20C and stored for
at least 12 hours. The plasma units are then allowed to thaw at
2-8C and the serum is collected. If the serum appears excessively
turbid, it may be desirable to clarify it by centrifugation,
e.g., at 9,000 rpm for 30 minutes at 2-8C.
Twenty grams of silica, e.g., AEROSIL-380, is added
per liter of serum and mixed for 2 hours at room temperature
to remove lipoproteins and stabilize the serum. The mixture is
then centrifuged and the precipitate is discarded. If desired,
* Trade Mark
- 23 -
'"'`'1 1
s ~ ,,
11~62~31
f the silica can be removed by filtrat:ion through appropriate
filter media.
The supernatant is then further processed by filtration
through, for example, MILLIPORE or HORM membranes and pads of
successively decreasing porosity, the last being a 0.45 micron
membrane. Before filtration through the 0.45 micron membrane,
sodium azide (NaN3) is added to the liquid in sufficient amount to
provide a concentration of 0.1% by weight. As is well known in
the art, sodium azide acts as a preservative. The final
filtration, through a 0.45 micron or smaller porosity filter,
should be done in a laminar flow environment using sterile
equipment and techniques.
The sterile solution may be then stored at 2-8C until
ready for subdivision into reagent sized containers for use in
the immunoassay. In the preferred, 100 test set, 7.5 ml of
negative control serum are provided.
The positive control serum is produced from recalcified
plasma from blood units which test positively for antigens
associated with hepatitis. From each positive unit, a 1%
sample is taken, and these are pooled together to form a trial
pool. The trial pool is first heat treated for 10 hours at
60C to inactivate any hepatitis causative agent in the sample.
When the pool has cooled to room temperature, a portion is
removed and titrated against a standard antibody using the CEP
technique to check that the antigen activity has been retained.
To the trial pool is then added a sufficient amount of silica
to provide a concentration of 20 g per liter of serum. The
serum is then stirred using a stirring bar at room temperature
for two hours after which it is centrifuged at 9,000 rpm for
30 minutes at 2-8C. The precipitate is discarded.
- 24 -
1~628~L
1 The supernatant is then titrated using CEP against a
standard reference antibody. If the titer has remained at
satisfactory levels, the total volume of all serum units may be
pooled together and subjected to the same process as just ~ -
described for the trial pool.
The trial pool and main pool are then combined and
diluted with a sufficient amount of negative control serum to
obtain optimum results with the positive control serum in the
immunoassay of the invention. Preferably, the reading of the
positive control serum in the test of the invention, in
absorbence units x 1000, should be greater than 2000. This
diluted positive control serum is then filtered through suitable
media as before described, using a successive range of decreasing
porosity. Before the final filtration through a 0.45 micron or
smaller membrane, 0.1~ by weight sodium azide is added. As with
the negative pool, the final filtration should be done under
aseptic conditions in a laminar flow environment.
The weakly positive control serum may be made by
diluting the strong positive control with negative serum. The
reading of the weakly positive serum as determined by the test
of the invention should be between 600 and 1000. In the 100
test set of the preferred embodiment of the invention, 2.5 ml of
strong positive control and 2.5 of weak positive control are
supplied.
D. Miscellaneous Reagents and Equipment
Two hundred or more glass, disposable, flat bottomed
vials are supplied which have a diameter slightly greater than
.5 inch, i.e., sized to match the 0.50 inch diameter of the
discs. One hundred of these vials are used for the initial
incubations and washings of the test, the other 100 are employed
- 25 -
;%8~.
1 for the final incubation with the enzyme substrate. Accordingly,
the test set enables 100 assays to be conducted simultaneously.
As set forth in more detail below, before one
insoluble solid disc is added to each of the 100 vials, a 0.05
ml portion of horse globulin test sample diluent is added to each
vial. This step is taken as a precaution to eliminate non-
specific reactions in the first incubation of the test samples with
the insoluble discs. Although the antibody coated on the disc
is purified and highly reactive with hepatitis antigens, there
is occasionally present in human serum or plasma a substance
capable of reacting with horse globulin per se that can thus form
a bridge between the disc and the enzyme labeled conjugate,
thus resulting in a false positive reaction. The horse globulin
added in the first step binds this substance so it is not free
to react with the disc. As disclosed above, the antibody
coated on the disc is produced by immunizing a horse with
hepatitis associated antigen collected from human blood. In
the 100 test set of the invention, a 5.5 ml solution comprising
330 mg of horse globulin dissolved in phosphate buffered saline
(PBS) is supplied.
F. Preparation of Substrate
The preferred substrate for the enzyme reaction in the
test is p-nitrophenylphosphate which is dissolved to a con-
centration of 1 milligram per ml in sodium carbonate buffer,
the concentration of which is 0.028 molar sodium carbonate and
0.001 molar magnesium (pH 9.8).
