U.S. patent number 4,298,685 [Application Number 06/035,619] was granted by the patent office on 1981-11-03 for diagnostic reagent.
This patent grant is currently assigned to Burroughs Wellcome Co.. Invention is credited to Pedro Cuatrecasas, Indu Parikh.
United States Patent |
4,298,685 |
Parikh , et al. |
November 3, 1981 |
Diagnostic reagent
Abstract
A sample, e.g. serum, containing an antigen, hapten or other
biological substance is mixed with antibodies raised against that
substance which have been tagged with biotin, and with a known
amount of that substance labelled with an enzyme. After the
competitive complexation of the antibody with the labelled
substance and the substance to be detected, avidin, immobilised on
an inert support is added. The avidin binds to the biotin and
causes the complex to be precipitated. The solid and liquid phases
are separated by centrifugation and the amount of biological
substance in the original sample is determined by measuring the
activity of the enzyme in either phase.
Inventors: |
Parikh; Indu (Chapel Hill,
NC), Cuatrecasas; Pedro (Chapel Hill, NC) |
Assignee: |
Burroughs Wellcome Co.
(Research Triangle Park, NC)
|
Family
ID: |
10100551 |
Appl.
No.: |
06/035,619 |
Filed: |
May 3, 1979 |
Foreign Application Priority Data
|
|
|
|
|
May 4, 1978 [GB] |
|
|
17749/78 |
|
Current U.S.
Class: |
435/7.5;
435/7.93; 435/188; 435/810; 435/975; 436/527; 436/529; 436/530;
436/531; 436/544; 436/547; 436/808 |
Current CPC
Class: |
G01N
33/538 (20130101); G01N 33/535 (20130101); G01N
33/537 (20130101); Y10S 436/808 (20130101); Y10S
435/81 (20130101); Y10S 435/975 (20130101) |
Current International
Class: |
G01N
33/536 (20060101); G01N 33/535 (20060101); G01N
33/537 (20060101); G01N 33/538 (20060101); C12N
009/96 (); G01N 033/54 () |
Field of
Search: |
;23/23B
;435/7,188,177,810,805 ;424/1,1.5,12,85,88 ;260/112R,121 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
3839153 |
October 1974 |
Schuurs et al. |
3852157 |
December 1974 |
Rubenstein et al. |
4017597 |
April 1977 |
Reynolds |
4134792 |
January 1979 |
Boguslaski et al. |
|
Other References
May et al. "NEO29-(t)-Biotinyl insulin and its Complexes with
Avidin", J. Biol. Chem., vol. 253, No. 3 (1978) pp. 686-690. .
Manning et al. "A Method for Gene Enrichment Based on the
Avidin-Biohn Interaction Application to the Drosophilia Ribosoma
RNA Genes", Biochem., vol. 16 No. 7 (1977) pp. 1364-1370. .
Heggeness et al. "Use of the Avidin-Biotin Complex for the
Localization of Actin and Myosin with Fluorescence Microscopy", J.
Cell Biol., vol. 73 (1977) pp. 783-788. .
Hofmann et al. "Avidin-Biotin Affinity Columns, General Methods for
Attaching Biotin to Peptides and Proteins", J. Am. Chem. Soc., vol.
100, No. 11 (1978) pp. 3585-3590. .
Bayer, et al. "Preparation of Ferriton-Avidin Conjugates by
Reductive Alkylation for Use in Electron Microscopic
Cytochemistry", J. Hist. Cytochem., vol. 24, No. 8 (1976) pp.
933-939..
|
Primary Examiner: Wiseman; Thomas G.
Attorney, Agent or Firm: Brown; Donald
Claims
We claim:
1. A process for the quantitative determination of a biological
substance in a test sample comprising,
(a) mixing said test sample, a soluble enzyme-labelled form of said
biological substance, and a soluble biotin-tagged antibody raised
against said biological substance,
(b) incubating the mixture under conditions suitable for forming an
antibody-biological substance complex,
(c) then adding isolubilized avidin, separating the resulting solid
phase from the liquid phase, and
(d) determining the enzyme activity of either of said phases.
2. A process as claimed in claim 1 wherein said enzyme of said
substance is selected from the group consisting of malate
dehydrogenase, staphylococcal nuclease, delta-5-ketosteroid
isomerase, yeast alcohol dehydrogenase, yeast glucose-6-phosphate
dehydrogenase, alpha glycerophosphate dehydrogenase, triose
phosphate, isomerase, horse radish peroxidase, alkaline
phosphatase, asparaginase, glucose oxidase, beta-galactosidase and
ribonuclease.
