Note: Descriptions are shown in the official language in which they were submitted.
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The present invention relates to a method for assaying the
concentration of antigen or antibody in a sample by an immunoassay
technique. More particularly, the present invention relates to
automated immunoassay techniques for measuring antigen or antibody
concentration via selective antibody-antigen reactions, under -
conditions which permit extremely rapid analysis.
In Canadian application Serial No. 273/303r filed
Marc~ 7, 1977, applicants disclosed and claimed a technique
and apparatus for radioimmunoassay of antigens wherein the antigen
containing solution was admixed with a solution of a known concen-
tration of an antigen tagged with a radioactive isotope and a
solution of a known titer of an antibody reactable with said antl-
gen. Further consideration of the apparatus and technique has now
led to the understanding of its applicability ~or the assay of
antibodies as well. Moreover, it is now understood that by modi~y~
ing the originally disolosed apparatus and proce~s, good results
can be attained either by radioactive tagging of the antigen or
antibody, or by tagging the antigen or antibody with one of a
multiplicity of suitable tagging agents, such as ultraviolet,
visible or infrared absorblng compounds, radio-opaque dyes, enzymes,
proteins, fluorescent, luminescent or radioluminescent compounds,
other antibodies or other antigens, etc. Moreover, it~is now
under~tood that the technique of making the ~irst measurement ;
which activates the timing mechanisms and which, thereby, aativateB
the control mechanism for the process, need not be a radioactivity `~
sensor but can be any of a multitude of suitable detection tech- ;
,: . .
niques.
~t is the purpose of the pre~ent disclo~ure to discus~
~, :
these important improvements to the original system.
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Accordingly, one object of the present invention is to
provide a fully automated and continuous assay method for the
rapid and accurate determination of antigen or antibody through
specific antibody-antigen interactions, using a novel immunoassay -
technique.
Another object of the present invention is to provide
apparatus which is designed for the rapid~ automatic immunoassay --
of antigen or antibody by specific antigen-antibody reactions.
Briefly, these objects and other object~ of the Lnvention
a~ hereinafter will become more readily apparent can be attained
by a process which compri~es incubating for a predetermined time
period, a multiplicity of sample solutions, each of which com-
prises a mixture of ~a) a sample possibly containing an antigen
intended to be assayed~ ~b) a solution of a ~nown aoncentration
o~ a tagged antigen; and when assaying or antigen, optionally,
(c) a solution containing a known titer of antibody reactable with
said antigen, the concentration of ~aid antibody being selectsd 80
. .
as to ~orm an inaubated sample contalning an antibody-antigen com-
plex, and possibly containing unreacted antigen and tagged antigen,
i~ said sample contains an antigen7
directing said incubat~d sample into an activating detector
whioh i8 capable of determining the presen¢e o said ~ample and
whlah is capable of activating a timing means upon detection of
~aid sample in sald detector;
isolating said incubated sample from any additional samples
,
being incubated on a predetermined ~ignal from sald timing means;
separating said incubatsd solution into a fir~t portion
containlng antigen or antibody which has not been complexed during
the said incubation and a ~econd portion containing antigen or
antibody which has been complexed during ~aid incubation;
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directing one o~ said portions into a measuring detector, .
detecting the amount of tagged material in said portion while
said portion is maintained in a virtually static condition, and
recording said measurement; ~:
evacuating said portion to waste; : :
directing a subsequent incubated sample into said activat-
ing detector on a second predetermined signal from ~aid timing
means so that the next subsequant incubated sample to be a~ayed
arrives in the activating detector ~or the next measurement after
the previous measurement is completed; and
comparing the measurement with precalibrated values to
determine the quantity o~ antigen being assayed.
~his pro¢e6s aan be e~featively carried out by the u~e o~
apparatus which comprises means for time controlled incubation of
a multiplicity o~ sample solutions, each of which comprise a
mixture o~ ~a) a sample, possibly containing an antigen or anti-
. body intended to be assayed; (b) a solution of a known concentra-
tion o~ a tagged antigen or tagJed an~ibody;.and when assaying
~or antigen, optionally; ~c) a solution of a known titer of an
antibody reactable with said antigen;
activating detector means for determining the presence of ;~
the incubated iample and or a¢tivating a timing m~ans upon detec-
tion of said sample at a detection station; :~
:, .
means ~or lntroducing said incubated sample into sald :~
::activating detector means; ~
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i~ timing means, which is activatable by said activating
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detector means when~sald detector meanE detects the presenc~ of ~ ;~
: the inaubat~d sample at a det~otion ~tatlon, ~or generating con~
.
