development of a rapid, sensitive, and specific-enzyme linked immunosorbent assay. (ELISA) suitable for measuring luteinizing hormone. (LH) in cattle, sheep,.
BIOLOGY
OF REPRODUCTION
(1987)
37, 595-605
Development for
of a Sensitive
Cattle,
Sheep,
Enzyme-Linked
Rat,
JIMMY
and
Immunosorbent
Mouse
L. SPEAROW2
Luteinizing
and
BETH
A.
Assay
Hormone1
TROST
Department
of Meat and Animal Science University of Wisconsin Madison, Wisconsin 53 706 ABSTRACT
assay
the
This manuscript reports (ELISA) suitable for
used
#15
LH
labeled
loss
96-well
serum-coated
of a rapid,
luteinizing
± 31 pg/mI,
diluted pg/ml.
and
a 50%
in
assay
buffer,
The
LH
EL ISA
times
more
was
validated
desired,
sensitive
plates
the
for LH
The
LH for
bLH
EL ISA
to
LH.
was
than
measurement
this LH ELISA RIA procedures.
specificity
displacement LH ELISA highly specific
the
for
Use of parable
and
and specific-enzyme in cattle, sheep, rats,
peroxidase-labeled
bovine
can
and
can can
a radioimmunoassay
of
LH
in
be conducted
offers The
EL ISA LH
Radioimmunoassays
(RIA)
buffer,
tissue
(RIAs)
by Niswender
with
be performed
with coated
other on
LH
96-well
antisera,
immunosorbent The LH ELISA
hormone
(bLH).
Bovine
linked for
the
et a!. (1968),
using
and (Nis-
iodinated
and Stabenfeldt, RIAs for LH
et
1982). use
Unfortunately, or ‘311-labeled
125!
the disadvantages of limited shelf tracers, licensing, safety, and waste associated with use of radioisotopes, expensive, large-capacity centrifuges. enzyme
immunoassay
this and tracers
and
provided
and
can easily
the
be automated.
and safety polyclonal
antisera
has
immunosorbent
a high
assay
in enzymatic of several
have
antibodies al.,
sensitive
life of iodinated disposal problems and the need for Recent develop(EIA)
waste,
economy, sensitivity, other monoclonal and
over comantibodies affinity
and
have 1985).
(ELISA)
alternatives hormones,
also
1981), 1979), 1984). been
chorionic gonadotropin and sandwich-enzyme
other have
The LH ELISA on the sensitivity
volumes.
(Hashimoto and Kawana, hormone (Kato et al., (Munro and Stabenfe!dt, procedures
that
sample
have resulted measurement
measurement
(rLH) used as the standard displacement point of 531 This LH ELISA was seven
plates.
ovine LH (oLH) and rabbit #15 anti-oLH serum, has proven to be suitable for the measurement of LH in mice, rats, cats, rabbits, sheep, cattle, any many other mammalian species (Millar and Aehnelt, 1977; Banks
in
linked mice.
and sera. Depending
no hazardous
in speed, convenience, was also conducted with
be uniformly
comparable
media,
culture
in 3 to 48 h, produces
improvements LH ELISA
of luteinizing hormone (LH) have been developed used extensively in many mammalian species wender et al., 1969). The LH radioimmunoassay
ments
and
luteinizing
point of 359 ± 69pg/ml. With rat LH-RP-2 had a sensitivity of 102 pg/mI and a 50% for LH from several mammalian species.
INTRODUCTION
developed
sensitive, (LH)
hormone
with peroxidase by the periodate method was stored at -15#{176}C for over 20 mo without appreciable activity. With bLH-B5 used as the standard diluted in assay buffer, the LH EL ISA had a sensitivity of
of
79.6
anti-LH
development
measuring
procedures
to RIA including
for the insulin
thyroid-stimulating and progesterone Competitive ELISA
developed
for
human
(hCG) (Hamada et a!., 1978), immunoassays utilizing labeled
been
developed
However,
competitive
to
for date,
the
or sandwich
human
LH
(Chow
development
ELISA
of a
suitable
for
the measurement of bovine, ovine, rat, and mouse LH has not been reported. The purpose of this paper is to describe the development and validation of a sensitive, competitive
LH
ELISA.
enzymeMATERIALS
AND METHODS
Reagents Accepted Received ‘Supported WIS-2848, 150510. 2 Reprint
March October and
31, 1987. 20. 1986.
in part by USPHS-NIH HD18889-02, by University of Wisconsin Graduate
Flat-bottomed, by
Hatch School
#3912)
Grant Grant
Chicago, plates
requests.
