CA2074590A1

CA2074590A1 - Process for the production of high affinity anti-hapten antibodies and composition for the determination of haptens

Info

Publication number: CA2074590A1
Application number: CA002074590A
Authority: CA
Inventors: Roswith Stenzel; Bernd Reckmann; Uwe Wuerzburg; Arnulf Heubner; Ulrich Schoenefeld; Winfried Linxweilen
Original assignee: Individual
Current assignee: Merck Patent GmbH
Priority date: 1991-07-26
Filing date: 1992-07-24
Publication date: 1993-01-27
Also published as: IL102635A0; CZ229092A3; JPH05255400A; ZA925598B; EP0524584A2; DE4124853A1; EP0524584A3

Abstract

ABSTRACT

The invention relates to a process for the production of high-affinity anti-hapten antibodies and to a composition for the determination of haptens with the aid of these antibodies.
The process for the production of these high-affinity anti-hapten antibodies is characterised in that in a first step an animal is immunised several times with a hapten-protein conjugate and in the second step the animal is immunised several times with a mixture of up to three hapten-protein conjugates, the conjugates con-taining the same hapten, but different proteins.

Description

2 ~

Merck Patent Gesellschaft mit beschr~nkter Haftung 6100 Darmstadt Process for the production of high-affinity anti-hapten antibodies and composition for the d termination of haptens The invention relates to a process for the production of high~affinity anti-hapten antibodies and a composition for the determination of haptens wi~h the aid of ~hese antibodies.
Haptens are low molecular weight substance~
without Lmmunogenic action, whose determination in clinical diagnosis has gained increasing importance. The hapten only become immunogenic as a result of binding to larger molecules and can then be determined with the aid of Lmmunological methods.
A known hapten is, for example, the very active cardiac glyco ide dlgoxin, which is used for the treat-ment of congestive heart failure and in certain cardiac arrhythmias. Since it has a low therapeutic breadth and the serum level cannot be foreseen, it is Lmportant to determine the concentration in the serum. This determina-tion i~ carried out with the aid of immunoassays. Since the digoxin serum concentration in comparison with other medicaments, for example anti-epileptics, is very low, the immunoas~ays must be very sensitive. The high sen-sitivity can be achieved, inter alia, by using high-~ffinity antibodies in immunoassays.
The production of high-affinity antibodies against haptenis is only possible with great difficulty using the immunisation methods known at present. In these, the hapten is covalently coupled ~o a protein such as bovine serum albumin (BSA~ or keyhole limpet haemocyanin (KLH). The resulting substance is designated an immunogen. Using this immunogen, an emulsion with ' ' : . .
: . j ~ ~ 71~ ) complete Freund's adjuvant is prepared which i5 in~ected at two to three week intervals into specific anLmals (sheep~ goats, mice, rab~its). Ater 3 to 6 immunisations antibodies are formed. Using this method, medium-affinity antibodies having affinity constants Of RA = 107 - 108 can be prepared; the titre is between 1/40 and 1/640. High-affinity antibodies (RA = 109 - 1012) are not formed by thiR method.
The present invention i9 based on the objec~ of making available a process for the production o high affinity anti-hapten antibodies haYing high titres, which make possible sensitive hapten determinations.
The invention relates to a process for the production of high-affinity anti-hapten antibodies, which 5 i8 characterised in that in a first step an animal i5 immunised several times with a hapten-protein conjugate and in the ~econd step the animal is immunised several tLmes with a mixture of up to three hapten-protein coniugates, the conjugates containing the same hapten, but different proteins.
The invention further relates to compositions for the determination of haptens which contain these high-affinity anti-hapten antibodies.
The hapten-protein conjug2te ~immunogen) is prepared by known methods (Clin. Inv. 47, 1035-1042 (19~8); Ann.Clin.Biochem 20, 227-232 (1983~).
5urprisingly, using the two step immunisa~ion method according to the in~ention, high-affinity anti hapten antibodies having high titres were obt~ined. These high-affinity antibodies are also obtained if in the second immunisation ~tep two instead of three con~ugates differing from one another in the protein are used or if the immunisation in the second step is carried out with a conjugate which differs by the protein from the con-jugate used for the immunisation in the first step.
Haptens in the hapten-protein conjugate which can be used in the second immunis~tion step are also hap~en derivati~es with the same immunogenic determinants or ~ 7~

