CA1273306A

CA1273306A - Process and reagent for the determination of the luteinising hormone and monoclonal antibodies suitable therefor

Info

Publication number: CA1273306A
Application number: CA000502915A
Authority: CA
Inventors: Christa Hubner-Parajsz; Hartmut Schetters; Helmut Lenz; Klaus Erler
Original assignee: Boehringer Mannheim GmbH
Current assignee: Roche Diagnostics GmbH
Priority date: 1985-03-06
Filing date: 1986-02-27
Publication date: 1990-08-28
Anticipated expiration: 2007-08-28
Also published as: AU5413086A; FI91975B; FI860936A0; ES8802262A1; EP0193881B1; EP0193881A3; FI860936A; DE3650561D1; KR860007547A; AU561726B2; EP0193881A2; DE3507848A1; ES552706A0; ES8802263A1; ZA861550B; FI91975C; JPS61218599A; KR890003591B1; ATE142023T1; ES552704A0

Abstract

ABSTRACT

An immunological process for the determination of the luteinising hormone (LH) employs at least one monoclonal antibody which is directed against LH and cross-reacts with other glycoprotein hormones to an extent of less than 3%; a reagent for the determina-tion of LH contains at least one such monoclonal antibody; monoclonal antibodies and a process and a hybridoma cell line for producing it are also pro-vided.

Description

7~
, The pre~ent invention i~ concerned ~ ch a proce~s and a re~g~nt for ~che dete~na~ion of ~he lu~eini ~ing hormolle, as well a~ with rnonoclonal antibodie~ suitable therefor.
The detennination of the luteini~ing honD~ne (LH~
in body fluid~, for exan~ple urin~ erlLm ~nd pla~ma, i 3 mainly em~loyed in order to be able to a~ he endocrinological ~a~us o:~ th~ hypthalamu~, hypophy~i~
and gonads. The~e inve~tis~ations ~erve, in particular9 for the differential diagno~i~ of hypogonadiam, infertility and the like., In addition, the L~I dater-mination i8 used in order to determine the ovulation time point in the case of an ~ nduction of pregnancy.
- - - In all th~se ca~e~, the ~erum values lie within 15 the physiological range. Furthermore, the concentration of ~H in ~he ~erum is also measured ~olely ~or the purpo-4e of obtaining evidence regarding the biological effectivene~s of ~hi~ hormone. Of dia~nostic import-ance i~, therefore, e~entially a knowledge of the serum level of the native hormone.
The physiological concentration of human LH in the 9erwm lie~ in the following ranges:
men ~ 4 - 24 mIU/ml.
women before menopause, cycle 10 ~ 20 mIU/ml~
women ovulation peak 80 - 100 mlU/ml.
w~men after menopau~e 80 - 150 mIU/ml.

~t73 `:

For the de~ermina~ion o~ h~, there are aspec-ially ~uitable in~nunological ~e~3t proce~e~ in whicll the hormone i~ determined a~ an~igen with one or more antibodie~ directed again~t it. The obtaining of antibodle3 wlth these polypeptide hormone~ lnvolve~
difficulties since all polypeptide honmone3 are poorly immunogenic. Becau2~e of ~he homology between LH and other glycopro~ein hormone~, for exampl2 the follicle-~timulating hormone (FSH), ~hyroid-3timulating honmone (TSH) and human chorionic gonado-tropin (hCG), it i9 very dif~icult to obtain specific antibodie3 against one of the3e hormone~. An a~tibody directed against one o~ these glycoprotein hormon23 usually display3 more or les~ cross-reactivity with the other glycoprotein hormones.
Clinica Chemica Acta 133, 263-274,/1983 descri}~
monoclonal antibodies again . t LH which cro~s-react with TSH and hCG up to 10% and with FSH up to 3%.
Briti~h Patent Specification No. 2,111,201 al~o de~-cribes a monoclonal antibody again~t LH~ However,this react ju~t a3 well with hCG. A monoclonal anti-body which i8 specifically directed against LH and display~ no cross-reactivity with the other glyco-protein hormones i~ hitherto not known. Therefore, at the moment, it i~ not po~sible to determine LH
immunologically without other glycoprotein hormone~
being mor~ or less included.

"0 ~7~

The present invention~seeks to provide a new in~unological proces~ and reagent with the help of which LH can be ~pecifically determined even in the pre~ence of other glycoprotein hormones~
Thu8, according to the pre~ent invention, there is provided an immunological proce~ for the determin-ation of the luteini~ing ho~mone (LH), ~herein at least one monoclonal antibody i9 u~ed which i5 specificalJy directed again~t LH and cross-reacts with other glyco-pxotein hormones at an extent of le~s than 3%.
The present invention al80 provide3 a reagen~
for the determination of the lllteini~ing hormone, wherein it contain3 at least one monoclonal antibody which is directed against LH and cro~s-reacts with other glycoprotein hormones to an extent of les~
than 3%.
Aq immunological determination methods, there can, in principle, be used all available immuno-a~says, quch a~ radio-immuno-assay, enzyme-immuno-as~ay, fluorescence-immuno-assay and the like. Furthermore, all process variants, such a~ competitive immuno-assay, sandwich proce3s and the like can be used. For labelling, there can be used the agents which are conventional for the particular determination methods.
Thus, in the case of a radio-immuno-as~ay, there are used radioi~otope~, for example 125I, ~or the labelling.
For an enzyme-immuno-a~ay, there can be used all .~,:;;. j, ~ ~73;~