3. A process as claimed in claim 1 or 2 wherein the avidin is
insolubilised by covalent binding to a macromolecular insoluble
carrier.
4. A process as claimed in claim 3 wherein said macromolecular
insoluble carrier is selected from the group consisting of agarose,
polystyrene, polyacrylamide, nylon, cross-linked dextran and filter
paper.
5. A process as claimed in claim 4 wherein said macromolecular
insoluble carrier is agarose.
6. A process as claimed in claim 1 wherein the avidin is
insolubilised by physical coupling to an insoluble carrier.
7. A process as claimed in claim 6 wherein said insoluble carrier
is selected from the group consisting of glass beads, plastic
objects, the inside of plastic test tubes, the inside of glass test
tubes and microtitre plates.
8. A process as claimed in claim 1; wherein said biological
substance is an antigen.
9. A process as claimed in claim 1 wherein said biological
substance is a hapten.
10. A process as claimed in claim 1 wherein said biological
substance is digoxin or codeine.
11. A kit for use in enzyme immunoassay comprising,
(a) a quantity of enzyme-labelled biological substance,
(b) a quantity of biotin-tagged antibody raised against said
biological substance,
(c) a quantity of insolubilized avidin, and
(d) a quantity of substrate for said enzyme,
wherein said quantity of biotin tagged antibody is sufficient to
bind a substantial quantity of the enzyme-labelled substance, said
quantity of insolubilized avidin is present in excess of the amount
required to precipitate the biotin tagged antibody, and said
quantity of said substrate is sufficient to react with said enzyme
label to produce a detectable signal.
12. A kit as claimed in claim 11 wherein the enzyme is selected
from the group consisting of malate dehydrogenase, staphylococcal
nuclease, delta-5-ketosteroid isomerase, yeast alcohol
dehydrogenase, yeast glucose-6-phosphate dehydrogenase, alpha
glycerophosphate dehydrogenase, triose phosphate isomerase, horse
radish peroxidase, alkaline phosphatase, asparaginase, glucose
oxidase, beta-galactosidase and ribonuclease.
13. A kit as claimed in claim 11 wherein the avidin is
insolubilised by covalent binding to a macromolecular insoluble
carrier.
14. A kit as claimed in claim 13 wherein said macromolecular
insoluble carrier is selected from the group consisting of agarose,
polystyrene, polyacrylamide, nylon cross-linked dextran and
filter-paper.
15. A kit as claimed in claim 14 characterised in that said
macromolecular insoluble carrier is agarose.
16. A kit as claimed in claim 11 wherein the avidin is
insolubilised by physical coupling to an insoluble carrier.
17. A kit as claimed in claim 16 wherein said insoluble carrier is
selected from the group consisting of glass beads, plastic objects,
the inside of a test tube made from plastic and the inside of a
test tube made from glass.
18. A kit as claimed in any one of claims 11 to 17 wherein said
biological substance is digoxin or codeine.
Description
The present invention provides a quantitative method for the
determination of biological substances and more particularly,
provides an enzyme immunoassay of such substances in biological
fluids.
There is a continuous need in medical practice and research for
rapid, accurate, quantitative determinations of biological
substances at extremely low concentrations. The presence of drugs
or narcotics in body fluids, such as saliva, blood or urine, has to
be determined in very small quantities with satisfactory accuracy.
In addition, in medical diagnosis, it is frequently important to
know the presence of various substances which are synthesised
naturally by the body or ingested. These include hormones, both
steroids and polypeptides, prostaglandins, and toxins as well as
other materials which may be involved in body functions.
To meet these needs, a number of ways have been devised for
analysing for trace amounts of materials. A common method for
isolating and detecting substances in biological fluid is use of
thin layer chromatography (TLC), for example, in combination with
mass spectroscopy or gas phase chromatography. However, TLC has a
number of deficiencies in being slow, being subject to a wide range
of interfering materials, and suffering from severe fluctuations in
reliability. Therefore, the absence of satisfactory alternatives
has resulted in intense research efforts to determine improved
methods of separation and identification.
