U.S. patent number RE29,474 [Application Number 05/598,141] was granted by the patent office on 1977-11-15 for method for the determination of proteins and polypeptides.
This patent grant is currently assigned to Pharmacia AB. Invention is credited to Rolf E. A. V. Axen, Jerker O. Porath, Leif Edvin Wide.
United States Patent |
RE29,474 |
Axen , et al. |
November 15, 1977 |
**Please see images for:
( Certificate of Correction ) ** |
Method for the determination of proteins and polypeptides
Abstract
A process comprising contacting particles of water insoluble
polymers to which have been bound antibodies, by means of covalent
bonds, against the protein or polypeptide to be determined, wherein
a certain quantity of the protein or polypeptide is labeled with a
radioactive isotope, whereupon the particles, subsequent to the
reaction between the protein or the polypeptide and the antibodies
attached to the particles are separated from the sample liquid and
the radioactivity of the particle material and/or in the liquid is
determined.
Inventors: |
Axen; Rolf E. A. V. (Upplands
Balinge, SW), Porath; Jerker O. (Uppsala,
SW), Wide; Leif Edvin (Uppsala, SW) |
Assignee: |
Pharmacia AB (Uppsala,
SW)
|
Family
ID: |
27354438 |
Appl.
No.: |
05/598,141 |
Filed: |
July 23, 1975 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
Reissue of: |
643190 |
Jun 2, 1967 |
03555143 |
Jan 12, 1971 |
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Foreign Application Priority Data
Current U.S.
Class: |
436/529; 436/533;
436/547; 436/804; 436/817; 436/818 |
Current CPC
Class: |
G01N
33/54313 (20130101); G01N 33/74 (20130101) |
Current International
Class: |
G01N
33/543 (20060101); G01N 33/74 (20060101); A61K
043/00 () |
Field of
Search: |
;424/1,1.5 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Munoz, John J., Some Newer Immunological Techniques, Analytical
Chemistry, vol. 31, No. 6, pp. 981-985, June 1959. .
Pressman et al., Computer Programs for Paired and Triad Radioiodine
Label Techniques, Chem. Abstracts, vol. 64, p. 5606G, 1966. .
Bennington, Radioautographic Analysis of Soluble Antibody-Antigen
Complexes, Chem. Abstracts, vol. 54, p. 17521A. .
Jagendorf et al., Biochem. Biophys. Acta, vol. 78, pp. 516 to 528
(1963). .
Drizlikh et al., Biokhimiya, vol. 29, No. 6, pp. 1054 to 1062
(1964)..
|
Primary Examiner: Sebastian; Leland A.
Attorney, Agent or Firm: Hubbell, Cohen, Stiefel &
Gross
Claims
What is claimed is:
1. A method for the determination of a member selected from the
group consisting of proteins and polypeptides in aqueous samples,
which comprises contacting particles of water insoluble polymers to
which have been bound, by covalent bonds, antibodies against the
said member to be determined, with the aqueous sample containing
said member and with the same member labelled with a radioactive
isotope to bind part of said labelled member and unlabelled member
to said antibodies to produce a two-phase system comprising a solid
phase comprising said bound part of labelled member and unlabelled
member and a liquid phase comprising unbound labelled member and
unlabelled member, separating the two phases from each other, and
measuring the radioactivity of at least one of the said solid and
said liquid phases, the value of said radioactivity being each a
function of the concentration of the said member in the aqueous
sample.
2. A method as claimed in claim 1, wherein the solid phase is
washed with an aqueous liquid before measuring the radioactivity of
said phase.
3. A method as claimed 1, wherein the determination is effected
quantitatively by comparing the measured value of the radioactivity
with a standard curve. .Iadd. 4. A method as claimed in claim 3,
wherein said antibodies have been covalently bound to said
particles under conditions sufficiently mild to ensure that the
reactivity of said antibodies does not substantially decrease.