At this point, it should be noted that other substrates
with suitable pH buffering agents, as appear in Table I below,
may be used.
- 26 -
~1~62~1
1 TABLE I
_
~-glycerol phosphate (serum), 7.4
~-glycerol phosphate barbital, 8.6
phenyl phosphate carbonate-bicarbonate, 9-10
~-naphthyl phosphate barbital, 9.1
p-nitrophenyl phosphate 2A2MlP, 10.25
phenolphthalein phosphate 2A2MlP, 9.90
p-nitrophenyl phosphate 2A2MlP, 10.17
thymolphthalein phosphate carbonate-bicarbonate, 10.0
p-nitrophenyl phosphate diethanolamine, 9.8
4-methylumbelliferyl phosphate carbonate-bicarbonate, 9. 2
The substrates indicated in the Table are all organic
phosphate esters. It should be apparent that other organic
phosphate esters could be used as substrates for the preferred
enzyme, alkaline phosphatase. Furthermore, it should be noted
that th~e skilled in the art will have little difficulty
selecting a suitable substrate if an enzyme other than alkaline
phosphatase is used in the antibody-enzyme conjugate.
G. Procedure
To perform the assay according to the invention, 100
vials are set out in racks and each is identified to correspond
to a test sample. To each vial is added 0.05 ml of the horse
globulin solution, then 0.5 ml of sample is added to 95 of the
vials. At the same time, three 0.5 ml samples of negative control
serum are placed in each of three vials, a 0.5 ml sample of
strong positive control serum is placed in one vial, and a
0.5 ml sample of weakly positive control serum is placed in
another vial. To each vial containing the horse globulin and
sample, including the control vials, is then added 1 antibody
coated disc. The vials, with contents, are incubated for 0.5
hours at 43C in, e.g., a shaking water bath. During this
- 27 -
11~62~31
1 incubation, hepatitis antigens present in the test sample or con-
trols will combine with the antibody on the disc.
Prior to the addition of the enzyme-tagged antibody
reagent to the vials containing the insoluble members, the super-
natant - from the first incubation must be removed and the
insoluble members must be washed to remove any unbonded antigen.
The wash solution is preferably a 0.85~ solution of sodium
chloride, pH 6.5 - 7.5. After two 2.5 ml washes using this
solution, 0.3 ml of the antibody-enzyme conjugate is added to
each vial, and the vials are again incubated at 43C for 1 hour
with shaking, during which time the enzyme tagged antibody will
react with the hepatitis antigens that were fixed to the antibody
coated disc during the first incubation.
After addition of the enzyme tagged antibody reagent
and a second incubation, the supernatants are aspirated off and
the discs in each vial are washed three times with 2.5 ml
aliquots of wash solution. This removes unreacted enzyme-antibody
conjugate. Each insoluble member is then transferred to a clean
vial and 2.5 ml of p-nitrophenylphosphate enzyme substrate-buffer
solution is added to each vial ~1 mg pNPP per ml). Since the
optimum operational pH of the alkaline phosphatase-p nitrophenyl-
phosphate system is 9.8, the enzyme substrate is dissolved in
carbonate-Mg++ buffer ~pH 9.8 + .1). This buffer as used
comprises an aqueous solution 0.028M in Na2C03 and O.OOlM in
Mg++. Forty ml of concentrate may be supplied with the set
which, when diluted to 400 ml with distilled water, may be
added directly to the 400 mg of pNPPo After addition of the
buffered substrate, the vials are subjected to a third incubation
for one hour at 43C with shaking. Different substrates must be
employed if a different enzyme antibody conjugate is used.
- 28 -
., : :
llG6Z81
1 Two drops (0.1 ml) of 3M NaOH solution are then added
to each vial to terminate the reaction. Each test set is supplied
with 15 ml of 3M sodium hydroxide for this purpose.
The enzyme substrate solution disclosed above changes
from a colorless liquid to one having a yellow colour in the
event enzyme is present on the disc, i.e., in those vials
containing samples positive fcr antigens associated with hepatitis.
The supernatant from the negative control are pooled
together in a suitable vial and their absorption is read at 405 nm
in a spectrophotometer against a distilled water blank. When
the negative controls read less than 600 (absorbance units x 1000),
they are considered as proper standards against which to com-
pare the test results. Using the pooled negative control samples
as a blank, test sample and positive control values are read,
and the results are recorded as absorbance units x 1000. With
some spectrophotometers, it is possible to insert a negative
control in the instrument, adjust the reading to zero, and
read the value of the test samples directly. An unknown test
sample whose optical density times 1,000 is greater than 100,
using the pooled negative controls as a blank, is considered
positive for antigens associated with hepatitis. This value
has been selected to limit nonrepeatable positives which, if
present, generally result from errors in laboratory technique.
