U.S. patent number 3,850,752 [Application Number 05/193,702] was granted by the patent office on 1974-11-26 for process for the demonstration and determination of low molecular compounds and of proteins capable of binding these compounds specifically.
This patent grant is currently assigned to Akzona Incorporated. Invention is credited to Antonius Hermanus Wilhelmus Maria Schuurs, Bauke Klass Van Weemen.
United States Patent |
3,850,752 |
Schuurs , et al. |
* November 26, 1974 |
**Please see images for:
( Certificate of Correction ) ** |
PROCESS FOR THE DEMONSTRATION AND DETERMINATION OF LOW MOLECULAR
COMPOUNDS AND OF PROTEINS CAPABLE OF BINDING THESE COMPOUNDS
SPECIFICALLY
Abstract
The invention relates to a process for the demonstration and
determination of a low molecular compound, such as a hapten, or a
protein or antibody capable of binding this compound specifically,
contacting a fluid containing such a compound or protein to be
determined with a given quantity of the coupling product of such a
low molecular compound and an enzyme, and with a given quantity of
the component to be determined, which component is brought in an
insoluble form, and determining the enzyme activity of the liquid
or solid phase of the resulting reaction mixture.
Inventors: |
Schuurs; Antonius Hermanus
Wilhelmus Maria (Oss, NL), Van Weemen; Bauke
Klass (Oss, NL) |
Assignee: |
Akzona Incorporated (Asheville,
NC)
|
[*] Notice: |
The portion of the term of this patent
subsequent to April 4, 1989 has been disclaimed. |
Family
ID: |
19811504 |
Appl.
No.: |
05/193,702 |
Filed: |
October 29, 1971 |
Foreign Application Priority Data
|
|
|
|
|
Nov 10, 1970 [NL] |
|
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7016396 |
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Current U.S.
Class: |
435/7.93; 435/25;
435/188; 435/964; 436/518; 436/547; 436/815; 436/817; 435/975;
436/530; 436/808 |
Current CPC
Class: |
G01N
33/543 (20130101); Y10S 435/964 (20130101); Y10S
436/808 (20130101); Y10S 435/975 (20130101); Y10S
436/817 (20130101); Y10S 436/815 (20130101) |
Current International
Class: |
G01N
33/543 (20060101); G01n 031/14 (); G01n
033/16 () |
Field of
Search: |
;195/13.5R
;23/253TP,23B |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Chemical Abstracts 65: 11124b, (1966). .
Spector et al., 168: 1348, 8, "Science," (June 12, 1970)..
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Primary Examiner: Tanenholtz; Alvin E.
Attorney, Agent or Firm: Weisberger; Hugo E.
Claims
What is claimed is:
1. Process for the demonstration and determination of a low
molecular weight organic compound by means of an antibody against
said low molecular weight compound, comprising the steps of:
a. providing a given quantity of the coupling product of said low
molecular weight organic compound with an enzyme;
b. providing a corresponding given quantity of an insolubilized
antibody against said low molecular weight organic compound;
c. contacting a sample of a fluid containing the low molecular
weight organic compound to be determined with said components (a)
and (b) to form a reaction mixture; and
d. determining the enzyme activity of the liquid or the solid phase
of the resulting reaction mixture, which activity is a measure of
the quantity of low molecular weight organic compound to be
determined.
2. The process of claim 1 in which said low molecular weight
organic compound is a hapten.
3. The process of claim 1 in which said enzyme is an
oxido-reductase.
4. Process for the demonstration and determination of a low
molecular weight organic compound by means of a protein capable of
reacting to bind said low molecular weight organic compound
specifically, comprising the steps of:
a. providing a given quantity of the coupling product of said low
molecular weight organic compound with an enzyme;
b. providing a corresponding given quantity of an insolubilized
specific binding protein having more than one valency capable of
reacting to bind said low molecular weight organic compound
specifically;
c. contacting a sample of a fluid containing the low molecular
weight organic compound to be determined with said components (a)
and (b) to form a reaction mixture; and
d. determining the enzyme activity of the liquid or the solid phase
of the resulting reaction mixture, which activity is a measure of
the quantity of low molecular weight organic compound to be
determined.
