AU640090B2

AU640090B2 - Storable protein solution

Info

Publication number: AU640090B2
Application number: AU26334/92A
Authority: AU
Inventors: Norbert Franken; Nicholas Hoyle; Gunter Pappert
Original assignee: Boehringer Mannheim GmbH
Current assignee: Roche Diagnostics GmbH
Priority date: 1991-10-28
Filing date: 1992-10-12
Publication date: 1993-08-12
Anticipated expiration: 2012-10-12
Also published as: ES2082326T3; KR930007973A; JPH06141860A; AU2633492A; ATE133262T1; DK0539923T3; EP0539923A1; CA2080971C; DE59205094D1; JP2615325B2; DE4135542A1; EP0539923B1; CA2080971A1

Abstract

A storage-stable POD conjugate solution is characterised by a pH of 5.0 to 6.5 which is adjusted by an organic buffer substance in a concentration of 40 to 100 mmol/l and/or 0.05 to 1.0 mol/l magnesium aspartate.

Description

640090

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION NAME OF APPLICANT(S): Boehrlnger Mannhelm GmbH ADDRESS FOR SERVICE: DAVIES COLLISON CAVE Patent Attorneys 1 Little Collins Street, Melbourne, 3000.

INVENTION TITLE: Storable protein solution The following statement is a full description of this invention, including the best method of performing it known to me/us:to t 6 s~---~au~bddl~ -"II I i a 0 «f« Os o 0 O 0 0 ,0 o 0 o Q 0.

0 tl S* 0 C C I a C 0c C oo C C C0 C CC o I 0 «4 Cs^ C I C C

L

t C C C The present invention is concerned with storable conjugates of proteins and horseradish peroxidase. Peroxidase is abbreviated herein "POD" and conjugates containing same are referred to as "POD conjugates".

In DD 237327 are described proteins, heterocyclic azines and polyalcohols in a medium with a pH value of from 6.0 to 7.5 for the stabilisation of horseradish POD (PODHRp) conjugates. In EP-A 0 303 062, hydrolysed chicken albumin and/or calf serum, polyvinylpyrrolidone and/or calcium propionate, ANS (anilinonaphthylsulphonic acid) in tris buffer of pH 7.2 are described for the stabilisation of POD conjugates; EP-A 0 197 447 describes aminopyrine, for example in phosphate, citrate or borate buffer; from DE-A 31 00 076 is known a medium containing ANS and serum prctein; EP-A 0 070 992 describes a medium containing 4-aminoantipyrine, ANS and serum protein; and US-A 4,.169,012 describes the use of polyvalent ions of Groups 2 and 5 of the Periodic System. However, POD conjugates in solution can only be stabilised for a limited period of time with thesa additives. Even after a few days, a distinct decrease of the POD activity or of the stability is observed in immunological tests.

It is an object of the present invention to provide a POD conjugate solution with improved stability which can be stored at ambient temperature for a comparatively long period of time and which can be produced in a simple and reproducable way.

Thus, according to the present invention, there is provided a storable POD conjugate solution, 930517,q:\oper\ejh,26334.175,2 characterised by a pH value of 5.0 to 6.5, adjusted by means of an organic buffer substance with a concentration of 40 to 100 mmol/1, and O.O~to 1.0 mol/1 magnesium aspartate.

As POD conjugates, there are preferably used the conjugates of POD as label and of a binder and especially immunological binder substance employed for immunological determination processes. As binder conjugate partners of POD, there are especially 10 preferably used antibodies, fragments thereof, antigens,, haptens,. biotin and streptavidin.

According to the present invention, as organic buffer substance, it is especially preferred to use Good buffer, citrate buffer and/or 2-(N-morpholino)-ethanesulphonic acid MES being preferably used.

On the basis of the pH values to be adjusted to one another,, of the concentration of the buffer substances and of the presence of magnesium aspartate, outstanding stability results are achieved with the POD conjugate solutions according to the present invention.

In additi6n, the solutions according to the present invention preferably contain stabilisers, preserving agents and/or inert proteins, namely, especially 0 to 0.4 g/l of stabilisers, 0 to 1 g/1 of preserving agents and/or 0 to 20 g/1 of inert protein., Inert proteins (supporting proteins) include, for example, albumins, casein and immunoglobulins. Stabi lisers which can be used according to the present -4invention include, for example,, ANS,, phenol and dimethylaminoantipyrine, Preservihg agents which can be used according to the present invention include, for example, Kathon, oxipyrion and methyl isothiazolone.

The solutions according to the present invention possess outstanding stabilities, For different POD conjugates, for example MAB-POD or hapten-POD, after storage for 3 weeks at 35 0 C, the peroxidase activity is still 80 to 99% of the initial value.

oo 10 The following Examples are given for the purpose o 0 o] of illustrating the present invention, without limiting o it thereto. If nothing otherwise is stated, the percentages and parts are by weight.

Example 1, S 15 Carrying out of an immunological determination process, The test was' carried out according to the ELISA test principle as a one-step sandwich test by means of solid phase-bound streptavidin, a biotinylated, monoclonal antibody (MAB-Bi) and a peroxidase-labelled second monoclonal antibody (MAB-POD).

100 p- of sample and 1000 kl of working solution were introduced into a polystyrene test tibe coated with streptavidin (production according to EP-A 0 269 092).

Composition of the POD storage solution.

1 i MAB-POD (POD activity: 20 U/ml) mmol/l buffer (4-morpholinoethanesulphonic acid, phosphate buffer or citrate buffer (buffer ion and pH value according to the following Table 1) 0,1 mol/1 magnesium aspartate 0.2 g/l 4-dimethylaminoantipyrine 0,.l g/1 oxipyrion g/L bovine immunoglobulin.