The readings of the test samples may also be compared
with the weak positive and strong positive control samples.
Thus, not only the presence, but an indication of the concen-
tration of hepatitis antigens in the sample may be obtained.
Examples
~ive controls should be assayed with each group of
unknowns--three negative controls, one strong positive control,
- 29 -
11~6;2~1
1 and one weak positive control. These should be subjected to
the same process and incubation times as the test samples.
Caution: Use a clean pipet or disposale tip for each transfer
to avoid cross-contamination.
1. Preset water bath at 43C.
2. Number two sets of vials to correspond to test sample
identification and controls, and place vials in vial holders.
The first set of these vials will be used for the incubation
of test specimens and controls with the antibody coated disc
and with the antibody-enzyme tagged reagent. The second
set of vials will be used in Step 12 for the substrate
reaction.
3. Pipet 0.05 ml (one drop) of horse globulin reagent into
the bottom of the first set of vials. Nonspecific false
positives resulting from antibodies present in certain
human sera which react with horse globulin, advantageously
are essentially eliminated by using horse globuline in the
test sample diluent.
4. Into this first set of vials pipet 0.5 ml of each test
sample into the bottom of the vial having the corresponding
sample identification; pipet 0.5 ml of the positive and
negative controls into the bottom of their respective
vials.
5. Transfer an antibody-coated disc to each vial of the first
set. Keep the surface of the discs clean. They should be
transferred with clean forceps or a suction-tipped cannula.
They should not be handled with the fingers.
6. Incubate the vials at 43C in a water bath with shaking
attachment set for mild agitation for 30 minutes.
7. After incubating the sample with the antibody disc, com-
pletely aspirate all supernatants from each vial. Wash the
- 30 -
1~36Z~31
1 discs by adding 2.5 ml of isotonic saline solution to
all the vials. Repeat this procedure so that each disc is
washed two times. To aid in removing all fluid, tilt the
vial holder while aspirating. Shake the vial holder
after each addition of wash solution. The liquid waste
collected in the container attached to the aspirator should
be autoclaved before disposal (minimum of one hour at 121C).
8. After final wash and aspiration, add 0.3 ml of the enzyme-
labeled antibody solution to each vial.
9. Incubate the vials for one hour at 43C in the water bath
with the shaking attachment set for mild agitation.
- 10. Prepare p-nitrophenyl phosphate substrate by rinsing the
contents of one vial of p-NPP (100 mg) into 100 ml of diluted
substrate buffer. (The latter is prepared by adding 10 ml
of concentrated sodium bicarbonate buffer to 90 ml of
distilled water.) Rotate gently to mix; solution should
occur immediately.
Note: This substrate solution must be prepared on the day
it is to be used. When not being used it should be
refrigerated. Any solution remaining after 24 hours
should be discarded.
11. Aspirate the supernatant and wash three times as in Step 7.
12. Transfer the discs to the second set of identically
numbered clean vials prepared in Step 2.
13. To each vial containing a disc, add 2.5 ml of the p-nitro-
phenyl phosphate substrate solution prepared in Step 10.
14. Incubate vials for one hour at 43C in a water bath with the
shaking attachment set for mild agitation.
15. Add two drops of 3M sodium hydroxide solution (approximately
0.1 ml total) to all the vials to terminate the reactions.
- 31 -
1~6281
1 Shake vials in holder to mix reagents well. The absorption
readings must be made within four hours after terminating
the reactions.
16. Pool the three negative controls and read the absorption
at 405 nm in a photometer against a blank of distilled
water. Record the result as absorbance units X 1000. If
the negative control reads greater than 600, the assay is
unsatisfactory and must be repeated. When a flow-through
photometer is used and strong positive samples are
encountered, the curvette should be rinsed with distilled
water before reading the absorbance of the next sample.
When nondisposable curvettes are used, the curvette must
be rinsed with distilled water following any positive
reading.
17. With the pooled negative control, adjust the instrument
to 0 absorbance. Determine the absorbance of each
reaction mixture, recording the results as absorbance units
X 1000. With some photometers, the instrument cannot be
adjusted to 0 with the pooled negative control samples.
In this case, the reading of the negative controls must be
subtracted from the reading of each sample.
Evaluation of Results
Unknown test samples whose absorbance units X 1000
values are greater than 100 are considered to be reactive, using
the pooled negative controls as a blank. It may be desired to
repeat the test on samples considered reactive. Before classi-
fying a reactive serum as positive for antigens associated with
hepatitis, confirmation must be obtained by testing with the
Cordis Confirmatory Test Set (Cat. No. 783-950). This assay
must be performed on all reactive samples. A reactive serum,
- 32 -
1~6~281
1 confirmed by neutralization with horse antiserum, must be
considered positive for hepatitis B antigen. As is noted above,
confirmation testing is required in order to evaluate the
results. Confirmation testing is required by the Food and
Drug Administration as well as by the laws of many states.