~, enzyme~ u~ually e~ployed fo~ this purpose, for exaT~ple pexoxida~ or ~-galacto~ida~3e. For a fluorescence-immuno-a~ay, the u8ual fluorsscing group~ can be u~ed as labels. Detail~ of the~e variou~ te3t method~
5 and process variants are well known. Te~t variant3 are advantageous in which at lea~t two %~onoclonal antibodie~ a~cording to the present invention ar~
employed which are directed against differ~nt antigenic determinant~ of the LH and at lea~t one o~ which cros~-10 reacts with o~her glycoprotein hormones to an extento~ less ~han 37~., For the determination of LH, it ha~ proved to be especially preferable to use the sandwich process in which the antigen to be determined i~ brought into contact with a carrier-bound and a labelled antibody.
For such a determination proces~, a 9pecific mono-clonal antibod~ according to the present invention can~ for example, be bound to the solid phase. In a ~ir~t incubation ~tep, this i~ incubated with the sample which contains the LH to be determined, a~ well aq, in gener~l, other ~lycoprotein honmone~, LH there-by being selectively bound by the ~pecific antibody.
After the u~ual washing step~ it i8 incubated with the labelled antibodyO Thi8 mu~t not necessarily be ~pecifically directed against LH but can al~o cross-react wi~h other glycoprotein hormones.

-`` 3L~7~

- Tbe procesa can al 80 b~ carried out wlth a non-~pecific carrier~bound antibody. ~owever, it i~ ~hen nece~ary that, aa labelled antibody, there ia employed an antibody according to ~he pre~ent invention ~hich directed ~pecifically against LH.
Variant~ of the sandwnch proces3 can al30 be u~ed for the determlnation of LH. Thus, for example, a soluble ~andwich complex can first be formed wi~h a non-labelled, ~oluble antibody and the labelled anti-body. Thi 8 i g subsequently made insoluble with thehelp of a carrier-bound anti~ody which is directed again~t the FCY part of the non-labelled soluble anti-body. In the ca~e of this proce3s variant, at lea~t the non-labelled, soluble antibody mu~t be an antibody accordiny to the present inventionO The labelled antibody is thereby preferably employed in exce~s.
The e3sence of the pre~ent invention is to be seen in that it i~, ~urprisingly, possible to make available for these immunological proces~es monoclonal antibodies which are specifically directed against LH
and which, therefore, maXe po3~ible a specific deter-mination of LH
Therefore, the present invention al~o provides monoclonal antibodies against hH, ~he cro~s-reactivity of which with other glycoprot~in honmones amounts to le3~ than ~%.

For obtaining the monoclonal anti~odie~ accord-ing to thP present invention, experimental animal~, for ex~mple mice~ are immunised with LH. For the immuni~atlon, the immunogen 1~ administered in the usual way, for example in combiIlation with an adjuvant.
A~ adjuvant, it i9 preferred to U~e aluminium hydroxide~
together with ~ ~ or Freund'~
adjuvant~ The immunisation preferably takes place over several months with at lea~t four ~mmuni3ation~
10 ~t 4 to 6 week intervala (intral?eritoneal injection3.
From thus i~muni ~ed animal 9 are obtained B-lymphocytes which are fu~ioned wqth a permanent myeloma cell line. ~he fusioning take~ place according to the known proce~s of ~ohler and Milstein (Nature 256, 49$-4~7/1975). ~he primary cultures of hybrid cellq thereby formed are cloned in the usual way, for example with the u~e of a commercially available cell ~orter or by "limiting dilution". In each ca~e, tho~e culture~ are further worked up which, in an appropriate te~t proces~, for example an enz~e-immuno-as3ay ~ELISA proce~s~, react positively against LH and negatively or only a little wlth the other gly~o-protein hormone~. ~here are thu~ obtained ~everal hybridoma cell lines which produce the monoclonal 25 antibodie~ according to the present invention. Accord ing to known method~, the~s cell line~ can be cultured and those of them producing monoclonal antibodi2s are i ~olated.

