Process for preparing a carrier useful in immunoassays by deposition of a complex of a specifically binding substance with hydrophobic protein, and the resulting carrier

The present invention provides a process for the preparation of a specifically bindable protein substance bound to an insoluble carrier material, especially for use in a heterogeneous analysis process, wherein a soluable protein with a molecular weight above about 500,000, which is more hydrophobic than the specifically bindable substance, is coupled to the specifically bindable substance and then the conjugate of reaction component and protein is adsorbed on a hydrophobic solid phase. The present invention also provides a carrier material for use in solid phase immunoassays including a hydrophobic solid phase which is adsorbed on a conjugate of a hydrophobic protein with a molecular weight above about 500,000 and of a specifically bindable protein or protein containing substance.

The present invention is concerned with preparing a carrier useful in an 
immunological assay. A complex is formed of a hydrophobic protein and a 
member of a specifically binding pair and this complex is bound to the 
carrier. The invention is also concerned with the carrier produced 
thereby. 
For the determination of a specifically bindable substance, there are 
frequently used processes according to the immunoassay principle. One of 
the components of a substance pair specifically bindable with another is 
thereby reacted with the receptor specific for it which is labelled in 
known manner. The conjugate of these two substances can then be reacted 
with a receptor which is specific for the conjugate or for one of the two 
parts of the conjugate. There are many variations for these immunological 
processes. It is thereby advantageous when one of the receptors is present 
bound to a solid phase. This makes easier the separation of reaction 
components present bound and non-bound. For the determination of the 
specifically bindable substance, one measures the amount of labelled 
reaction component bound to the solid phase or of labelled reaction 
component present in the solution and relates it in a known manner to the 
amount of reaction component to be determined. 
In standard immunological processes, when a solid phase is used it is 
generally a test tube or a microtiter plate made of a synthetic material. 
The inner surface of these materials have reaction components fixed 
thereto. If spheroids are used, then the outer surfaces of these have the 
reaction components bound thereto. These synthetic resin test tubes, 
microtitre plates or spheroids usually consist of a relatively inert 
synthetic resin material so that the binding of the reaction component 
gives rise to difficulties. Furthermore, the binding of the specific 
reaction component to the surface in question must take place in such a 
manner that it does not lose the ability of specific binding to the 
substance specifically bindable with it. For this reason, the binding of 
the reactive component to the solid phase mostly takes place adsorptively. 
Therefore, it has already been suggested to bring about the fixing of the 
reaction component to the solid phase via a coupling agent which brings 
about the binding. Care must thereby again be taken that the binding of 
the reaction component to the binding agent does not destroy the 
specifically reacting region of the molecule and that the reaction 
component is so bound that its reacting region is facing away from the 
solid phase and toward the complementary member of the specifically 
binding pair. 
Furthermore, in Federal Republic of Germany Patent Specification No. 25 33 
701, it is suggested, in order to achieve a better binding, to cross-link 
the individual immunologically effective proteins and then to absorb them 
on polystyrene spheroids. A further possibility given in this literature 
reference is simultaneously to cross-link an inert protein with the 
protein with immunological properties so that a cross-linked product 
results of inert and active protein which is then again adsorbed on 
polystyrene spheroids. However, depending upon chosen reaction conditions, 
this type of crosslinking leads to differing, non-reproducible 
crosslinkages with variable proportions of non-cross-linked protein, as 
well as of protein which has become insoluble. Furthermore, due to the 
differing degree of crosslinking, products result with differing binding 
properties. 
A similar process is described in European Patent Specification No. 
0,122,209 and it also displays the same disadvantages. Thus, all these 
known processes are still not satisfactory, still do not give an optimal 
adhesion of the specifically bindable substance and are of little 
suitability for the reproducible preparation of coated solid phases. 
Therefore, it is an object of the present invention to provide a process 
which reproducibly improves the adhesion of the specifically bindable 
substance to the solid phase and provides a carrier material suitable 
therefor. Since many immunological processes are carried out with the 
addition of detergents in order to avoid turbidities, it is also an object 
of the present invention to improve the adhesion to such an extent that, 
even in the presence of detergents, the bound, specifically bindable 
substance is not dissolved off. 