alternatively conjugates which differ in the hapten derivative and in the protein.
For example, digoxin or succinyldigoxigenin were coupled to BSA, RLH and horse IgG. A mixture was prepared from these three immunogens. The animals wera Lmmunised in two steps. In the first step, they were i~munised several times with digoxin-BSA or digoxin-KLH or digoxin-IgG, in the second skep they were immunised several times with the Lmmunogen mixture. After the 3rd to 6th immunisation with the mixture, a 30 to 200% increase in the rise and a strong increase in the titre to 1/5120 ts 1/10240 occurred. Without change of the immunogen, the titre and affinlty of the antibody would ha~e constantly remained low.
The same immunisation process was also used with analogous succes~ and good reproducibility for other haptens such as phenytoin, phenobarbital, carbamazepine, primidone, valproic acid, ethosuximide, theophylline, gentamicin, tobramycin, amikacin, netilmicin, vancomycin, kanamycin, streptomycin, dibelcacin, methotrexate, cyclosporin, digitoxin, disopyramicle, flecainide, procai-namide, N-acetylprocainamide, lidocaine, quinidine, corti~ol, testosterone, progesterone, oestriol, tetra iodothyronine~ triiodothyxDnine9 amphetamine, metamphet-amine~ opiates, morphine, benzodiazepine, cocaine~
barbiturate, phencyclidine, methadone and cannabis. On the basis of the method according to the invention, it was possible to develop very sensitive Lmmunoassays for the determination of haptens.
The determination of the antibody titres was carri~d out with the aid of fluorescence polarisation immunoassay (FPIA). In this, the hapten is labelled with a fluorescent dye. When thi~ reagent (tracer) is activated with polaxised light, the emitted light is polarised, in the case of large molecules which move slowly. In the FPIA, the light emitted by the tracer, however, i5 not polarised, as the tracer has a small molecular weight. The bound tracer irradiates polarised .

.

light, sinee a large eomplex is formed by the binding to ~he antibody. This Lmmunoassay can therefore be carried out homogeneously, i.e. the fxee tracer does not have to be separated from the bound tracer. To detsrmine the hapten concentrationt the test is carried out competi-tively, i.e. hapten and tracer compete for binding to the anti~ody. The higher the concentration o the hapten, the less tracex i~ bound. The measured signal is thus inversely proportional to the hapten concentration, Example 1 1. Immunogen preparation 1.1 Coupling of succinyldigoxigenin to BSA
50 mg of BSA are di~solved in 25 ml of water (pH 5.5). 30 mg of EDC (1-ethyl-3-(3-dLmethylamino-propyl)carbodiLmide hydrochloride) are added to this.20 mg of succinyldigoxigenin are dissolved in 3.5 ml of dimethylformamide and added dropwise with stirring to the above solution. The pH is adjusted to 5.5. After 10 minutes, a further 10 mg of EDC are added, The reaction solution is stirred at 25C for 18 hours and then dialysed against running water for 5 days.
1.2 Coupling of succinyldigoxigenin to RIH
The reaction is carried out analogously to Example 1.1, 20 mg of KLH being employed instead of 50 mg of BSA.
1.3 Coupling of succinyldiqoxigenin to IgG
The reaction i~ carried out analogously to Example 1.1, 25 mg of IgG being employed instead of 50 mg of B5A.
1.4 Coupling of digoxin to BSA
210 mg (U.27 mmol) of digoxin are dissolved in 10 ml of ethanol. 10 ml of a 0.1 M sodium metaperiodate solution are added dropwise to this solution. After 25 mi~utes, 0.3 ml of a 1 M ethylene glycol solution is added.
In another ve~sel 280 mg of ~SA are dissolved i~
8 ml of watex which is adjusted to a pH of 9.5 with 5~
pota68ium carbonate ~olution. The activated digoxin is :;

