Note: Descriptions are shown in the official language in which they were submitted.
131L3r5~2~
FIELD OF INVENTION
This invention relates to the field of immunoassay techniques for
the detection and quantification of antibodies and antigens (including haptens)
and other substances present in small amounts in body fluids. More specifically,it relates to chemiluminescent immunoassay procedures and a novel conjugate
for use in those procedures.
DESCRIPTION OF THE PRIOR ART
Various test systems have been developed for the detection and
quantification of antibodies, antigens and other substances present in small
amounts of body fluids. The most sensitive are immunoassay procedures which
take advantage of the fact that an antibody binds specifically to its antigen
and the reaction obeys the mass action law, Ab + Ag = AbAg.
Several immunoassay techniques are currently employed which can
be classified by their binding characteristics. The most common utilize competi-tive and sandwich binding. Regardless of the technique a conjugate tagged for
detection must be employed. The most widely used tags are radioisotopes and
the procedure is called radioimmunoassay (RIA). Other procedures using
different tags include enzyme immunoassay (EIA) and fluorescent immunoassay
(FIA).
Radioimmunoassays using as tags the radioactive isotopes 125I,
14C or 3H presently command the major share of the market because of their
extremely high sensitivities, that is, their abîlity to detect 10-15 moles or less
of antigen or antibody. Despite their high sensitivity, RIA's suffer from the
following disadvantages: (1) the beta and gamma counters required for the
readout system are expensive and require expensive upkeep by highly trained
service persons, (2) supervisory personnel must be highly trained and licensed by
the government for work with radioisotopes, (3) because of the radioactivity
RIA's are effectively prohibited in many countries outside of the United States,(4) the high radioactivity required for an appropriate signal requires the use of
short-lived isotopes, thus requiring the frequent resynthesis of tagged compounds,
and (5) the short lives means that the signal to concentration of tag ratio is
continually and rapidly changing requiring frequent recalibration for each assay.
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Enzyme immunoassays require the use of complex biological
materials with retention of their native activity. The procedures themselves
are time consuming due to the slow nature of the readout reaction and do not
always yield requisite high sensitivities.
Fluorescent systems have slightly lower sensitivity limits than
radioimmunoassay systems and require long incubation periods. In addition, high
and variable blanks are often encountered.
Chemiluminescence refers to light produced as a direct result of
chemical change. It involves the transformation of the free energy of a chemicalreaction into light energy. The chemical reaction must release sufficient energyto populate an excited energy state; the reaction pathway must favor the
formation of the excited state product; and the excited state product must be
capable of emitting a photon itself for transferring its energy to another mole-cule that can emit.
A well-known, highly efficient chemiluminescent reaction is the
oxidation of luminol (5-amino-2-,3-dihydrophthalazine-1,4-dione) in a basic solution.
The most frequently used oxidant is hydrogen peroxide in the presence of a
catalyst such as Fe(CN)6~3, Cu(II), and Co(II). Other oxidants include perborate,
hypochlorite, iodine, permanganate and oxygen.
Chemiluminescence has been utilized in immunoassay procedures.
Maier United States Patent No. 4,104,029 discloses a procedure for the assay of
pharmacologically, immunologically and biochemically active compounds in
biological fluids in which a ligand is labeled with a chemiluminescent material
such as luminol. In addition, it is known that iron and specifically iron proto-porphyrin compounds react with luminol in the presence of hydrogen peroxide or
sodium perborate and a base to give an excited species which emits light with
high efficiency, see Ewetz and Thore's Factors Affecting the Specificity of the
Luminol Reaction with Hematin Compounds, Analytical Biochemistry, 71, 564-570
(1976).
SUMMARY OF THE INVENT_
This invention provides a fast, sensitive and objective immunoassay
for antigens or antibodies. It eliminates the radioisotope hazards and the need
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for highly trained persolmel. It further eliminates the need for special facilities
associated with isotope work. In addition, it utili~es only simple instrumentation
and provides fast readout and high sensitivity (up to 10-15 mole detection). The
invention provides a long-lived tagged immunoreagent which is of relatively low
cost and of ready availability. Because of its universal reactivity, it is applicable
to a number of different assay procedures.
The invention is directed to a conjugate for use in an immunoassay
procedure which is detectable by chemiluminescence. The conjugate comprises
an immunologically active compound to which a metallo porphyrin tag is attached.
10 Any antibody or binding partner for an antibody including antigens and haptens
may be used. Antibodies raised in animal species such as goats and rabbits are
particularly suitable. Antigens such as hormones and blood plasma proteins and
haptens are suitable. Immunoglobulin and particularly IgG is particularly useful
because of its availability and because it acts as both an antigen and antibody.
Antigens which naturally contain iron porphyrin such as hemoglobin, myoglobin
and cytochrome C may be used as the entire conjugate.
