U.S. patent number 3,646,346 [Application Number 04/786,959] was granted by the patent office on 1972-02-29 for antibody-coated tube system for radioimmunoassay.
This patent grant is currently assigned to Pharmacia AB. Invention is credited to Kevin J. Catt.
United States Patent |
3,646,346 |
Catt |
February 29, 1972 |
**Please see images for:
( Certificate of Correction ) ** |
ANTIBODY-COATED TUBE SYSTEM FOR RADIOIMMUNOASSAY
Abstract
A radioimmunological method and means for the determination of
proteins in aqueous samples which includes adsorbing on the
internal surface of a test tube of water-insoluble polymeric
material the antibody against the protein to be determined.
Suitable proteins which can be determined according to this method
are plasma proteins, enzymes, and many hormones.
Inventors: |
Catt; Kevin J. (Melbourne,
AU) |
Assignee: |
Pharmacia AB (Uppsala,
SW)
|
Family
ID: |
25140050 |
Appl.
No.: |
04/786,959 |
Filed: |
December 26, 1968 |
Current U.S.
Class: |
436/531; 206/223;
206/524.3; 206/828; 250/303; 250/304; 435/7.4; 436/804; 436/808;
436/810; 436/817; 436/818; 422/527 |
Current CPC
Class: |
G01N
33/545 (20130101); Y10S 436/81 (20130101); Y10S
206/828 (20130101); Y10S 436/804 (20130101); Y10S
436/817 (20130101); Y10S 436/818 (20130101); Y10S
436/808 (20130101) |
Current International
Class: |
G01N
33/544 (20060101); G01N 33/545 (20060101); G01t
001/16 () |
Field of
Search: |
;250/83SA,16T ;23/23B
;195/103.5 ;424/12 ;206/84 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Borchelt; Archie R.
Claims
What I claim is:
1. A method for the determination of proteins in aqueous samples
wherein said proteins are capable of acting as antigens which
comprises:
a. coating by adsorption at least part of the internal surface of a
test tube of water-insoluble polymeric material with antibodies
against the protein to be determined;
b. said water-insoluble polymeric material being capable of
adsorbing said antibodies;
c. contacting the internal surface of said test tube with the
aqueous sample containing the protein;
d. contacting the internal surface of said test tube with the same
protein in labeled form capable of emitting radiation;
e. contacting steps (c) and (d) causing the bonding of part of the
labeled protein and part of the unlabeled protein to said
antibodies adsorbed on the surface of the polymeric material;
f. producing a two-phase system;
g. said two-phase system comprising a solid phase and a liquid
phase;
h. said solid phase comprising the bound part of the labeled and
unlabeled protein;
i. said liquid phase comprising the unbound part of the labeled and
unlabeled protein;
j. separating said two phases from each other;
k. and measuring the emitted radiation of at least one of said
phases,
wherein the value of said radiation being a function of the
concentration of the protein in the aqueous sample.
2. The method of claim 1 wherein the protein is labeled with a
radioactive isotope of iodine.
3. The method of claim 2 wherein said radioactive isotope of iodine
is I.sup.125.
4. The method of claim 1 wherein said water-insoluble polymeric
material is selected from the group consisting of polystyrene,
polyethylene, polypropylene, nitrocellulose, and copolymers of
acrylonitrile with styrene.
5. The method of claim 1 wherein said concentration of the protein
is obtained by direct comparison with a standard curve.
6. The method of claim 1 wherein said protein is selected from the
group consisting of human placental lactogen, human growth hormone,
luteinizing hormone, human chorionic gonadotropin, tetanus toxin,
and insulin.
7. Means for the determination of proteins in aqueous samples,
comprising:
a. a first reagent and a second reagent;
b. said first reagent including a test tube of a polymeric material
which is capable of adsorbing antibodies against the protein to be
determined;
c. the internal surface of the test tube being at least partially
coated with the antibodies;
d. said second reagent comprising labeled protein capable of
emitting radiation;
e. the first reagent being intended to be contacted with the sample
containing the protein and with the second reagent to bind part of
the labeled and unlabeled protein to the adsorbed antibodies to
produce a two-phase system;
f. the two-phase system comprising a solid phase and a liquid
phase, wherein said solid phase includes the bound part of the
labeled and unlabeled protein and the liquid phase includes the
unbound labeled and unlabeled protein; and
g. the emitted radiation of each phase being a function of the
concentration of the protein.