Thus, according to the present invention, there is provided a process for 
the preparation of a specifically bindable protein or protein containing 
substance bound to an insoluble carrier material, especially for use in a 
heterogeneous analysis process, according to the immunoassay principle, 
wherein a soluble protein with a molecular weight above about 500,000, 
which is more hydrophobic than the specifically bindable substance, is 
coupled to the specifically bindable substance and then the conjugate of 
reaction component and protein is adsorbed on a hydrophobic solid phase. 
The specifically bindable substance fixed in this way to a solid phase 
displays an improved adhesion. The binding is also stable with regard to 
detergents. In the case of the production of calibration curves which are 
necessary for the evaluation in the case of many immunological processes, 
the solid phase-bound, specifically bindable substances according to the 
present invention give steeper calibration curves, which results in an 
increase of the exactitude. 
A further advantage of the process according to the present invention is 
that it is possible to control the bound amount more exactly. Since the 
adhesion is significantly better than in the case of the previously known 
processes, the amount of specific protein which must be used is also 
smaller. 
For the choice of soluble proteins which are suitable according to the 
present invention, there must be determined the molecular weight, as well 
as the hydrophobicity, in comparison with the corresponding value for the 
specifically bindable substance. The molecular weight is determined 
according to known methods. 
A comparison of the hydrophobicity between soluble protein and specifically 
bindable substance can also take place by conventional methods. Suitable 
methods are, for example, a comparison of the fluorescent extinction after 
binding to coloured materials (Biochem. Biophys. Acta, 624, 13-20/1980); 
the elution behaviour in the case of hydrophobic chromatography (Biochem. 
Biophys. Acta, 576, 269-279/1979); the surface tension (Biochem. Biophys. 
Acta, 670, 64-73/1981); and the retention times in the case of hydrophobic 
interaction chromatography (HIC) (Angew. Chemie, 98, 530-548/1986; J. 
Chromat., 296, 107-114/1984; and Anal. Biochem., 137, 464-472/1984). 
A comparison of the hydrophobicity of substances suitable according to the 
present invention is to be found in Sep. Sci. Technol., 14, 305-317/1979. 
According to that, the hydrophobicity increases, for example, in the 
following series: .alpha..sub.2 -macroglobulin (M.W. 820,000), bovine 
serum albumin/human serum albumin (M.W. about 70,000), egg albumin, 
.alpha..sub.2 HS-glycoprotein (M.W. about 49,000), .beta..sub.1c 
/.beta..sub.1A -globulin, immunoglobulin (M.W. about 150,000) and 
transferrin (M.W. about 90,000). 
Thus, if an immunoglobulin is used as specifically bindable substance, 
then, for example, human serum albumin or .alpha..sub.2 HS-glycoprotein 
are not suitable as soluble proteins in the meaning of the present 
invention without further pre-treatment. 
When human serum albumin or .alpha..sub.2 HS-glycoprotein is used, they 
must be rendered hydrophobic and must be treated to produce a polymeric 
molecule with a higher molecular weight. When transferrin is used, 
cross-linking suffices to render this substance useful in the invention. 
When .alpha..sub.2 -macroglobulin is used, the molecule only needs to be 
rendered hydrophobic. 
Proteins which are suitable for the coupling with immunoglobulin as 
specifically bindable substance without pre-treatment include, for 
example, .beta.-lipoproteins (M.W. about 3.2 million) and .alpha..sub.2 
-lipoproteins (M.W. about 5-20 million). 
Hydrophobing can take place, for example, by the use of heat, treatment 
with acids, denaturing agents and/or chaotropic ions and/or by chemical 
coupling with a hydrophobic compound. 
Increasing of the molecular weight can take place, for example, by the use 
of heat, treatment with acids, denaturing agents and/or chaotropic ions 
and/or by cross-linking with a bi- or polyfunctional protein reagent. 
The treatment is carried out until a protein polymer is obtained with a 
molecular weight of 500,000 or more. It is especially preferred to use a 
protein polymer with a molecular weight of from 500,000 to 20 million. 
When cross-linking is also to take place, the hydrophobing can be carried 
out before, during or after the cross-linking but not in the presence of 
the specifically bindable substance. 