Because the tag functions as a catalyst rather than a reactant,
each tag produces a plurality of photons whereas a luminol tag would only
produce a single photon. This results in two advantages, namely greater sensi-
20 tivity and prolonged emission.
The metallo porphyrin tag may be a molecule containing a metalloporphyrin and particularly a protein containing iron porphyrin. It may be iron
porphyrin itself such as hemin. Hemoglobin, myoglobin, cytochrome C and
catalase are suitable.
The metallo porphyrin tag is attached to the antigen or antibody
by means of known bifunctional linking agents such as cyanuric chloride, acrylyl
chloride, and glutaraldehyde.
The conjugate is useful in various im munoassay procedures to
determine the concentration of an unknown in an analyte. Typically such pro-
30 cedures use a reagent, an analyte and a conjugate. In a competitive assay thereagent which is generally attached to a solid phase such as nylon or polystyrene
must be a binding partner for the conjugate. The analyte may be a binding
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partner for the conjugate or the reagent but not both. In a sandwich type assay
the conjugate and the reagent cannot be binding partners for each other. Both
are binding partners for the analyte. The binding reactions can be shown
graphically as follows:
A. Competitive Assay
Reagent Analyte Conjugate
(Solid Phase)
~ ~- Ab + Ag-Tag
(1) ~ - Ab ~ Abs + Ag-Tag ~ ~ +
AbS + Ag-Tag
~- Ab- Ag-Tag
(2) ~ - Ab + Ags + Ag-Tag ~' +
~ - Ab- Ags
B. Sandwich Assay
~- Ab-Ags-Ab'-Tag
(3) ~ - Ab + Ags + Ab'-Tag ~
Thus in a competitive assay the greater the amount of the unknown (AbS or Ags)
in the analyte, the less conjugate which can react with the reagent. In a
sandwich assay the opposite is true. As the amount of the unknown in the
analyte increases the amount of conjugate which reacts with the reagent
increases.
To determine the amount of the unknown in the analyte, the
reagent is separated from the remaining reactants. It is for ease in separation
that the reagent is generally attached to a solid phase. After separation including
thorough washing to make certain no unreacted conjugate remains, the reagent is
mixed with a chemiluminescent reactant. Luminol is preferred because of its
efficiency. Other chemiluminescent substances which may be used include
tetrabis ~dimethylamino) ethylene, luciferin, lucigenin (dimethyl diacridinum nitrate)
and oxalyl chloride. As an oxidant sodium perborate is preferred. However,
other known catalysts may be used.
The metallic component of the tag in the conjugate will catalyze
the chemiluminescent reaction. The amount of light emission recorded in a
standard manner gives a quantitative determination of the amount of conjugate
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which is bound to the reagent and, hence, the amount of unknown in the
analyte.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The following examples serve to illustrate the practice of the
invention but are not to be regarded as limiting:
Example I
Competitive Assay of Human Immunoglobulin G.
A. Preparation of Reagent (Solid Phase).
Nylon mesh was partially hydrolyzed by treating in 2NHCl at 37
overnight. The nylon was washed free of acid with 0.01 M Na2CO3, pH 10, and
washed with water. The nylon was placed in a solution OI 400 mg of 1 ethyl-3
(3-dimethylaminopropyl)-carbodiimide in water, pH 3.7. The pH was maintained
between 3.5 and 3.8. The activation reaction was continued at room temperature
until the consumption of acid ceased. The nylon was rapidly washed two times
with dilute HCl (pH 3.4 to 3.8).
Human immunoglobulin, dissolved in 50 mM NaCl, pH 3.4, to 10 mg
of protein per ml, was added to the washed activated nylon and the pH was
gradually raised to pH 7 over 60 minutes. The attachment reaction was allowed
to proceed for 60 minutes at pH 7. The pH was adjusted to 7.2 to 7.4 and the
20 composition was incubated for 4 hours.
The nylon was washed according to the following sequence:
2 M NaCl, two times; water; 10 mM phosphate buffer, pH 7.35, containing
0.3 nl NaCl, 0.2% bovine serum albumin and 0.1% Tween 80, two times;
phosphate buffered saline, two times; and water. The washed nylon-immunoglobulin
reagent was air dried and used in 1 cm2 pieces as the solid phase in immunoassays.
B. Preparation of Conjugate.
An iron porphyrin-antibody conjugate was prepared as follows:
Goat anti-human serum was partially purified by precipitation with ammonium
sulfate, and dialyzed against saline. To 2 ml of this solution (100 mg protein),
30 30 mg of sodium bicarbonate and 2 ml of water was added. A solution of
cyanuric chloride in dioxane, 4 mg/ml, was added to the rapidly stirring solution
of globulin. After 2 minutes a solution of 100 mg of hemoglobin in 4 ml of
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water was added and the binding reaction was allowed to proceed for 18 hours
at 25C.