8. Means according to claim 7 in the form of a test pack.
Description
The present invention relates to a method and means for the
determination of proteins, for instance protein hormones, in
aqueous samples, e.g., from body fluids such as blood serum or
urine, but also from other sources such as different types of gland
extracts. An essential factor of the method is that the substance
to be determined is capable of acting as an antigen, i.e., is
capable of causing the formation of antibodies against itself in
animals.
In the following specification and in the annexed claims the term
"protein" is intended to include proteins, polypeptides and
peptides.
The invention is based partly upon the knowledge that under certain
circumstances proteins are generally able to act as antigens, i.e.,
able to cause the formation of antibodies, and partly on the fact
that radioimmunological methods are very sensitive and well suited
for determining different proteins present in a very low
concentration in body fluids.
Radioimmunological methods are in general based on the ability of
an antibody to bind its protein antigen irrespective of whether the
latter is labeled with, for example, a radioactive isotope, or not.
The binding of labeled and unlabeled protein antigens takes place
in proportion to the concentration of labeled and unlabeled,
respectively, proteins. The radioactivity of the labeled protein
which is bound to the antibodies, and/or of the free, labeled
protein in the sample liquid is measured. The amount of unlabeled
competing protein can be determined from the obtained values by
calculation or by direct comparison with a standard curve.
In principle, radioimmunological methods can be applied to proteins
which are antigenic, capable of being purified and labeled with a
radioactive isotope or a fluorescent group. The antibody bound
protein has to be separated from the unbound protein.
The present invention is based on the ability of a polymeric
material coated, by adsorption, with antibodies against the protein
to be determined to bind specifically the same protein labeled with
a radiation emitting atom or group such as a radioactive isotope.
The use of antibodies in this form allows rapid removal of the free
labeled protein by washing of the solid phase with water on
completion of the immune reaction. The solid-phase material, that
is the labeled protein bound to the antibodies on the polymer, can
then be counted for quantitation of the bound labeled substance,
which varies inversely with the total quantity of protein in the
original sample. This simple and sensitive procedure can be used to
measure very small quantities of the protein in, for example,
plasma.
In connection with the conception of the present invention it has
been found that various polymeric materials present applicability
to the above determination method based on solid-phase
radioimmunoassay. It has been apparent that certain polymers may
adsorb antibodies that can then bind an adequate quantity of
labeled protein or polypeptide for use in the assay. In contrast,
adsorption of antibodies to glass is negligible. The adsorption of
antibodies by polymeric surfaces, from antiserums preassume that
the antiserum is of moderately high titer.
The adsorption process has been applied to the immunoassay above
mentioned by coating of the interior of plastic tubes with uniform
quantities of specific antibodies. The excellent practical results
and advantages obtained with the invention is ascribed to the use
of the above plastic tube for adsorbing the antibody material.
The method according to the invention comprises contacting the
internal surface of a test tube of water-insoluble polymeric
material capable of adsorbing antibodies against the protein to be
determined, at least part of said surface being coated with a layer
of said antibodies by adsorption, with the aqueous sample
containing the protein and with the same protein in labeled form
capable of emitting radiation to bind part of said labeled and
unlabeled protein to said antibodies adsorbed on the surface of the
polymeric material to produce a two-phase system comprising a solid
phase comprising said bound part of labeled and unlabeled protein
and a liquid phase comprising unbound labeled and unlabeled
protein, separating said two phases from each other, and measuring
the emitted radiation of at least one of said solid and said liquid
phases, the value of said radiation being each a function of the
concentration of the protein in the aqueous sample.
The labeled protein can be labeled with, for example, a radioactive
isotope or a fluorescent group.
The method can be used for qualitative and quantitative
determination.
A method is previously known in which the antibodies are attached
to particles of a water-insoluble carrier by covalent bonds. The
labeled protein reacting with the antibodies in the determination
can thus be readily separated from the unbound labeled protein, the
separation being insensitive to variations in the salt and protein
concentration of the liquid within physiological limits.
In comparison with the above-mentioned excellent method the present
one offers several additional advantages. Thus, it will be very
easy to prepare the reagent comprising antibodies in that a
solution of said antibodies only need to be contacted with the
inside of the tube of polymeric material. After the removal of the
liquid containing the antibodies from the surface of the test tube
the antibodies will be bound by adsorption with sufficiently high
firmness to render it possible to transport the tube without any
special precautions. (If particles are used as in the previously
described method steps must be taken so that they retain in the
tube during transportation.) The steps of the determination will be
very easy to carry out which is of value in routine analysis
carried out in hospital laboratories. The separation of the solid
and liquid phases, which is the essence of the method before
measuring the radiation can be carried out by simply removing the
liquid from the test tube rather than separating by such methods as
filtration and centrifugation, said latter methods being also
connected with risks that the separation be incomplete.
