Note: Descriptions are shown in the official language in which they were submitted.
CO~PETITIVE IMMUN FLUORE~SCEl~CE ASSAY AND TEST KIT
Description
Technical Fle~d
The invention is in the field of immuno-
5 diagnostics. More particularly it involves an
improved com~etitive immunofluorescence test for
antigens.
t
Competitive immunoassays for anti~ens or
10 haptens are known in the immunodiagnostic art. They
are based on competition for specific antibody against
the antic~en between labeled anti~en (known) and unla-
beled antigen (unknown) in the sample being assayed.
Immune complexes that form between the antigen/hapten
15 and antibody are separated and the amount of label
therein detected by appropriate label detection means.
The concentration of unknown (unlabeled) antigen/hap-
ten in the sample is determinecl by comparison with the
effect of standards.
US Pats Nos 3,981,982 and 4,298,592 describe
a type of competitive radioimmunoassay called a
"double antibody separation" assay. In this technique
the sample suspected of containing antic3en is first
incubated with a first specific antibody and known
radiolabeled antigen. Followin~ tllis a second anti-
body th~t is specific to the first antibody is added
`i"
5~ -
together with polyethylene gly~ol. The second anti-
body and polyethylene gIycol form a complex with the
antigen-first antibody reaction product that agglom-
era~es and precipitates from the incubation mixture.
The precipitate is separated from the supernatant
containing free antigen. The radioactivity of the
separated precipitate is read directly with a scintil-
lation counter and the concentration of antigen in the
sample is determined by compariny the reading to a
standard radioactivity-antigen concentration curve.
These radioimmunoassays are sensitive but have the
disadvantage of all radioimmunoassays of involving
radioactive reagents that must be handled, used, and
disposed of with extreme care by highly trained
personnel. Corresponding immunofluorescence assays in
which the fluorescence of a solid phase is read are
known. Such assays suffer from hi~h background
fluorescence from the solid phase itself or from
scattered light.
Chard, T., and Syl~es, A., Clin Chem (1979)
26/6: 973-976 teach a fluoroimmunoassay for human
choriomammotropin in which a plasma sample is incu-
bated with fluorescein-labeled human choriomammotropin
and sheep antiserum to the hormone. Following the
incubation immune complexes are separated from free
hormone by addition of aqueous polyethylene glycol.
The mixture is centrifuged a~d the fluorescence of the
supernatant is read and compared to a standard. Such
assays in which the fluorescence of the supernatant is
read have the problem of fluoresence due to interfer-
ing materials in the supernatant (plas~a or serum).
Pourfarzaneh, M., et al, Clin Cllem (1980)
26/6: 730-733, describes a competitive immunofluores-
cence assay for cortisol in serum. The serum sample
is incubated with fluorescein-^labeled cortisol and
anti-cortisol antibody coupled to magnetizable cellu-
lose/iron oxide particles. After the incubation the
solid ph~se is separated, washed to remove free,
labeled antigen, and eluted with an equivolume mixture
of methanol and 0.02 M NaOH. The fluorescence of the
eluate is read with a fluorometer and compared to a
standard curve to determine the concentration of
cortisol in the sample.
Japanese Patent Application no 15n547/79
describes direct and indirect immunofluorescence
assays for various antigens. The sample is first
incubated with antibody (fluorochrome-labeled in the
direct assay, unla~eled in the indirect assay). The
mixture is then incubated with an immunoadsorbent.
The indirect assay involves an additional incubation
of the immunoadsorbent ~ith a fluorochrome-labeled
antibody against the first antibody. In both types of
assay, immune complexes are eluted from the immuno-
adsorbent with 0.02-0.04 ~ NaOH and the fluorescence
of the eluate is read and compared to a standard.
These prior assays involving immunoadsorption and
immobilization have several shortcomings. Firstly,
the reproducibility of the immobilization is often
inconsistent and affects the precision of the assay
adversely. The adsorption may also affect the sta-
bility of the immune complex~s adversely and cause
artificially low immunofluorescence intensity read-
ings. Elution inefficiency and/or interference ~rom
spurious eluted materials may also cause artificially
low fluorescence intensity readings.