- 5 - 2 ~ 7 ~
added dropwise to this B5A solu~ion. After 45 minute~, the pH is adjusted to 9.3 and the solution is treated with a sodium borohydride solution (0.15 g/10 ml lf water). The solution is stirred at 25C for 3 hours. The pH i~ ad~usted to 6.5 using 1 M formic acid; the solution is incubated for a further hour. The pH is then adjusted to 8.5 using a 1 M ammonia solution and the solution is dialysed against runniny water for 5 days.
1.5 Coupling of digoxin to RLH
The coupling is carried out analogously to Example 1.4, 100 mg of RLH being employed instead of 280 mg of BSA.
1.6 Coupling of digoxin to IgG
The coupling is carried out analgously to Example 1.4, 150 mg of IgG being employed instead of 280 mg of of BSA.
1.7 Coupling of digoxigenin bi~digito~oside to BSA
The reaction is carried out analogously to Example 1.4J lR5 mg (0.27 mmol) of digoxigenin bisdigi-toxoside being employed instead of 210 mg (0.27 mmol) of digoxin.
1.8 Coupling of lanatoside to ~H
The reaction is carried out analogously to Example 1.5, 266 mg of lanatoside being employed instead of 210 mg (0.27 mmol) of digoxin.
1.9 Coupling of 4-aminophenylprimidone to BS~
20 mg of solid sodium nitrite are added to a solution of 4-aminophenylprimidone in 2 ml of Ool M
hydrochloric acid and the mixture is stirred at 0C for 10 minutes. ~he excess nitrous acid iæ destroyed by addition of 20 mg of urea. The pH of the solution is ad~us~ed to 8 - 9 u~ing a 1 M sodium bicarbonate solu-tion. ~he diazotised aminoprimidone is ~hen added drop-wise and with stirring to a ~olution of 2 ml of BSA
(30 mg/ml). The solution is stirred at O~C for 30 minute~
and then dialy~ed against running water for 2 days.
1.10 CouplLng of triiodothyronine (~3) to BSA

~ ....... : . ~, ... .

- 6 - ~ 7~ qg 50 mg of BSA are dissolved in 25 ml of water (pH 5.5) and 3U mg of EDC are added. 20 mg of triiodothyrnine (free acid) are dicsolved in 2 ml of dLmethylformamide and added dropwise wi~h stirring to the above solution. ~he pH is adjusted to 5.5. ~fter 10 minutes, a further 10 mg of EDC are added. The reaction solution is stirred at 25C for 18 hours and then dialysed against running water for 5 days.
1.11 Coupling of amikacin to thyroglobulin 100 mg of thyroglobulin are dissolved in 5 ml of water and mixed with 5 ml of amikacin solution (8 mg/ml).
334 mg of EDC are dissolved in 10 ml of water and added dropwise with vigorous stirring ~n ~he course of 30 minutes. The solution is incuba~ed at room temperature for one hour and at 4C Por 24 hours~ The immunogen is purified by column chromotography using a SephadexR G25 column.
1.12 Coupling of cortisol-3-CMO to BSA
130 mg of cortisol-3-CMO are dissolved in 23 ml of dioxane. 2.5 ml of triethylamine and 2.5 ml of butyl chloroformiateare added to the solukion and it i8 stirred at 20C for 30 minutes. 100 mg of BSA are dis~ol~ed in 10 ml of water and the p~ is adjus,ted to 9 using 0.1 M
NaOH.
~he cortisol solution is added to the BSA solu-tion and after 5 minutes the p~ is ad~u~ed to 8.5. ~ha mixture is stirrQd at 4C for 4 hours and then dialysed again~t water for 3 days.
1.13 Preparation of the Lmmuno~en mixtures 1.130 1 5 mg of succinyldigoxigenin-BSA, 5 mg of succinyldigoxigenin-~hH and S mg of ~uccinyl-digoxigenin-IgG are mixed 1 * 1 + 1.
1.13.2 10 mg of amikacin-IgG and O mg of amikacin-RL~
are mixed 1 + 5.
1.13.3 1 mg of succinyldigoxigenin-BSA, 3 mg of lanatoside-RLH and 7 mg of digoxin-IgG are mixed 1 + 3 + 70 .. . . .