The method requires access to the protein to be determined for
producing antibodies and for preparing radioactive labeled
proteins, and suitably also for obtaining standard solutions, for
instance, for obtaining standard curves.
Examples of proteins against which antibodies can be obtained are
plasma proteins, enzymes and many hormones. Examples of such
hormones are insulin, gonadotropins, growth hormone, ACTH,
thyrotropin and parathormone.
The antibodies against the protein can be prepared by any method
known per se, by immunizing animals used for experiments, by, for
instance, repeated subcutaneous injections of small amounts of the
antigenic protein possibly combined with a so-called adjuvant such
as Freund's mineral oil emulsion, into the animal. The antibodies
produced in the animals can be recovered from the blood serum of
the same. The protein fraction, which contains the antiserum, can
be recovered by conventional methods, e.g., by precipitating the
serum with suitable amounts of a saturated aqueous solution of
ammonium sulphate.
Labeling of the protein with a radioactive isotope can be effected
in a conventional manner, a suitable isotope for the purpose being
selected, e.g., I.sup.125, I.sup.131, C.sup.14 or H.sup.3. A
particularly suitable isotope is a radioactive isotope of iodine
such as I.sup.125, as labeling with this isotope is simple and as
many hospital laboratories now have the equipment necessary to
measure this isotope.
As test tubes of polymeric materials may be mentioned ordinary
plastic test tubes for laboratory purposes. As polymeric materials
may be mentioned polystyrene, polyethylene, polypropylene,
nitrocellulose, copolymers of acrylnitril with styrene such as
poly(styrene-co-acrylonitril).
The radioactivity determinations can be effected by common methods,
e.g., by means of scintillation detectors.
The amount of labeled protein, e.g., I.sup.125 hormone, added in
the reaction is chosen so that, for instance, 20-60 percent of the
labeled hormone can be bound to the antibodies when no competing
unlabeled hormone is present. The incubation is preferably made at
temperatures between +4.degree. and 37.degree. C. and commonly at
room temperature. It is not necessary for the reaction between the
antigen and the antibodies to go to completion. The reaction is
interrupted after, for instance, 24 hours, but may also be stopped
earlier, for instance, after 2-4 hours. It is important that the
reaction time and temperature are the same for the sample solutions
and standard solutions.
Because the method is simple, rapid and practical, and gives
accurate analysis results it is well suited for quantitative
determinations also for routine usage and permits determination of
even very small amounts of sample substances.
The invention also encompasses a means for carrying out the
above-mentioned method. This means comprises a first reagent
comprising a test tube of polymeric material capable of adsorbing
antibodies against the protein to be determined, the internal
surface of said test tube having at least part thereof coated by
adsorption with said antibodies, and a second reagent comprising
labeled protein capable of emitting radiation, said first reagent
being intended to be contacted with the sample containing the
protein and with the second reagent to bind part of the said
labeled and unlabeled protein to the adsorbed antibodies thereby to
produce a two-phase system comprising a solid phase comprising said
bound part of labeled and unlabeled protein and a liquid phase
comprising unbound labeled and unlabeled protein, the emitted
radiation being each a function of the concentration of the
protein.
According to a preferred embodiment of the invention the means is
in the form of a test pack.
In the accompanying drawings there are shown examples of standard
curves which were used in connection with practicing the present
invention. The following is a brief description of the various
figures:
FIG. 1 shows a standard curve obtained by plotting radioactivity
(measured as counts per minute .times.10 .sup..sup.-3) along the
ordinate against amount of human placental lactogen in nanograms
along the abscissa (see Example 1 below),
FIG. 2 shows a standard curve obtained by plotting radioactivity
(measured as counts per minute .times.10.sup. .sup.-3) along the
ordinate against amount of human growth hormone in nanograms along
the abscissa (see Example 2 below), and
FIG. 3 shows a standard curve obtained by plotting percentage of
bound .sup.125 I-insulin (based on the total amount of .sup.125
I-insulin added) along the ordinate against concentration of
insulin in .mu.U per ml. along the abscissa (see Example 6
below).
