All materials contaminated with patient specimens or !

*+,% should be
!" inactivated by validated procedures (autoclaving or chemical treatment)
in accordance with applicable regulations.
ELISA Test for the Quantitative !02-3% irritates eyes, skin and mucous membranes. Upon contact,
Determination of Luteinizing Hormone (LH) rinse thoroughly with copious amounts of water and consult a doctor.
in Human Serum */$@+5+/D
The reagents are stable up to the stated expiry dates on the individual
#$%&$'()*+,( labels when stored at 2...8°C.
!"#$% 53010 96 Tests Complete Test Kit After opening reagents have to be stored at 2...8°C and used within 60
days (see also "Note").
!&'(%
!)&*%
-./(.0(0)12(
- sealed in an aluminium bag with a desiccant.
Luteinizing hormone (LH) is a glycoprotein hormone of approx. 30 kD,
- before opening, the strips must be at 3==E)/(E4(3$/G3(.
which is synthesised in the hypophysis. Physiologically this hormone
- unused: return to the zip-lock bag with the desiccant. Strips stored
acts on the gonads and is essential for their development and function.
in this way at 2...8°C can be used until the expiration date (see also
During the first phase of the menstrual cycle LH stimulates the
"Note").
progress of the follicle to the Graaf’s follicle. Ovulation is induced by
- Do not touch the upper rim or the bottom of the wells with fingers.
peak levels of LH. The clinical and diagnostic usefulness of LH
measurement for ascertaining the homeostasis of fertility regulation via ;($'(./)#3(4$3$/+=.
the hypothalamic-pituitary-gonadal axis has been well established. LH Bring all reagents to 3==E)/(E4(3$/G3( (15...25°C) before use.
determination is indispensable for monitoring subjects who undergo !" Reagents not in use should always be stored at 2...8°C.
#!$%& fertilisation.
!"#$%&'(!)*+(,"-./%"&(!/+04%
#3+.%+45( 6)*$.07+%8)9-:)6 - ?$+./)/G3@+0+/DK)78+%8)E$D)$44($3) +.) /8() %=.%(./3$/() !/0%K) 7+55
The LH ELISA is based on the classical sandwich ELISA technique. %=E45(/(5D)0+22=5I()=.)0+5G/+=.L
As a second generation assay, it makes use of the extremely high - dilute !/0% to 1000 ml with fresh, deionised water in a suitable
affinity of the system Biotin-Streptavidin. Streptavidin has been coated container. Rinse vial several times.
on the surface of microtiter wells. In the first incubation step, - Stability:)G4)/=)MN)0$D2)$/)OPLLLQPR<L
specimens, calibrators or controls, enzyme conjugate (peroxidase-
labeled anti-LH) and a second biotinylated monoclonal anti-LH are ,.0*/#)/1(!"#$%&'(,"-./%"&(!051%
mixed to form the sandwich complex which is bound to the surface of " for longer periods of usage: prepare needed amount by mixing equal
the wells by the interaction of biotin with the immobilised streptavidin. portions of !0+%6 and !01%. Use only a clean plastic vial previously
At the end of the incubation excess enzyme conjugate and monoclonal rinsed with deionised water.
antibodies are washed out. TMB/Substrate is added (step 2) and the - for use within 30 days: pour contents of the vial !0+% in vial !01% mix
resulting colour, which turns into yellow after stopping the reaction with and store at 2...8°C.
the stop solution, is measured photometrically. The intensity of colour - handle !051% carefully and $I=+0) %=./$E+.$/+=. ! Do not use, if it
is directly proportional to the LH concentration in the sample. looks blue!
The absorbance of calibrators and specimen is determined by using - Store protected from bright light.
ELISA microplate readers or automated ELISA systems (like - Stability: SN)0$D2)$/)QLLLTR<
HUMAN´s HUMAREADER or ELISYS line).! The concentration is
evaluated by means of a calibration curve which is established from *4(%+E(.
the calibrators supplied with the kit. Serum
Do not use highly lipemic or hemolysed specimens.
;($'(./2)$.0)<=./(./2 Specimens may be stored for 5 days at 2...8°C, up to 30 days at
!)&*% 12 >+%3=/+/(3)*/3+42)(in 1 strip holder) -20°C. C3((,() $.0) /8$7) =.%() =.5D. Thawed specimen must be
homogenised. Eliminate particulate matter by centrifugation or
8-well 2.$46=??)strips, coated with streptavidin
filtration.
!*+,% A-F <$5+@3$/=32)(white cap)
6x2.0ml Ready to use, in human serum #3=%(0G3(
LH level: 0 (:), 5 (A), 25 (<), 50 (B), 100 (9) and C=55=7)/8()43=%(0G3( (U$%/5D as described.
200 (C))IU/l 2#"314.#)-(5"/1*