1',2~33~

By wsy of example, for- cell lines obtained in thia way, there are mentioned:
clone 369 (NCACC 84122001) and clone 799 (NCACC 84122005).
The cell lines have beèn deposited under the given number at the NCACC depository (National Collection of Animal Cell Cultures).
The ~o obtained noclonal antibodies have a very high affinity (affinity constants of the order of magnitude of 109 to 101 l/mol) against LH and croaa-react with other glycoprotein hormone~ to an extent of leoJ than 3%. Preferred monoclonal anti-bodieJ diJplay a crosJ-reactivity toward~ other glyco-protein hormoneJ, Juch as hCG, FSH and TSH, of leaJ
than 1% and eapecially of le~ than 0.1%. For the determination of the affinity and of the croaJ-reactivity with other hormones, there can be used the proce~JeJ known for thia purpose.
The noclonal antibodies according to the preaent invention are outstandingly useful for the apecific determination of the hormone LH in the preaence of other glycoprotein honmones in a sample, for example aerum or plasma. For these determination proceJJea, there can be used the monoclonal antibodies aJ Juch or fragment~ thereof which poJaeaa the corres-ponding immNnological propertieJ, for example Fab fragmentJ, Therefore, the expre~sion ~monoclonal .;. ,, , .-. , :

~ ;~7~ 6 antibodies" is to be understood to Mean not only the complete antibodies but also the fragments thereof.
In accordance with another aspect of the invention a kit for determining the presence of luteinising hormone comprises separate components of reagents at least one of which is a monoclonal antibody of the invention.
The following Examples are given for the purpose of illustrating the present invention and in which reference will also be made to the accompanying drawings in which:
FIGURE 1 illustrates schematically a rotor insert element which may be employed in the deter-mination of LH, and FIGURE 2 is a calibration curve for sera of known LH content.

... .

~3 Balb/c mice, 8 - 12 week~ old, are i~uni sd intraperitoneally with 100 ~g. hLH (obtain2hle from Immunex), ad~orbed on alumlnium hydroxide and pertu~8i9. After 6 week~, three further immunisations are carried out at 4 week intervals. 50 ~g~ LH, adsorbed on aluminium hydroxide and ~ pertussi~, are thereby, in each case, administered intraperitoneally.
~bout four month~ after the last immuni-Qation, fusioning is carried out. Four day~ and three day~
bef~re fusioning, in each ca~e, immuniQatiOn i3 carried out again with 100 ~g. LH/PBS (phosphate buffered ~aline~ intraperitoneally or intravenou~ly.
For the fusioning, with referen~e to the method deQcribed by Galfre (Method~ in ~nzymology, 73, 3/1981) 108 ~pleen cells of an immunised mouse are mixed once with 2 x 10 myeloma cells (P3x63Ag8-653, ATCC-CRL 8375) and subsequently centrifuged for 10 minute-~ ~300 g, 4C.).
20 The cells are again washed once with B5S (balanced 3alt solution) and centrifug~d at 400 g. The supernatant is r~moved. The cell 3ediment i~ mixed wqth 1 ml. 5~%
P~G solution (M.W. 4000, Merck)0 Thereafter, at ambient ~;~'7;~

t~mperature, 5 ml. ~PMI 164Q medium (RPMI ~ ~osew~ll Parker Memory Institu~e) wi~hout fo~tal calf ~erum (FCS) and ~ub~equently once again 5 ml. RPMI 1640 medium with 10% FCS, are ~lowly added dropwise thereto, the mixture iq made up with medium to 50 r~. and centrifuged for 10 minute~ at 400 g. The aedimented cells are taken up in RPMI 1640 m~dium with 1~% FCS.

2 x 105 spleen cellq are each ~eeded into 24 ~11 cell culture plate3 ~obtainable from ~unc)O To each culture are added 1 x 105 ~pleen cells or 5 x 104 peritoneal -exudate cells as feed cells. On the following day, hypoxanthine-azaserine selection medium (100 mM
h~poxanthine, 1 ~gc/ml. azaserine) i~ added thereto.
After akout 7 - lO days, many clones are already vi~ible. The supernatent of the primary culture~ i~
tested according to the ELISA proces~ de~cribed in Example 2. Primary culture~ which contain antigen-specific antibodies are further cloned with the help of a fluorescence-activated cell sorter on 96-well culture plates (obtainable from Nunc). As feed cells, there are used l x lO peritoneal exudate cells or 2 x 104 spleen cells per 96-well of the culture.
In thi~ way, there can be isolated the two hybridoma cell lines of clone 369 and clone 799, which have been depo~ited at the NCACC depoqitory (National Collection of Animal Cell Cultures) under ~he deposit numker~:

~CACC 84122001 (cl~ne 369) and NC~CC 84122005 (clone 799~.
For ~he production of ascite3, 5 x 106 hybrid cell~ are injected ~ntraperitoneally to muce which had previously been pre-treated 1 to 2 time~ with O.S ml.
pristane. One to three week~ thereafter, ascite~ fluid can be obtained from the mice and the antibodie~ can be isolated herefrom in the usual way. Theae monoclonal antibodies are ~pecifically directed against LH and ~how no or only a ~mall cro~q-reactivity wqth other glycoprotein hormone~. In the followQng, they are de~ignated a~ MAB 369 (from clone 369) and MAB 799 ~from clone 799).
The tw~ monoclonal antibodies belong to the sub-clas~ IgGl/~. They po~sess the following affinities:
MAB 369 = 7.9 x 101 l/mol MAB 799 = 9.5 x 1011 l/mol.
For the determination of the affinitie~, compet-ition curves are determined wnth the homologou~ antigen according to the method of Scatchard (Ann~ ~.Y. Acad.
Sci. 51, 660/1949) and evaluated. The mea~urement~
necessary therefor are carried out analogou~ly to Example 2.