In contrast to the above described prior
competitive immunofluorescence assays, the invention
assay does not involve solid phase readings or read-
ings o supernatants or eluates that may containinter~erinc3 materials. Instead the invention assay
involves precipitating the immune complex with a
nonfluorescent, non-light scattering immunoprecipi-
tant, dissolving the resulting immunoprecipitate witha solvent t'nat does not add background fluorescence or
light scatter, and reading the solution. Compared to
the prior competitive immunofluorescence assays, the
invention assay is highly sensitive and has improved
reproducihility and dose-response and precision
eharaeteristics,. and requires less antibody reagent.
Disclosure of the Invention
One aspect of -the invention is a competitive
assay Eor determining the amount o an antigen in a
sample suspected of eontaining the antigen eomprising:
(a) incubating the sample with a sol~tion of a fluo-
rescent-labeled antigen, anti-antigen antibody, and an
antibody against the anti-antigen antibody; (b) adding
a nonfluorescent nonlight-scatterinc3 immunopreeipitant
to the incubation mixture to form an immunopreeipi-
tate; (e) separatinc3 the immunopreeipitate and dissol-
ving the immunoprecipitate in a nonfluorescent solvent
that has a low ionic strength and maintains the p~ of
the resulting solution substantially eonstant; and
(d) ~easurinc3 the fluoreseence intensity of the solu-
tion of step (c) and compari~ng said fluorescence
intensity to a standard curve..
Another aspeet of the invention is a test
kit or carrying out the above-described competition
immunofluorescence assay comprisin~ in association:
(a) a fluorescent-labeled anti~en reagent; (b) anti-
antigen antibody reagent; (c) an antibody against the
anti-anti~en antibody; and (d) a nonfluorescent sol-
35~6
vent for dissolvin~ immunoprecipitates of the immuno-
precipitant that has a low ionic stren~th and the
ability to maintain the p~ of the immunoprecipitate
solution substantially constant.
~od_s ~or Carryiny Out the Invention
The sample that is analyzed by the invention
method is a body fluid such as blood, blood serum,
~J~ ~/g5 blood plas~, urine, lymph Eluid, bile, spinal fluid or
the like. The particular body fluid analyzed may vary
with the antigen being assayed. In most instances
blood serum will be used. About 0.1 to about 500 ~1
of 1uid will be used per assay. Samples may be cryo-
prescrved pendin~ analysis if necessary.
Substances that may be assayed by the inven-
tion method include anti~ens (molec~les that elicit animmune response when introduced into the b~oodstream
of a vertebrate host) and haptens that are not immuno-
~enic per se but may be conju~ated to a protein car-
rier to form a conjugate that is immuno~enic and
capable of raisin~ antibodies against the hapten. The
term "anti~en" is use~ herein to ~enerically denote
both antigenic and haptenic compositions. Such sub-
stances include drugs, hormones, pesticides, toxins,
vitamins, human, bacterial, and viral proteins, and
the like. Examples of anti~ens that may be assayed by
the invention method are thyroxine (T4), triiodo-
thyronine (T3), di~oxin, ~entamicin, amikacin, tobra-
micin, kanamycin, netilmicin, cortisol, luteinizing
hormone, digitoxin, vitamin B12, progesterone, human
chorionic gonadotropin, theophylline, an~iotensin,
human ~rowth hormone, and the like.
-3~2-~5.~
The reagents that are incubated with the
sample suspected of containing antigen to form immune
complexes are (1) fluorescent-labeled antigen,
(2) anti-antigen antibody, (3) antibody against the
anti-antigen antibody, and (4) a nonfluorescent non-
light-scattering immunoprecipitant. The fluorescent-
labeled antigen may be made by coupling the antigen
with a reactive derivative of a fluorogen such as
fluorescein (eg, fluorescein isothiocyanate (FITC) and
fluorescein amine), rhodamine or dansyl using
multifunctional coupling a-Jents such as aldehydes,
carbodiimicles, dima]eimides, imidates, and
succinimides. Fluorescein is a preEerred label.