5. The process of claim 4 in which said enzyme is an
oxido-reductase.
6. Process for the demonstration and determination of a specific
binding protein having two or more binding sites by means of a low
molecular weight organic compound capable of reacting to bind said
protein specifically; comprising the steps of:
a. providing a given quantity of the coupling product of said low
molecular weight organic compound and an enzyme;
b. providing a corresponding given quantity of an insolubilized
specific binding protein having two or more binding sites capable
of binding said low molecular weight organic compound;
c. contacting a sample of a fluid containing the protein to be
determined with said components (a) and (b) to form a reaction
mixture; and
d. determining the enzyme activity of the liquid phase of the
resulting reaction mixture, which activity is a measure of the
quantity of the specific binding protein to be determined.
7. The process of claim 6 in which said protein is an antibody.
8. A diagnostic pack for the demonstration and determination of a
low molecular weight organic compound by means of an antibody
against said low molecular weight organic compound, comprising:
a. a given quantity of the coupling product of said low molecular
weight organic compound with an enzyme;
b. a corresponding given quantity of an insolubilized antibody
against said low molecular weight organic compound; and
c. a substrate for the determination of the enzyme activity of said
enzyme.
9. The diagnostic pack of claim 8 in which said enzyme is an
oxido-reductase.
10. A diagnostic pack for the demonstration and determination of a
low molecular weight organic compound by means of a protein capable
of reacting to bind said low molecular weight organic compound
specifically, comprising:
a. a given quantity of the coupling product of said low molecular
weight organic compound with an enzyme;
b. a corresponding given quantity of an insolubilized specific
binding protein capable of reacting to bind said low molecular
weight organic compound specifically; and
c. a substrate for the determination of the enzyme activity of said
enzyme.
11. The diagnostic pack of claim 10 in which said enzyme is an
oxido-reductase.
12. A diagnostic pack for the demonstration and determination of a
hapten by means of an antibody against said hapten, comprising:
a. a given quantity of the coupling product of said hapten with an
enzyme;
b. a corresponding given quantity of an insolubilized antibody
against said hapten; and
c. a substrate for the determination of the enzyme activity of said
enzyme.
13. The diagnostic pack of claim 12 in which said enzyme is an
oxido-reductase.
Description
For the determination of low molecular substances occurring in low
concentrations, such as steroid hormones in body fluids, methods
have been developed using proteins capable of binding the substance
to be determined specifically. These methods are based on the
competition between the substance to be determined in the sample
and a known quantity of the same substance which is labelled
radioactive, for a limited quantity of specific binding protein.
The unknown quantity of bindable substance then determines what
portion of the radioactive labelled substance is bound by the
specific binding protein.
It is also possible to determine by these methods an unknown
quantity of specific binding protein by reacting a sample
containing an unknown quantity of specific binding protein with a
given quantity of bindable radioactive labelled substance.
In the literature it is common practice to distinguish these
determination methods according to the nature of the specific
binding protein employed, although the underlying principle of all
determinations is identical. Thus, for example, they speak of
"competitive protein binding assays" when they employ receptor or
transport proteins occurring in the body, and of
radio-immunological determinations when they employ
anti-substances.
For both types of determinations radioactive labelled substances
are required. The work with these substances requires the presence
of precious measuring apparatuses, good laboratory facilities and a
highly qualified staff. These high requirements prevent a general
application of these determination, especially in smaller
laboratories.
A process has now been found for the demonstration and
determination of a component of the reaction between a low
molecular compound and a protein capable of binding this compound
specifically using the binding affinity of such components for each
other, characterized in that the determination is performed with a
given quantity of the coupling product of the low molecular
compound and an enzyme, and with a given quantity of a component of
the said reaction which has been made insoluble, and the enzyme
activity of the liquid or the solid phase of the resulting reaction
mixture is determined, which activity is a measure for the quantity
of the reaction component to be determined.
The method found can often by employed for the determination of
haptens, which may be regarded as a special group of low molecular
compounds, and their anti-substances. These substances mostly occur
in low concentrations. According to the original definition of K.