595
were
IL. (Nunc
96-well, from
American
Flat-bottomed, Immuno Plate
polyvinyl Scientific
96-well I #439454)
plates
(Falcon Products,
polystyrene were from
SPEAROW
596
Scientific Department program growth prolactin Medical 1072-7.
TX)
Supply, Schiller of Agriculture supplied USDA hormone (bPRL)-B-1.
and USDA Reichert
Leo
(PMSG), IU
per
2610 mg,
were
Co., St. Louis, MO. Other the National Hormone NIADDK, Bethesda, MD. Number Gordon
15 anti-oLH Niswender
State
Univ.,
the
bovine (Albany
Antibodies, purified Antibody
on
from
serum
was
Dr.
obtained
Terry
CO).
0220-518B7 was obtained Animal Science, CA, compliments
et a!. (1980), precipitation. other from used
Mode!
EL
Mouse
phosphate, acetate,
administering
a booster
pH pH
of 250
antibodies were to the procedures
including
the
final,
cold
iig
studies decreased
was
purchased
Burlington, VT. from Vanguard
from
Nunc-Immunowash International,
12 Neptune,
Conjugate periodate
methods
of Nakane
and Kawaoi (1974) and Kato et a!. (1979) were used to label bLH-B-5 with Sigma Type VI horseradish peroxidase (peroxidase). The major modifications consisted of 1) the addition of activated peroxidase to bLH-B5 (0.8:1 munoglobu!in G conjugate on a 90
molar (IgG), X 1.5-cm
in 50%
in 10%
sorbitol, acid)
and 0. 005% snap-frozen
and
0.02 M (MOPS),
chiorhexidine and stored
showed that with long-term
preparation
eliminate activity,
3)
in a Biotek conjugate
at
bLHstorage
of dilutions
of
losses of immunothe conjugate was
glycerol,
0.02
M MOPS,
0.12
M
diluted
into
the
ELISA.
LH
Stock
assay
buffer
immediately
before
use
in
Solutions
The
following
ELISA 1) 0.02
solutions
were
used
in
the
LH
system. Phosphate-buffered M NaPO4, 0.12
saline M NaCl,
pH
Wash
(PBS) coating buffer: 2.5 ig/ml preimmune
(cIgG), pH buffer:
7.2; 0.05
M
sodium
9.6; M
NaPO4,
0.025% polyoxyethylene (Tween 20), pH 7.2; 4) Assay buffer: 0.02 M 0.1% gelatin, 0.05% Tween
sorbitan
monolaurate
NaPO4, 20,
0.12 M NaCI, 0.01 M EDTA,
0.005% pH
buffer:
0.02
chlorhexidine
diacetate,
0.12
M
NaC1,
0.002%
phenol
red,
0.5
ml of 3,3’,5,5’-
7.2;
Peroxidase
substrate
buffer:
tetramethylbenzidine (TMB) stock solution (20 mg TMB/ml dimethyl sulfoxide [DMSO]), 0.160 ml 0.5 M H2O2, 49.34 ml 0.05 M Na acetate, pH 4.8 (prepare
Storage
of the
and to enzymatic
bicarbonate,
Bio-tek
solution,
NaC1, 0.01 M ethylene diamine tetraacetic acid (EDTA), 0.1% gelatin, and 0.005% chlorhexidine diacetate, pH 7.2 (glycerol buffer), and stored unfrozen at -15#{176}C. Conjugate in glycerol buffer was
5) and
to ease
chicken immunoglobin 2) Bicarbonate-coating
ethanol
stop
was diluted propanesulfonic
1/100
determined
activity at 450 nm Bovine LH-peroxidase
Preliminary activity
the conjugate logical and
was
10 pl of diluted eluant buffer, 2) terminating
substrate
-15#{176}C. peroxidase
3)
310,
50 il
gelatin, were
5.
purified of Poison
40%
eluants
0.12 M NaCl, 0.1% diacetate; aliquots
diluted
monoclonal
anti-bLH 13-subunit from Dr. Jan Roser, University of Caliof Monoclonal
Chicken according
of bLH-Peroxidase Modifications
Dr.