Antibod~ against the antic~en may be made by
inoculating a host vertebrate with the antigen,
typically repeatedly at several day intervals, bleed-
ing the host and separatiny Ig from the blood. Anti-
body against the antibody to the antigen may be made
by immunizing another host vertebrate species with
said Ig and collecting Ig rom tl~e second host. The
non luorescent, nonlight-scattering immunoprecipitant
is a compound that causes an immune complex to
precipitate or increases the rate of immune complex
precipitation. Compounds that are conventionally used
to precipitate proteins and that have the required
nonfluorogenic properties may be used. Examples of
such materials are polyethyl~ne glycol having a
molecular weight in the range of about 3000 to about
12000, preferably about 4000 to 10000, and inorganic
salts such as ammonium sulfate. Polyethylene glycol
is a preferred immunoprecipitant.
~ he absolute amounts of the reagents used
will, of course, depend upon the volume of the sample.
A known amount of labeled antigen will be used. The
labeled antigen may be diluted- to provide a reagent
that is stoichiometrical]y ideal for the antiserum
used. Common aqueous buffers such as Tris (an aqueous
solution of tris (hydroxymethyl) amino methane), bar-
bital, borate, phosphate, and the li~e may be used todilute the labeled antigen. The antiserum against the
antigen will normally be added in excess of the total
amount of antigen in the reaction medium. It, too, may
be diluted with an aqueous buffer. The second anti-
lG serum will be also added in excess relative to theantiserum against the anti~en. The second antiserum
sho~ld be one that does not c-ross react with the anti-
gen. The immunoprecipitant will normally be added as
a dilute aqueous solution (about 5~ to 8~ by weight)
in amo~nts in the ran~e of about 2% to about 20%,
preferably 5~ to about 8% by weight of solution based
on the second antiserum.
The sequence in which the reagents are added
to the sample is generally not critical. Vsually the
labeled antigen, first antiserum, and second antiserum
will be incubated with the sample separately and the
immunoprecipitant added later. The incubation will be
carried out at a temperature and pH that permits for-
mation of immune complexes. Moderate temperatures in
the range of about 15C to about 55C, conveniently
room temperature, and pHs in the range of about 6 and
9, preferably 7 to 8, will n~rmally be used. The
duration of the incubation will depend to some extent
upon the sequence in which the reagents are added to
the sample. In the case where the labeled antigen and
two antisera are added to~ether at the above condi-
tions, incubation times of about one min to two days
will be sufficient time to allow the antigen and anti-
sera to react and form immune complexes. The com-
,
~ ~1$~36
plexes formed by the antigen ~labeled and unlabeled inthe sample) and the two antisera remain in solution
and may not ag~lomerate and precipitate until the
immunoprecipitant is added. In instances where the
immunoprecipitant is included in the incubation,
immunoprecipitation occurs concurrently with the
formation of the immune complexes. When immunopreci-
pitation is carried out in a separate step by adding
the precipitant later, about one min to one hr are
allowed for the precipitant to be incorporated into
the complex and form a precipitate.
Following the immunoprecipitation, the prec-
ipitate is separated from the supernatant, such as by
centri~uging, and the supernatant is discarded. The
precipitate may be washed with buffer if desired. The
precipitate is then dissolved in a nonfluorescent sol-
vent that has a lo~ ionic strength (ie, typically less
than about 0.2 and more usually between about 0.001
and 0.06) and that maintains the pH of the resulting
solution substantially constant (ie, l0.005). Pre-
ferably the pH is maintained above 8, more preerably
above 9. Examples of such solvents are aqueous solu-
tions of alkali metal (eg, Na, K) hydroxides and
borates, barbital, and Tris. Preferably the solvent
has the capacity to buffer the solution at the speci-
fied p~. The ionic strength that provides maximum
fluorescence intensity will ~ary depending upon the
particular solvent involved. For instance, in the
case of sodium hydroxide the ionic strength is less
than about 0.04, usually 0.02 to 0.04, and preferably
approximately 0.03. For sodium borate the ionic
strength should be less than about 0.2, usually 0.01
to 0.1, and preferably approximately 0.06. A minimum
volume of the solvent (ie, that amount that just dis-
.,
121~3S~$~
solves the precipitate) is pre^ferably used. That
amount will usually range between about lO0 and 500 ~1
for samples of the above described volumes. The
dissolution may be done at moderate temperatures,
conveniently at ambient temperature. The resulting
solution is then transferred to an appropriate
container, eg, a flow cell, for fluorescence intensity
reading. The fluorescence intensity is compared to a
standard curve of fluorescence intensity vs antic3en
concentration derived by assaying a series of samples
containing known amounts of antigen. From this com-
parison, the quantity of antic3en in the sample is
determined.