~rol slgnals at predetsrmined tlme ~equence~7 .:
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means for separating said measured solution into a fir~t ~ -
portion containing antigen or antibody which has not been com-
plexed during the said incubation, and a second portion containing
antigen or antibody which has been complexed during said incuba-
tion;
mean~ for introducing one of said portions from said
separating means into a measuring detector means;
measuring detector means for detecting the amount of
tagged material in said portion while said portion is maintained
in a virtually static condition;
recording means coupled to said detector means for record-
ing the amounts o~ tagged material in said measuring detector
meana T
means for isolating said incubated sample from additional
sample solutions introduced into said apparatus for assay, wherein
said i~olating means is activatable by a control ~ignal emitted
from said timing means, whereby said isolating means lsolate~
the system at a first predetermined ~ignal from said timing means,
and whereby said isolating means reconneats the system to ~ald
inaubation means at a second predetermined signal from ~ald timing :
means, wherein the second predetermined time period i~ ~et 80 as
to enable suffi¢ient time to complete said measurement beore the
next ~ncubated sample arrives in the said detector means for the
next sequential measurement; and - ~:
means for flushing said sample from said system af~er said
measurement thereof 80 a~ to prepare ~aid detec~or for the next
sequential ~ample for said measurement.
- Figure 1 is a schematic diagram of one type of apparatue
according to this invention;
Figure 2 i~ a ~tandard curve for cyclic AMP;
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Figure 3 is a ~tandard curve for cyclic GMP;
Figure 4 is a standard curve for Digoxin;
Figure 5 is a comparison of commercial Digoxin standards
with standards determined by the present invention;
Figure 6 is an Angiotensin I standard curve;
Figure 7 is an insulin standard curve; ~ ~ -
Figure 8 is a thyroxine standard curve;
Figure 9 i~ a measurement of anti-¢yclic AMP antibodles;
Figure 10 is a standard curve for Digoxin using anti-digoxin ;~
separating column;
Figure 11 is a standard curve for Digoxin using a rabbit,
anti-sheep IgG column.
In the ~ollowing description, the reference numerals refer
to the numerals in the ~everal Figures
According to thls invention, immunoassay is carried out
continuously and automatically, including reagent addition, sepa-
ration o~ bound ligand from the total lncubation mlxture, and
~inal measurement determinations on line without any human inter-
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- vention. The first results become available within 3-30 minute~,
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20 and ln some instances, ~a~ter, and a new sample i9 theXea~tflr ;:
processed every one and one-hal~ to three minutes. One system
according to the present lnventlon can process over 400 sample~
per day, a rate which would be di~icult to achieve by several ~ ;~
~echnicians using presently available conventional equipment. ~ ' -
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n accordan¢e with~the present embodiment of thls inven~ion ~:
immunoassay of a multiplicity of ~amples is achieved. ~he sample
whlch is suspeated to contain an antigen, o~r an antibody to be
,
~ ~ assayed, i8 first mixed wlth a solution of a detectabl~ antigen :
,
~ or antibody. The antigen or antibody can be made detectable by
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~ reacting a detectable llgand therewlth, such a~ a radioactiv~
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isotope, a fluorescent compound, a luminescent compound, a bio-
luminescent compound, an enzyme, another antibody or another
antigen or by a variety of known techniques. i'
The mixture is incubated so that the detectable antigen or
antibody is complexed with the antibody or antigen, if any,
in the sample being assayed. It is possible, for instance, to
use a detectable antigen to complex with poss:ible antibodies in
the sample. Alternatively, it is possible to use a detectable
antibody to complex with possible antigens in the sample. Still
alternatively, it is possible to mix and incubate an antibody
solution and a solution of detectable antigen with the sample
being assayed, which is suspected of containing antigen, so that
the detectable antigen competes with any sample antigen or the
available antibody.
The measurement is e~fected by reliance on the tagging. In
U.S. Patent Number ~,022,577, the "tagged" materials consisted
only of radioactive isotopes. To the extent that the sample
being assayed contained antigens, the antigen would compete
proportionately with the radioactively tagged antigen for the
available antibody. Separation of the incubated sample into two
portions, one containing antigen-antibody complexes and the other
containing unreac~ed antigen, and measurement of the quantity of
radioactivity present in one of the portions gave a proportional
measurement of the quantity of antigen present in the sample
being assayed.
Although tagging with a radioactive isotope is an effective
means of making the measurement, it is now understood that there
are many other tagging means that might also be used. For instance,
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suitable tagging techniques which can be used include tagging with -
ultraviolet, visible or infrared absorbing compounds, enzymes,
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enzyme substrates, fluorescent compounds, lumineccent or bio-
luminescent compounds, proteins, other antibodies or other
antigens. If the sample being assayed is suspected of contain- ~;
ing an antigen, then the tagged antigen solution which is used
in conjunction with the solution containing a known titer of
antibody, may be tagged wi~h a dye, ~or in~tance. In this case,
after separation of the complexed antibody-antig~ns from the
unreacted antibody and antigen, a proportional measurement can
be made using a colorimeter of a photometer to determine the
a ratio of color intensity in one of the samples compared with the ~ ;
maximum or minimum color intensity, as the case may be, if the
sample being assayed contained no antigen.
I~ the antigen is tagged with an en~yme, then the mea0ure-
ment aan be made by permitting the en$ymatic activi~y o~ one o~
the portions to act on a substrate to which the particular enzyme
is specific. The resulting product is then measured.
Tagging with fluorescent compounds enables measurement of
the proportioning of the complexed rom the non-complexed portions
by use of a fluorometer. Tagging with other antibodies enables ;~
measurement bylmeans capable of detecting qaid antibodie~. Tagging
with other antigens enables measurement by means capable o~ detect-
ing~said antigens.