7 -- h ~ . 7 ,~
1.13.4 10 mg of triiodothyYDnine BSA, 5 mg of triiodo-thyronine-KLH and 20 mg of triiodothyrmine-IgG
are mixed 1 + 0.5 + 2O
Example 2 S Detenmination of the antibody ti~re To determine the antibody titre an antibody titration is carried out according to the following pipetting scheme:
50 ~1 of antibody dilution 900 ~1 of buffer Blank measurement 50 ~1 of tracer 5 minutes incubation at 37C
8 different antibody dilutions are employed.
The same experiment is performed by simulta-neously carrying out a displacement reaction with the highest concentration of hapten which is used in the standard curve.
50 ~1 of antibody dilution 50 ~1 of standard 850 ~1 of buffer Blank measuremenk 50 ~1 of tracer 5 minute~ incubation at 37C
Figure 1 shows the result of an antibody titra-tion for digoxin. ~he titre is the antibody dilution at which the difference in the measured signal between the measurement without standaId and the measurement with standard [rise) is large~t. The titre in Figure 1 is 1/1280.
Example 3 Mea~urement of a Rtandard curve To plot a tandard curve, a specific ~mount of antibody and 6 different s~andard concentrations are employed. The pipetting scheme corre~ponds to the scheme with standard described in Example 2. Figure 2 shows a standard curve for digoxin.

, 2 ~ ,r~
~xample 4 One-s~ep Lmmunisation 0.8 mg of suc,cinyldigoxigenin-BSA i5 emulsified with an adjuvant (complete or incomplete Freund's ad-juvant, aluminium hydroxide, water-oil emulsion or muramyl dipep~
tide). Using this emulsion, the animal is immunised twice at intervals of 21 days. Thereafter, it is im-munised 4 - 12 times with an emulsion of 0.8 mg of succinyldigoxigenin-B5A with incomplete Freund's adjuvant at intervals of 21 days.
Table 1 shows the titre course in a sh~ep which has been immunised by this method.

Table 1 15 Boost Rise Titre 4 ~9 80 42 ~0 6 29 16~
7 ~0 320 1~ 39 160 After the 5th boost, the rise is con~tantly low between 30 - 40 mP and the titre is constantly low at 1/160.
Example 5 Immunisation in two steps 5.1 First step: Immunisation with succinyldigoxigenin-BSA, Second step: Immunisa~ion with a mixture of succinyl-digoxigenin-BSA, succinyldigoxigenin-KLH

~ ' ' `' , , and succinyldigoxi~enin-Ig& in the sheep.
O.8 mg of an Lmmunogen mixture prepaxed aocording to 1.13.1 is emulsified with complete Freund's ad~uvant.
~s described in Example 4, after the animal has b~en immunised ei~ht times it is then immunised twice at intervals of 21 days with 0.8 mg of the mixture in complete Freund's adjuvant. It is then Lmmunised 3 times with 0.8 mg o the mixture in incomplete Freund's ad-juvant each time at intervals of 21 days.
The titre is de~ermined as in ~xample 2. Table 2 shows the rPsult of such an immunisa~ion. After the xise after the 8th immunisation with succinyldigoxigenin-BSA
remains almost cons~ant, it increases by 2-fold after the 2nd to 5th immunisation with the mixture. The titre remains constant up to the lO~h immunisation and in-creases from 1/640 to 1/5120 from the 11th to 13th Lmmunisation.