For hydrophobing by heating, one usually uses temperatures of from 40 to 
95.degree. C. over a period of time of 1 minute to 10 hours, for example 
as described in Biochem. Biophys. Acta, 624, 13-20/1980. 
As acids, there are used, for example, acetic acid, propionic acid, lactic 
acid or hydrochloric acid. The usual concentrations are 1 to 100 
mMole/liter with a period of action of from 10 minutes to 16 hours. 
Suitable chaotropic ions include, for example, thiocyanates, iodides, 
fluorides, bromides, perchlorates and sulphates. Suitable denaturing 
agents include, for example, guanidine hydrochloride and urea. 
Concentrations of 10 mMole/liter to 6 mole/liter are usually here used. 
For the derivatisation of hydrophobic compounds, there are preferably used 
soluble fatty acids, lipoids in low and high molecular weight form, as 
well as synthetic polymers, such as polypropylene glycol, or soluble 
copolymers of polystyrene. The derivatisation takes place according to 
well known methods. 
Cross-linking by way of bi- and polyfunctional compounds is carried out 
with known protein binding reagents. These are compounds which contain at 
least two functional groups, which can be the same or different and can 
react via these functional groups with functional groups of proteins. 
Compounds are preferably used which consist of an alkyl chain on the ends 
of which are present, for example, succinimide, maleinimide and/or 
aldehyde groups. 
The protein is cross-linked in the usual manner with the bi- or 
polyfunctional compounds by reacting together the soluble protein and the 
bi- or polyfunctional compound. 
For hydrophobing and/or cross-linking, there are preferably used precursor 
proteins with a molecular weight of from 10,000 to 700,000, bovine serum 
albumin, lipase and immune .gamma.-globulin being especially preferred. 
The specifically bindable protein substance to be bound is then coupled to 
the protein in known manner. Suitable coupling methods are described, for 
example, by Ishikawa in J. Immunoassay, 4, 209-327/1983. Proteins such as 
antibodies, antibody fragments, antigens and haptens can be used as 
specifically bindable substances. 
The conjugate obtained of specifically bindable protein substance and 
protein is then adsorbed adsorptively on the synthetic resin surface 
serving as solid phase. The adsorptive binding to the solid phase takes 
place via strong and weak exchange actions, hydrophobic forces, 
dipole-dipole and ion-dipole interactions. As hydrophobic solid phases, 
there can be used carrier materials with a surface tension which is 
smaller than the surface tension of the hydrophobic soluble protein, i.e. 
are more hydrophobic than protein. Carrier materials with a surface 
tension of &lt;40 erg/cm.sup.2 are preferably used. Polystyrene, 
polymethacrylate, polytetrafluoroethylene (Teflon), polyamide, copolymers 
of styrene and acrylonitrile, glass and cellulose products are especially 
preferred. They can be present in any desired form, for instance, in the 
form of a film, plate, powder, granules or fibre fleece, preferably in the 
form of a glass fibre fleece or a fleece from cellulose-/cellulose ester 
fibres and polymer fibres. 
Hydrophobed proteins display an especially good adsorptive binding. Due to 
the hydrophobing, intramolecular bridge bonds of the protein are possibly 
opened so that hydrophobic parts of the protein reach the surface and 
there better adhere to the hydrophobic synthetic resin surface than the 
hydrophilic parts which are particularly to be found on the surface in the 
non-hydrophobed protein. 
The process according to the present invention can be used for the 
determination of a specifically bindable substance. As substance pairs, 
one reaction component of which is present bound to the solid phase, there 
can be used, for example, antigen-antibody, hapten-antibody and other 
proteins capable of specific binding to one another, such as, in 
particular, the system streptavidin or avidin and biotin, which is 
preferred. 
Before the conjugate of protein and specifically bindable substance is 
adsorbed on to the hydrophobic solid phase, it is also possible to 
pre-treat the solid phase physically or chemically. Thus, for example, a 
synthetic resin surface can be pre-swollen or activated in some other 
known way. 
The carrier material according to the present invention for use in solid 
phase immunoassay is characterised in that it consists of a hydrophobic 
solid phase on which is adsorbed a protein with a molecular weight of 
above about 500,000 to which is bound a specifically bindable substance. 