- Thus, whereaq 1n the parent application, tagging was recog-
nized a~ belng limited to use of radioactive isotopes, and that
.. ..
such type of taggLng~provided quite superior re~ults, it is now
understood that a wlde variety of di~ferent tagging techniques
could also be suitably usèd.
The qelection of the tagging material i9 made on ths basis
o~ itq ability to react with the parti¢ular antigen or antibody -~
.
being tagged, and on the availability of instrumentation which
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10884z~ UVA-3
can distinguish between varying concentrations of the tagged
material. Moreover, of course, the tagging material selected
must not adversely interfere with the antibody-antigen reaction.
Suitable tagging materials which can be used include
enzymes, such as ca~alase, glucose oxidase, alkaline phosphatase,
hexokinase; fluorescent compounds, such as fluoresceine,
umbelliferone, quinine, NADTH, Naphthalene; proteins, such as
histone, gelatin, casein; and antigens, such as ATP or dinitro-
phenol. Al~o useable are radioluminescent compounds, ~uch as
2,5-diphenyloxazole; radioopaque dyes such as iodopaque, and
the like.
Following the incubation of the samp]e with at least a
solution containing the tagged antigen or tagged antibody, it
is neces~ary to separate out the antigen-antibody complex
formed, or to separate out the unreacted antibody, or the
unreacted antigen. Alternatively, i~ the incubatlon i8 of a
mixture of sample, detectable antigen solution, and a known
quantity of antibody, then the separation might be of the
combination of unreacted antibody and antigen-antibody complex.
If the incubation i~ of a mixture of detectable antigen solution
and a sample po~ibly containing an antibody, the incubated
mixture will contain the antigen-antibody complex and unreacted
antibody and unreacted antigen. One might separate out the
antibody, and the antigen-antibody complex, or one might
separate out the antigen and the antigen-antibody complex.
The separation of the desired component is conventionally
carried out by adding an adsorbent to the components, or by
precipitation or filtration technique~. The ad~orbent might
be specific for the antigen, or it might be ~pecific for the
antibody. A very wide range of ad~orbent materials may be
used for this purpose, as is well known in the art.
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Following a separation of one of the components of the
incubated mixture, the remainder is measured for the presence
of detectable antigen or detectable antibocly. The quantity in
the measurement is then a measure of the relative quantity
of antigen or antibody contained in the sample.
In U.S. Patent Number 4,022,577, it was considered
: . :
necessary to use a solution of known concentration of antibody ~;
in combination with a solution of the tagged antigen and the
sample solution. It is now understood that while very high ;~-
10 sansitivity is attainable with such systems, the use of a - ;~
solution of known concentration of tagged antigen can be
dispensed with for specific systems. Specifically, if the
sample is being assayed for speciEic antigens, it is sometimes iJ'"'
superior to use a tagged antibody alone. The antigen solution
could be dispensed with if tagged antibody is available and
by use of a means for separating antibody from antigen-
complexed antibody (e.g., a column containing immobilized ;;
antigen).
In U~S. Patent Number 4,022,577, it was disclosed that
: .. ... .
radioactive isotope tagging can be used for the dual purposes
of measuring the proportions of complexed and unreacted anti-
bodies and antigens, and for process control o~ the system. ~ ;
Thus, the total incubated mixture, including total amount of
tagged antigens, the solution of the antibody and the sample
being assayed, were moved into a radioactivity detector means.
. . .
The appearance of the incubated mixture in the crystal detector
I activated a timing mechanism which in turn activated certain
¦ valves which isolated the measuring and incubation sections of
the system. The timing mechanism also controlled other
processing conditions
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The same radioactive isotope tagged material was also
used to measure the proportioning of the complexed antibody-
antigens, from the unreacted materials. Thus, after the
incubated mixture was passed through a radioactivity detector
to set the system controls, it was then passed into a separation
means to separate the complexed from the uncomplexed antibody-
antigen combinations. One of the portionls was then tranRferred
to the radioactivity detector means in or~er to determine the
degree of proportioning. In contrast, it is now understood
that for specific systems, different criteria can be relied
upon to measure the presence of the sample for purposes of
activating the timing mechanism, and for measuring the degree
of proportionation.
A detecting station is loaated subsequent to the
lncubation apparatus. When the sample arrlves at the detection
station, its presence mu~t be sensed and aontrol signals
activated. IncZtead of relying on radioactive isotope detection
to determine the presence of a sample at the detection station,
it is possible to rely on any of a number of alternate criteria
for making a similar determination and system activation. For
instance, since each incubated sample i9 separated by a space
containing a buffer solution, it i8 possible to measure the
dierence in conductivity between the buffer and the incubat~d
mixture. Thus the arrival of the incubated mixture at the
detection system is announced by an abrupt change in electrical
condùctivity or by an abrupt change in pH, since the pH of the
incubated sample will be different from the bufer ~olution
spacers between samples. Alternatively, the differing polari-
zation of the sampIes and bu~fer can be utilized. Other
suitable techniques include the concept of admixing a meaYurable
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cation into the sample or into the buffer, or by the addition
of various absorbing compounds, fluorescent materials, lumine-
scent materials, into the sample or buffer, or by reliance
on the differing refractive indices, differing dif~raction
patterns, which differences can be enhanced by the addition to
either the buffer or the sample of inert particles, emulsifiers,
or even small air bubbles. Alternatively, the same tagged
material which is used to measure the proportioning of the
incubated sample between complexed and non-complexed antibodies
and antigens, can be used to measure the presence of the
incubated sample at the detection station. Thus, the activating
detector means may be a con~uctivity detector, p~ meter, a
photodetector, a re~ractometer, fluoroscope, a lum:Lnescent
detector, or the like.