The invention will be more closely illustrated in the following
with reference to detailed examples.
EXAMPLE 1
Determination of Human Placental Lactogen (HPL) in plasma
a. Preparation of antibodies
Rabbits were injected with 5 mg. quantities of purified human
placental lactogen. The placental lactogen was emulsified in a 1:1
mixture of saline and complete Freund's adjuvant, and injected
subcutaneously at intervals of 3 weeks for a period of 3-6 months.
After this time, two of four rabbits were found to have a
satisfactory titre of antibodies to placental lactogen, showing
reactions of partial identity with human growth hormone on gel
diffusion. These antisera were used to coat tubes for the assay
without further fractionation.
b. Preparation of antibody-coated tubes
Polystyrene blood collection tubes were coated with 1.0 ml. of a 1
in 500 dilution of anti-HPL serum in 0.05 M bicarbonate buffer pH
9.6. After incubation for 2-16 hours at room temperature, the
contents were aspirated and the tubes washed with saline and 10
percent aged human serum as described above.
c. Preparation of labeled HPL
Purified HPL was labeled with .sup.125 I by the chloramine-T
procedure as described above for human growth hormone. The
resulting iodinated peptide had a specific activity of 100-120
m.mu.c/m.mu.g, and was stored frozen in 0.5 percent bovine serum
albumen in 0.15 M NaCl until used for the assay. This material was
stable for up to 6 weeks when stored frozen.
d. Measurement of HPL in plasma and urine
Standard HPL solutions in 20 percent horse serum and a final volume
of 1.0 ml. were incubated in the coated tubes with 0.25 ml. of
buffer containing 100,000 counts per minute of .sup.125 I human
placental lactogen. Plasma or urine samples were similarly diluted
with a solution of 20 percent horse serum in 0.15 M NaCl containing
0.01 M phosphate buffer pH 7.4 and 0.01 percent merthiolate. An
identical aliquot of .sup.125 I - human placental lactogen was
added to the sample tubes. After incubation of standards and
samples at 37.degree. for 16 hours, the contents of the tubes were
aspirated and washing was performed twice with tap water followed
by counting for 1 minute in an automatic gamma counter. A standard
curve was constructed by plotting bound counts versus added HPL,
and as with the HGH assay described above, a linear form of the
standard curve could be obtained by plotting a reciprocal function
of the bound count against added HPL. The concentration of HPL in
plasma and urine samples was estimated by reading off the standard
curve, which was performed with each assay. HPL was not detected in
the plasma or urine of normal subjects, and was found to be present
at a level of 36 nanograms/ml. at the ninth week of pregnancy,
rising to 33,000 ng./ml. at the 26th week.
On accompanying drawing FIG. 1, there is shown a standard curve
obtained with tubes coated with 2.0 ml. of antiserum to human
placental lactogen (HPL) diluted 1:250. Incubation has been
performed in quadruplicate for 64 hours at 37.degree. C. with 0 to
10 ng. of HPL and 118,000 counts per minute .sup.125 I - HPL in 2.2
ml. of 20 percent horse serum.
In this connection, the tube count (counts per minute)
.times.10.sup. .sup.-3 has been plotted along the ordinate as a
function of the amount of HPL measured in nanograms along the
abscissa to obtain the standard curve as shown.
The curve can be used as a basis for determining the amount of HPL
in unknown samples in the range of from 0 to 10 nanograms.
EXAMPLE 2
Determination of Human Growth Hormone (HGH) in blood plasma
a. Preparation of antibodies
Rabbits were injected subcutaneously with 2.5 mg. human growth
hormone in saline: complete Freund's adjuvant: 1:1 at intervals of
3 weeks for a total period of 3-5 months. Blood from the animals
with the highest antibody titre was then harvested over the
following several weeks and the serum stored frozen. These antisera
were used to coat plastic tubes without further fractionation.
b. Preparation of antibody-coated tubes
Polystyrene blood collection tubes (90.times.15 mm.) were used for
most of the assays. These polystyrene tubes were coated with
antibody by adding 1.0 ml. of a 1 in 500 dilution of anti-HGH serum
in 0.05 M bicarbonate buffer pH 9.6, then standing the tubes for
2-24 hours. After this period, the contents were aspirated and the
tubes washed out three times with 0.15 M NaCl and once with 10
percent aged human plasma in 0.15 M in NaCl containing 0.01 percent
merthiolate.