.Iaddend..Iadd. 5. A method as claimed in claim 4, wherein said
covalent bonds are formed through a group of the formula
--NH.multidot.CS.multidot.NH--, --NH.multidot.CO.multidot.NH--, or
--N=N--. .Iaddend..Iadd. 6. A method as claimed in claim 5, wherein
said group is --NH.multidot.CS.multidot.NH--. .Iaddend..Iadd. 7. A
method as claimed in claim 3, wherein said water-insoluble polymers
contain at least one reactive group selected from the group
consisting of amino, hydroxyl and carboxyl. .Iaddend..Iadd. 8. A
method as claimed in claim 7, wherein said reactive group is
hydroxyl. .Iaddend..Iadd. 9. A method as claimed in claim 7,
wherein said reactive group is amino. .Iaddend..Iadd. 10. A method
as claimed in claim 3, wherein said polymer is obtained by
crosslinking a material selected from the group consisting of
carbohydrates, sugar alcohols, and polyvinyl alcohol, with a
bifuctional compound of the formula X--R--Z, wherein X and Z are
each independently halogen or epoxy, and R is an aliphatic radical
containing from 3 to 10 carbon atoms. .Iaddend..Iadd. 11. A method
as claimed in claim 10, wherein said material is dextran and said
bifunctional compound is epichlorohydrin. .Iaddend..Iadd. 12. A
method as claimed in claim 3,
wherein said member is a plasma protein. .Iaddend..Iadd. 13. A
method as claimed in claim 3, wherein said member is an enzyme.
.Iaddend..Iadd. 14. A method as claimed in claim 3, wherein said
member is a hormone. .Iaddend..Iadd. 15. A method as claimed in
claim 3, wherein said member is insulin. .Iaddend..Iadd. 16. A
method as claimed in claim 3, wherein said member is a
gonadotropin. .Iaddend..Iadd. 17. A method as claimed in claim 3,
wherein said member is growth hormone. .Iaddend..Iadd. 18. A method
as claimed in claim 3, wherein said member is ACTH. .Iaddend..Iadd.
19. A method as claimed in claim 3, wherein said member is
thyrotropin. .Iaddend..Iadd. 20. A method as claimed in claim 3,
wherein said member is parathormone. .Iaddend..Iadd. 21. A method
as claimed in claim 3, wherein said aqueous sample is from blood
serum. .Iaddend..Iadd. 22. A method as claimed in claim 3, wherein
said aqueous sample is from urine. .Iaddend..Iadd. 23. A method as
claimed in claim 3, wherein said radioactive isotope is I.sup.125,
I.sup.131, C.sup.14, or H.sup.3..Iaddend.
Description
The present invention relates to a method for the determination of
proteins and polypeptides, for instance protein and polypeptide,
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.
The invention is characterized in that particles of water insoluble
polymers to which have been bound antibodies, by means of covalent
bonds, against the protein or polypeptide to be determined, are
contacted with the sample and with a certain quantity of the
protein or polypeptide labeled with a radioactive isotope,
whereupon the particles, subsequent to the reaction between the
protein or the polypeptide and the antibodies attached to the
particles having taken place, are separated from the sample liquid
and the radioactivity of the particle material and/or in the liquid
is determined.
The method can be used for qualitative and quantitative
determination.
The invention is based partly upon the knowledge that under certain
circumstances proteins and polypeptides 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 and
polypeptides, 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 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
and polypeptides which are antigenic, capable of being purified and
labeled with a radioactive isotope. The antibody bound protein has
to be separated from the unbound protein. This separation process
has previously been effected by a large number of different
methods, such as paper chromotography, electrophoresis,
precipitation with a salt or ethyl alcohol, precipitation of
antibodies by antibodies against the latter or gel filtration.
These methods are complicated, time consuming, unpractical and
unreliable for use in routine tests, e.g. in an ordinary hospital
laboratory.
The great advantage of the present method is that the antibodies
are firmly attached to an insoluble carrier and that the labeled
protein, which reacts with and is bound to the antibodies in the
determination, can thus be easily separated from the unbound
labeled protein, e.g. by simple centrifugation, or filtration, the
separation being insensitive to variations in the salt and protein
concentrations of the liquid within physiological limits. The test
is easy to perform as known amounts of particles together with
antibodies attached thereto, can be predispensed in test tubes, for
instance, and stored without loosing the binding ability. The whole
procedure, including the separation of the free labeled proteins
and antibody bound labeled proteins, can be made in one and the
same test tube without any further addition of precipitants or the
like.
The method requires access to the protein or the polypeptide to be
determined for producing antibodies and for preparing radioactive
labeled proteins or polypeptides, and suitably also for obtaining
standard solutions, for instance, for obtaining standard
curves.