This carrier material is outstandingly suitable for use in solid phase 
immunoassays since the specifically bindable substance adheres very well 
and is also not desorbed in the case of the addition of detergents. 
The carrier material is used, for example, in the form of test tubes, 
microtitre plates or spheroids which are coated with a cross-linked 
protein to which is bound a specifically bindable substance. 
On the solid base is adsorbed a conjugate consisting of a cross-linked 
protein and a specifically bindable substance. The protein is preferably 
bovine serum albumin, lipase or an immune .gamma.-globulin which has been 
cross-linked in the described manner. 
According to the present invention, there is provided a process and a 
carrier material in order to fix a specifically bindable substance with 
good adhesion and longlastingly to a hydrophobic solid phase. The adhesion 
could thus be improved to such an extent that even an addition of 
detergents does not lead to a dissolving off of the substance. The process 
according to the present invention is also simple to carry out.

EXAMPLE 1 
Binding of Fab Fragments of a Monoclonal Antibody Against TSH to 
Polystyrene Test Tubes 
a) Preparation of Fab Fragments (Fab &lt;TSH&gt;) 
Monoclonal antibodies (MAB &lt;TSH&gt;) are obtained by the method described by 
Galfre and Millstein (Meth. in Enzymology, 73/1981). For further 
purification, the ascites liquid is subjected to an ammonium sulphate 
precipitation and to a passage over DEAE-cellulose. 
A papain fission is subsequently carried out by the method described in 
Biochem. J., 73, 119-126/1959. The Fab fragments hereby formed are 
separated from the non-digested IgG molecules and the Fc fragments by 
means of gel filtration over Sephadex G100 and ion exchanger 
chromatography over DEAE-cellulose according to Meth. in Enzymology, 73, 
418-459/1081. 
b) Crosslinking of the Fab Fragments Without Protein Addition (Comparison) 
50 mg. of Fab fragments are dissolved in 2 ml. 0.05 mole/liter potassium 
phosphate buffer (pH 7.5) and 0.4 ml. disuccinimidyl suberate 
(manufacturer Pierce) dissolved in dioxan (7.4 mg./ml.) is added thereto, 
with stirring. After incubating for 2 hours at 25.degree. C., the reaction 
is broken off by the addition of 0.2 ml. of 0.1 mole/liter lysine 
hydrochloride. The reaction batch is diluted with 0.2 ml. potassium 
phosphate buffer (v. supra) and centrifuged. The supernatant is 
desalinated over an Ultrogel AcA 202 column (LKB, Grafelfing, Federal 
Republic of Germany), 11.3 ml. being obtained with 45 mg. of protein. A 
part of this preparation is fractionated on a Superose-6-column (Deutsche 
Pharmacia GmbH) at a flowthrough rate of 0.5 ml./minute in 0.05 mole/liter 
potassium phosphate buffer (pH 7.0) and the fractions with a molecular 
weight of about 500,000 to 5 million are further used. 
c) Crosslinking of the Fab Fragments with Untreated .gamma.-Globulin 
(Comparison) 
Fab fragments and bovine .gamma.-globulin (Serva, Heidelberg, Federal 
Republic of Germany) are mixed in a weight ratio of 1:1. The crosslinking 
is carried out as described under b). 
d) Binding of Fab Fragments to Pre-Crosslinked .gamma.-Globulin (According 
to the Present Invention) 
1.25 g. .gamma.-globulin are dissolved in 10 ml. of 0.05 mole/liter 
potassium phosphate buffer (pH 7.8) and centrifuged clear in a Sorvall 
cooled centrifuge for 10 minutes at 5000 r.p.m. 1.75 ml. Disuccinimidyl 
suberate are added to this solution which is then diluted with 2.5 ml. 
water. After stirring for 4 hours at 25.degree. C., 10 ml. of 0.1 liter 
lysine are added thereto and the pH value adjusted to 6.8 and centrifuged. 
The supernatant is separated on a preparative gel filtration column (TSK 
3000, LKB Grafelfing, Federal Republic of Germany), concentrated by 
ultrafiltration and stored at 4.degree. C. 