The antisera obtained from various rabbits were diluted over a
range 1:500-1:25,000 to determine the maximum dilution which could
satisfactorily be used for the assay. As expected, the most diluted
solutions gave somewhat more sensitive assays, though the
relationship between dilution and sensitivity was much less direct
than that observed in the conventional liquid-phase assay. For most
assays, a standard antiserum was used at a dilution of 1:10,000,
giving very satisfactory results over the range 0.4-10
nanograms/ml. plasma.
c. Preparation of labeled human growth hormone
Human growth hormone was labeled with .sup.125 I by a modification
of the technique of Hunter & Greenwood. In this procedure, 5-10
.mu.g. of purified HGH was reacted with 2 millicuries of .sup.125 I
by the Chloramine T method, using a specially designed disposable
radioiodination pipette. The labeled HGH was then isolated by
fractionation of the reaction mixture on a column of Sephadex G 75,
or on a column of cellulose. This procedure uniformly gave labeled
HGH of specific activity of 100-150 microcuries per microgram, a
level found to be satisfactory for use in this form of
radioimmunoassay. The labeled hormone was collected into 5 percent
bovine serum albumen in 0.15 M NaCl, and stored frozen for up to 3
weeks for use in the radioimmunassay.
d. Measurement of HGH in plasma
To estimate HGH levels in human plasma, specimens were diluted 1 in
5 with a diluent solution consisting of 5 percent aged human plasma
in 0.15 M NaCl containing 0.01 M phosphate buffer pH 7.4 and 0.01
percent merthiolate. Standard HGH solutions were prepared in a
solution consisting of 20 percent serum in a similar diluent
solution, and levels of 0.25-5 nanograms/ml. were set up in each
standard curve. The total volume of standards and diluted serums
was 1.0 ml., to this was added 0.25 ml. of diluent containing
10.sup. 5 c.p.m. of .sup.125 I HGH. The final incubation volume
then exceeded the coated volume of the tube by 0.25 ml. The tubes
were incubated overnight at 37.degree. C., then the contents were
aspirated and each tube was washed out twice with tap water and
counted for 1 minute in an automatic gamma counter. The counts
obtained for the standard HGH solutions were used to construct a
standard curve from which the quantities of HGH present in the
unknown samples could be read. It has been shown that the
relationship between any reciprocal function of the bound count,
i.e., the count attached to each tube is proportional to the
quantity of growth hormone present in the tube, so that a straight
line can be obtained by plotting the levels of the standard growth
hormone versus any reciprocal function of the bound count.
Alternatively, the bound count can be plotted directly against
added HGH concentration, giving a hyperbolic standard curve.
The FIG. 2 shows a standard curve obtained in analogy with FIG. 1
with tubes coated with 1.0 ml. of antiserum to HGH, diluted 1:500.
Incubation has been performed for 20 hours at 37.degree. C. with 0
to 10 ng. of HGH and 95,000 counts per minute .sup.125 I - HGH in
1.2 ml. of 20 percent horse serum.
By this method, the levels of HGH present in normal plasma were
found to be between 1 and 10 nanograms/ml. The level fluctuates
considerably in normal subjects, sometimes reaching spontaneous
peaks of up to 25 nanograms per ml. In hypopituitary individuals,
such peaks are not observed, and the basal level of less than 1
nanogram/ml. does not rise during insulin hypoglycaemia as it does
in the normal subject. In acromegalic subjects, the level of HGH
was between 12 and 200 nanograms/ml., and these levels were not
suppressed following glucose ingestion, unlike the elevated levels
sometimes observed in the normal individual.
EXAMPLE 3
Measurement of Luteinizing Hormone (LH) and Human Chorionic
Gonadotropin (HCG) in human plasma and urine
Tubes were coated with anti-HCG serum (1:2000) and used to measure
LH and HCG in human plasma and urine, employing .sup.125 I labeled
LH as tracer and purified LH as standards. Levels of 1-2 ng./ml.
were found in plasma in normal males and females rising to 7-13
ng./ml. at the time of ovulation, and following the menopause.
EXAMPLE 4
Estimation of Luteinizing Hormone (LH) in the plasma of the sheep
and cow
Antisera to purified ovine and bovine LH prepared in the horse and
rabbit were used to coat polystyrene tubes, at dilution of
1:2,000-1:10,000. Such coated tubes were used to establish an assay
for estimation of LH in sheep and cow plasma. Basal levels in
female animals were 1-4 ng./ml., rising to high levels (up to 200
ng./ml.) at the time of ovulation.