Examples of proteins and polypeptides 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 or the polypeptide can be
prepared by any method known per se, by immunising animals used for
experiments, by, for instance, repeated subcutaneous injections of
small amounts of the antigenic protein or polypeptide possible
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 or polypeptide 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.sub.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, for instance, many hospital laboratories now have
the equipment necessary to measure this isotope.
Particles of water insoluble polymers are used as carriers for the
antibodies. The polymer is selected so that it contains, or can be
provided with, suitable reactive groups such as amino groups,
hydroxyl groups and carboxylic groups, to readily make possible the
binding of the antibodies to the polymer by bridges with covalent
linkages.
Particularly suitable is the choice of polymer particles consisting
of a three dimensional network, held together by covalent linkages.
Such particles even though they are swellable in water, are
completely insoluble therein and are thus unable to release any of
the polymer material or of the substance bound thereto by covalent
linkages, e.g. during washing procedures. Examples of such polymer
particles are grains of copolymers obtained by cross linking
substances containing a plurality of hydroxyl groups, such as
carbohydrates and sugar alcohols, such as dextran, starch, dextrins
and other polysaccharides, and polyvinyl alcohol with a
bi-functional substance, e.g. bi-functional substances of the type
X--R--Z, wherein, for instance, X and Z are each halogen or an
epoxy group and R is the residue of the bi-functional substance,
e.g. an aliphatic radical containing from 3 to 10 inclusive carbon
atoms.
Grains of the commercially accessible product Sephadex which is
dextran cross-linked with glycerine ether-bridges, obtained by
treating dextran with epichlorohydrin, for instance, can be used
for the purpose. Sephadex and products obtained in a similar manner
are gel grains capable of swelling in water, but insoluble therein.
They contain hydroxyl groups and can thus easily be substituted
with other groups, e.g. groups containing amino groups or carboxyl
groups, and are thus well suited for forming bridges by covalent
bonds to the antibodies.
Suitably, small particles are chosen so that a wide contact area is
obtained.
The antibodies are bound to the said carrier particles with
covalent bonds under mild conditions so that the immuno-chemical
reactivity of the antibodies does not substantially decrease.
Because of the covalent binding the antibodies cannot loosen and
become washed away from the particles. Reactive groups, such as
amino groups, hydroxyl groups, and carboxyl groups, are used for
chemically binding the antibody protein with the polymer particle,
a bridge having covalent bonds being established between the
antibody protein and the polymer particle, e.g. of the type:
Antibody--NH.CS.NL.Polymer particle
Antibody--NH.CO.NH.Polymer particle
Antibody--N=N-- Polymer particle.
Further, in the analysis a solution of the protein or polypeptide
of known concentration is suitably used as a standard.
The radioactivity determinations can be effected by common methods,
e.g. by means of scintillation detectors.
The quantity of particles with antibodies is selected, among other
things, with thought to the sensitivity level required in the
test.
The amount of labeled protein or polypeptide, e.g. I.sup.125
hormone, added in the reaction is chosen so that, for instance,
approximately 40-60% 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 and
37.degree. C. and usually 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 will be more closely illustrated in the following
with reference to detailed examples and the annexed drawings.
In the annexed drawings,
FIG. 1 is a curve showing counts per minute plotted as a function
of the concentration of gonadotropin (in international units) in a
series of standard solutions, said count being obtained according
to Example 1 and said curve being possible to use for the
determination of gonadotropin in unknown samples as indicated by
the dashed lines;
FIG. 2 is a curve showing counts per 10 minutes plotted as a
function of the concentration of growth hormone (in nanograms per
ml.) in a series of standard solutions, said counts being obtained
according to Example 2 and said curve being illustrated as used for
the determination of growth hormone in unknown samples obtained
from a patient after glucose loading at 0 min; at 0 plus 17 min.;
and at 0 plus 80 min., respectively, and
FIG. 3 is a curve showing counts per 5 minutes plotted as a
function of the concentration of insulin in micro units, .mu.u, in
a series of standard solutions per ml., said counts being obtained
according to Example 3 and said curve being illustrated as used for
the determination of insulin in unknown samples obtained from a
patient after glucose loading at 0 min.; at 0+7 min.; at 0+12 min.;
at 0+17 min.; at 0+22 min.; and at 0+40 min. respectively.