Landsteiner haptens are protein-free substances whose chemical
configuration is such that they can react with specific antibodies,
but not such that they are capable of causing the formation of
anitbodies. In order to be able yet to make antibodies against
haptens, the haptens must be coupled to polypeptides before being
injected into the test animal. In the determination of a low
molecular compound the substance to be determined and its coupling
product with an enzyme enter into competition for a given quantity
of the insoluble specific binding protein. The more low molecular
compound the sample contains, the less opportunity the soluble
enzyme conjugate of that compound has to combine with the insoluble
specific binding protein and the more of the conjugate will remain
in the liquid phase, in which the enzyme activity can be measured
in a simple manner.
In the determination of a specific binding protein with the same
reagents the soluble protein to be determined and the insoluble
protein enter into competition for a given quantity of the
conjugate of the corresponding low molecular compound and an
enzyme. If the content of specific binding protein of the sample is
higher, the insoluble protein will bind less of the enzyme
conjugate, and consequently more enzyme remains in the liquid
phase.
A specific binding protein having two or more binding possibilities
can also be demonstrated and determined according to the invention,
i.e. with the enzyme conjugate and with the low molecular compound
in an insoluble form. The liquid phase of the reaction mixture then
contains the conjugate bound to the specific binding protein and in
the solid phase the complex of enzyme conjugate - specific binding
protein - insoluble low molecular compound. The more of the protein
to be determined there is in the sample, the more enzyme activity
the liquid phase will possess.
By means of an assay curve for a certain system in which increasing
contents of substance to be determined have been set off against
the enzyme activity found, preferably in the liquid phase, the
quantity of substance to be determined in the sample can be derived
from a value of enzyme activity found.
The most important reagents of this determination method is the
coupling product of the low molecular substance and an enzyme, for
convenience hereinafter also called enzyme conjugate, which on the
one hand can react with the specific binding protein, via the low
molecular component and on the other hand possesses enzyme
activity. This reagent is prepared by a method described for
similar products. The second reagent, the insoluble component in
the reaction system, serves to facilitate the separation between
various enzyme-containing fractions of the reaction mixture.
Addition of this reagent causes the formation of a solid phase in
addition to a liquid phase. The present determinations can in
principle be performed without an insoluble component of the
reaction system. In that case the various fractions with enzyme
activity will have to be separated by, for example, chromatographic
or electrophoretic route or gelfiltration. For practical reasons
such a determination is less attractive, however,
The enzyme activity of a fraction of the reaction mixture is
demonstrated or measured by incubating that fraction with a
substrate and other substances required for processing the relative
enzyme reaction. For preference a reaction is used in which a
coloured compound is formed or removed whose absorption can also be
measured quantitatively in an easy manner.
Low molecular substances which are eligible for demonstration by
the new method and have a molecular weight of up to about 1500 are
for example: steroids, vitamin B.sub.12, folinic acid, thyroxine
and triiodothyronine, releasing factors, histamine, serotonin and
other biogenic amines, digoxine, digitoxin, prostaglandins,
adrenalin, nor-adrenalin, vegetable hormones such as auxin, kinetin
and gibberellic acid, and antibiotics such as penicillin.
The method of demonstrating specific binding proteins for low
molecular substances can be employed for, for example, the
determination of antibody against penicillin or for the
determination of intrinsic factor.
The preparation of conjugates of enzymes and low-molecular
substances can take place in various manners. Some low molecular
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 stands to reason that the original binding
properties of the low molecular compound and the activity of the
enzyme may not change essentially 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 low
molecular substance also possesses such groups, the coupling can be
performed by, for example, reactions known from the peptide
synthesis. Furthermore such substances as glutaraldehyde,
difluorodinitrodiphenylsulfon, toluene diisocyanate, di- and
trichloro-s-triazine can be employed for the coupling reaction.
Specific examples of the coupling of haptens to proteins are
described in, for example, Methods in Immunology and
Immunochemistry, vol. I. The methods described are used for the
preparation of conjugates for immunisation but they can also be
used for the preparation of conjugates of the low molecular
substance and an enzyme which are essential in the invention.