Composition of the working solution, s.g/ml MAB-Bi 1/100th POD storage solution Incubation was carried out for 1 hour at ambient temperature, followed by washing twice with 0.9% aqueous sodium chloride solution, whereafter 1000 1l of substrate solution were added thereto.

S bstrate solution; 100 mmol/1 phosphate-citrate buffer (pH 1.4.7 mmol/1 sodium perborate 9.1 mmol/l 2,2'-azino-di-(3-ethylbenzthiazoline-6sulphonic acid) diammonium salt (ABTS After incubation for 30 minutes, the colour formed was determined photometrically at 420 nm.

Example 2, Determination of a peroxidase activity in MAB-POD conj ugates, 2,9 ml of substrate solution and 0.1 ml of dilute sample (MAB-POD) were mixed in a cuvette with 1 cm path length and, after mixing, the absorption U _930517,q:\oper\ejh,26334.175,5 -6change was monitored at ambient temperature for minutes at 405 nm.

An average value of the extinction change per minute was determined. The POD activity was determined according to the following equation: activity 0.8152 x A/min (U/ml of sample solution) Example 3, Stability of anti-TSH antibody-POD conjugate in various buffer solutions.

Anti-TSIL antibody-POD conjugate was stored in POD storage solution (without the addition of MAB-Bi) at different pH values in different buffer substances for 3 weeks at 40C and 350C.

The function, of the conjugate was tested 15 according to the process described in Example 1 in the presence of buffer and magnesium aspartate. The POD activity was also tested according to Example 2.

The following Table 1 shows the results obtained: 4i 4i j

F-

-7- Table 1 activity after storage for buffer ion, pH 3 weeks at 4°0 5 weeks at 55 0

C

immune POD immune POD activity activity activity activity acetate, pH 6.0 100% 100% 87% phosphate,: pH 6.0 100% 100% 78% 100% MES, pH 6.5 100% 100% 87% 100% MES, pH 6..0 100% 100% 87% 100% MES, pH 5.0 100% 100% 82% 100% ES, pH 4.0 100% 100% 45% n.d.

hosphate,, pH 8.0 100% 100% 62% 100% e I4 0 *o 4 o a 0 o 00 a B <*o 0 01 000 0 0d ft 6t O 0968* n.d. not determined Table 1 shows that admittedly the POD activity is 15 still present after storage for 3 weeks at 350C but small losses in the activity are only seen when the pH value of the buffer is from to Example 4.

Influence of magnesium aspartate.

As described in Example 3, anti-TSH antibody-POD conjugate (activity 20 U/ml in 40 mmol/ml 4-MES buffer at pH 6.0) was stored in the presence of different magnesium aspartate concentrations.

The stability of the solutions was tested after 3 weeks storage in a test according to Example 1. The -8following Table 2 shows the results obtained: TabJI 2 magnesium aspFrtate activity after storage for concentration 3 weeks at 40°C' 3 weeks ait 0.00 mol/1 100% 64% 0.05 mol/1 100% 84% 0.10 mol/1 100% 77% 0.25 mol/1 100% 81% 6 na a a o o a o 0 o ««a o *~m Table 2 shows that magnesium aspartate has a 10 positive influence on the performance of the anti-TSH antibody conjugates.

Example Influence of buffer solutions with a pH value of from to 6.0 on the stability of different POD conjugates.

15 The POD-hapten and antibody conjugates mentioned in Table 3, with an average enzymatic activity of 0.4 to 20 U/ml, were stored for 3 weeks at 40°C and in a solution of 40 mmol/1 4-MES (pH 5.0 to 0.1 g/1 oxipyrion and 10 g/1 bovine immunoglobulin.

Subsequently, an appropriate function test for the antigen was carried out analogously to Example 1.

The following Table 3 shows the results obtained: Z; or or* 9 -9- Table POD conjugate with activity after storage for 0.1 inol/ aspartate 3 weeks 4 0 C 5 weeks 350Q anti-TSH MAB-POD 100% anti-TSH-PAB-POD 1,00% 89% anti-LH MAB-POD 100% 86% anti-FSH NAB-POD 100% 84% anti-HCG NAB-POD 100% 79% anti-CEA MAB-POD' 100% 77% 0 3 0 3

O

3

O

3 O ~I 0 It 1)010 3 3 Oi~O~il 3 OaDd 3 O ill i) 0 0 D Pr O i) 3

U

~881 r) r i IfII

Claims

1. Storable POD conjugate solution, characterised by a pH value of 5.0 to adjusted by means of an organic buffer substance with a concentration of 40 to 100 mmnnol/1, and 0.05 to 1.0 mol/l magnesium aspartate.

2. Solution according to claim 1, wherein it contains MES as buffer.

3. Solution according to any of the preceding claims, wherein it additionally contains at least one stabiliser, preserving agent and/or inert protein.

4. Solution according to claim 3, wherein it contains 0 to 0.4 g/1 of stabiliser.

Solution according to claim 3 or 4, wherein it contains 0 to 1 g/l of preserving 4 agent. 4

6. Solution according to any of claims 3 to 5, wherein it contains 0 to 20 g/1 of inert protein. 4a 4

7. Storable POD conjugate solution according to claim 1, substantially as hereinbefore described and exemplified. 4 DATED this 20th day of May, 1993 BOEHRINGER MANNHEIM GmbH By its Patent Attorneys DAVIES COLLISON CAVE 1 l 930520,q:\oper\cjh,26334.175,10 Abstract Storable protein solution The prresent inventioi, provides a storable PO0D conjugate solution, characterised by a pH value of 5.0 to 6.5, adjusted by means of an organic buffer substance with a concentration of 40 to 100 mmol/l, and 0.05-- to 1.0 mol/l magnesium aspartate.