50 mg. of this crosslinked .gamma.-globulin are dissolved in 5 ml. of 0.05 
mole/liter potassium phosphate buffer and the pH value adjusted to 9.5 by 
the addition of solid sodium carbonate. 50 mg. N-acetylhomocysteine 
thiolacetone (Serva, Heidelberg, Federal Republic of Germany) are then 
added thereto and stirred for 5 hours at 25.degree. C., while gassing with 
nitrogen. The batch is subsequently desalinated over an Ultragel AcA 202 
column in a buffer of 0.1 mole/liter potassium phosphate (pH 7.0), 0.001 
mole/liter magnesium chloride and 0.05 mole/liter sodium chloride. 
Fab fragments prepared according to a) (10 mg. in 1 ml. of 0.01 mole/liter 
potassium phosphate buffer (pH 7.0)) are activated with 0.002 ml. 
maleinimidohexanoyl-N-hydroxysuccinimide ester (Boehringer Mannheim GmbH) 
in dimethyl sulphoxide (33 mg./ml.) for 2 hours at 25.degree. C., 
subsequently centrifuged and desalinated over an AcA 202 column. 
These fragments are subsequently combined with the .gamma.-globulin (weight 
ratio of the proteins 1:1) and incubated for 1 hour at 25.degree. C. and 
at pH 7.0. Subsequently, it is dialysed against desalinated water 
overnight at 4.degree. C. and at a protein concentration of 2.5 mg./ml. 
This conjugate can be used directly for the adsorption on to a solid phase. 
e) Preparation of Fab Fragments Coupled to Thermo-BSA (According to the 
Present Invention) 
1 g. BSA-I is dissolved in 100 ml. of 50 mMole/liter potassium phosphate 
(pH 7.0), heated to 70.degree. C. and kept at this temperature for 4 
hours, with gentle stirring. The solution is cooled, filtered and adjusted 
in an ultrafiltration cell (exclusion limit: 30,000 Dalton) to a 
concentration of 50 mg./ml. Subsequently, it is dialysed against a 30 fold 
volume of double distilled water and subsequently lyophilised. The product 
has a molecular weight of about 700,000. 
Before coupling to the Fab fragments, the thermo-BSA is activated. For this 
purpose, 68 mg. thermo-BSA are dissolved in 2 ml. 0.1 mole/liter potassium 
phosphate (pH 7.8) and slowly mixed with a solution of 3.8 mg. 
S-acetylmercaptosuccinic acid anhydride (SAMSA). After a reaction time of 
3 hours, it is dialysed against 2 litres of 50 mmMole/liter potassium 
phosphate (pH 6.5). This thermo-BSA is incubated for 1 hour at 25.degree. 
C. and pH 7.0 with the Fab fragments activated according to d) in a weight 
ratio of 1:1, and subsequently dialysed against desalinated water 
overnight at 4.degree. C. and at a protein concentration of 2.5 mg./ml. 
This product is used directly for the coating. 
f) Preparation of Fab Fragments Coupled to .beta.-galactosidase (According 
to the Present Invention) 
Fab fragments are activated as described in d) and coupled to the SH groups 
of .beta.-galactosidase according to J. Immunoassay, 4, 209-327/1983. The 
.beta.-galactosidase used has a molecular weight of 500,000 to 2 million. 
For another experiment, there is used crosslinked .beta.-galactosidase 
(M.W. about 5 million). 
g) Loading of Polystyrene Test Tubes with Fab Fragments or Conjuqates 
Thereof 
50 mg. of a lyophilisate of the Fab fragment or of the conjugate are 
dissolved in 10 ml. double distilled water. 1 ml. of this solution is 
diluted in 1000 ml. of a loading buffer of 5.25 g./liter sodium dihydrogen 
phosphate and 1 g./liter sodium azide and stirred for 30 minutes at 
ambient temperature. 