~ hus, the sample i8 mixed, incubated and pa~3ed inko a
detection station. The pre~ence of the incubaked ~ample is
detected by change in electrical conductivity, photometrically,
diffraction, etc. or by the presence of the tagged material.
The detector activates the timing means which controls the
system valves. The incubated mixture i8 then passed to a
separator where the complexed and non-complexed materials are
~eparated into two portions, and one portion is passed into a
measuring detection means wherein the quantity of tagged material
i8 detected.
A wide variety of antigens and antibodies and complexe~
: ; of the same can be treated by the methoas of this invention.
For in3tance, suitable antigens and their corresponding anti-
~ bodie and complexe~ which can be separated include:
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Pituitary hormones Calcitropic hormones
Growth hormone Parathyroid hormone (PTH) ~ :
Adrenocorticotropic hormone (ACTH) Calcitonin (CT) .:.
Melanocyte-stimulating hormone (MSH) Gastrointestinal hormones
a-MSH Gastrin
~-MSH Secretin
Glycoproteins Cholecystokinin-
pancreozymin (CCK-PZ) -
Thyroid-stimulating hormone (TSH) ..
Follicle-stimulating hormone (FSH) Enteroglucagon :
Luteinizing hormone (LH) Vasoactive tissue hormones ~.;
Prolactin Angiotensins ` `
Lipotropin (LPH) Bradykinins
Vasopressin Hypothalamic-releasing factors .
Oxytocin Thyrotropin-releasing
~actor (TRF)
Chorionia hormones
~Iuman chorionic gonadotropin (HCG) Steroids
Human chorionic somatomammotropin Aldosterone
(HCS) Testosterone
Pancreatic hormones Dihydrotestosterone
Insulin Estradiol
Proinsulin Estrone ~
C-peptide Estriol
Glucagon 2-Hydroxyesterone
Prostaglandins Enzymes ;
Thyriodal hormones Clesterase .
Triiodothyronine Fructose 1,6-diphosphatase
Thyroxine
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DrugsVirus
Digoxin Australia antigen (HAA)
Digitoxin Tumor antigens -
Morphine Carcinoembryonic antigen ~ ;
LSD a-Fetoprotein
Cyclic nucleotides Serum proteins ~ :
cAMP Thyroxine-binding ::
globulin
cGMP Immunoglobulin G (IgG)
cIMP
Albumin
cUMP ~ :
Other
Intrinsic factor
Rheumatoid factor .
Folic acid
Naurophysin
Instead of a single antibody or a single antigen, a
plurality of different antigens or antibodies can be
simultaneou~ly assayed by using two or a plurality of diffarent
tagged antigens or tagged antibodies.
The ~ample intended to be assayed may have been obtalned
from such biological 30urces as blood, sera, urine, plasma, ..
ascities or the like which is suspected of containing specific
antigens or antibodies.
The antibody or antigen solution of known concentration
which is tagged by the tagging material can be ob~ained from
known, commercial sources, or can be prepared by techniques
whiah axq known in the a~t. The concentration o~ tha antibody
is selected so as to be insuf~ic1ent to react with thls total
quantity of the antigen in the ~ample being assayed.
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Alternatively, if the antibody is being assayed, then the
tagged antigen solution is provided in excess of the expected
antibody concentration. In the case of tagged antibody it is
provided in excess of the antigen concentration being measured
(See Miles, L.E.M. & Hales, C.N. 1968 Nature 219, 186-9).
Referring now to the drawing of this application for
further understanding of the present invention: The invention
will now be described from the point of view of assaying for
an antibody using a radioactively tagged antigen. The
detection of the sample is measured by conductivity changes
and the measurement of the sample after separation of the
complexed from non-complexed antigen and antibodie~, is made
by radioactivity detectian. It will be under~tood, however,
that the apparatus and procedure i8 e~ually appllcable to
assaying ~or an antigen using a tagged antibody, or assaying
for an antigen using a tagged antigen and a solution of a
known concentration of an antibody.
A tagged antigen containing solution is maintained in
container 1. Isotopic tagging of the antigen can be effected
by conventional techniques such as by reacting the antigen
wlth moieties that wlll leave residual amounts oE 125I or 131I,
or any other suitable radioaative isotope a~ is known ln ths
art guah as 75Se, 3H, 14C or 32p. Alternatively, non-radio- ;
active "tags" may be used provided that there are suitable
; detection means. Examples are luminescent, biolumine~cent, -
ultraviolet, visible and infrared adsorbing compounds, etc.