~.
~
In order to recognise the pre3ence and specificity of antibodies again~t LH in the serum of immuni3ed mice or in ~he culture ~upernatant of ~he hybrid cell~ or in a~cite~, th~re i~ u~ed an ELISA pro~e~s a~ test princ~ple: Microtitre plates are coa~ed oYernight with 1 ~g. hH/ml. of coa~ing buffer (0.2 M ~odium carbonate/bicarbonate, pH 9.3 ~ 9 7 5) at 37C. and then after-treated for 10 minute~ with 0.~% ~odium c~loride solution and 1% albumin solution and subsequently wa3hed with 0.~% ~odium chloride ~olution. Sub~e-quently~ incubation i~ carried out at 37C. for one hour wnth 100 ~1. of ~2mple and again waRhed with 0.~%
~odium chloride solution. There follow~ a further incubation for one hour at 37~. with 100 - 150 mU/ml.
of a 3heep anti-mou~e-IgG~peroxidase conjugate. After a further wa~hing ~tep with 0.9% ~odium chloride qolution, the peroxidase activity i~ determined in the usual way (for example wqth AB~S, 30 minutes at ambient temperature, ~here being read off the extinction difference, ~ m~, at 405 nm).
The ELISA te~t can al90 be carried out as followq:
~ he microtitre plates are first coated with a ~heep anti-mou~e IgG (20 - ~0 ~g./ml. coating buffer, one houx to overnight, 37C.). Thereafter, further treatment i8 carried out a~ describ2d above, the sample solution i8 add~d and again wa3hed. Finally, incubation i3 carried out with 250 mU/ml. of an LH-peroxida~e con~ugate for 1 hour at 37C. After again washing, ~ ~7;3;~

th~ peroxida~e activity i~ de~ermined, or example wi~h ABTS~

~.

~
The procedure de3cribed in Exa~ple 2 i9 u~ed.
The reactivity of LH is fir~t determined. Then, to the particular monoclonal antibodies, there iS3 in each ca~eO added the antigens (hCG, TSH, FSH) to be te~ted for cro~q-reaction, in increa~ing concentration.
~ he cros~-reactions are subsequently calculated according to the following equation~

x 100 = % cro~-reaction C (cros~-reac~ing antigen) C = concentration of the antigen which 1~ necessary for the achievement of 5~ of the maximum ~ignal In the following Table 1 are ~et out the mea~ured value3 for MAB 369 and MAB 799:

~ 3~;
.

Table _ ~___ __ ___ glyco- origin max.conc. conc.range MAB 369 MAB 799 hormone ~Firm) in the in the te~t % cross % cro3s ~erum to cro~9- react. react.
__ ~_ . .. . . ....... ......... _ 5 hCG UCB 200 0 - 250 ~ 0,l < Ool ~elgium IU/ml. IU/ml.
_~ __ ~ _~ . . .
FSH UCB 400 0 - 9 ~ O.l ~ O.l . ~elgium mIU/ml. IU/ml.
____ ~_ __ ~ _ -TSH Boehringer lO00 0-9000 ~ O.l ~ O.l Mannheim ~IU/ml. ~IU~ml.
_ ___ ~_~ _ ~-TgH Boehringer lO00 0-9000 ~ O.l < O.l MaTmheim ~, IU~ml. ,~IU/ml.
.. . . ~ , ~ ~ ~

Example 4.
~ ~ .A microtitre plate i~ coated for 2 hours at 37C.
or ove~night at 4C. with 10 ~g./ml. ~heep antibody again~t the Fc~ region of a mou~e antibody in 0. 2M
carbonate buffer (pH 9.6). Thereafter, it i~ wa~hed with PBS~0.1% Tween~20 (pH 7.35). Sub~equently, there are added thereto 100 ~1. of a monoclonal antibody (MAB 1~ ~onc~ntration lO ~g./ml.~ in incubation buffer ~PBS, 0.1% bovine serum albumin (BSA), 0.1% Tween 20) and incubated for 2 hours ~t 37C.
An L~-peroxida~e conjugate (lO0 mU/ml.) i pre-incubated overni~ht with lO ~g./ml. of a #econd mono-trade mark clonal antibody (MAB 2) in solution at 4C.
After the incubation of the plate with MA~ 1, exce~ M~B 1 i~ removed by wa~hing with PBS-Tween 20.
The plate iA then after-coa~ed for 30 minutea at ambient te~perature with 1% mou~e normal serum in F~S-~SA. 100 ~1. of the pre-incubated ~AB 2/L~
peroxidase complex are applied to th~ plate and incubat~d for 2 hours at 37C. ~he bound peroxidase activity i~ made vi~ible with ~BTS ~8 ~ubstrate. The mea~ured colour inten~ity i~ directly proportional to ~he MAB 2~LH-peroxida~e conjugate bound to MAB 1. For MAB 799 as MAB 1 and MAB 369 as MAB 2, the bound activity amounts to 34% of the non-competing LH-POD
activity.
The re~ults found show that the tw~ noclonal antibodie~ are directed against various epitope3 of ~he antigen LH.
~52a~ -Determination of LH