The choice of the enzyme that is to be a component of the conjugate
(low molecular substance-enzyme) depends on properties such as the
specific activity (a high conversion rate enhances the sensitivity
of the test system) and the simplicity of the determination of the
enzyme. The determination of an enzyme which catalyses a conversion
involving coloured reaction components, is simple. Such
colorimetric determinations can be automatized in a simple
manner.
According to the invention it is also possible to use enzymes which
catalyse conversions involving reaction components that can be
determined spectrophotometrically or fluorimetrically. These
determinations can also be automatized.
For the preparation of the conjugates enzymes such as catalase,
peroxidase, .beta.-glucuronidase, .beta.-D-glucosidase,
.beta.-D-galactosidase, urease, glucose-oxidase and
galactose-oxidase are preferred, particularly the group of the
oxido reductases.
The insoluble specific binding protein or the insoluble low
molecular compound to be used in the present determination can be
prepared by a known method, for example, by cross-linking with
chloroformic acid-ethylester, by covalent binding with insoluble
carriers such as agarose, cross-linked dextran or filter paper, or
by physical coupling to insoluble carriers such as plastic
objects.
The form in which the reagents can be used are manifold. The
component of the reaction system conjugated with an enzyme can be
freeze-dried or dissolved in a buffer. Furthermore a solid carrier,
for example, a strip of paper impregnated with the conjugate, can
be employed.
The insoluble component can be brought in the form of particles of
various sizes, such as grains, platelets and rods, or in the form
of a strip of some or other carrier material.
For the performance of the process according to the invention a
test pack in preferably employed, chiefly composed of:
a. a given quantity of the coupling product of the low molecular
compound and an enzyme;
b. a corresponding quantity of one of the components of the
reaction system in an insoluble form;
c. a substrate for the determination of the activity of the enzyme
employed.
If required, the test pack can also contain the necessary
auxiliaries for making a dilution series of the sample to be
examined for a quantitative determination, such as test tubes,
pipettes and flasks of diluent. For the determination of a hapten
or its antibody the test pack contains at least:
a. a given quantity of the coupling product of this hapten and an
enzyme;
b. a corresponding quantity of a component of the reaction system
in an insoluble form, hapten-antibody;
c. a substrate for the determination of the enzyme activity.
The invention is illustrated further by the following examples,
which are not to be construed as limiting.
Example I
Determination of testosterone
A. preparation of testosterone-3-HRP
One hundred milligrams of testosterone-3-(O-carboxymethyl)-oxim and
0.143 ml of tri-n-butylamine were dissolved in 5 ml of dioxan. The
solution was cooled down to 2.degree.C and then 0.03 ml of
isobutylchlorocarbonate were added. After 30 minutes the solution
was added to 100 mg of HRP (horse radish peroxidase) in a mixture
of 9 ml of water and 6 ml of dioxan, and adjusted to pH 9 with 0.1
N NaOH. This solution was stirred for 4 hours at 2.degree.C and
dialysed overnight. The precipitate obtained after the dialysate
had been adjusted to pH 4.6 was centrifuged after having been left
to stand overnight, suspended in 10 ml of water and dissolved by
means of caustic soda solution. The material was precipitated three
times with 15 ml of acetone at pH 4.5, dissolved in 15 ml of water
which had been adjusted at pH 7.8 with caustic soda solution,
dialysed and finally lyophilized.
B. preparation of testosterone-3-BSA
This conjugate was prepared in the same manner as the
testosterone-3-HRP, but the starting materials were 50 mg of
testosterone-3-(O-carboxymethyl)-oxim and 150 mg of BSA (bovine
serum albumin).
C. preparation of antibodies against testosterone-3-BSA
Five rabbits were injected intramuscularly with increasing dosages
of testosterone-3-BSA in complete Freund's adjuvant (0.5, 1 and 2
mg) at intervals of 3 weeks. Two weeks after the last injection the
animals were injected intravenously with 2 mg of antigen dissolved
in physiological salt. One week after that blood was taken from the
animals. The antibodies formed against BSA were removed by treating
the serum portionwise with BSA-m-aminobenzyloxymethyl cellulose,
prepared in accordance with Gurvich's method (see D).