EXAMPLE 1
Determination of Gonadotropin in Urine
(A) Preparation of antibodies: Rabbits were injected subcutaneously
with 0.5 mg. of human gonadotropin in 2 ml. of Freund's adjuvant.
Immunization was repeated every week for four weeks. Subsequent to
the passing of a further week, blood was drawn off from the rabbits
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 a saturated aqueous solution of ammonium sulphate,
2.5 ml. of the saturated solution being added to 5 ml. of
serum.
The precipitate was separated by centrifugation. The precipitate
was dissolved in water and the precipitating process with ammonium
sulphate solution was repeated twice. Subsequent to the third
precipitating process the precipitate was dissolved in 0.1 ml. of
an aqueous solution of sodium hydrogen carbonate after which
dialysis took place against 0.1 M sodium hydrogen carbonate
solution. This antibody fraction was used for the coupling.
(B) Preparation of particles with covalently bound antibodies;
Finely grained particles of the product Sephadex (G 25, superfine)
were used as a starting material, the product being dextran
cross-linked with glycerine ether-bridges and substituted with
p-nitrophenoxy-hydroxy-propyl-ether groups to a substitution degree
of 200 .mu.mol nitro groups per gram of dry substance. (The product
had been obtained by reacting Sephadex G 25, superfine, with
2,3-epoxy-1-(4-nitrophenoxy)-propane in alkaline milieu.) 10 grams
of the substituted Sephadex product were introduced together with
50 ml. of water, into a two-necked flask, after which the
temperature of the mixture was maintained at 35.degree. C. The
mixture was agitated and at the same time there were supplied 25
ml. of a 5 N aqueous solution of sodium hydroxide and 6 grams of
sodium dithionite for reducing the nitro groups into amino groups.
After approximately 30 minutes a further 5 grams of sodium
dithionite were added. The reduction process was interrupted after
approximately 1 hour whereupon neutralization took place with
diluted hydrochloric acid, the solid substance being removed by
filtering and washed with distilled water on a suction filter.
10 grams of the above obtained Sephadex product substituted with
p-amino-phenoxy-hydroxy-propyl groups were introduced into a
reaction flask together with 100 ml. of a 10 percent solution of
thiophosgene in carbon tetrachloride. The flask was sealed with a
plug and the mixture agitated for approximately 2 hours. The
obtained mixture was cooled in an ice bath whereupon the flask was
opened and the contents filtered. The residue of filtration was
washed with a 0.1 mol aqueous solution of sodium hydrogen
carbonate, distilled water and acetone. The residue was then dried
in a drying oven at 60-80.degree. C. The Sephadex product, obtained
according to the above, substituted with
p-isothiocyanato-phenoxy-hydroxypropyl groups was swollen in 30 ml.
of a 0.1 M aqueous solution of sodium hydrogen carbonate. The
agitator was connected, whereupon 5 ml. of the dialysed antibody
solution according to (A) was added in a drop-wise manner. The
mixture was agitated for 24 hours at 20.degree. C, whereupon it was
filtered. The filter residue was washed with 0.5 M sodium hydrogen
carbonate solution to remove the unbound substances. The product
can be dried carefully, e.g. by lyophilization.
(C) Preparation of labeled gonadotropin: Human gonadotropin was
labeled with I.sup.125 according to the following method: 2 mc.
I.sup.125 in the form of NaI was oxidized with Chloramine T in the
presence of 5 .mu.g. of gonadotropin 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 gonadotropin labeled with I.sup.125 was
separated from low molecular weight products by gel filtration on a
copolymer of dextran with epichlorohydrin (Sephadex G-50). The
gonadotropin labeled in this way has a specific activity of 200-300
mc. per mg. 1 ml. of the labeled protein fraction was collected in
a small vessel containing 1/2 ml. of a solution of bovine
plasma-albumin containing 50 mg. per ml. The labeled hormone was
stored in cold surroundings and diluted before being used.
(D) Determination: The analyses are suitably effected in glass or
plastic tubes 50 .times. 10 mm. in size.
(1) 1 ml. of a suspension of polymer particles (e.g. 1 mg./ml.) to
which the antibodies have been found was introduced into each of
eight tubes designated, respectively, A, B, C, D, E, F, G and
H.