~) 13 _~L~ bY~S~:
15 mM sodium phosphate buffer, pH 7.4 15 m~ ~odium chloride S n~ EDTA
0.~% bovine serum albumin, pH 7.4 5 mM o-nitrophenylgalactoside 2) Rece tor l solution~
A~ receptor 1, there i~ employed a monoclonai mouBe anti-hLH antibody according to the pre~ent invention. The ascite~ fluid containing thi~ antibody 5 i~ mixed ad 1.~ M with anmlonium gulphate. The precipit-ate i8 taken up in a bu~fer of 15 mM sodium phosphate (pH 7.0) and 50 mM sodium chloride. ~he solution ~o obtained is ~ubjected to a pa~3age over D~A3-cellulo~e~

3) RecePtor 3 solution:
As receptor 3, there i3 also employed a monoclonal mouse anti-hLH antibody according to the pre~ent invention whi~h, however, recogni~es a dif~erent anti-genic determinant than receptor l. ~he ascites fluid containing thi 9 antibody i 9 purified as described in 2) aboveO m e complete antibody is split up in known manner into the Fab fragment. The Fab frag~nt3 obtained are coupled wnth ~-galactosidase according to the method of R.R. Porter (Biochem. J., 73, ll9/1959~.
43 Activated recePtor 2 olution:
Sheep anti-mou3e FC~ antiserum is mixed ad 1.8 M
wnth ammonium sulphate. The precipitate is taken up in a buf~er of 15 mM sodium pho~phate (pH 7.0) and 50 mM sodium chloride. The solution ~o obtained i~
subjected to a pas~age over DEA~-cellulose.
B) Prod 1) ~L~

3~:3 _17-40 ~1. of a solution which contain~, per ~.
100 ~mol sodlum phosphate (pH 7.3, 37 ~.), 2 ~mol magne~ium chloride, O.9% sodium chloride, .5 0.~ bovine ~erum albumin, 5 ~g.~ml. anti-hLH monoclonal antibodie3 from u~e ~receptor 1 ~olution) 100 mU anti-hLH antibody (mou~e) Fab ~ragment-~-galacto~idase conjugate ( receptor 3 ~olution 3 i~ applied dro~wise to a fleece which ~on i~t~ of commercial polyester paper. Sub~equen~ly, it i~ dried at ambient t~mperature. Until used, the~a fleece are 3tored at 4C. and at a relative atms~spheric humidity of 209~.
~) ~D~ ' Sheep antibodies againAt the Fc'~ part of mou~e antibodies (activated receptor 2 colution) are fixed on to cellulose fleece according to the known cyanogen bromide activation proces~ (see Federal Republic of Germany Patent Specification No. 1768512), ~ereby, per g~ of fibre material, there are provided 10 ~g.
of ~ntibody for f ixiny . Uncoupled antibody i 9 removed by washin~ and the fleece iB gently dried at ambient temperatuxe~ The 50 obtained 1eece i~ 3tored analogou~ly to r~gent carrier 1.

L2~73~

The determination with the help of these two re-agent carriers l and 2 is further described with reference to Figure l and takes place with the use of the device for carrying out analytical determinations described in Federal Republic of Germany Patent Speci-fication No. 34250080 This describes a rotor insert element for centrifugal automatic analysers comprising a formed body which contains a sample application chamber, which is in connection with a plurality of reagent fields, each of which contains an absorbent carrier material impregnated with a particular reagent, at least one mixing valve chamber and a measurement chamber which together form a sample liquid transport path which leads from radially inwardly to radially further outwardly when the insert element is fixed on the rotor and also has at least one further chamber for the reception of a liquid and a transport path which leads from this chamber to the measurement chamber and is at least partly identical with the sample liquid transport path. With reference to Figure l the sample liquid transport path thereby leads from a sample application chamber (Sc) via a chamber (a) filled with absorbent material containing buffer, a chamber (c) and a first valve chamber (VCl) arranged between the chambers ta)- and tc), to a second valve chamber tVC2) and from this, _ a chamber (d) and via a collection chamber (AC), to a measurement chamber (M). For the reception of a further liquid, there is provided a substrate chamber .. . .