An alternative to TLC has been radioimmunoassay (RIA). Here
antibodies for specific haptens or antigens are employed. By mixing
an antibody with solutions of the hapten or antigen, and with a
radioactive hapten or antigen analogue, the radioactive analogue
will be prevented from binding to the antibody to an extent
directly related to the concentration of hapten or anitgen in the
solution. By then separating and assaying the free radioactive
analogue from the antibody bound radioactive analogue, one can
indirectly determine the amount of hapten or antigen in the
original solution. However, the use of radioisotopes in such an
assay could be a potential health hazard and, furthermore, the
instrumentation generally required for radioimmunoassay is
relatively sophisticated and generally too expensive too allow
small hospitals and physicians to routinely perform, for example, a
patient's blood or urine analysis. Enzyme immunoassay overcomes the
above problems and in addition, has the unique advantage of
potential amplification of the measured parameter.
In essence this method replaces the radioactive biological
substance analogue with an enzyme labelled biological substance
(hapten or antigen). Such modified enzyme molecules retain their
enzymatic activity and the enzyme-labelled biological substance
will compete for antibody complex formation with the unknown amount
of free biological substance in the system. The complexes may be
separated (cf. U.K. Pat. No. 1,348,935) in view of their
insolubility in certain instances and the activity of this, or the
part remaining in solution is used as a measure of the amount of
antigen originally present. The same principle may be applicable to
a reverse system, using enzyme-labelled antibodies whenever the
unmodified version of the same antibody present in biological
fluids has to be determined.
It has now been found that covalent attachment of biotin (Vitamin
H) to the antibody molecule, resulting in a soluble biotin-tagged
complex, facilitates convenient separation of all antibody forms
including all enzyme-labelled and unlabelled biological
substance-antibody complexes. The separation process can then be
performed by the use of the biotin-specific receptor protein,
avidin, which is immobilised in an insoluble form. The very strong
affinity between avidin and biotin, which approaches covalent bond
character, results in insolubilisation of all antibody forms and
consequently allows an extremely efficient and easy removal of all
biotin-tagged antibodies, and complexes formed by such
antibodies.
According to one aspect of the present invention, therefore, there
is provided a process for the detection and/or determination of a
biological substance in a test sample, which comprises admixing the
test sample, a predetermined quantity of a soluble enzyme-labelled
form of the biological substance, and a predetermined quantity of a
soluble biotintagged antibody raised against the biological
substance, allowing to come to equilibrium, adding insolubilised
avidin, separating the resulting solid phase from the liquid phase
and determining the enzyme activity of either of these phases.
For the purposes of the present invention, any biological substance
may be detected and/or determined for which an appropriate antibody
may be produced having satisfactory specificity and affinity for
the biological substance. The recent literature contains an
increasing number of reports of antibodies for an increasingly wide
variety of biological substances. Compounds for which antibodies
can be provided range from simple phenylaklyl amines, for example
amphentamine, to very high molecular weight polymers, for example
proteins.
The biological substances for detection and/or determination in the
process of the present invention may be divided into three
different categories, based on their biological relationship to the
antibody. The first category is antigens, which when introduced
into the blood stream of a vertebrate, result in the formation of
antibodies. The second category is haptens, which when bound to an
antigenic carrier, and the hapten-bound antigenic carrier is
introduced into the bloodstream of a vertebrate, elicit formation
of antibodies specific for the hapten. The third category of
biological substances includes those which have naturally occurring
antibodies in a living organism and the antibodies can be isolated
in a form specific for the biological substance.
The most important group of biological substances for the purposes
of the present invention are those of the second category, the
haptens. Methods for the production of antibodies to the three
different categories of biological substances are well known in the
art.
Selection of the enzyme for use in the present invention is
governed by a number of criteria. Thus it should possess
potentially reactive groups to which the biological substance can
be coupled without destroying enzyme activity and should not occur
naturally to an appreciable extent in the type of tissue to be
assayed for the said biological substance. In addition, the enzyme
should have a relatively long shelf life, a high specific activity
and also be capable of being easily assayed, for example with a
visible light spectrophotometer.
Examples of enzymes which may conveniently be employed in the
process of the present invention are, malate dehydrogenase,
staphylococcal nuclease, delta-5-ketosteroid isomerase, yeast
alcohol dehydrogenase, yeast glucose-6-phosphate dehydrogenase,
alpha glycerophosphate dehydrogenase, triose phosphate isomerase,
and horse radish peroxidase, more preferably, alkaline phosphatase,
asparaginase, glucose oxidase, beta-galactosidase and ribonuclease.