(Colorado
hormones and reagents were purSigma Chemical Co. The 96-well plate in all experiments, EIA Autoreader
Instruments, was purchased NJ. Preparation
from
Nett
Dl 1-61 (chicken anti-LH) hens with 750 g oLH
fl-subunit immunizing
then
fl-subunit. egg yolk
All chased reader
supplied by Program,
Inc. Mouse monoclonal antibody was protein A-agarose (Sigma Chemical Co.). was loaded onto the protein A-agarose
and
with
(bLH-peroxidase) (3- [N-morphoiino]
and bovine Sigma Chemical
column and rinsed with 0.1 M sodium 8.5, and eluted with 0.1 M sodium
fl-subunit
reaction
at 4#{176}C. Therefore,
Collins,
Chicken anti-oLH was produced by
in column
determining peroxidase EL 310 autoreader.
mg,
hormones were and Pituitary
and
Ft.
antibody clone (518B7 anti-LH) Department of fornia, Davis,
IU per
activity
by 1) a 10-mm incubation of in 100 jil peroxidase substrate
College, Albany, NY) supplied bLH-LERDr. Darrel Ward (Univ. of Texas, Houston, supplied oLH 13-subunit. Pregnant mare’s serum
gonadotrophin insulin, 25.3
oLH from
Peroxidase
Park, IL. The United States (USDA) animal hormone bLH-B-5, USDA bovine
(bGH)-B-1, Dr.
AND TROST
ratio) rather than to imand 2) purification of Sephadex G-100 column.
immediately before use); 6) Substrate stop solution: LH
ELISA
Procedure
Using
0.5 #15
M H2 SO4. Antiserum
Falcon #3912 microtiter plates were coated with 50 zl per well of 1/25,000 rabbit #15 antiserum in PBS coating buffer. However, Well one was filled with only PBS coating buffer for use as a machine blank.
OF
DEVELOPMENT
Plates
were
dried
and stored were rinsed assay buffer
completely
at 37#{176}C in desiccated
desiccated at 4#{176}C. Antibody-coated 8 times with wash buffer, and was immediately added to prevent
air
plates 25 ! of drying
of the wells. Fifty microliters of sample or standard diluted in assay buffer were then added. To ensure that most serum samples would be on the standard curve to
0.5
to
5 j.zl of serum
be added
mixed
on
to each an orbital
in a humidified However, the tions
of
sample
to
24
h.
conjugate
in assay
well.
shaker,
container data shown
2
peroxidase
diluted
Plates
and
buffer
were
routinely
After (50
the
zD,
addition
plates
had
to
bLH-
conducted
ELISA
with
#15 rinsed
Briefly, with wash
antiserum. 8 times
518B7-coated buffer, and
each well. overnight
well.
50
Plates
bLH-peroxidase
p!
were
4#{176}C.The
final
washed
then incubation
8 times
j.zl substrate
was
mixed
and
with
at 20#{176}Cwas
cold
was assay
added
added
incubated
volume to
buffer, each
solution, to each mined EIA LII
50 p1 of substrate stop solution well. Optical density (OD) was at 450
nm
or 450-600
nm
was then
in a Model
added deterEL
Autoreader. ELISA
Procedure
Using
Chicken anti-oLH Falcon #3912 plates used with
with #15 chicken
same
manner
Anti-oLH
(D11-81) was with a procedure
antiserum. anti-oLH as
Chicken
The LH was then
ELISA
procedures
coated similar
onto to that
ELISA conducted performed in the utilizing
before washing plates with cold wash buffer adding substrate. After a 0.5- to 2-h incubation peroxidase substrate solution, 50 p1 of substrate solution was added. OD was then determined 45 0-600
Mouse
Procedure
Monoclonal
125
After
a
substrate was added at 45 0-600
in
bovine
et
a!. rabbit and
pituitary
to
the
(1985), using #15 antiserum, NIAMDD-bLH-4
Pituitary
Cell
bovine
was
procedures
of
200 pl of ‘251-bLH (2.4
per-
Hinshe!-
1:40,000 Nis(LER 1072-7)
X NIH-LH
Si)
as
Culture
pituitary
cells in 3 mg/rn! 2 pg/ml DNase,
cultures
was dissociated
collagenase, 1 mg/mi and 10 mg/mI bovine
into
individual
hyaluronidase, serum albumin
(BSA) in Dulbecco’s Modified Eagle’s Medium in (DMEM). Pituitary cells were plated at 500,000 cells LHper 35-mm plate and cultured for 6 days in DMEM 4#{176}C with 10% horse serum and 2.5% fetal calf serum. and The medium was removed and pituitary cells were with challenged with no hormone, or 10b0 to 10 M stop gonadotropin-releasing hormone (GnRH) in DMEM at (no serum) for 6 h. The final incubation was removed
Using
frozen
Mouse
Anti-bLH anti-bLH was coated a concentration of buffer. After adding
LH
according
Each
and
Mouse monoclonal 5 18B7 Nunc Immuno I Plates at ng/ml in bicarbonate coating
of
formed
Bovine
and
#15
nm.