The basic ingredients of the test kit for
carryinc3 out the invention assay are: (l) the fluores-
cent-labeled antigen, (2) the first antiserum against
the anti~en, (3) the antiserum against the first anti-
serum, (4) the immunoprecipitant, and (5) the solvent.
These ingredients are preferably packaged separately
in the kit and ~lill typically be provided in amounts
sufficient to carry out multiple assays. The test kit
may also include appropriate buffers, unlabeled anti-
gen reagent for preparinc3 standard and control
samples, equipment or vials for performing the assay,
and instructions for carryinc3 out the assay. The kit
components may be packaged in manners conventionally
used in diagnostic kit manu~acturin~. The kits will
be stored at reduced temperatures, preferably 2C to
6C.
The following examples further illustrate
the invention and its advantages over alternative
assay techniques. These cxamples are not intended to
limit the invention in any manner. Unlcss indicated
otherwise, percentages are by weight.
lZ~5 ~
--1 o--
Example 1: T4 Assay by _ vention Method and Prior Art
Twenty-five ~1 aliquots of serum containing
Xnown amounts of T4 were each incubated at 25 C for 20
min with 25 ~1 of FITC-labeled T4 in carbonate buffer,
O.OlM, pH 8.6, 25 ~1 of a mixture of rabbit anti-T4
seru~ and goat anti-rabbit Ig serum in phosphate buf-
fered saline. Following incubation, one ~1 of a cold
6.5~ aqueous solution of polyethylene glycol (8000 mw)
was added to the mixture. The resulting precipitates
were separated by centrifu-~ation and the supernatants
were discarded. The precipitates were each dissolved
in 200 ~1 of 0.03 N aqueous NaOH. The solutions were
transferred to flow cell cuvettes and their fluores-
cence intensities were read with a fluorometer.
A du~licate set of T4-containing aliquots
~ere assayed by prior art procedure.
The results of both assays are reported in
Table 1 below.
Table 1
20T4/TubeRelative Fluorescence Intensity (RFI)
(ng)Normalized for Comparison
Prior Art Invention
0 45 340
46 342
251.25 42 278
41 282
2.50 31 - 217
34 217
7.25 34 151
- 28 141
:
As indicated by the data of Table 1, the
effective assay range in RFI is about ten-fold greater
in the invention method relative to the prior art
method. The coefficient of variation in RFI for the
invention method was less than 2% as compared to about
10% for the prior art method. This indicates that the
precision of the invention T4 assay is substantially
better than the precision of the comparison prior art
T4 assay.
l~xam~le 2: T3 Assay
T3 assays were carried out on standards and
unknowns using the general procedure described in
Exam~le 1. The reagents and incubation conditions
were:
15 Item Description
Serum sample size 100 ~1
FITC-labeled T3 reagent 25 ~1
rabbit anti-T3 antibody -
goat anti-rabbit Ig antibody 25 ~ll
20incubation temp 37C
incubation time 1 hr
The results of these assays are reported in
Table 2 below.
Table 2
Concentration (ng %)
Standard~Sample RFI- Obtained Actual
1. Standard 1 731 - 0.0
2. Standard 1 762 - 0.0
3. Standard 2 712 - 50
4. Standard 2 713 - 50
5. Standard 3 684 - 100
6. Standard 3 697 - 100
J~
-12-
^ Concentration (ng %)
Standard/Sample RFIObtainedActual
7. Standard ~ 583 - 400
8. Standard 4 601 - 400
5 9. Standard 5 528 - 800
10. Standard 5 526 - 800
11. Sample a 705 68 62
12. Sample a 712 52 62
13. Sample b 678132 172
1014. Sample b 672143 172
15. Sample c 569523 486
16. Sample c 574470 486
Example 3 DicJo in Assa~
Digoxin assays were carried out on standards
and unknowns using the general procedure described in
Example 1. The reagents and incubation conditions
were:
Item Description_
Sample size 100 ~1
FITC-labeled digoxin reagent 25 ~1
20 rahbit anti-di(Joxin antibody -
goat anti-rabbit Ig antibody 25 ~1
incubation temp 37C
incubation time 1 hr
~8~96
The results of these assays are reported in
Table 3 below.