Normally it is preferred to purify the enzyme, for example by
dialysis against saline, before use.
The preparation of the enzyme-labelled biological substances for
use in the present invention can take place in various ways known
per se in the art. Some biological substances may already possess
groups that can be cross-linked with reactive groups at the surface
of the enzyme, while other substances will have to be provided with
such groups by organic chemical reactions. It should be emphasised
that neither the original immunological properties of the
biological substance nor the activity of the enzyme ought to change
appreciably during this process. The groups of the enzyme which are
particularly suited for coupling reactions are amino and carboxyl
groups. If the modified or unmodified biological substance also
possesses such groups, the coupling can be performed by, for
example, reactions known from peptide synthesis. Furthermore, such
substances, as for example, glutaraldehyde,
difluorodinitrodiphenylsulphone, toluene diisocyanate, di- and
trichloro-s-triazine and others can be employed for the coupling
reaction.
Examples of the coupling of biological substances to enzymes are
described in, for example, L. A. Steinberger, Immunocytochemistry,
Prentice Hall, New Jersey (1974).
Specific examples of the coupling of haptens to proteins are
described in, for example, C. A. Williams and N. W. Chase, Methods
of Immunology and Immunochemistry Vol. 1 Academic Press, New York,
1967. The methods described are used for the preparation of
conjugates for immunisation but they can also be used for the
preparation of the enzyme-labelled biological substances which are
essential in the process of the present invention.
Biotin-tagged antibody is conveniently prepared by reaction of a
biotin derivative, for instance a biotin ester derivative such as
the N-hydroxysuccinimide ester of biotin, with the antibody. The
biotin ester derivative is dissolved in a polar, aprotic solvent,
for example dimethylformamide, and is then added in a 20 to 300
molar excess to the antibody in 0.01 M to 1.0 M, preferably 0.05 M
to 0.5 M, most preferably 0.1 M phosphate buffer, for example
potassium phosphate buffer at from pH 6.5 to pH 8.5, preferably pH
7.5. After admixture of the reactants, the reaction is allowed to
proceed at a temperature of from 2.degree. to 10.degree. C., more
preferably at 4.degree. C. for a time sufficient for its
completion. Normally this takes of the order of 10 hours. After
completion of the reaction, the biotin-tagged antibody may be
separated from the reaction mixture by standard methods well known
in the art, for example by gel permeation chromatography on, for
instance, a cross-linked dextran.
Insolubilised avidin, that is, avidin immobilised by attachment to
a solid support, may be prepared by a method in actual use or
described in the literature, for example, by covalent binding with
macromolecular insoluble carriers such as agarose, polystyrene,
polyacrylamide, nylon, cross-linked dextran or filter paper or by
physical coupling to insoluble carriers such as glass beads or
plastic objects, or to the inside of test tubes made from either
plastic or glass or to microtitre plates.
Specific examples of coupling haptens and other biological
molecules to agarose and polyacrylamides are described by
Cuatrecasas in J. Biol. Chem., 245, 3059-3065, (1970). W. B. Jacoby
and M. Wilcheck; Methods in Enzymology; Vol. 34--Academic Press,
New York 1974. These methods may also be used, in principle, to
prepare (a) avidin-solid support, (b) hapten-protein conjugates and
(c) biotin-antibody conjugates. Conveniently either avidin or the
carrier is activated before covalent binding occurs, but most
conveniently the carrier is activated before covalent binding. In
one form of the invention the carrier used is agarose since this
exhibits excellent coupling of avidin and retention of
biotinbinding capacity. Most preferably, benzoquinone-activated
agarose is used due to its ease of preparation, and its lack of
non-specific absorption of enzyme. In other forms of this invention
avidin is coupled to nylon rings or rods or to the inside of
polystyrene test-tubes.