ELISA
RIA wood wender
each 4 h at
Procedure
RIA
as trace, standard.
antisera. Specifically, bLH standard was incubated antibody-coated wells for 4 h at 20#{176} C. Bovine peroxidase was added and incubated 20 h at
LH
310
a
and to
p1. Plates
well.
LH
added at 20#{176} C. After peroxidase substrate
to for
150
4#{176}C, and 100 0.5 to 2 h
was with
a
of then
Plates were covered, mixed, at 20#{176} C. Plates were cooled
0.5to 1-h incubation with peroxidase solution, 50 pl of substrate stop solution to each well. OD was then determined nm in a Model EL 310 EIA Autoreader.
substrate incubation
in utilizing
plates were 50 p1 of assay
mixed on an orbital shaker, and routinely incubated for 16 to 24 h at 4#{176}C. However, the data shown in Table 1 involved incubations for 1 to 24 h at either 4#{176} or 20#{176} C. The final incubation volume was 125 j.il. Plates were then washed 8 times in wash buffer at jii
518B7
was immediately added. Fifty microliters or standard diluted in assay buffer was
were
covered,
LH
plates was then performed to the LH ELISA procedure
4#{176}C, and
24 h. incuba-
of
were
4#{176}C.The
added to incubated
covered,
20#{176}Cfor 16 in Table 1 involved
at
597
anti-bLH-coated manner similar
buffer sample
incubated
at
ELISA
LH
on 250 100
pl of Mouse 518B7 anti-bLH to each well, plates were covered and incubated overnight at 20#{176} C. Plates were rinsed with wash buffer and either used immediate!y or stored for no more than one week in assay buffer
until
Pituitary
assays
for
LH by ELISA
and
by RIA.
Superfusion
ICR mice were obtained from Sasco King Labs, Oregon, WI. Eight-wk-old females were ovariectomized and implanted with a 10-pg estradiol-17fl implant. One week later, mice were killed by decapitation. Pituitaries were removed, dissected into halves, and superfused with 1 ml of medium per h. 0.1%
The
culture gelatin
medium, and
25
DME:Hams pg/ml
gentamycin
F-12
1:1
sulfate,
with was
598
SPEAROW
gassed without
with 95% GnRH,
until
assayed
Serum
for
for 15 collected for
LH
mm per h for another in 96-well plates and
samples
that
were
cows
also
were
cannu!ated
after
start
administered obtained Blood
ovariectomized from
sampling,
fraction 0.5-5
p!)
was was
80
frozen assayed
vein
samples were and one-half h g
until after
GnRH
was
samples were et a!., 1985). 800 X g, and assay. dilution
Serum into
buffer.