D. preparation of anti-testosterone cellulose
This substance was prepared in accordance with Gurvich's method as
described in Biokhimiya 26, 934 (1961):
1. Preparation of "aminocellulose":
Fifty grams of Whatman cellulose, which had been frequently washed
and decanted, were suspended in 100 ml of a 0.7 % sodium acetate
solution containing 2 gm of N(m-nitrobenzoxy)-methylpyridine. The
mixture was dried at 60-80.degree.C and heated for 40 minutes at
125.degree.C. The resulting product was thoroughly washed with
distilled water, dried at 80.degree.C, washed with benzene and
dried again. Fifty grams of the dried product were reduced by
suspension in 300 ml of a 15% Na.sub.2 S.sub.2 O.sub.4 solution and
stirred for 30 minutes at 50-60.degree.C. The product was filtered
and washed with successively distilled water, 30% acetic acid and
again with distilled water.
2. Treatment with ammoniacal copper solution:
Forty millilitres of 10% sulphuric acid, 20 ml of 50% nitric acid
and 140 ml of distilled water were heated, while stirring, to
90.degree.C, after which 5.9 gm of CuO were added in small
portions. The solution was boiled for 2 hours and completed to 500
ml with distilled water. Eighty millilitres of this solution were
transferred into an icebath and added to 160 ml of cold 4 N NaOH,
while stirring. After being stirred for 30 minutes, the precipitate
was washed twice with distilled water and dissolved in 80 ml of 25%
ammonia. To this solution was gradually added 1 gm of
aminocellulose. The mixture was stirred for 1.5 hours, after which
40 ml of boiling water were added, whereupon the solution was
quickly cooled down to 0.degree.C. The solution was neutralized
with 10% sulphuric acid, after which the aminocellulose
flocculated. It was washed with cold distilled water.
3. Preparation of .gamma.-globulin:
To rabbit anti-testosterone serum were added 180 mg of Na.sub.2
SO.sub.4 per ml of serum. The mixture was stirred for 1 hour at
room temperature, after which the resulting precipitate was
centrifuged, washed twice with an 18% Na.sub.2 SO.sub.4 solution
and taken up in so much 0.05 M sodium borate of pH 8.6 that the
protein concentration was about 10 mg/ml.
4. Binding .gamma.-globulin to aminocellulose
Aminocellulose (350 mg) was suspended in distilled water (50 ml).
The suspension was cooled down to 0.degree.C. Ten millilitres of a
36% hydrochloric acid were added and after that dropwise 10 ml of a
10% NaNO.sub.2 solution. The suspension was centrifuged, washed
with cold distilled water and then with 0.05 M sodium borate of pH
8.6. The cellulose was suspended in 43 ml of 0.05 M sodium borate
of pH 8.6. To this suspension were added 7 ml of the
.gamma.-globulin solution prepared. The mixture was stirred for 26
hours at 4.degree.C, centrifuged and washed with 0.02 M phosphate
buffer of pH 6.0. From the antiserum of each of the 5 immunized
rabbits a cellulose suspension was prepared (A - E
respectively).
E. determination of testosterone by means of testosterone-3-HRP and
anti-testosterone cellulose
The following test system was built up:
I Immunoreaction
0.5 ml of a sample containing testosterone, 0.2 ml of
testosterone-3-HRP (100 ngm/ml) and 0.3 ml of an
anti-testosterone-cellulose suspension were rotated at room
temperature for 2 hours and then centrifuged for 5 minutes at 1000
g.
The immunoreaction took place in 0.02 M phosphate buffer of pH 6.0
and containing 2% sheep serum.
Ii enzyme reaction
0.5 ml of the supernatant liquid was incubated at room temperature
with 1.5 ml of substrate for 30 minutes. The extinction was
measured at 460 nm.
The enzyme substrate contained 10 .mu.l of 30% hydrogen peroxide
and 20 mg of 5-aminosalicylic acid in 150 ml of 0.02 M phosphate
buffer of pH 6.2.