A schematic representation of the assay is presented in FIG. 1. To
the vessel in which the reaction is to take place are added at room
temperature and at near physiological pH, preferably at pH 7,
successively, with a minimum time interval between the additions:
free biological substances for example, contained in a serum
sample, an aqueous solution of enzymelabelled biological substance,
and a quantity of previously tagged antibody sufficient to
neutralise (ie. complex with) 50%-80% of the enzyme-labelled
biological substance. These three components are mixed, and
immediately insolubilised avidin is added to the reaction mixture
to create a heterogeneous system and the mixture shaken until a
predetermined equilibrium point is reached. The quantity of avidin
used is normally several hundred fold in excess of that
theoretically required. The solid phase removes the free antibody
as well as the antibody bound to the biological substance. The
supernatant contains enzyme-labelled biological substance in direct
proportion to the level of free biological substance, whilst the
deposited solid phase has enzyme-labelled biological substance in
inverse proportion to level of the free biological substance.
Either the deposited solid phase or the supernatant can then be
assayed for enzymatic activity to determine the amount of
biological substance present in the unknown sample.
The enzyme activity measurement of the solid and/or liquid phase of
the reaction mixture resulting from the process of the present
invention may be carried out by methods already known in
themselves. See, for example, H. U. Bergmeyer, Method for Enzymatic
Analysis, Academic Press, New York (1965). The assay of the
carefully separated and rinsed solid phase may take place after
removal of the supernatant by, for example, aspiration.
The various forms in which the reagents of the present invention
can be used are manifold. For instance, the enzyme labelled
biological substance can be freeze-dried or dissolved in a buffer.
Furthermore, a solid carrier for example, a strip of paper
impregnated with the enzyme-labelled biological substance, can be
employed.
For carrying out the process for a single test according to the
present invention, use can be made of a pack comprising, in
separate containers:
(a) a preselected quantity of enzyme-labelled biological
substance;
(b) a preselected quantity of biotin-tagged antibody raised against
the said biological substance;
(c) a preselected quantity of insolubilised avidin; and
(d) a substrate or substrates and co-factors for the determination
of the activity of the enzyme employed, together with instructions
to admix the enzyme-labelled biological substance, the
biotin-tagged antibody raised against the biological substance, and
a test sample, allowing the mixture to come to equilibrium, then
adding the insolubilised avidin, separating the resulting solid
phase form the liquid, and determining the enzyme activity of
either of these phases using the enzyme substrate or substrates and
co-factors.
If required, it may also contain the necessary auxiliaries for
making a dilution series of the test sample to be examined for a
quantitative determination, such as test tubes, pipettes and flasks
of diluent.
The invention is illustrated further by the following examples
which are not to be construed as limiting the invention to the
specific procedures described in them.
EXAMPLE 1
Coupling of digoxin to enzymes
Digoxin was coupled to enzyme in the following manner:
.sup.3 H-digoxin (250 .mu.Ci/mg) in ethanol was reacted with a
slight molar excess of sodium metaperiodate for two hours. An
aliquot of oxidised .sup.3 H-digoxin (50 nmoles) was added to
enzyme (5 nmoles) in sodium acetate buffer (1 ml; 200 mM; pH 7).
The final concentration of ethanol was usually 10-20%. Sodium
cyanoborohydride (10-20 mg) was added and the reaction was allowed
to proceed for 3 days at 4.degree. C. Labelled enzyme was separated
from the reactants by gel exclusion chromatography on Sephadex
G-50. The table below shows the degree of substitution by digoxin
(calculated by means of radioactivity measurements) and the
retention of enzymatic activity for four enzymes after reaction
under the described conditions.
______________________________________ Digoxin substitution
Specific activity (M/M) recovered as % Enzyme .sup.3 H counting RIA
of control ______________________________________ Malate
dehydrogenase 0.50 0.10 36.7 Alkaline Phosphatase 1.10 0.15 92.3
Glucose Oxidase 1.70 0.30 100.0 Asparaginase 1.00 0.60 100.0
______________________________________
Conjugation of .sup.3 H-digoxin with malate dehyrdogenase was
investigated more fully (FIG. 2). Oxidised digoxin was reacted at
various concentrations (molar excess varied from 5 to 50) with
aliquots of enzyme, and the retention of enzyme specific activity
was determined for each point. In addition, the degree of
substitution of digoxin was determined both by tritium counting to
determine the absolute number of haptens and by RIA to determine
the number of immunologically reactive digoxin residues. The data
shows that malate dehydrogenase is very sensitive to the degree of
labelling by digoxin and that only 20% of the groups attached are
functionally available to interact with antibody.
Insolubilisation of Avidin
Avidin was coupled directly to a solid support of
benzoquinone-activated sepharose. The avidin-gel was diluted, as
desired, with underivatised sepharose.