The concentration of the tagged antigen solution can vary
~rom one millimole to the limits of concentrations of isotope
.
detectable by the radioactivity detection, usually one femtomole.
~ . . .
Instead of a single antibody or a single antigen, ~wo or a
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plurality of different tagged antigens and different antibodie~
can be used in combination to give a simultaneous multiple
assay. Representative of the tagged antigen systems which
might be used include: 125I-Digoxin, 131I-Thyroxine,
125I-secretin, 32P-cyclic AMP, 131I-Insulin, 125I-Glucagon,
75Se-Cortisol, 125I-Angiotensin I, 125I-Carcinoembryonic
antigen, 125I-Somatostatin, 131-I-Insulin, 131I-Triiodo-
~hyronine, 125I-Thyroxine, 125I-Growth Hormone, 125I-cyclic
AMP, 131I-cyclic GMP, 125I-Morphine, 125-Vasopressin,
131I-Aldosterone derivatives and their respective antibodies.
The antigens and antibodies could be contained in sera, urine
or buffers commonly u~ed in the presently known art of
radioimmunoassay such as sodium acetate, Tris-HCl, Barbital,
Phosphate, MES, TE~, etc. The samples pos~ible containing
the antibody are piaked up by pipette 3. The ~ample being
assayed could be measured at several dilutions such as 1
1:2, 1:5, 1:10, 1:100, 1:1000, etc. A buffer so1ution i~
maintained in cup 5. Intermittently, ~amples of antibodie~
to be assayed are picked up at point 8 and brought into the
system by the peristaltic pump 17. The solution of tagged
antigen i9 picked up from 1 and mixing occurs in conduit 13.
Each intermedient sample is separa~ed by a ~pace filled with
a buffer solution which is taken ~rom 5.
After the solutions are mixed by the pumping action,
the mixture is passed through conduit 13 and into an incubation
chamber 19 where the mixture will be held under standardlzed -
conditions for a fixed predetermined incubation time period.
The incubation cham~ber 19 may take many forms, however,
one of the most advantageou~ is a long coil-like conduit, not
shown, which i~ held at a predetermined incubation temperature.
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The flow rate of the sample solution through the incubation
coil may be adjusted so that the reaction is sufficiently
completed, or at a constant fraction of the maximal reaction,
by the time the sample solution traverses the length of the
conduit. A multitude of sample solutions can be present in
the incubation conduit at the same time, each separated by
a buffer solution space. The buffer space not only separates
sample solutions, but al~o picks up tailings so that the next
succeeding sample will not be contaminated. Since the
solutions in the incubation coil are moving in laminar flow,
there is, of course, a potential flow problem with the fluid
in the center of the tube moving at a faster rate than at the
edges. Thls potential problem can be quite easily dis~ipated
by intermitten~ly introducing bubbles in regularly 3paced
intervals into conduit 13, through bubbler 14, which serves
to move the fluid along at a more uniform rate. This technique
is disclosed in Skeggs, U.S. Patents 3,797,149 and 2,879,141.
The incubation temperature will, of course, depend upon `
the particular antibody-antigen system under study. In general,
however, the incubation temperature may vary from 0 to 60C.,
and often incubation can be effected at room temperature.
The 1exibility o~ this ~ystem is quite excellent and
the system may be used for continuous assay of different
antibody-antigen systems. Thus, each sample solution extracted
and introduced into conduit 13 may contain a different antigen-
antibody system. It is~not even neces~arily required to
readjust the incubator conditions each time the antibody-
antigen system i4 changed. It is only necessary that the
calibration o~ the equipment for standardized samples be made
under the same conditions as thoq~ u~ed for the unkrlotm.
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That is to say, it is not necessary that the incubation
period be suf~icient for the reaction to go to completion.
It is only necessary that the incubation period for the samples
be the same as the samples used for calibration. This is in
sharp contrast to prior systems which, in general, required
the reactions to go to completion for success. The length of
incubation may vary from 1 minute to 30 minutes and even up
to 1 day, depending upon the particular system. In general,
if the incubation time iB unacceptably long, it i~ pos~ible
to speed incubation by the addition of more antibody or antigen
or altering the temperature.
Alternatively, the sample can be moved into one of a
plurality of containers which is held under predetermined
incubation conditions, after which a pipette or similar
device removes the incubated sample from the container and
moves it through conduit 29 toward the isolating valve 31.
At the termination of the incubation period, the sample
is passed through conduit 29 into an isolation va}ve or by-
pass valve 31. The driving orce or the movement of the
s ample through the sy3tem to this point ie u~ually due to the
pressure created by the peristaltic pump.