Fig. 1 shows the measure in which testosterone-3-HRP is bound by
the anti-testosterone-cellulose preparations made. In this case
only buffer was added as a sample in the test system. If cellulose
is added instead of anti-testosterone-cellulose, more than 95% of
the enzyme activity will remain behind in the supernatant liquid.
The preparations B, D and E proved to bind hardly any
testosterone-3-HRP, but A and C did.
Fig. 2 shows the results of incubation of a testosterone dilution
series with testosterone-3-HRP and four different concentrations of
anti-testosterone-cellulose C : 1 mg/ml (I), 2 mg/ml (II), 4 mg/ml
(III) and 16 mg/ml (IV). It is obvious that with this system a
quantity of about 10 ngm of testosterone can be demonstrated.
EXAMPLE II
Determination of oestradiol
A. oestradiol-17-succinyl-HRP was prepared by the mixed anhydride
method described in example I A, using 50 mg of
oestradiol-17-hemisuccinate and 50 mg of HRP as starting
materials.
B. oestradiol-17-succinyl-BSA was prepared by the mixed anhydride
method described in example I A, using 100 mg of
oestradiol-17-hemisuccinate and 150 mg of BSA as starting
materials.
C. for the preparation of antibodies against
oestradiol-17-succinyl-BSA five rabbits were immunized in
accordance with the scheme described in example I C. The sera were
absorbed with BSA-m-amino-benzyloxymethyl-cellulose.
D. anti-oestradiol-cellulose was prepared in the same manner as the
anti-testosterone-cellulose described in example I D. From each of
the immunized rabbits a cellulose preparation was made, numbered 16
up to 20 inclusive.
E. the test was performed analogous to that for testosterone as
described in example I E.
Fig. 3 and 4 show a few results. FIG. 3 shows that the immunization
yielded 3 usable antisera, of which As 17 has the highest
titre.
Fig. 4 shows the test system in which anti-oestradiol-cellulose 17
is used in a concentration of 8 mg/ml. The system does not
discriminate between oestron and 17.beta.-oestradiol.
17.alpha.-oestradiol and especially oestriol show a smaller
cross-reaction. Testosterone and progesterone influence the system
only in very high concentrations.
Example III
Determination of antibodies against penicillin
Penicilloyl-catalase
Thirty milligrams of benzyl penicillinic acid were dissolved in 5
ml of 96% ethanol and added dropwise to 200 mg of catalase in 45 ml
of 0.1 M phosphate buffer of pH 7.5. The reaction process was
continued for 2 hours, the pH being maintained between 7.2 and 8.2
with 0.5 N NaOH. The reaction mixture was dialysed against 6
.times. 3 l of 0.02 M phosphate buffer of pH 7.0.
In the same manner 250 mg of benzyl penicillinic acid were coupled
to 5 gm of m-aminobenzyloxymethyl-cellulose prepared by Gurvich's
method, Biokhimiya 26, 934 (1961). The coupling product was not
dialysed however, but washed on a glass filter.
It proved to be possible to demonstrate over -sensitiveness to
penicillin in humans in the following manner.
0.2 ml of a sample of non-haemolysed serum were mixed with 0.5 ml
of a solution of penicilloyl-catalase (1 : 800). After 30 minutes
10 mg of the penicilloyl-m-aminobenzyloxymethyl-cellulose were
added. The mixture was rotated, for 30 minutes after which the
enzyme activity in the supernatant liquid was determined by adding
0.02 ml of it to 2.8 ml of 0.05 M phosphate buffer of pH 6.8, which
contained 1.2 .mu.l of 30% H.sub.2 O.sub.2, and following the
decrease in the extinction at 240 nm. In the serum from patients
who were hyper-sensitive to penicillin less enzyme activity was
found in the liquid than in checking with rabbit serum. The serum
from normal human beings in these tests did not deviate
considerably from normal rabbit serum.
Example IV
Determination of folic acid
A. preparation of folate-glucose-oxidase
Two hundred milligrams of glucose-oxidase (140 IU/mg) were
dissolved in 10 ml of PBS (phosphate buffered saline, a
phosphate-containing physiological salt solution) of pH 7.0. Thirty
milligrams of 1-cyclohexyl-3-(2-morpholino-ethyl)-carbodiimide
(MCDI) were added and then 24 mg of folic acid. The reaction
process lasted 2 hours, after which a thorough dialysis was
performed against PBS of pH 7.0.