A 50% suspension of agarose in sodium phosphate buffer (100 mM; pH
8.0) was added in a 4:1 volume/volume ratio to p-benzoquinone (250
mM) in ethanol. After stirring the suspension for 1 hour at room
temperature, the activated gel was washed by suction filtration
using equal volumes of, successively, ethanol (20%), aqueous sodium
chloride (1.0 M), water, and sodium phosphate (pH8, 100 mM). The
gel (1 volume) was then added to avidin (1 volume; 10 mg/ml)
dissolved in sodium phosphate buffer (pH8, 100 mM), and the
resulting suspension was shaken for 15 hours at 4.degree. C. After
this time, the avidin-gel conjugate was washed successively with 5
volumes each of sodium acetate (0.1 M, pH 4.0) containing sodium
chloride (500 mM), sodium bicarbonate (0.1 M, pH 9.0) containing
sodium chloride (500 mM), and water. The substitution of avidin on
the gel was in the range of 4-5 mg/ml packed gel. The substituted
avidin was found to have 100% of its biotin binding capacity.
Substitution of Antibody with biotin
Sheep antidigoxin antibody was treated variously in potassium
phosphate (0.1 M; pH 7.5) with a 20 to 300 molar excess of the
N-hydroxysuccinimide ester of biotin in dimethylformamide (DMF).
The final concentration of DMF was 50%. After incubation overnight
at 4.degree. C., the biotinised antibody was separated from
reactants by gel permeation chromatography on Sephadex G-50. The
titre of the biotinised antibody was assessed using .sup.125
I-digoxin and dextran-coated charcoal. The sensitivity of the
biotinised antibody to avidin-gel was assessed using previously
iodinated antibody and measuring the take-down of .sup.125 I by
avidin-gel. At levels of biotinisation above 3 biotin molecules per
antibody molecule, the antibody retained its titre for digoxin and
was completely absorbed by avidin-gel. The antibody-biotin
preparation chosen for the enzyme immunoassay empirically met these
criteria.
Glucose oxidase-digoxin conjugates, with various levels of
substitution of digoxin, were titrated with various dilutions of
biotinised antibody and excess avidin-gel (FIG. 3). The enzyme
derivatives has been previously shown not to be inhibited by even
very high concentrations of anti-digoxin antibody. The data shows
that at between 1:100 and 1:1000 dilution of the stock antibody
solution, the digoxin-enzyme and antibody are at approximately
stoichiometric levels such that essentially all enzyme activity is
precipitated by the gel. The data also shows that with increasing
digoxin substitution less antibody is needed to precipitate 50% of
the enzyme activity, indicating that the immunoreactivity of the
enzyme conjugate is increasing with substitution.
The anti-digoxin antibody and digoxin interaction
Using appropriate levels of glucose oxidase-digoxin, biotin-tagged
antibody, and excess avidin-gel, the time course to reach
equilibrium between the three components of the assay was followed.
Glucose oxidase-digoxin and avidin-gel were preincubated at room
temperature. At t=0, biotin-tagged antibody was added and the
mixture was shaken at room temperature. At various time intervals,
aliquots of the assay mixture were centrifuged and the supernatants
assayed for enzyme activity. In general, maximum takedown of
enzyme-labelled digoxin by the antibody-avidin gel complex occured
within 90 minutes, while the binding of biotin-tagged antibody by
avidin-gel is essentially complete within 10 minutes.
Enzyme Immunoassay for digoxin
Serum digoxin (50 .mu.l), glucose oxidase-labelled digoxin (50
.mu.l) which contained 1 ng/ml of bound digoxin, and appropriately
diluted biotin-tagged antidigoxin antibody (50 .mu.l) were mixed in
a test tube (FIG. 1). Immediately a 50% aqueous suspension of
avidin-sepharose (50 .mu.l) which had been previously diluted 1:10
with native sepharose was added. All assay reagents were in
phosphate buffer (50 mM; pH 7) containing bovin serum albumin
(0.1%). The reaction mixture was then incubated for 2 hours at room
temperature with shaking, and was subsequently suspended in
ice-cold buffer (5 ml) containing bovin serum albumin (0.1%). The
mixture was centrifuged and the supernatant aspirated away to leave
the gel pellet. After washing the pellet, assay buffer (1.0 ml)
containing glucose (100 mM), o-dianisidine (0.1 mg/ml) and horse
radish peroxidase (7.5 .mu.g/ml) in sodium phosphate buffer (100
mM; pH 6) was added, and shaking was performed for 1 hour.