The isolation or by-pass valve 31 connects the conduit
29 with conduit 33 and, alternatively, with conduit 41, to
waste. At the start of the flow system, valve 31 is opened to ~
conduit 33 and the incubated sample is directed into a con- ~ -
ductivity detector 3. The conductivity of the fluid passing
through the conductivity detection station 3 is continuously
monitored. Since each sample is separated by a buffer solution,
the conductivity of the sample being assayed is neae~sarily
different from th~ buffer spacer. By detecting abrupt change~
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in conductivity, the detector 3 can determine the pre~ence
of a new ~ample. The conductivity detector comprises a
chamber containing a pair of platinum electrodes and has an
ohmmeter placed across the electrodes. As the solution passes
through the chamber, a change in resistance occurs which i~ `
detected by the electrodes. Upon detection of the new ~ample,
the detector triggers a predetermined timing sequence which
begins scaling the sample for a predetermined period of time,
usually 1 minute or less. This timing means 48 is set after
a comparator circuit 47 indicates that the threshold con-
ductivity is present. Control signals from the timing means
4a thereafter set off variou~ valves in a sequence nece~sary
to cause the sample solution to be brought through conduit 2
to a separator 51, where a portion of ree isotope tagged
antigen wl~ll be separated from a portion o~ antigen-antibody
and tagged antigen-antibody complexe~. Thereafter, one of the
portions will be ~rought through conduit 52, through fill valve
53 and conduit 56 into a ~mall reservoir cup 55 in a radio-
activity detector 35 where the amount of radioacti~ity i~ ~ ;
measured for a predetermined time period, about one minuteor les~.
The amount o~ radioactivity detected is compared to
standard curves which had been previously constructed ~rom
known titers of ~olutions. The present system assume3 that
flow characteristics are ~ufficiently constant and stable.
The timing for each sample is exactly the same and i5 not
influenced by small variations in the pumping rate, which can
occur over long periods of time.
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10884Z~ UVA-3
The timing means 48 measures predetermined time
sequences, sending out control signals through line 39 to
activate the isolation valve 31, which thereby isolates
the measurement portion of the system from the incubation
portion of the system. The time sequence measured before
valve 31 i5 activated, is sufficient to at least enable the .:
entire sample to pass into the detector 35 before the measure- ~:
ment system is isolated. In other words, the valve 31 will . `
usually close at some point in time as buffer solution spacsr~
which separate adjacent sample solutions to be measured are
passing through the val~e 31.
A recording means 49 is provided which is coupled to
the comparator circuit 47 and the timing mean~ 48, which is
used for recording the amount o~ radioactivity in the ~tatia
measurements. These mea~urement~ will be u~ed to determine
the amount of specific antibody in the unknown sample using a
computer 80 which is interfaced to the recording means 49 and
timing means 48.
After the isolation valve 31 is closed, thereby cliverting
the feed from conduit 29 into waste conduit 41, a mean~ can be
provided for increasing the ~peed o the sample solu~ion
already in conduit 33. In FI~URE 1, thi~ means takes the
orm o~ a high flow rate buffer solutlon inlet valve 43 which
is opened simultaneously as the isolation valve 31 ls closed,
by a predetermined signal from timer 48 from line 45. A -
: ~ solution at relatively high speed enters through conduit 44
into the conduit 33. The high flow rate buffer solution pu~hes :~
ths sample ~olution through conduit 2 into and through a
eparator column 51. This high flow rate may be needed becau~e ::
the re~istance of the 3eparator column might otherwi~e prevent
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lOB~Z~ ~
free flow of the solution therethrough. This high speed
turbulent buffer solution also acts to wash any tailings
from the sample solution into the separator column 51, so as -
to prevent contamination of subsequent sample solutions. ~n
general, the high flow line can feed buffer at a rate of 0.5
ml/min to 50 ml/min and preferably 3 ml/min to 10 ml/min.
The flow rate through the separator column 51 is controlled
by the absorbent properties of the column and the hydrostatic
head created by the outlet of conduit 54 when the air trap
valve 85 is open to connect conduit 86 with conduit 84 which
is at atmospheric pressure. The air trap valve 85 is energized
simultaneously as the high flow buf~er inlet valve 43 is
actuated and the isolation valve 31 is closed. This causes
the hydrostatic pressure to increase in the column 51 and
thus the rate of 1Ow through the column 51 increases. The
flow rate now clearly equals the flow rate of the high flow
buffer line 44.
The separator column 51 serves to separate the sample
solution into two portions: a irst portion containing
unreacted antigen which has been tagged with the isotope, and
a second portion containing the antibody-tagged antigen complex.
This separation can be accomplished by a variety of means, some
conventional and some not conventional, such as that described
in U.5. Patent Number 4,022,577.
,: ... ~: . - :.
The residual sample is eluted from separator 51 by the
high flow rate buffer and passes through conduit 52 into fill
valve 53 which had been opened by a predetermined signal from
circuit 90 from timing means 48. Once the entire eluted
portion passes through the fill valve 53, it is again closed,
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thereby divertiny additional buffer coming from the separation
column 51 to waste through conduit 54. The column is now
ready to receive the next sample. The po:rtion being measured ~.
is thus passed into the static measurement section 55 of the
radioactivity measurement detector 35 through conduit 56. ~ ~-
The static measurement section 55 is a cup having an inlet
apperture 57 situated at the base thereof. The residual
sample flows from valve 53 through line 56 and into the cup
55 through lower aperture 57. After the separated portion
of the sample fills the cup, a radioactivity level Coullt ~or
a predetermined time which is controlled by timing mearl~ 48
i5 made and recorded by recorder 49.