After reaction, the mixture was charged onto a 1.5 x 100 cm
., ` ~
column of polyacrylamide gel, Bio-Gel P-200 (100-200 mesh) and elution performed
with 0.002 N phosphate buffer, pH 7.0, containing 0.05 M sodium chloride and
0.02% sodium azide. In this way any unreacted hemoglobin could be removed
from the conjugate. The fractions containing both hemoglobin and antibody
activity were pooled and used as the conjugate.
C. Preparation of Chemiluminescent Reactants.
The chemiluminescent readout measurements were performed on
an Aminco Chem-Glo Photometer.
The following solutions were used for CL analyses:
Luminol Stock Solution. To 2.00 mg of lurninol and 32 mg of
glucose was added 4 ml of 0.1 N sodium hydroxide. After complete dissolution
of the luminol, the solution was made up to 100 ml with water. The solution
was stored at 4.
Luminol Working Solution. Each working day, 1.00 ml of the
above solution and 10 ml of 0.1 N sodium llydroxide was made up to 100 ml
with water.
Perborate Solution. Each working day, 77 mg of sodium perborate
was dissolved in 100 ml of water.
Analyses of chemiluminescence were carried out as follows:
In a shell vial was placed 0.5 ml of the test solution, made up in
the luminol stock solution as described above. This was placed in the sample
compartment of the Chem-Glo Meter. To this was then added 0.1 ml of the
perborate solution. The light output was taken as the maximum pen deflection
shown on the recorder. In most cases, an average of the results of three
sample injections was used.
D. Assay Procedure.
An immunoassay was performed with the human immunoglobulin-
nylon solid phase and the iron porphyrin-antibody conjugate.
Nylon discs (surface area = 1 sq. cm.) were incubated with 0.4 ml
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of conjugate at various dilutions plus 0.1 ml of phosphate buffered saline or
0.4 ml of conjugate at various dilutions plus 0.1 ml of a solution of human
immunoglobulin. All dilutions of conjugate and human immunoglobulin were
made with phosphate buffered saline. After incubation for 60 minutes at room
temperature, the solutions were aspirated and the nylon discs were washed
.~
successively with 4.5 ml of 0.5% BSA-0.05% Tween 20, 2 M NaCl-pH 9.0,
BSA-Tween-20 and two times with 0.01 M phosphate pH 6.1 buffer.
After washing, individual discs were eluted with a luminol working
solution. For this purpose, 3.0 ml of the luminol working solution was added
10 to each tube and the tube plus contents was sonicated for five minutes. A
500 yl aliquot of the solution was introduced into a vial, placed in an appropriate
instrument such as the Aminco Chem-Glo Photometer, reacted with a solution of
sodium perborate and the resultant light output was recorded.
E. Results.
The results obtained are presented in Table I. Inhibition of
conjugate uptake onto the insoluble nylon-human immunoglobulin is seen over a
wide range of conjugate concentration and competing immunoglobulin concen-
trations.
TABLE I
Soluble
Nylon ConjugateImmunoglobulinRelative
Sheet Conc. (mg/ml) Added ~llg) Light Intensity
#A 0.1 0 4,300
B 0.1 0.1 2,885
C 0.04 0 1,995
D 0.04 0.1 1,245
E 0.04100.0 202
F 0.02 0 1,490
G 0.02 0.1 364
H 0.01 0 1,125
0.01 0.1 182
J 0.000 0 52
Example II
Preparation of Hemoglobin-Antibody Conjugates.
30A series of hemoglobin-antibody conjugates were prepared as
shown in Table II.
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TABLE II
Activation SteP _ Binding Step Product Composition
Cyanuric ~ a
VolumeChlorideTime Temp.HemoglobinHemoglobin
Example ml mg sec. C. mg Immunoglobulin
II-A 4 4 120 25 400 0.83
B 4 8 120 25 400 0.65
C 4 4 120 25 100 0.63
D 4 4 600 25 100 0.74
E 4 8 120 25 100Insoluble products
F 4 2 120 25 100 0.49
G 4 1 120 25 100 0.19
H 10 4 10 0 100 1.34
4 1 0 100 1.07
J 10 2 10 0 100 0.90
K 20 4 10 0 100 1.05
L 4 4 10 0 100 1.73
a Molar ratio, determined from the amount of free hemoglobin and the
amount of hemoglobin in the oligomer fraction as shown by analysis of
gel filtration column as in Example I.
Conjugates were tested and had both CL catalyzing and immuno-
logical properties. Results similar to Example I were obtained in the immuno-
assay with conjugates II-G and II-L and nylon bound antibody from Example I.
Example III
Competitive Assay of Immunoglobulins in Human Serum.