Briefly, confirmation testing in accordance with the present
invention is accomplished as follows:
The positive test sample is tested in duplicate. After
the first step in which the sample is exposed to the disc in
each vial, following washing, one disc is exposed to antibodies
specific for the hepatitis antigens; and, the other disc is
exposed to normal horse serum. After one half hour of
incubation, the subsequent procedures are identical to those
carried out in the routine screening assay. If a sample is
positive for hepatitis, the specimen which was exposed to the
horse antibody will have a very low value; whereas, the disc
exposed to the normal horse serum will have a high value
equivalent to that found in the routine screening procedure.
Samples which have low concentrations of the hepatitis
antigen, tend to have low final read out values. Those which
have a high concentration of hepatitis antigens will have
maximum values. Within a narrow range of concentration differences,
the resulting read out optical densities will tend to give
quantitative information concerning the concentration of
hepatitis antigens present in the sample.
Limitations to the Procedure
l. Nonrepeatable reactives: If repeat testing on a reactive
sample shows the value is less than the lO0 cutoff value,
the test is presumed to be a nonrepeatable reactive and
is considered negative for antigens associated with hepatitis.
- 33 -
6Z~
1 The original result may be due to errors in technique, such
an inadequate washing.
2. Nonspecific positives: The nonspecific false positives
resulting from antibodies present in certain human sera
that react with horse globulin are essentially eliminated
by using horse globulin in the test sample diluent.
3. Plasma from blood collected in EDTA should not be used.
It should be noted that the incubation periods for
various steps and the temperature at which incubation is
performed can be accomplished over a wide range of times and
temperatures. Thus, this invention is not intended to be
limited in any way to the time and temperature of incubation.
For example, the time of the incubation of the insoluble
member with the sample may range between 10 minutes and 24 hours,
with the temperature of incubation ranging between 2C and
50C. On the other hand, the time of incubation of the
insoluble member with the enzyme tagged binding partner ranges
between 30 minutes to 24 hours, with the temperature of
incubation of the insoluble member with the enzyme tagged binding
partner ranging between 2C and 50C.
It should also be noted that the process of the
present invention can be used to determine the presence of
antigens associated with hepatitis in any body fluid where
the antigens are present. Thus the process can be used to
determine the presence of these antigens in serum, plasma,
components of plasma, components of serum, urine, saliva,
and cerebrospinal fluid.
Results
A representative showing of the results of tests
performed in accordance with the present invention appear below.
- 34 -
~6281
1 The results shown in Table A are the results of tests in
accordance with the present invention for samples which test
positive for hepatitis by the CEP method. The values given in
Table B are the values for test samples which test positively
for hepatitis by the radioimmunoassay method yet negative
for hepatitis by the CEP method and the values given in
Table C test negative for both the RIA and the CEP method.
The numerical values given in Tables A, B and C for the various
- test samples are the values obtained by testing with the
procedure of the present invention.
TABLE A TABLE B TABLE C
CEP+ RIA+ CEP- Neg
.
Test (O.D. x 1000) Test (O.D. x 1000) Test (O.D. x 1000)
Sample oD405 SampleoD405 Sample oD405
72 2807 65 2745 88 0
73 2802 68 2675 89 0
79 2815 69 2266 90 0
82 2822 75 1354 9I 23
121 2831 77 265 92 42
201 2768 81 953 93 0
202 2780 87 1383 94 0
20205 2749 1231688 97 38
206 2758 1242739 98 0
208 2795 1251741 99 0
209 2800 204 285 100 0
210 2764 2432466 101 0
213 2776 2612636 102 0
214 2804 103 0
217 2795 104 o
218 2800 203 o
219 2746 207 o
220 2718 211 8
226 2695 212 o
228 2782 215 o
30232 2760 216 o
234 2774 224 0
235 2764 225 o
- 35 -
1 TABLE A (contd.) TABLE C (contd.)
.
CEP+ Ne~
. _ _ _ _ _
Test (O.D. x 1000) Test (O.D. x 1000)
Sam~le OD405 Sam~le OD405
. . . _ . .
238 2760 227 0
239 2755 229 0
244 2730 230 0
250 2707 231 0
251 2761 233 45
252 2778 240 0
253 2783 241 0
254 2742 242 0
255 2743
256 2784
260 2775
The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The present embodiment is therefore to be considered
in all respects as illustrative and not restrictive, the scope
of the invention being indicated by the appended claims rather
than by the foregoing description, and all changes which come
within the meaning and range of equivalency of the claims are
therefore intended to be embraced therein.
- 36 -
~., - -