73;306 (PC) constructed as pump chamber which is connected with the second valve chamber (VC2) via a dosing device comprising a dosing chamber (DC) and a capillary (Cap), and an overflow chamber (OC). In operation reagent carrier 1 is placed on field c of the disposable insert element and reagent carrier 2 in field d. 40~1. of sample are thereby pipetted through an opening on the upper edge directly on to the field a. The sample is undiluted. 270 ~1. of substrate solution are pipetted into chamber PC. By means of an appropriate programme where high speeds of rotation alternate with stopping, sample and substrate solution are then conveyed in the - direction of the separation matrix and cuvette. In the course of the programme, the receptors 1 and 3 are thereby eluted by the sample liquid from the field c and the homogeneous mixture is subsequently brought to reaction. On field d, the complexes formed are bound to the receptor 2. The tran~fer of the sample from field c to field d takes place within a very short period of time.
The substrate solution is divided into portions by the dosaging chamber DC, the first of which serves for washing out excess, non-complexed conjugate.
The ~-galactosidase activity bound to d via complex formation is proportional to the amount of hLH
contained in the sample. This activity is determined ~'~7~3~

with a further substrate portion, the substrate thereby being reacted in a 5 minute reaction to give coloured products. The colour formed is measured in the cuvette at 410 nm.
A chamber (b) connected to chamber (a) and first valve chamber (VCl) is optional and extends the area of use of the insert element.
The insert element further includes an entry S _ to substrate chamber PC.
The calibration curve according to Figure 2 of the accompanying drawings is obtained from calibra-tion sera of known LH content which cover the range from 0 to 25 mU hLH/ml. (standardised according to the first IRP standard for hLH 68/40) and makes possible a sufficiently sensitive measurement of hLH
in serum or plasma. On the basis of this calibration curve, there can be determined the unknown content of hLH in body fluids, for example serum or sample. The finding again of standards made up with 100 IU/ml.
hCG (lst IRP = 1st international reference prepara-tion of WHO) amounts to 96 to 104~.

~q ~;~'73;~

The ordinate in Figure 2 xepresents the extinc-tion which is measured in a cuvet-te at 410 nm accor-ding to Example 5; the ex-tinc-tion has no units but is based on the Beer-~ambert Law:

S E log _o in which E is the extinction, Io is the incident intensity of light and I is the intensity of emerging light. E varies between 0 and 1; if E is 0 there is no interference between the sample in the cuvette and a light beam, whereas if E is 1 no light passes through the sample.
Example 6.
Determination of LH according to the sandwich prin-cipl~ ~
100 ~ul. of a solution which contains a mono-clonal antibody against LH according to the present invention in a coating buffer (0.2 M sodium carbonate/bicarbonate, pH 9.4) in a concentration of 50jug./ml., are introduced into each recess of a microtitre plate and incubated for one hour at ambient temperature. Subsequently, it is after-coated with incubation buffer (1% bovine serum albumin, 0.9% sodium chloride) and ineubated for 30 mlnute~ at ambient temperature. After washing with wa~h buffer (0.9YO ~odium chloride, 0.1%
Tween 20), there are introduced into each recesR
100 ~1. of ~ample which contains the hH to be deter-mined and incubated for 30 mlnutes at ~bient temper-ature. After again wa~hing with wash buffer, it i~
loaded with 100 ~1. of a conjugate o peroxida~e (activity 100 mU~ml.~ and a further monoclonal antibody according to the pre~ent invention which, however, i~
directed against a different epitope of ~ and incubated for 1 hour at ambient temperature.
For the preparation of the conjugate, there i B
used horseradish peroxida~e (EC 1.11.1.7). The con-jugate i8 prepared by oxidation with periodate and Qub3equent reduction with boron hydride according to the procedure of P.K. Nakane (M.B. Wil~on and P.X. ~akane, in W. Knapp ed. "Immunofluorescence and Related Staining Techniques", 1978, Elsevier/North Holland, Biomedical Press, pages 215 - 224).
After wa~hing with wa~h buffer, it i~ loaded with 100 ~1. ABTS sub~trate solution and, after a one hour colour reaction~ the extinction i~ measured at 405 nm in an ELISA reader (~ynatec).
For the production of a calibration curve, in ~ 25 the case of the above-described proces~, instead of t the ~ample~ olutions are used which contain LH in different, definite concentrations.

* trade mark The Patent publications referred to herein are more fully identified below:
Fed. Rep. of Germany Patent 1,768,512, published (Offenlegungstag) October 14, 1971, R.E.A.W. Axen et al, Pharmacla AB.
Fed. Rep. of Germany Patent Publication ~Offenlegungsschrift) 3,425,008 filed July 6, 1984, - published (Offenlegungstag) February 6, 1986, Sigmar Klose et al, Boehringer Mannheim GmbH.
U.K. Patent Application 2,111,201, filed December 9, 1982, published June 29, 1983, W.M. Hunter et al, Celltech Limited.

~ ' ~"`' '' , ,'

Claims

The embodiments of the invention in which an exclu-sive property or privilege is claimed are defined as follows:

1. An immunological process for the deter-mination of the luteinising hormone (LH), wherein at least one monoclonal antibody is used which is directed against LH and cross-reacts with the glyco-protein hormones: follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and hurnan chorionic gonadotropin (hCG), to an extent of less than 3%.

2. A process according to claim 1, wherein at least one monoclonal antibody is used which is directed against LH and cross-reacts with the glyco-protein hormones: follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and human chorionic gonadotropin (hCG), to an extent of less than 1%.