B. preparation of folate-MBSA (methylated bovine serum albumin)
Folate-MBSA was prepared by Ricker and Stollar's process described
in Biochemistry 6, 2001 (1967). Twenty-five milligrams of MCDI were
added to 50 mg of MBSA in 5 ml of water and then 20 mg of folic
acid. Two hours later a yellow precipitate had formed. Finally the
whole reaction mixture was dialysed against physiological salt for
a considerable time.
C. preparation of anti-serum against folate-MBSA
On the days 0, 21 and 42 four rabbits were each injected
intramuscularly with 2 mg of folate-MBSA in complete Freund's
adjuvant and on day 35 intravenously with 2 mg of folate-MBSA in
physiological salt. On day 49 the animals were exsanguinated.
D. anti-folate cellulose was prepared by the process described in
example I D.
E. test system for folic acid
One hundred .mu.l of sample and 700 .mu.l of anti-folate cellulose
suspension were rotated for 3 hours. Two hundred .mu.l of
folate-glucose-oxidase (1 : 1500) were added. The mixture was
rotated again for 3 hours and centrifuged, after which the enzyme
activity in the supernatant liquid was determined. This was done by
mixing 0.5 ml of it with a solution of 50 mg of glucose, 10 .mu.gm
of HRP and 1 mg of 5-aminosalicylic acid in 2.5 ml of 0.05N
phosphate buffer of pH 6.0 and measuring the extinction after 30
minutes at 460 nm.
Fig. 5 shows the percentage of enzyme bound against the
concentration of an anti-folate-cellulose.
Fig. 6 shows the sensitivity of the test system in an
anti-folate-cellulose concentration of 2 mg/ml and the effect of
glycine, asparagine, alanine and glutamic acid.
Example V
Determination of digoxin
A. preparation of digoxin-HRP
To 22 mg of digoxin, suspended in 1 ml of absolute ethanol, was
added dropwise, while stirring, 1 ml of 0.1 M sodium metaperiodate.
After 25 minutes 0.3 ml of 0.1 M ethyleneglycol was added. Five
minutes later this mixture was added dropwise, while stirring, to a
solution 32 mg of horse radish peroxidase (HRP) in 1 ml of
distilled water, which had been adjusted to pH 9.5 with a 5%
K.sub.2 CO.sub.3 solution. During the reaction the pH was
maintained at 9-9.5 with 5% K.sub.2 CO.sub.3. When the pH had
become stable 15 mg of NaBH.sub.4 in 1 ml of distilled water was
added. After 3 hours the pH was adjusted to 6.5 with 1 M formic
acid. One hour later 1 M NH.sub.4 OH was added till a pH of 8.5 had
been reached. The mixture was dialysed overnight against cold
running water. Finally the pH was adjusted at 4.5 with 0.1 N
hydrochloric acid. The mixture was left to stand at room
temperature for 1 hour and 4 hours at 4.degree.C to obtain a
precipitate which was centrifuged for 1 hour at 1000 g. The
precipitate was dissolved in 5 ml of 0.1 M NaHCO.sub.3, thoroughly
dialysed and freeze-dried.
B. preparation of digoxin-BSA
Digoxin bovine serum albumin (BSA) was prepared in the same manner
as the above digoxin-HRP, but the starting materials were 436 mg of
digoxin and 560 mg of BSA, the quantities of the other reagents
having been raised in the same ratio as the digoxin.
C. preparation of antibodies against digoxin
Five rabbits were each injected with 400, 800 and 1600 .mu.gm of
digoxin-BSA respectively, at fortnightly intervals. The immunogen
was mixed with complete Freund's adjuvant and administered
intramuscularly. A fortnight after the last injection the animals
were injected intravenously with 800 .mu.gm of digoxin-BSA in
physiological salt. Ten days later the animals were bleeded. The
serum was adsorbed with BSA-m-aminobenzyloxymethyl-cellulose.
D. preparation of anti-digoxin-cellulose
Anti-digoxin-cellulose was prepared by Gurvich's method as
described in example I D.