Statistical
LH immunoassay parameter Packard
logistic 9816
Breme! regression
and was
squares. Slopes were compared between calculated
titers
of
at 4#{176}C with The
binding
plates
was
with zero
data were curve fitting Microcomputer
analyzed with a four program on a Hewlett (Rodbard, 1974;
Spearow, unpublished determined by the of standard by t-tests assay from
curves and (Schefler,
coefficients the estimated
LH among
all sample respectively.
results). method
replicates
of and
sample 1969).
variation concentration quality
of
Linear least
no of tested
coating. of #15 enough unlabeled
standard
binding. under
immunoprecipitable of total peroxidase of 0.05% to obtain
#15
anti-oLH
in the stored
LH ELISA desiccated
detectable
Tween low
serum
only
a
to
with
in binding to
insure
good
to achieve an OD 0.7 to 1.0 resulted sensitive where
and LH about
bLH-peroxidase activity) was bound. 20 in the nonspecific
of
p1 of 1/25,000 by incubation
sensitivity
The most conditions
activity.
uniformity
with 50 followed of
bLH-
antibody-coated
reasonable
Coating antibody,
and
were determined. for at least 1 mo
decline
bLH-peroxidase
bLH-peroxidase hormone
curves
specific performed
well Analysis
with
Conditions
antibody dilution
cattle.
via the jugular Blood One
via the cannula, and blood for another 8 h (Hinshelwood samples were centrifuged at
the serum (routinely assay
been obtained
days postpartum. at 30-mm intervals. of
from mice by deat 1200 h diestrus
had
at 75-80 withdrawn the
Assay
The
obtained females
females
Nonsuckled
unfrozen at -15#{176}C in 50% glycerol buffer slight loss of activity over a 6-mo period.
peroxidase needed Dried plates were
10-wk-o!d
means,
7.5 h. frozen
TROST
by ELISA.
samples were of 10-wk-o!d
1 wk. Blood
and were
a 2-h superfusion were pulsed with
Samples
Blood capitation and
After
hemipituitaries
M GnRH was
108
Perifusate
CO2.
5%
02,
AND
low
with in non-
assays were 28% of the
activity (12% The inclusion
assay buffer was necessary binding of conjugate to
surfaces.
The effect of duration of each incubation on sensitivity and 50% point of the bLH ELISA summarized in Table 1. It was possible to conduct LH ELISA in as little as 3 h (1 h with standard unknown, substrate). with
1 h with conjugate, rinse, Even lower sensitivities
longer
incubations
with
standard
and were or
the are the or
1 h with obtained unknown
curves Within (CVs) of control
>-
I‘-I
In
z I*i
RESULTS
bLH-Peroxidase
Conjugate
-J
As shown in Figure 1, chromatography of bLHperoxidase on Sephadex G-100 resulted in a broad peak with peroxidase activity. The leading shoulder of this peak was saved for use as bLH-peroxidase conjugate. Immunoprecipitation with excess anti-oLH resulted tion of 43% of peroxidase conjugate was stored frozen
.6
of the conjugate in the immunoprecipitaactivity. bLH-peroxidase at -15#{176} C for over 20 mo
with no apparent loss of activity. For solution, bLH-peroxidase conjugate
a working was
stock stored
a:
#{149}
U I-I
I-
2
0 0
FRACTION FIG. 1. Chromatography of bovine luteinizing on Sephadex G-100. Optical density of aliquots incubation in peroxidase substrate solution.
NUMBER of
hormone-peroxidase diluted eluant
after
DEVELOPMENT TABLE linked
1. Effect immunosorbent
of
duration assay
1st
of incubations on (ELISA) procedures
sensitivities using #15
incubation
and 50% antiserum.
2nd
OF points
LI-I of
599
ELISA
luteinizing
hormone
incubation (add trace)
Method
Duration
T
Duration
T
Total duration of assay
RIA ELISA ELISA ELISA EL1SA ELISA ELISA
24h 1 h 2h 4h 4h
20#{176} 20#{176} 20#{176} 20#{176} 20#{176} 20#{176} 20#{176}
24h 1 h 2h 16-24h 16-24h 3h 16-24h
4 20 20 20 4 4 4
2days 3 h 5h lday lday lday 2days
(sample
or standard)
16-24h 16-24h
aTemperature,
before
degrees
adding
(79.6
RP2/ml) incubating
wells
adding
pg
for
16-24
101.8
at
conjugate,
good
Specificity
rLH-
The effects gonadotropins
pg
LH ELISA by in antibody-
room and
temperature, then
incubating
16-24 h at 4#{176}C.While a lower 50% LH ELISA was obtained by decreasing of the incubation with bLH-peroxidase
standard hormone LH ELISA was LH was
Since the performance, remainder
or
h
conjugate to 3 h at 4#{176}C, the was greater. Using the same
bLH than procedure
Very
in the 2-day and unknowns
bLH-peroxidase
for another point in the the duration
conjugate.