3. A process according to claim 1 or 2, wherein at least two monoclonal antibodies directed against LH are used of which at least one cross-reacts with the glycoprotein hormones: follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and human chorionic gonadotropin (hCG), to an extent of less than 3%.

4. In an immunological process for the deter-mination of the luteinising hormone (LH) in a body fluid, which comprises contacting a sample of the body fluid with an antibody to said hormone, the improvement wherein said antibody is at least one monoclonal antibody to LH which cross-reacts with the glycoprotein hormones: follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and human chorionic gonadotropin (hCG), to an extent of less than 3%.

5. A process according to claim 4, wherein said at least one monoclonal antibody cross-reacts with the glycoprotein hormones: follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and human chorionic gonadotropin (hCG), to an extent of less than 1%.

6. An immunological process for the deter-mination of the luteinising hormone (LH) in a body fluid comprising:
contacting a sample of said body fluid containing luteinising hormone (LH) and glycoprotein hormones selected from follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSEI) and human chorionic gonadotropin (hCG) with at least one monoclonal antibody to LH, which cross-reacts with the glycoprotein hormones; follicle-stimulating hormone (FSH) r thyroid-stimulating hormone (TSH) and human chorionic gonadotropin (hCG), to an extent of less than 0.1%, said monoclonal antibody having an affinity constant against LH of magnitude 109 to 1011 l/mole.

7. A process according to claim 6, wherein said at least one monoclonal antibody is derived from a cell line designated clone 369 deposited in NCACC
under No. 84122001.

8. A process according to claim 6, wherein said at least one monoclonal antibody is derived from a cell line designated clone 799 deposited in NCACC
under No. 84122005.

9. A reagent for the determination of the luteinising hormone, comprising at least one mono-clonal antibody which is directed against LH and cross-reacts with the glycoprotein hormones:

follicle-stimulating hormone (FSH), thyroid-stimu-lating hormone (TSH) and human chorionic gonadotropin (hCG), to an extent of less than 3%.

10. A reagent according to claim 9, wherein said at least one monoclonal antibody is bound to a carrier.

11. A reagent according to claim 9, wherein said at least one monoclonal antibody is labelled with a labelling agent.

12. A reagent according to claim 9, 10 or ll, wherein said at least one monoclonal antibody is directed against LH and cross-reacts with the glyco-protein hormones: follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and human chorionic gonadotropin (hCG), to an extent of less than 1%.

13, A reagent according to claim 9, 10 or ll, comprising at least two monoclonal antibodies directed against LH, at least one of which cross-reacts with the glycoprotein hormones: follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and human chorionic gonadotropin (hCG), to an extent of less than 3%.

14. A reagent for the determination of the luteinising hormone (LH) bound to a carrier, said at least one monoclonal antibody cross-reacting with the glycoprotein hormones: follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and human chorionic gonadotropin (hCG), to an extent of less than 0.1%, said monoclonal antibody having an affinity constant against LH of magnitude 109 to 1011 l/mole.

15. A reagent for the determination of the luteinising hormone (LH) comprising at least one monoclonal antibody to LH labelled with a labelling agent, said at least one monoclonal antibody cross-reacting with the glycoprotein hormones: follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and human chorionic gonadotropin (hCG), to an extent of less than 0.1%, said monoclonal antibody having an affinity constant against LH of magnitude 109 to 1011 l/mole.

16. A reagent according to claim 15, wherein said labelling agent is a radioisotope.

17. A reagent according to claim 14, 15 or 16, wherein said at least one monocional antibody is derived from a cell line designated clone 369 deposited in NCACC under No. 84122001.

18. A reagent according to claim 14, 15 or 16, wherein said at least one monoclonal antibody is derived from a cell line designated clone 799 deposited in NCACC under No. 84122005.

19. A monoclonal antibody against the luteinising hormone (LH) which cross-reacts with the glycoprotein hormones: follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and human chorionic gonadotropin (hCG), to an extent of less than 3%.

20. A monoclonal antibody according to claim 19, which cross-reacts with the glycoprotein hor-mones: follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and human chorionic gonado-tropin (hCG), to an extent of less than 1%.

21. A monoclonal antibody according to claim 19 or 20, derived from a cell line desiynated clone 369 deposited ln NCACC under No. 84122001.

22. A monoclonal antibody according to claim 19 or 20, derived from a cell line designated clone 799 deposlted in NCACC under No. 84122005.

23. A process for obtaining a monoclonal antlbody agalnst the luteinising hormone (LH) which cross-reacts with the glycoprotein hormones:
follicle-stimulating hormone (FSH), thyroid-stimu-lating hormone (TSH) and human chorionic gonado-tropin (hCG), to an extent of less than 3% comprising:
immunizing animals with LH, obtaining B-lymphocytes from the spleen of the immunised animals, fusing the B-lymphocytes with myeloma cells to form hydridoma cells, cloning the hybridoma cells, selecting clones whlch produce antibodies directed specifically against LH, and forming antibodies with said clones.