EXAMPLE 5
Estimation of Tetanus Toxin by radioimmunoassay
Tubes were coated with anti-tetanus serum diluted 1:1000, and
purified tetanus toxin was labeled with .sup.125 I by the
chloramine-T method. By this procedure, small concentrations of
tetanus toxin, down to the range of 10-100 nanograms/ml. could be
estimated.
EXAMPLE 6
Determination of insulin in blood plasma
a. Preparation of antibodies
Guinea pigs were each injected subcutaneously with 0.1 mg. of pig
insulin in 1 ml. of Freund's adjuvant. Immunization was repeated
every week for 4 weeks. After further 2 weeks, blood was drawn off
from the guinea pigs and antiserum recovered from the blood by
allowing the same to coagulate, and removing the clots of
blood.
The antibody fraction was precipitated from this antiserum by
treatment with 18 percent sodium sulphate.
The precipitate was separated by centrifugation. The precipitate
was washed two times with a 18 percent sodium sulphate solution.
Subsequent to the last washing the precipitate was dissolved in
original serum volume of an aqueous solution of sodium hydrogen
carbonate, after which dialysis took place against 0.05 M sodium
hydrogen carbonate solution. This antibody fraction was used for
the preparation of antibody coated tubes.
b. Preparation of antibody coated tubes
Polystyrene test tubes (55.times.10 mm.) were used as the starting
material. To the tubes was added 1 ml. of a 5,000 times diluted
gammaglobulin solution of the anti-insulin serum, in 0.05 M
carbonate-bicarbonate buffer of pH 8.3. The tubes were incubated at
37.degree. C. for 1 hour and then carefully washed two times with
0.9 percent sodium chloride solution containing 1 percent human
serum albumen and finally with 0.05 M phosphate buffer, pH 7.4
containing 0.05 percent NaN.sub.3, 0.9 percent NaC1, 0.3 percent
human serum albumen and 0.05 percent Tween 20. (This buffer will be
called "incubation buffer.")
c. Preparation of labeled insulin
Pig insulin was labeled with .sup.125 I according to the following
method: 5 mC .sup.125 I in the form of NaI was oxidized with
Chloramine T in the presence of 5 .mu.g. of insulin in accordance
with a method described by Hunter and Greenwood (ref.
Nature/London/ , volume 194/1962/, page 495). Subsequent to the
labeling sodium dithionite was added to convert the remaining
amount of iodine to soluble iodide. The obtained insulin labeled
with .sup.125 I was mixed with human plasma-albumen and separated
from low molecular weight products and from denaturation products
of insulin bound to the plasma-albumen by gel filtration on a
copolymer of dextran with epichlorohydrin (Sephadex G-50). The
insulin labeled in this way has a specific activity of 100-200 mC
per mg. The second peak of the labeled protein fraction was
collected in a small vessel containing 1/2 ml. of a solution of
human plasma-albumen containing 50 mg. per ml. The labeled hormone
was stored in cold surroundings and diluted before being used.
d. Determination
The analyses were performed in the antibody coated test tubes.
1. 0.5 ml. of "incubation buffer" was added to the tubes.
2. 0.1 ml. of the plasma to be tested was added to quadruplicate
tubes.
3. 0.1 ml. of a solution containing different concentration of the
hormone, e.g., 200, 100, 50, 25, 10, 5 and 2.5 .mu.U insulin/ml.
and 0 .mu.U insulin/ml. were distributed to different tubes in
quadruplicates.
4. 0.1 ml. of a solution containing .sup.125 I-insulin (approx. 1
nanogram per ml.) was added to all tubes.
5. 0.5 ml. of "incubation buffer" was added to each tube.
6. Incubation took place at + 4.degree. C. for 64 hours.
7. The tubes were either washed two times with the "incubation
buffer" or twice with distilled water. After the last removal by
suction of the washing liquid, the tubes were placed in a counter
for estimating the gamma radiation from the antibody bound labeled
hormone.
The number of "counts" per time of standard tubes was determined
and converted into per cent of bound .sup.125 I - insulin based on
the total amount of .sup.125 I - insulin added to the tubes. The
percentages obtained were plotted along the ordinate against the
concentration of insulin in .mu.U per ml. along the abscissa to
form the curve as shown in diagram 3. From this diagram the
concentration of insulin in the unknown sample could be easily
determined.
* * * * *