At a predetermined time during the time interval, when
the radiatlon level i8 being determlned, timing means ~8
sends a control signal to by-pass valve 31 which reconnects
conduit 29 with conduit 33 and permits the next sequential
sample ~olution to begin its transit to the detector 35. At
a predetermined time after conduit 29 is reconnected with
conduit 3.3, the timing means 48 sends a signal and valve 59
is opened, whioh connects conduit 61 with conduit 63 which 1
connected to a vacuum source developed by the peri~taltia
pump pumping.
The solution in the cup 55 is thereby rapidly evacuated
through exit port 65 and is disposed of. The control
mechanism can be set, if desired, such that at the completion
of one control sequence, and hence completion of analysis of
one sample, the indication level must return to a predetermined
minimum base line before the mechanism can be reaativated to
. begin the analysis o~ the next sample.
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10884Z~ WA 3
In the above-discussed arrangement, the inside walls of
cup 55 may be made of a non-adhering material such as Teflon
or polyphenylenesulfide. Moreover, the clesign of the cup with
the inlet and outlet apertures situated at the base of the
cup, functions to avoid splashing so that: the cup will empty
quite cleanly even without the introduction o~ the buffer wa~h
solution. Simultaneous with the activation of valve 59, the
timing means sends a signal to stop the recording means 49 and
causes the recording means to transfer the accumulated counts
to computer 80 for data processing as determined by the soft-
ware programs. The recording mean~ 49 then resets and is
ready to record data for the next sample.
Before the isolation valve 31 i8 reopened, the high flow
rate bufer inlet port 44 i8 closed by a control signal from
the timer. When the isolation valve 31 i~ reopened, connec~ing
conduits 29 with 33, the pressure in conduit 33 approximates
that in conduit 29 so that there is no sudden backwash through
the incubation system. Similarly, when the by-pass valve 31
is in its closed position, thereby connecting conduits 29 with
41, the pressure in conduit 41 approximates that in conduit
~ 33 since the outlets of conduit 41 and conduit 54 are physiaally
; held at the same hydrostatic pxessure head. Thus, the flow
rate of sampleo is not altered when valve 31 is energized or
j deenergized. A timing sequence provides for a difference in
time between the shut off of the isolation valve 31 and the
shut off of the high flow stream valve 43 and energizing of
the column air trap valve 85 sufficient to allow conduit 33
to reach the ~ame hydrostatic pre~ure as conduit 29 so that
backflow or alteration in flow rata does not occur when the
conduits 29 and 33 are reconnected.
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The major advantage of the present method i6 tha~ it
provides a fully automated procedure for the measurement of
antibody or antigen concentration in a test sample, in only a
fraction of the time previously required by conventional
immunoassay techniques. The present metho~d makes it possible
to run a series of tests of widely varying antigen-antibody
interactions without any disruption in continuous operation
of the apparatus. Consequently, the system does not require
the control of a skilled operator, and even unskilled labor
can be used to perform the relatively simple taskc required
to set up the present system. Since there are litera~ly
hundreds o~ drugs, hormones and biochemically important
aompounds currently measured by manual immunoassay such aa
analyses of digoxin, insulin, angioten3in I, thyroxine, cyclic
AMP, and the like, the present method provide3 a means for
rapidly and accurately conducting these analyses.
This system also allows the simultaneous detection of
several antibodies in the same sample. In this instance,
several different isotopes are used with the different antigens.
The radioactivity detector isotopes are used with the different
antigens. The radioac~ivity detector will thu~ detect the
different levels of radioactivity emitted by each o~ the
isotopes and by computerized selectivity, simultaneous deter-
minatlon of two or more antibodies can be made.
Having generally described the invention, a more complete
understanding can be obtained by reference to certain specific
exampl~q, whiah are incluaed ~or puxpose~ o~ illustrati~n ~nly,
and are not intended to be limiting unless otherwise specified.
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Assays for digoxin, cyclic AMP, cyclic GMP, insulin,
angiotensin I and thyroxine are easily performed with this
process. In these cases, the sample, isotope solution and
antisera were drawn for 30 seconds with a 2 1/2 minute wash
between samples. The timing means was preset. Air bubbles
were introduced at the rate of 0.32 ml/min into conduit 14.
The conduit 63 was pumped at 3.9 ml/min to create ~uction to
rapidly evacuate the static counting cell 55 when valve 59
was opened. Table I provides the essential details o~ the~e -
assays with regard to reagents, and flow rates. The
coefficient of variation for these assays was about 2%.
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EX~MPLE 1
FIGURE 2 ~hows a standard curve for cyclic AMP after the
standard~ had been acetylated (500 ml standard ~ 10 ml
triethylamine + 5 ml acetic anhydride~. This data and all
following data for standard cuxve~ is plotted with the con-
centration of ligand being measured on the abscissa ~log)
vs the ratio of radioactivity found for standards to that
when only the radioligand was present and i~ presented as the
~/Bo ratio. Whether or not the results are normalized by
correction using the irst aount (count 1) the end re~ult i~
the ~ame, since the fixst aounts would be the same i~ per~ect
pumping reproduclbllity occurred, Thi~ compound is thought
to be an important mediator o~ hormone action.