_able 3
Concentration (ng/ml)
Standard/Sample RFI Ohtained Actual
1. Standard 1 2920 - 0.0
2. Standard 1 2937 - 0.0
3. Standard 2 2736 - 1.0
4. Standard 2 2801 - 1.0
5. Standard 3 2463 - 2.0
10 6. Standard 3 2543 _ 2.0
7. Standard 4 :L979 - 4.0
~. Standard 4 2020 - 4.0
9. Standard 5 1803 - 8.0
10. Standard 5 1792 - 8.0
1511. Sample a 1821 7.05 6.7
12. Sample a 1833 6.6 6.7
13. Sample b 2532 1.90 2.0
14. Sample b 2541 1.86 2.0
15. Sample c 1922 4.82 5.1
2016. Sample c 1910 5.00 5.1
~Z1~5~
-14-
Exam~le ~: 5entamicin Assay
_ .
Gentamicin assays were carried out on stan-
dards and unknowns using the general procedure des-
cribed in Example 1. The reagents and incubation
conditions were:
Item Description
sample size 25 ~1
FITC-labeled reagent 10 ~1
rabbit anti-gentamicin
10antibody -
goat anti-rabbit Ig antibody25 ~1
incubation temp 25C
incubation time 10 min
The results of these assays are reported in
Table 4 below.
~i85~6
-15-
Table ~
- Concentration (~g/ml)
Standard/Sample RFI ObtainedActual
1. Standard 0 7086 - 0.0
2. Standard 1 6793 - 0.5
5 3. Standard 2 4453 - 2.0
4. Standard 3 2822 - 4.0
5. Standa~d 4 1234 - 16.0
6. Sample 1 1902 7.9 8.0
7. Sample 2 2791 4.15 4.0
10 8. Sample 3 120n 17.0 16.0
9. Sample 4 1853 8.2 8.0
10. Sample 5 6480 0.66 0.5
11. Sample 6 3828 2.45 2.0
12. Sample 7 5814 0.99 1.0
1513. SaMple 8 6921 0.5 0.0
14. Sample 9 5666 1.08 1.0
15. Sample 7 5800 1.00 1.0
16. Sample 7 5821 0.99 1.0
17. Sample 7 5762 1.03 1.0
2018. Sample 7 5748 1.03 1.0
l9. Sample 7 5819 0.99 1.0
20. Sample 7 5709 1.05 1.0
21. Sample 7 5798 1.00 1.0
22. Sample 7 5810 0.99 1.0
2523. Sample 4 18~70 8.1 8.0
24. Sample 4 1854 8.2 8.0
25. Sample 4 1900 7.9 8.0
26. Sample 4 1888 8.0 8.0
27. Sample 4 1909 7.9 8.0
3028. Sample 4 1844 8.3 8.0
29. Sample 4 1865 8.2 8.0
30. Sample 4 1866 8.2 8.0
s~
-16-
Example 5: Amikacin As say
Amikacin assays were carried out on stan-
dards and unknowns using the general procedure
described in Example l. The reagents and incubation
5conditions were:
Item Description
Ser~m sample size 25 ~l
FITC-labeled amikacin lO ~1
rabbit anti-amikacin antibody -
10goat anti-rabbit Ig antibody 25 ~ll
incubation temp 25C
inc~bation time lO min
The results o~ these assays are reported in
Table 5 below.