Ta~le 2 _ 20 Boost Ri~e Titre ~ 35 640 6 36 640 1st step B 3g 640 11 70 2560 2nd step 13 ~3 5120 35 5.2 First step: immunisation with succinyldigoxi-genin-RLH, -- 1 0 ~ L~
Second step: immunisation with digoxin-BSA in the mouse Succinyldigoxig2nin-RLH is emulsified with complete Freund's adjuvant. The anLmal is Lmmunised f iYe times with this emulsion, which in each ca~e contains 0.2 mg of the Lmmunogen. After that, it is Lmmunised five times with 0.2 mg of digoxin-BSA each tLme. The rise and titre, which are constant up to the 6th boost, slowly increase after the immunisation with digoxin-BSA until the rise on the 9th boost has incre~ ed by 2.5-fold and the ti re has quadrupled, as Table 3 below shows.

Table 3 Boost Rise Titre 4 42 320 1st step ` 20 5 41 320 ; 7 50 320 2nd step 9 103 1~80 10~ 1280 .

5.3 First step: Immunisa~ion with amikacin-~SA, Second s~ep: Immunisa~ion with a mixture of amikacin-KLH and amikacin-IgG in the sheep The sheep was imm~nised five times with a solu-tion of incomplete Freund's adjuvant and amikacin-BSA
(0.8 mg per boost)~ It was then L~munised five times with ~he immunogen mixture (0.8 mg per boost) as in 1.13.2. A~
can be seen from Table 4, rise and titre remain constant up to the 1st boost with the Lmmunogen mixture. After 4 : 35 boosts with the immunogen mixture the rise has increased : by 4 fold and the titre by 8-fold.

, Table 4 Boost Rise Titre 3 20 ~
4 30 80 1st step 3~ 80 1 29 ~0 3 100 320 2nd step 4 12~ 640 5.4 First step: Immunisation with prLmidone-BSA, Second step: Immunisation with prLmidone IgG in the r~bbit The rabbit was immunised 5 tLmes wikh the solution o aluminium hydroxide and prLmidone-BSA. It was then immunised 5 tLmes with prLmidone-IgG. As can be seen from Table 5, rise and ~itre increase slowly after the 2nd Lmmunisation with primidone-IgG. On the 4th immunisa-tion with primidone-lgG, the ri~e has tripled and the titre ha3 increa~ed eight fold.

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Table 5 ~7~ s Boost Rise ~itre 4 50 40 1st step 53 4~
.
1 ~0 40 3 80 160 2nd step ~ 1~1 320 __ _ 5.5 First step: Immunisation with triiodothy~nine BSA, Second step: Immunisation with a mixture of triiodothyronine-BS~, triiodothyr-onine-RLH and triiodothyrmine-IgG in the goat.
The goat was f irs~ irmnunised 5 times with 0.8 mg of triiodothyronine BSA in each case cmd then 8 times with the i~unogen mixture a~ in 1.13.4. A~ a result of the immunisation with the Lmmunogen mixture, the ris~
25 increa3es by 50~ and the titre by 4-fold, as Table 6 below ~how~.

1:. . , "` '~ :

Table 6 .
Boost Rise Titre : .
3 2~ 160 4 50 640 l~t step 4 70 640 2nd step 1~80 __ _ __

Claims

1. Process for the production of high-affinity anti-hapten antibodies, characterised in that in a first step an animal is immunised several times with a hapten-protein conjugate and in the second step the animal is immunised several times with a mixture of up to three hapten-protein conjugates, the conjugates containing the same hapten, but different proteins.

2. Process according to Claim 1, characterised in that a mixture of two conjugates which differ from one another in the protein are used.

3. Process according to Claims 1 and 2, charac-terised in that the immunisation in the second step is carried out with a conjugate which differs by the protein from the conjugate used for the immunisation in the first step.

4. Process according to Claims 1 to 3, characterised in that hapten derivatives having the same immunogenic determinants are used as hapten in the hapten-protein conjugate of the second immunisation step.

5. Process according to Claims 1 to 4, characterised in that in the second immunisation step conjugates which differ in the hapten derivative and in the protein are used.

6. Composition for the determination of haptens, containing the high-affinity anti-hapten antibodies prepared by the method according to Claims 1-5.