EXAMPLE 2 ;
FIGURE 3 demonstrates a standard curve for cyclic GMP
a~ter the standard~ have been acetylated. It has been sugge~ted
that cyclic GMP could be an important regular of processes
controlled by the parasympathetic nervous system. In addition,
some workers eel that cyclic GMP oould be an important
indicator of cell growth and it~ presence in urine could be
used to detect certain organ~ malignancy. The sensitivity
shown here is su~fiaient to measure cyclic ~MP in less than
1 microliter of human urine.
EXAMPLE 3
FIGURE 4 reveals a standard curve for digoxin. It takes
less than 4 minutes to do a single determination. Digoxin
i8 an important cardiac glycoside taken by between 3--5 million
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people in the USA alone. The drug markedly stimulates the
heart in people with congestive heart failure. However, the
drug i~ also very toxic to the heart causing rhythm disorders.
Serum digoxin levels of 1.4 Ng/ml are considered therapeutic
while toxic levels are considered when the serum level rises
about 2.5 - 3 Ng/ml. As can be seen, the assay methocl has
sufficient sensitivity to make this dist:Lnction. When
commercial serum dlgoxin standards were repetitively assayed,
an excellent correlation was found as shown in FIGURE 5.
In addition, this FIGURE demonstrates the excellent instrument
stability over a long period of time.
EXAMPLE 4
FIGURE 6 illustrates a standard curve for Angiotensin I.
While plasma levels are very low, the radioimmunoassay for
Angiotensin I is very useful to measure pla~ma renin activity
.. .
~PRA). Normal PRA is about 1-6 Ng/ml Angiotensin I/hour and
abnormal from lO-100 Ng/ml Angiotensin I/hour. It can be seen
that the method can easily make this distinction.
EXAMPLE 5
Insulin is an important hormone in glucose homeostasls.
Mea~urement of serum or plasma insulin can be o~ aid in the
diagnosis and management of patients with diabetes. The assay
of insulin normally takes several days uslng conventional
- techniques. FIGURE 7 demonstrates a standard ~urve for
insulin. The to~al assay time for an individual in~ulin
sample in the present system is only 21 minutes. This could
- be useful in cases where it is critical to know the serum
ineulln concentration in order to develop a therapeutic plan
or a diabetic patient in insulin imbalance. The l~nsitlvlty
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UVA-3
o~ this assay is sufficient to monitor insulin in the concen-
trations normally encountered in clinical medicine.
EXAMPLE 6
FIGURE 8 demon~trates a standard curve for thyroxin.
The sensitivity of the automated assay is comparable to other
radioimmunoassay~ for thyroxine.
EXAMPLE 7
The automated radioimmunoassay system o~ the pre~ent
invention is especially versatile being able to alternately
sequentially assay or different substances. The following
nine substances at the concentrations indicated were placed
in the sampler tray and their respective isotope solutions
were drawn as each sample was processed. Incuba~ion wa~ for
21 minutes at 39C. No delay between samples occurred and it
took 27 minutes for all nine samples to be drawn into the
instrument. Notice the excellent reproducibility and the
ability to switch between different antigens without any
equilibration time needed.
TABLE II
.
20 Sample No~ Compound ~ ml B/Bo
1 Angiotensin I 0 1.00
2 Angiotensin I 25 0.23
3 In ulin 0 1.00
4 - Thyroxin 0 1.00
5 ~ Insulin 25 0.45
6 Thyroxin 0 1,05
7 Insulin 25 0.42
B Angioten~in I 25 0.20
30 ~ 9 In~ulin 0 0.9S
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EXAMPLE 8
FIGURE 9 demonstrates the mea~urement of anti-cycli~ AMæ
antibodies in goat ~era harvested from go,ats immunized with
cyclic AMP chemically coupled to human serum albumin. The
present automated immunoassay system was used to assay ~or
anti-cyclic AMP antibodies using 125I-lablslled succinyl cyclic
AMP tyrosine methyl ester. Curve A i~ the dilution curve of
antiserum bled from a goat 17 days after a boo~ter immunizatio~.
B is the curve of antiserum bled 37 days after the same boost.
Similarly, curves C and D repre~ent anti-cyclic AMP antibody
binding in antLsera obtained ~rom a second goat bled 17~C)
and 37(D) days after boosting. E represents control serum , ,
obtaine~ ~rom an un-immunized goat.
EXAMP~E 9 ~ ,
FIGURE 10 demonstrates an immunoassay te~hnique or digoxln ~ ,
wherein the st~ndard curve ia obtained using a separation ~-
mean~ wherein antisera to digoxin ~raised in'~heep) i8
covalently linked to 50-100 mesh agaro~e beads.
EXAMPLE 10
~IGURE 11 demonstrate~ an immunoAs~ay technique for
digoxin. The initial inaubat:Lon utLlizes sheep anti-digoxin
serum. Separation is achieved on a aolumn containing im-
- mobilized Rabbit anti-sheep IgG. ''
Having now fully described the inven~ion, it will be '~, ,,
apparent to one o~ ordinary,skill in the art that many changes ' '-
.
; and modiiaations aan be,made thereto without departing ~rom
the spirit or scope oe the invention as set forth herein.
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