Nylon bound human immunoglobulin, as prepared in Example I,
20 and Hemoglobin conjugated to anti-immunoglobulin, Example II-C, were incubated
in the presence and absence of human serum. ~educed light output was observed
in the presence of human serum.
Example IV
Direct Assay of Human Hemoglobin.
Antibody to human hemoglobin was partially purified by ammonium
sulfate precipitation; dissolved to 12~ug protein/ml of 0.05 M sodium carbonate,
pH 9.6. The antibody was adsorbed onto polystyrene tubes (Falcon Number 2054)
for 1 hour at 37C. The solid phase antibody tubes were treated with 0.5%
bovine serum albumin for 30 minutes at 37C. and washed 2 times with 0.2%
30 bovine serum albumin containing 0.5% Tween in buffered saline. The antibody
tubes were incubated in the absence and in the presence of human hemoglobin
for 30 minutes at room temperature and washed with the albumin-Tween buffered
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saline 3 times and chemiluminescence was measured as in Example I. Light
emission was higher when hemoglobin was added to the tubes and proportional
to the amount of hemoglobin present.
Example V
Immunoassay of Myoglobin.
Antibody to myoglobin was adsorbed to polystyrene and immuno-
assays were performed as in Example IV with similar results.
Example VI
Sandwich Assay of Chorionic Gonadotropin.
A. Preparation of Reagent~
Goat antibody to human chorionic gonadotropin (HCG) bound to
nylon beads: Partial hydrolysis of the nylon was accomplished with N HCL for
2 hours. The beads were washed and additional carboxyl groups were introduced
by allowing the beads to react with an excess of maleic anhydride at a constant
pH between pH 8 and 9 for 1 hour. The beads were washed and the carboxyl
groups were activated with 2 g of water soluble carbodiimide at a constant pH
of 4.5 to 5.2 for 30 minutes. Excess carbodiimide was removed and goat
anti-HCG was bound to the beads for 3 hours at room temperature followed by
18 hours at 4C. The antibody beads were treated with 0.2% bovine serum
20 albumin in buffered saline, washed and stored in bovine serum albumin in buffered
saline at 4C. prior to use.
B. Preparation of Conjugate.
An iron porphyrin containing peptide derived from cytochrome C
was prepared by pepsin digestion of cytochrome C, followed by acid precipitation
and dialysis. This peptide, demonstrated a higher chemiluminescent yield per
porphyrin molecule than cytochrome C itself. Goat antibody to HCG was partially
purified by ammonium sulfate precipitation and digested with pepsin to produce
E~ (Fab')2 fragments which were purified by gel filtration on Sephadex G-75 column.
Thiol groups were introduced onto the antibody fragment using N-acetyhomocys-
30 teine thiolactone and the thiolated antibody fragment was separated from excessthiolating reagent by gel filtration over a Sephadex G-25 column. The iron
porphyrin peptide was activated for conjugation to the thiolated antibody fragment
7~ad~
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by introduction of maleimide moieties using m-maleimidobenzoyl N-hydroxy-
succinimide ester followed by Sephadex G-25 filtration. The conjugate was
prepared by mixing thiolated antibody fragment and maleimido-iron porphyrin
peptide for one hour at room temperature followed by gel filtration on a
Sephadex G-75 column.
Immunoassays using antibody to HCG bound to nylon beads and
iron porphyrin conjugated to antibody fragments derived from antibody to HCG
were performed.
C. Assay Procedure.
Nylon beads conjugated with antihuman IgG were placed in glass
tubes and incubated 30 minutes at 37C. with 2.5 I.U. of HCG in a solution of
0.2% bovine serum albumin in phosphate buffered saline or phosphate buffered
saline alone. The solutions were then aspirated and the beads were washed with
~` 0.05 M HEPES, 0.15 M NaCl, pH 7.2 and then incubated for 30 minutes at 37C.
~3 with various dilutions of the conjugate in 0.05 M HEPES, 0.15 M NaCl, pH 7.2
buffer. After incubation at 37C. for 30 minutes, the beads were once again
washed and the beads were eluted with 0.5 ml of 0.1% sodium dodecyl sulfate
for five minutes. Luminol was added and light emission was measured as in
Example I. Table VI shows results typical of this double antibody sandwich
20 assay for an antigen.
TABLE VI
Dilution ofRelative
Tube HCG Added Conjugate Light Sensitivity
+ 1: 5 61.1
2 + 1:10 39.0
3 + 1:25 16.1
4 + 1:50 3.25
+ 1:100 2.40
6 + - 1.21
7 - 1:5 19.5
8 - 1:10 4.81
- 1;25 3.56
- 1:50 2.34
11 - 1:100 2.21
12 - - 1.63
The data shows increased CL indicating increased uptake of
conjugate in the presence of HCG over a wide range of conjugate dilutions.
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