12~
Table 5
Concentration (~g/ml)
Standard/Sample RFIOhtainedActual
1. Standard 1 2815 - 0.0
2. Standard 2 2128 - 3.0
5 3. Standard 3 1644 - 10.0
4. Standard 4 1357 - 20.0
5. Standard 5 1011 - 50.0
6. Sample 1 164010.1 10.0
7. Sample 2 114236.0 35.0
10 8. Sample 3 25623.0 0.0
9. Sample 4 100851.0 50.0
10. Sample 5 118032.5 35.0
11. Sample 6 21692.7 3.0
12. Sample 7 140018.7 20.0
1513. Sample 2 115035.2 35.2
14. Sample 2 116134.3 35.0
15. Sample 2 115435.1 35.0
16. Sample 2 120538.0 35.0
17. Sample 2 117233.3 35.0
2018. Sample 2 113137.0 35.0
19. Sample 2 114136.0 35.0
20. Sample 2 113936.0 35.0
21. Sample 1 16529.9 10.0
22. Sample 1 163910.1 10.0
2523. Sample 1 16~910.0 10.0
24. Sample 1 1646,10.0 10.0
25. Sample 1 16619.7 10.0
26. Sample 1 163210.3 10.0
27. Sample 1 164210.0 10.0
3028. Sample 1 16589.8 10.0
29. Sample 1 164010.1 10.0
lZ185~
-18-
Fxample 6- Tobramicin Assay
-
Tobramicin assays were carried out on stan-
dards and unknowns using the general procedure of
Example 1. The reagents and incubation conditions
were:
Item Description
Sample size 25 ~1
FITC-labeled reagent 10 ~1
rabbit anti-tobramicin antibody -
10goat anti-rabbit Ig antibody 25 ~1
incubation temp 25C
incubation time 10 min
The results of these assays are reported in
Table 6 below.
--19-
~able 6
Concentration (~g~ml)
Standard/Sample RFI OhtainedActual
1. Standard 0 1957 - 0.0
2. Standard 1 1882 - 0.5
53. Standard 2 1502 - 2.0
4. Standard 3 1057 - 4.0
5. Standard 4 610 - 16.0
6. Sample 1 801 8.00 8.0
7. Sample 2 1752 0.99 1.0
108. Sample 3 621 15.7016.0
9. Sample 4 782 8.40 8.0
10. Sample 5 1872 0.55 0.5
11. Sample 6 1733 1.07 1.0
12. Sample 7 1452 2.22 2.0
1513. Sample 8 ln95 3.73 4.0
14. Sample 9 1949 0.5 0.0
15. Sample 2 1748 1.00 1.0
16. Sample 2 1753 0.99 1.0
17. Sample 2 1740 1.03 1.0
2018. Sample 2 1739 1.03 1.0
19. Sample 2 1751 0.99 1.0
20. Sample 2 1745 1.01 1.0
21. Sample 2 .1748 1.00 1.0
22. Sample 2 1749 1.00 1.0
2523. Sample 1 79~ 8.0 8.0
24. Sample 1 820 7.4 8.0
25. Sample 1 819 7.4 8.0
26. Sample 1 806 7.9 8.0
27. Sample 1 788 8.3 8.0
3028. Sample 1 793 8.1 8.0
29. Sample 1 825 7.3 S.0
30. Sample 1 800 8.0 8.0
.~
-20-
Example 7: Tobramicin Assay Using Sodium Borate
_ _ _
Tobramicin assays were carried out on
standards using the procedure of Example 6 except that
0.03 M sodium borate was used as a solvent instead of
0.03 N sodium hydroxide. Eight hundred ~1 of the
borate solution was used for each precipitate. For
comparison purposes assays using 0.03 N sodium
hydroxide were also carried out. The results of these
assays are reported in Table 7 below.
Table 7
Concentration RFI
Standard~g/ml NaOH Borate
1 0 2500 3094
2 1.0 2230 2513
3 2.0 1950 1653
4 4.0 1360 151g
8.0 990 1106
6 16.0 760 793
F,xample 8
The usefulness of the sodium borate solution
of Example 7 as a solvent in assays of T4 and genta-
micin was shown as follows. Known amounts of FITC-
labeled antigen in the carbonate buffer of Example 1
were incubated with rabbit s~rum against the antigen
and goat anti-rabbit serum as in Example 1. The
labeled T4 solution contained 0.82 ng T4 whereas the
labeled gentamicin solution contained 0.5 ng genta-
micin. The resultin~ immune complexes were precipi-
tated with aqueous polyethylene glycol, the precipi-
tates were separated, and the supernatants were dis-
carded as in Example 1. The resulting precipitates
3S$6
-21-
were dissolved in 0.03 N sodium hydroxide or 0.03 M
sodium borate and the fluorescence intensities:were
read with a fluorometer. These tests were run in
duplicate an~ are reported below~
Anti~ NaOH Borate
1658 1835
T4 1690 1882
1962 2377
Gentamicin 1973 2418
Modifications of the above described modes
for carrying out the invention that are obvious to
those of ordinary skill in the immunodia~nostics art
and/or related arts are intended to be within the
scope of the followin~ claims.