E. determination of digoxin
A dilution series was prepared of digoxin in 0.1 M phosphate buffer
of pH 7.5 containing 0.9% NaCl, 0.5% Tween-20 and 1.0% of BSA. The
dilution series was of from 0.1-100 ngm/ml. One millilitre of a
digoxin solution was mixed with 0.1 ml of digoxin-HRP in a suitable
dilution, after which 2 mg of antidigoxin-cellulose suspended in
0.4 ml of buffer were added. The mixture was rotated for 6 hours at
room temperature, after which it was centrifuged and the enzyme
activity in the supernatant liquid determined.
Addition of 0.8 ngm of digoxin proved to cause a measurable
increase of enzyme activity in the supernatant liquid. Digitoxin
only showed a slight cross-reaction in the system whereas
cholesterol, cortisol, oestradiol, testosterone and progesterone
did not show any cross-action in the system.
Example VI
Determination of cortisol
A. preparation of cortisol-21-galactose-oxidase
Fifty mg of cortisol-21-hemisuccinate and 100 mg of
galactose-oxidase were coupled by the mixed anhydride technique as
described in example I A.
B. preparation of insoluble transcortine
One hundred milligrams of transcortine, purified by DEAE,
cellulose, and hydroxyl apatite chromatography respectively, were
coupled to 3 gm Sepharose 4 B by the CNBr method, as follows. Three
grams of Sepharose 4 B suspension were activated by mixing it with
4 ml of a 2.5% (w/v) CNBr solution in distilled water, after which
the pH was adjusted between 10 and 11 with 1 N NaOH, at which value
it was maintained for 6 minutes. The Sepharose was washed with
ice-water and with 0.1 M NaHCO.sub.3. Then 100 mg of transcortine
in 20 ml of 0.1 M NaHCO.sub.3 were added and the suspension was
shaken for 24 hours at 4.degree.C. After being washed with
successively 0.5 M NaHCO.sub.3, 0.05 M citrate buffer of pH 1.1 and
0.05 M phosphate buffer of pH 6.0, the Sepharose was kept in the
last buffer, to which 0.1% merthiolate had been added.
C. determination of cortisol
Of a cortisol-containing sample (standard, plasma or urine) 0.5 ml
were extracted twice with methylene chloride (2 .times. 3 ml). The
combined extraction liquids were evaporated to dryness. The residue
was taken up in 0.5 ml of physiological salt solution and mixed
with 0.2 ml of cortisol-21-galactose-oxidase in a suitable
concentration and 0.3 ml of a transcortine-Sepharose suspension (5
mg/ml). The mixture was rotated for 15 minutes at 4.degree.C and
centrifuged, after which the enzyme activity in the supernatant
liquid was determined by adding 0.5 ml of it to 1.5 ml of substrate
consisting of: 100 mg of D-galactose, 20 mg of 5-aminosalicylic
acid and 10 .mu.gm of peroxidase in 150 ml of 0.02 M phosphate
buffer of pH 6.0. Thirty minutes later the extinction was measured
at 460 nm.
Five ngm/ml of cortisol in the sample caused a measurable increase
of the enzyme activity in the supernatant liquid. Corticosterone
and progesterone only influenced the system when larger quantities
were added. Testosterone and aldosterone had hardly any
influence.
Example VII
Determination of transcortine
The reagents used for the determination of cortisol, as described
in Example IV, were also employed for the determination of
transcortine.
Of a dilution series of transcortine of from 0 - 1280 ngm/ml 0.5 ml
was incubated for 15 minutes at 4.degree.C with 0.2 ml of
cortisol-21-galactose-oxidase in a suitable dilution. To this
dilution series 0.3 ml of transcortine-Sepharose (15 mg/ml) were
added and the mixture was rotated for 15 minutes at 4.degree.C. The
activity in the supernatant liquid was measured as described in
example VI.
A sample containing 40 ngm/ml of transcortine proved to cause a
measurable increase of enzyme activity in the supernatant liquid,
whereas in the presence of 320 ngm/ml all the enzyme activity was
found in the supernatant liquid.
* * * * *