bLH-B5/ml
were obtained standards
coated
variability antiserum
diluted in assay about seven times RIA procedures. more sensitive
2-day this
LH ELISA
the
studies
of
Validation
Sensitivity (bLH B-5)
558 420 126 364 115 33 79
RIA)
and
50% pg/mi
(bLH
LH
point B-5)
enzyme-
pg/mi
2800 2218 1179 1300 787 197 359
in the results and 50 p1 of
buffer, the 2-day more sensitive for
(2.1
showed was
conducted
the
best for the
#15
anti-
used
with
and
Si)
X
in the
LH ELISA ovine, and
showed rat LH.
cross-reactivity
concentrations ELISA are shown
for
LH
ELISA
of several in Figure
gonadotropins of USDA
is shown
in Table
a high cross-reactivity The LH ELISA also follicle-stimulating
and bLH-B5 2. The
for bovine, showed a low
hormone
and PMSG, a very low cross-reactivity thyroid stimulating hormone (rTSH), detectable cross-reactivity for hCG or
In fact, a 3-h ELISA than a 2-day LH RIA.
ELISA procedure
of increasing in the LH
3. The cross-reactivity of several other hormones relative to that
(FSH)
for bGH, rat bPRL, and no bovine insulin.
100
80
serum. Assay
(LH
Centigrade.
bLH-peroxidase
sensitivities
radioimmunoassay
Characteristics
60 A typical standard curve using #15 rabbit anti-oLH antiserum and rat LH-RP2 as standard is shown in Figure 2. The 2-day LH ELISA had a sensitivity of 101.8
±
point of ± 0.042,
15.4
pg/rn!
21.2%. Using had a sensitivity point
of
for
rLH-RP2,
50%
531 ± 34 pg/ml for rLH-RP2, intrassay CV of 7.3% and
359
bLH-B5 of 79.6 ±
69
as ±
pg/ml,
standard, 31 pg/ml, slope
displacement
slope interassay
of 1.248 CV of
the LH ELISA 50% displacement of
1.287,
intraassay
CV of 12.0%, and interassay CV of 22.4%. A 96-well plate-reader was needed to accurately quantify ODs. Nevertheless, it was possible to distinguish semiquantitatively with eye.
high
the or low
differences LH
concentrations
S... .S.
in OD
between
samples
with
unaided
the
0 .001
.01
NG FIG. 2. Luteinizing hormone (LH ELISA) standard curve using #15 anti-ovine LH as antiserum, Percent bound = % BO.
.1
rLH
1
PER
10
ML
enzyme-linked immunosorbent assay bovine LH-peroxidase as trace, rabbit and rat LH-RP-2 (rLFI) as standard.
SPEAROW
600
AND
a
TROST
Parallelism Purified bovine and
100
assayed
80
in the
the following significantly: oLH-24
(b
20
.1
NG
1
10
GONADOTROPIN
100
PER
ML
FIG. 3. Effects of increasing concentrations of gonadotropins in the luteinizing hormone enzyme-linked immunosorbent assay (LH ELISA) conducted with #15 antiserum. bLH = bovine LH, 0LH = ovine LH, rLH rat LH, bCG = human chorionic gonadotropin, bFSH = bovine follicle-stimulating hormone, 0FSH = ovine FSH, %BO = percent bound.
Furthermore,
other
studies
in
the chicken anti-LH revealed of gonadotropin preparations, ELISA
= 1.58
or in LH RIA
procedures
our
laboratory
(not
of several hormones’ in the luteinizing immunosorbent assay (LH ELISA) conducted LH (oLH) and bovine LH (bLH)-peroxidase.
Cross-reactivity
USDA bLH-B5 oLH (NIAMDD-oLH-24) rLH-RP2 (AFP5666C) oFSH-RP1 (AFP5679C) bGH-B1 PMSG bFSH-B-1 bPRL-B-1 rTSH-RP-2 hCG CR121 blnsulin
100 92 81 5 1.9 0.9 0.7 0.1