Test tubes of polystyrene or Luran (manufacturer BASF) are each filled with 
1.5 ml. of the solution and loaded overnight (about 22 hours). Thereafter, 
the test tubes are completely emptied and the function test described 
hereinafter is carried out. 
h) Function Test Via TSH Determination 
The polystyrene and Luran test tubes loaded according to g) are used in a 
TSH determination reagent analogously to TSH-Enzymun test (Boehringer 
Mannheim GmbH, order No. 736 082) and a calibration curve measured 
according to the test procedure. There are hereby obtained the measurement 
values shown in the following Table 1 and in FIG. 1 of the accompanying 
drawings. 
It can be seen (FIG. 1) that with Fab fragments which have been immobilised 
without the addition of protein, only a very flat calibration can be 
obtained (curves 1 and 2). By coupling to proteins with a molecular weight 
below 500,000 and low hydrophobicity, the calibration curve (curve 3) is 
somewhat steeper but satisfactory results still cannot be achieved. On the 
other hand, with the conjugates prepared according to the present 
invention, there can be achieved a sufficiently steep calibration curve 
(curve 4). 
TABLE 1 
__________________________________________________________________________ 
Standard values for various Fab &lt;TSH&gt; conjugates 
measurement signal of TSH 
hydrophobicity 
M.W. of the 
standard in mE/ml. 
conjugate* of the protein** 
protein 
0.1 .mu.U TSH.sup.1) 
12.5 .mu.U TSH.sup.2) 
__________________________________________________________________________ 
Fab &lt;TSH&gt; -- -- 102 107 
crosslinked 
-- -- 126 134 
Fab &lt;TSH&gt; 
without protein 
(Example 16) 
Fab &lt;TSH&gt;/.beta.-Gal 
39.6 500,000 
110 260 
(Example 1f) 
crosslinked 
40.2 5 million 
110 260 
Fab &lt;TSH&gt;/.beta.-Gal 
(Example 1f) 
Fab &lt;TSH&gt;/.gamma.- 
32.0 150,000 
117 253 
globulin 
(Example 1c) 
Fab &lt;TSH&gt;/.gamma.- 
not 5 million 
75 480 
globulin elutable 
(example 1d) 
__________________________________________________________________________ 
*total amount of protein in the case of adsorption: 1.5 ml. with 5 
.mu.g./ml. of protein per test tube 
**Tp [min], cf. Example 3 
.sup.1) lowest value of a measurement curve (should be as low as possible 
.sup.2) highest value of a measurement curve (should be as high as 
possible) 
EXAMPLE 2 
Immobilisation of Streptavidin 
a) Preparation of Crosslinked Streptavidin 
Streptavidin (manufacturer Boehringer Mannheim GmbH) is crosslinked 
analogously to Example lb. 
b) Preparation of Streptavidin Bound to BSA or to .beta.-galactosidase 
The binding to non-crosslinked BSA or to .beta.-galactosidase takes place 
analogously to Example 1f. 
c) Preparation of Streptavidin Coupled to Thermo-BSA 
Activation of Streptavidin 
60 mg. Streptavidin are dissolved in 6 ml. of 50 mMole/liter potassium 
phosphate/100 mMole/liter sodium chloride (pH 7.0) and stored at 4.degree. 
C. 6.16 mg. maleinimidohexanoyl-N-hydroxysuccinimide ester are dissolved 
in 620 .mu.l. dioxan and stirred into the streptavidin solution. After a 
reaction time of 2 hours at 4.degree. C., it is dialysed twice against 1 
liter of 50 mMole/liter potassium phosphate/100 mMole/liter sodium 
chloride (pH 5) at 4.degree. C. 
Preparation of a Conjugate of Streptavidin and Thermo-BSA 
66 mg. Streptavidin are dissolved in 10 ml. of 50 mMole/liter potassium 
phosphate (pH 5.0) and 100 mMole/liter sodium chloride and 72 mg. of 
activated thermo-BSA-SAMBA (preparation according to Example 1e) in 5 ml. 
of 50 mMole/liter potassium phosphate/100 mMole/liter sodium chloride (pH 
6.5) are added thereto. After mixing, 50 .mu.l. of 1 mole/liter 
hydroxylamine (pH 7.0) are added thereto in order to stop the reaction. 