Alternatively, 0.1 mM; 2,2-azino-bis-(3-ethylbenzthiozoline
sulphonic acid) (ABTS) can be substituted for the o-dianisidine as
an enzyme substrate.
After this time the mixture was cooled, and the optical density of
the supernatant was recorded at a wavelength of 450 nm and the
values of the patient sera compared to a standard curve constructed
with control sera.
FIG. 4 shows the effect of addition of various amounts of free
digoxin (plotted as the logarithm of the digoxin concentration in
ng/ml versus the percentage of total enzyme activity on the gel) to
the incubation mixture. The relationship between free digoxin
levels and the enzyme activity is linear over 0.15 to 10.00 ng/ml
of free digoxin.
EXAMPLE 2: Codeine
To illustrate the general applicability of the principle of the
herein described enzyme immunoassay, the narcotic drug codeine was
assayed by this method.
Coupling of Codeine to Glucose Oxidase
Codeine hemisuccinate (20 .mu.moles) dissolved in dry, redistilled
dimethylformamide (600 .mu.l) was added to 200 .mu.moles
N-hydroxysuccinimide dissolved in dry, redistilled dioxane (300
.mu.l) and 20 .mu.moles dicyclohexylcarbodiimide in dry,
redistilled dioxane (30 .mu.l). The mixture was allowed to react a
room temperature for 5-6 hours in a tightly stoppered test tube.
The codeine hemisuccinate N-hydroxysuccinimide thus prepared in
situ was used without purification to couple to the enzyme. To 5
nmoles of enzyme (glucose oxidase) dissolved in 600-900 .mu.l of
sodium acetate buffer (100 mM, pH 7.0) was added 100-500 .mu.l
(preferably 300 .mu.l) of the codeine N-hydroxysuccinimide ester
solution as prepared and described above. After 12-24 hours at room
temperature the enzyme (codeine conjugated together with any
unconjugated enzyme) was separated from the reactants by gel
exclusion chromatography. The degree of codeine substitution to
glucose oxidase was determined by radioimmunoassay. As in the case
of digoxin the degree of substitution of codeine varies depending
on the reaction conditions. A substitution of 3-5 codeine molecules
per enzyme (glucose oxidase) molecule was found to be ideal for the
present assay method.
Coupling of biotin to anti-codeine antibody
This reaction was performed as described in Example 1 for coupling
of biotin to anti-digoxin antibody. The titer of biotinized
antibody was essentially unaltered as compared to the underivatized
antibody. Antibody preparations with 3 or more convalently bound
biotin molecules were completely precipitable by solid-phase avidin
and were found to be ideally suited for the present assay.
Enzyme Immunoassay for Codeine
With appropriate, predetermined quantities of enzyme-codeine
conjugate, biotinized anti-codeine antibody and solid-phase avidin
the time course to reach equilibrium between the three components
of the assay was determined. Usually after 45-60 minutes the system
is at equilibrium. Codeine standard (0.1 to 10,000 ng/ml solutions
(50 .mu.l), glucose oxidase-codeine conjugate (50 .mu.l, containing
1-20 fmoles codeine) and appropriately diluted biotin tagged
anti-codeine antibody were mixed in a test tube. A 50% aqueous
suspension of solid-phase avidin (50 .mu.l) was added to the above
mixture. All assay reagents were prepared in 50 mM phosphate
buffer, pH 7 containing 0.1% bovine serum albumin. The reaction
mixture after incubation for 2 hours at room temperature was
diluted with 5 ml of ice-cold buffer containing 0.1% bovine serum
albumin. The mixture was centrifuged and the supernatent aspirated
away. After washing the pellet the assay buffer (1 ml) containing
glucose (100 mM, o-dianisidine (0.1 mg/ml) and horse radish
peroxidase (7-5 .mu.g/ml) in phosphate buffer (100 mM, pH 6) was
added. The assay mixture was incubated for 30-90 minutes
(preferably 60 minutes) at room temperature, cooled in ice bath and
optical density at 450 nm was recorded. Thus a standard curve with
various codeine concentrations was constructed (FIG. 5).
* * * * *