(2) 0.25 ml. of the urine sample to be tested was added to one of
the tubes (tube A).
(3) 0.25 ml. of standard solutions containing 100, 50, 25, 10, 5,
2.5 and 0.1E per liter was added to, respectively, tubes B, C, D,
E, F, G and H.
(4) Incubation took place for 20 hours at room temperature, the
tubes being slowly rotated during the incubation period.
(5) 0.1 ml. of the solution containing I.sup.125 gonadotropin
(approximately 1 nanogram per ml.) was added to each of the tubes
A-H.
The abbreviation "IE" means "international units" (E=German
Einheiten). In this connection, reference is made to World Health
Organization Technical Report Series No. 293 WHO Expert Committee
on Biological Standardization 17 e report Geneve 1964, page 12.
(6) Incubation as in item (4) but only for four hours.
(7) The particles were centrifuged down at 3000 revolutions per
min. for 5 minutes.
(8) The particles were washed twice with a 0.9 percent aqueous
solution of sodium chloride. After the last removal by suction of
the supernatant the tubes were placed in counter tubes for
estimating gamma radiation from the antibody bound labeled
hormone.
(9) The number of "counts" for a certain time for standard tubes
were entered on a counts-dose diagram on a lin-log scale, from
which the amount of gonadotropin in the unknown test samples could
then be calculated. (See separate FIG. 1).
Alternatively subsequent to the centrifuging in item (7) 1 ml. of
transferred supernatant can be transferred into counter tubes,
whereupon the gamma radiation from the free labeled hormone can be
estimated. "Counts" from these standard tubes can, in the same way,
be entered into a count-dose diagram in lin-log scale and the
amount of gonadotropin in the unknown test samples can then be
estimated graphically from the connecting points in the same way as
above.
EXAMPLE 2
Determination of Growth Hormone in Blood Plasma
(A) Preparation of antibodies: Guinea pigs were injected
subcutaneously with 0.5 mg. of human growth hormone in 2 ml. of
Freund's adjuvant. Immunization was repeated every week for four
weeks. Subsequent to the passing of a further week, 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 an aqueous solution of saturated ammonium sulphate,
2.5 ml. of the latter being added to 5 ml. of serum.
The precipitate was separated by centrifugation. The precipitate
was dissolved in water and the precipitating process with ammonium
sulphate solution was repeated twice. Subsequent to the third
precipitating process the precipitate was dissolved in 0.1 ml. of
an aqueous solution of sodium hydrogen carbonate after which
dialysis took place against 0.1 M sodium hydrogen carbonate
solution. This antibody fraction was used for the coupling.
(B) Preparation of particles with covalently bound antibodies: The
preparation takes place in the same manner as under Example
1(B).
(C) Preparation of labeled growth hormone: Human growth hormone was
labeled with I.sup.125 according to the following method: 2 mc.
I.sup.125 in the form of NaI was oxidized with Chloramine T in the
presence of 5 .mu.g. of growth hormone 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 growth hormone labeled with I.sup.125 was
separated from low molecular weight products by gel filtration on a
copolymer dextran with epichlorohydrin (Sephadex G-50). The growth
hormone labeled in this way has a specific activity of 200-300 mc.
per mg. 1 ml. of the labeled protein fraction was collected in a
small vessel containing 1/2 ml. of a solution of bovine
plasma-albumin containing 50 ml. per ml. The labeled hormone was
stored in cold surroundings and diluted before being used.
(D) Determination: The analyses are suitably effected in glass or
plastic tubes 50 .times. 10 mm.
(1) 1 ml. of a suspension of polymer particles (e.g. 1 mg./ml.) to
which the antibodies have been bound was introduced into each of
all tubes.
(2) 0.1 ml. of the plasma to be tested was added to one tube.
(3) 0.1 ml. of standard solution having different concentration of
the hormone, e.g. 20, 10, 5, 2, 1, 0.5 and 0.2 nanogram per ml. and
0 nanogram per ml. were each added to 1 tube.
(4) Incubation took place for 20 hours at room temperature, the
tubes being slowly rotated during the incubation period.
(5) 0.1 ml. of the solution containing I.sup.125 -growth hormone
(approx. 2 nanogram per ml.) was each added to all tubes.