24. A process according to claim 23, whereln said animals comprise mice.

25. A process according to claim 23, whereln said clones are selected from clone 369 (deposited in NCACC under No. 84122001) and clone 799 (deposited in NCACC under No. 84122005).

26. A process for obtaining a monoclonal antibody against LH which comprises culturing a cell line selected from clone 369 deposited in NCACC under No. 84122001 and clone 799 deposited in NCACC under No. 841,22005.

27. A process for obtaining a monoclonal antibody against LH which comprises:
injecting appropriate animals with a cell line selected from clone 369 (deposited in NCACC
under No. 84122001) and clone 799 (deposited in NCACC
under No. 84122005), recovering ascites fluid from said animals, and isolating the monoclonal antibodies from said ascites fluid.

28. A hybridoma cell line which produces monoclonal antibodies directed against luteinising hormone (LH) and which cross-react with the glyco-protein hormones: follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and human chorionic gonadotropin (hCG), to an extent of less than 3%.

29. A hybridoma cell line designated clone 369 deposited in NCACC under No. 84122001.

30. A hybridoma cell line designated clone 799 deposited in NCACC under No. 84122005.

31. Monoclonal antibody of claim 19, comprising MAs 369.

32. Monoclonal antibody of claim 19, comprising MAB 799.

33. A process for the determination of the presence of luteinising hormone (LH) comprising contacting a sample with at least one monoclonal antibody which specifically binds to LH and cross-reacts with the glycoprotein hormones: follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and human chorionic gonadotropin (hCG), to an extent of less than 3% under conditions favor-ing formation of monoclonal antibody-antigen com-plexes between said monoclonal antibody and LH and determining the presence of said complexes.

34. A process of claim 33, wherein at least one monoclonal antibody is used which specifically binds to LH and cross-reacts with the glycoprotein hor-mones: follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and human chorionic gonado-tropin (hCG), to an extent of less than 1%.

35. A process according to claim 33, wherein at least two monoclonal antibodies are used and at least one of said monoclonal antibodies cross-reacts with the glycoprotein hormones: follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and human chorionic gonadotropin (hCG), to an extent of less than 3%.

36. A process of claim 33, wherein said mono-clonal antibody is radioactively labelled.

37. A process of claim 33, wherein said mono-clonal antibody is enzymatically labelled.

38. A process of claim 35, wherein said process comprises a sandwich immunoassay.

39. A process of claim 33, wherein said mono-clonal antibody comprises a Fab fragment.

40. A process of claim 36, wherein said label is 125I.

41. A process of claim 37, wherein said enzyme label is peroxidase or .beta.-galactosidase.

42. A process of claim 33, wherein said anti-body is fluorescently labelled.

43. A reagent of claim 9, comprising at least two monoclonal antibodies one of which cross-reacts with the glycoprotein hormones: follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and human chorionic gonadotropin (hCG), to an extent less than 1%.

44. In a kit for determining presence of luteinising hormones comprising separate components of reagents, the improvement in which at least one of said components is a monoclonal antibody which specifically binds to luteinising hormone and cross-reacts with the glycoprotein hormones: follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and human chorionic gonadotropin (HCG), to an extent less than 3%.

45. A kit as in claim 44, comprising an anti-body which specifically binds to luteinising hormone and cross-reacts with the glycoprotein hormones:
follicle-stimulating hormone (FSH), thyroid-stimulat-ing hormone (TSH) and human chorionic gonadotropin (hCG), to an extent less than 1%.

46. A kit as in claim 44, comprising at least two monoclonal antibodies which specifically bind to luteinising hormone at least one of which cross-reacts with the glycoprotein hormones: follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and human chorionic gonadotropin (HCG), to an extent less than 3%.

47. A process of claim 33, wherein said at least one monoclonal antibody comprises MAB 369 or MAB 799.

48. A process of claim 35, wherein said at least one monoclonal antibody comprises MAB 369 and MAB 799.

49. A process of claim 9, wherein said at least one antibody comprises MAB 369 or MAB 799.

50. A reagent of claim 43, comprising mono-clonal antibodies MAB 369 and MAB 799.

51. A kit of claim 44, comprising at least monoclonal antibody MAB 369 or MAB 799.

52. A kit of claim 46, comprising monoclonal antibodies MAB 369 and MAB 799.

53. In a kit for determining presence of luteinising hormone comprising separate components of reagents, the improvement in which at least one of said components is a monoclonal antibody which specifically binds to luteinising hormone and cross-reacts with the glycoprotein hormones: follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and human chorionic gonadotropin (hCG), to an extent less than 3% and an antibody which specifically binds to the Fc? part of said mono-clonal antibody against luteinising hormone.

54. In a kit for determining presence of luteinising hormone comprising separate components of reagents, the improvement in which at least one of said components is an unlabelled monoclonal antibody which specifically binds to luteinising hormone and cross-reacts with the glycoprotein hormones:
follicle-stimulating hormone (FSH), thyroid-stimulat-ing hormone (TSH) and human chorionic gonadotropin (hCG), to an extent less than 3% and another is labelled antibody against luteinising hormone.