!*-.% 13 ml 9.,DE()<=.FG'$/()(white cap) #OV Do not mix or use components with different lot numbers. Do not
ready to use, coloured yellow pH 7.45 ! 0.1 mix caps of vials (risk of contamination). Do not use reagents after
anti-LH (goat), HRP-labelled, anti-LH their expiration date.
(monoclonal, mouse), biotinylated 1.0 µg/ml #QV Do not use reagents that could be contaminated or look or smell
!/0% different than usual.
20 ml H$28)*=5G/+=.)(black cap)
#SV Record !*+,%, specimens and controls carefully on the spread
Concentrate for ca. 1000 ml pH 8.8 ! 0.4 sheet supplied with the kit.
MOPS buffered saline 5 mmol/l #WV !)&*% -6select the required number and place firmly in the holder.
!0+% 7.0 ml *G@2/3$/();($'(./):)(yellow cap) pH 3.5 ! 0.1 #PV ;G.)0G45+%$/(2 for !*+,%, controls and specimens. #+4(//()/8(E
3,3', 5,5'-tetramethylbenzidine (TMB) 4 mmol/l =.)/8()@=//=E in the microwells.
Sodium acetate buffer 0.05 mol/l #MV :57$D2) $00) 3($'(./2) +.) /8() 2$E() =30(3) $.0) /+E+.') /=
!01%
E+.+E+2() 3($%/+=.) /+E() 0+??(3(.%(2) @(/7((.) 7(552L) This is
7.0 ml *G@2/3$/();($'(./)A)(blue cap) pH 4.5 ! 0.1 important for reproducible results. Pipetting of specimens should not
Urea hydrogen peroxide 10 mmol/l exceed 10 minutes. Otherwise pipette the calibration curve in the
Sodium acetate buffer 0.05 mol/l indicated positions at half way time of the series. If more than 1 plate is
!02-3% 7.5 ml */=4)2=5G/+=.)(red cap) used, repeat the dose response curve for each plate.
#XV Avoid/remove air bubbles prior to incubations and reading
Sulphuric acid 0.5 mol/l
absorbance.
1 :08(2+I()2/3+4 #TV !051% initiates and !02-3%6 terminates a kinetic reaction. :I=+0
#3(2(3I$/+I(2: Total concentration < 0.04%. @3+'8/)5+'8/ during colour development.
#YV !)&*%6 -6 3=%& '(./5D) ?=3) QN6SN) 2(%L $?/(3) ($%8) 4+4(//+.') 2/(4
*$?(/D)J=/(2 without spilling the solutions to ensure thorough mixing. If available mix
Do not swallow the reagents. Avoid contact with eyes, skin and on a plate shaker.
mucous membranes. All patient specimens and !*+,% should be
handled as potentially infectious. !*+,% have been checked on donor
level for HCV and HIV-1/2 antibodies and HBsAg and found negative.
Wear protective clothing and disposable gloves according to Good
Laboratory Practices.
!)*+(2#"314.#1 #(3?=3E$.%()<8$3$%/(3+2/+%2
Z8() 7$28) 43=%(0G3() +2) %3+/+%$5. Insufficient washing will result in The LH ELISA test has an analytic sensitivity of about 0.8 IU/l LH.
poor precision or falsely high absorbance. Specimens with LH concentrations above 200 IU/l may be diluted with
HOV Remove adhesive strips, aspirate off the contents, add !/+04%, normal male serum and reassayed. To obtain the sample's
aspirate off after 30 sec. soak time and repeat washing twice. concentration multiply by the dilution factor.
HQV In case of automatic washers fill and prime with !/+04%. The assay is standardised in accordance with WHO IRP for LH
Subsequently wash strips 3 times. Ensure the washer fills all wells (68/40).
completely and aspirates off efficiently after 30 sec. (remaining liquid:
< 15 µl). Typical performance data can be found in the Verification Report,
HS After washing, 3(E=I() 3(E$+.+.') 5+[G+0) @D) /$44+.' the plate accessible via
upside down on tissue paper. www.human.de/data/gb/vr/el-lh.pdf or
www.human-de.com/data/gb/vr/el-lh.pdf
2%61//%&'(,3+171
Reagents and specimens should be at room temperature before J=/(
use. The components of the kit are stable until the expiry date even after
*/(4)O H(55))\]5^ opening. However, a potential contamination is directly related to the
9QLLL number of samplings. The 60 days limit after first use is set for safety
:OLLLBQ
Calibrators Specimen
reasons.
The handling should always be in compliance with common GLP
!*+,% A-F; in duplicate 50 -- requirements (*)! The validation criteria must be met!
Specimens, Controls; in duplicate -- 50 (*This includes: Proper caps being replaced on the vials and firmly tightened / Remove
!*-.% 100 100 only reagents required for a run from stock solutions if they could come into contact with
other contaminating solutions like patient specimens etc. / Stock solutions always
Mix and cover !)&*% with Adhesive Strip returned to 2...8°C when not in use.)
Incubate 60 min. at 20...25°C
Wash 3 times as described (see W1 - W3)
2 ,5