(6) Incubation as in item (4) but only for four hours.
(7) The particles were centrifuged down at 4000 revolutions per
min. for 1 min.
(8) The particles were washed twice with a 0.9 percent aqueous
solution of sodium chloride. After the last removal by suction of
the supernatant the tubes were placed in counter tubes for
estimating the gamma radiation from the antibody bound labeled
hormone.
(9) The number of "counts" for a certain time for standard tubes
were entered on a counts-dose diagram on a lin-log scale, from
which the amount of growth hormone in the unknown test samples
could then be calculated. (See separate FIG. 2).
Alternatively subsequent to the centriguing in item (7) 1 ml. of
supernatant can be transferred into counter tubes, whereupon the
gamma radiation from the free, labeled hormone can be estimated.
"Counts" from these standard tubes can, in the same way, be entered
into a count-dose diagram in lin-log scale and the amount of the
growth hormone in the unknown test samples can then be estimated
graphically from the connecting points in the same way as
above.
EXAMPLE 3
Determination of Insulin in Blood Plasma.
(A) Preparation of antibodies: Guinea pigs were each injected
subcutaneously with 0.5 mg. of pig insulin in 2 ml. of Freund's
adjuvant. Immunization was repeated every week for four weeks.
Subsequent to the passing of a further week, blood was drawn off
from the guinea pigs and anti-serum recovered from the blood by
allowing the same to coagulate, and removing the clots of
blood.
The antibody fraction was precipitated from this anti-serum by
treatment with a saturated aqueous solution of ammonium sulphate
solution, 2.5 ml. of the latter being added to 5 ml. of serum.
The precipitate was separated by centriguation. The precipitate was
dissolved in water and the precipitating process with ammonium
sulphate solution was repeated twice. Subsequent to the third
precipitating process the precipitate was dissolved in 0.1 ml. of
an aqueous solution of sodium hydrogen carbonate, after which
dialysis took place against 0.1 M sodium hydrogen carbonate
solution. This antibody fraction was used for the coupling.
(B) Preparation of particles of covalent bound antibodies: This
preparation took place in the same manner as under Example
1(B).
(C) Preparation of labeled insulin: Pig insulin was labeled with
I.sup.125 according to the following method: 2 mc. I.sup.125 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 I.sup.125 was mixed with bovine plasma-albumin
and separated from low molecular weight products and from
denaturation products of insulin bound to the plasma-albumin by gel
filtration on a copolymer 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
bovine plasma-albumin containing 50 mg. per ml. The labeled hormone
was stored in cold surroundings and diluted before being used.
(D) Determination: The analyses are suitably effected in glass or
plastic tubes 50 .times. 10 mm.
(1) 1 ml. of a suspension of polymer particles (e.g. 1 mg./ml.) to
which the antibodies have been bound was introduced into each of
all tubes.
(2 ) 0.1 ml. of the plasma to be tested was added to one tube.
(3 ) 0.1 ml. of a standard solution having different concentration
of the hormone, e.g. 200, 100, 50, 25, 10, 5 and 2.5 .mu.E/ml. and
0 .mu.E/ml. were each added to 1 tube.
(4 ) 0.1 of a solution containing I.sup.125 -insulin (approx. 1
nanogram per ml.) was added to all tubes.
(5 ) Incubation took place for 20 hours at room temperature or
+4.degree. C., the tubes being slowly rotated during the incubation
period.
(6) The particles were centifuged down at 4000 revolutions per min.
for 1 minute.
(7) The particles were washed twice with a 0.9 percent aqueous
solution of sodium chloride. After the last removal by suction of
the supernatant the tubes were placed in counter tubes for
estimating the gamma radiation from the antibody bound labeled
hormone.
(8) The number of "counts" for a certain time for standard tubes
were entered on a counts-dose diagram on a lin-log scale, from
which the amount of insulin in the unknown test samples could then
be calculated. See separate FIG. 3.
Alternatively subsequent to the centrifuging in item (7) 1 ml. of
supernatant can be transferred into counter tubes, whereupon the
gamma radiation from the free, labeled hormone can be estimated.
"Counts" from these standard tubes can, in the same way, be entered
into a count-dose diagram in lin-log scale and the amount of
insulin in the unknown test samples can then be estimated
graphically from the connecting points in the same way as
above.
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