!/+04% 300 300 2

*/(4)Q
!051% 100 100 1 ,5
:)WPN

Incubate 15 min. at 20...25°C (see P8) 1

!02-3% 50 50 *

Mix carefully 0 ,5

Measure the absorbance at WPN).E as soon as possible or 7+/8+. 0

ON)E+.L)after terminating of reaction, using a reference wavelength 0 50 100

! " ) < = . % ( . / 3 $ / + = . )\ - 1 ` 5^ a
150 200

of 630-690 nm (if available).

_$5+0$/+=.)=?)/8()Z(2/
The test results are valid provided the following criteria are met:
Maximum absorbance (!*+,% C) O.D. " 1.5
LH concentration at 80% of max. absorbance = 120 ! 25 IU/l
LH concentration at 50% of max. absorbance = 60 ! 10 IU/l
LH concentration at 20% of max. absorbance = 19 ! 4 IU/l
<$5%G5$/+=.
A dose response curve is used to extrapolate the concentration of LH
in unknown specimens.
1. !*+,% - Plot the absorbance for each duplicate versus the
corresponding LH concentration in IU/l on linear graph paper (do
not average the duplicates of the calibrators before plotting).
2. Draw the best fit curve through the plotted points.
3. To determine the concentration of LH for an unknown sample (S),
locate the average absorbance of the duplicates on the vertical
axis of the graph, find the intersecting point on the curve, and read
the concentration (in IU/l) from the horizontal axis of the graph.
;(?(3(.%(2
1. Kosasa T. S., Measurement of Human Luteinizing Hormone,
Journal of Reproductive Medicine QM, 201-6 (1981)
2. Danzer H. '$()*+, Maternal Serum Human Chorionic Gonadotropin
Concentrations and Fetal Sex Predictions, Fertility and Sterility SW,
336-40 (1980)
3. Braunstein G. D. '$( )*+, Serum Human Luteinizing Hormone
Levels through Normal Pregnancy, American Journal of Obstetrics
and Gynecology OQM, 678-81 (1976)
4. Goldstein D. P. and Kosasa T. S., The Subunit Radioimmuno-
assay for LH Clinical Application, Gynecology M, 145-84 (1975)
5. Batzer F., Hormonal Evaluation of Early Pregnancy, Fertility and
Sterility SW, 1-12 (1980)
6. Braunstein G. D. '$( )*+, First-Trimester Luteinizing Hormone
Measurements as an Aid to the Diagnosis of Early Pregnancy
Disorders, American Journal of Obstetrics and Gynecology OSO,
25-32 (1978)

-./(343(/$/+=.)=?);(2G5/2
LH depends upon diverse factors other than pituitary homeostasis.
Thus, the determination of LH alone is not sufficient to assess the
clinical status.
LH is suppressed by estrogens, still in women under oral contra-
ception the LH levels may be normal.
Excessive dieting and weight loss may lead to low LH concentrations.

9U4(%/(0) _$5G(2) ?=3) !") !(I(52) /83=G'8=G/) J=3E$5) >(.2/3G$5

<D%5(
<D%5()48$2( !")\-1`5^
Follicular phase 0.8 - 10.5
Midcycle 18.4 - 61.2
Luteal phase 0.8 - 10.5
Postmenopausal 8.2 - 40.8
In men, LH values of 0.7 - 7.4 IU/l have been reported. EL-LH
Each laboratory should establish its own Expected Values utilising
instrumentation, blood collection methods and testing techniques
INF 5301001 GB
11-2005-13 !
commonly used in that laboratory.

Human Gesellschaft für Biochemica und Diagnostica mbH

Max-Planck-Ring 21 - D-65205 Wiesbaden - Germany
Telefon: +49 6122 9988 0 - Telefax: +49 6122 9988 100 - eMail: human@human.de