After 3 hours, the reaction product is purified via gel chromatography 
(Superose 6, 50 mMole/liter potassium phosphate/100 mMole/liter sodium 
chloride; pH pb 7.5). There is obtained a conjugate with a molecular 
weight of from 1 to 5 million. 
d) Loading of Polystyrene and Luran Test Tubes with Streptavidin or 
Streptavidin Conjuqate 
The loading takes place in the manner describes in Example 1g). 
e) Measurement of Standard Values Via a TSH Determination 
The test tubes loaded according to d) are used in a TSH Enzymun reagent (4 
fold conjugate activity). However, in variation of the there-described 
procedure, instead of the immobilised antibody, there is used a 
biotinylated MAB &lt;TSH&gt;. The preparation of this biotinylated MAB takes 
place according to J.A.C.S., 100, 3585-3590/1978. The antibody is used in 
the test in a concentration of 400 ng. per test tube, together with the 
other reagents. 
The results obtained are shown in FIG. 2. It can be seen therefrom that, 
with increasing molecular weight and with increasing hydrophobicity, the 
gradient of the calibration curve and thus of the achievable exactitude 
increases. 
EXAMPLE 3 
A T3 test is carried out with the test tubes loaded according to the 
present invention. For this purpose, 200 .mu.l. of sample or of standard 
solution are pre-incubated together with 500 .mu.l. of a polyclonal 
antibody conjugate against T3, which is labelled with POD, in a test tube 
which had been coated with streptavidin coupled to thermo-BSA. After 5 
minutes, 400 ng. of polymerised T3 biotinylated in known manner are 
incubated for 30 minutes in 500 .mu.l. of buffer. As buffer, there is used 
a solution of sodium hydrogen phosphate with a pH of 8.65 which contains 
0.20% BSA and 0.04% 8-anilino-1-naphthalenesulphonic acid (ANA). After 
incubation, washing is carried out three times and subsequently 1 ml. ABTS 
substrate solution is added thereto. After further incubation for 30 
minutes, measurement is then carried out at 405 nm in a photometer. The 
measurement values obtained are given in the curve shown in FIG. 3. 
EXAMPLE 4 
An HBsAg test is carried out with test tubes coated according to the 
present invention. For this purpose, in a test tube coated according to 
Example 1g), there are simultaneously dissolved 400 ng. biotinylated 
monoclonal antibody against HBs-antigen and 50 mU of the same antibody 
which are labelled with POD and 1 ml. of the buffer of the same 
composition as described in Example 3 added thereto, together with 200 
.mu.l. of standard solution. As standard solution, there is added, on the 
one hand, purified HBsAg sub-type ay and, on the other hand, purified 
HBsAg sub-type ad. Incubation is then carried out for 4 hours at ambient 
temperature. After washing the test tubes three times, 1 ml. ABTS 
substrate solution is added thereto. After 20 minutes, the reaction is 
substantially ended and the extinction is measured at 405 nm in a 
photometer. The measurement values are to be seen from FIG. 4, in which 
the unbroken curve gives the values for HBsAg sub-type ad and the broken 
curve gives the values for HBsAg sub-type ay. 
EXAMPLE 5 
The dissolving off of the test tubes coated according to the present 
invention is compared with test tubes coated according to known processes. 
For this purpose, on the one hand, test tubes are loaded with 1.5 ml. of a 
streptavidin-thermo-BSA solution (4 .mu.g./ml.) in 40 mM sodium hydrogen 
phosphate buffer (pH 7.4) at 20.degree. C. for 18 to 24 hours. After 
sucking out the test tubes, there takes place an after-treatment with 1.8 
ml. of a 2% saccharose solution which contains 0.9% sodium chloride and 
0.3% BSA, the after-treatment being carried out for 30 minutes at 
20.degree. C. Subsequently, the test tubes are dried for 24 hours at 
20.degree. C. and 40% relative humidity. These test tubes are ready for 
use for carrying out tests. Furthermore, test tubes are loaded in known 
manner with streptavidin. The dissolving off behaviour in the case of the 
action of detergents is tested with these test tubes. The results obtained 
are given in the following Table 2: 
TABLE 2 
__________________________________________________________________________ 
% dissolving % dissolving 
% dissolving 
off % dissolving 
off off 
1% Pluronic 
off function test 
function test 
test tube F 68 (30 min., 
0.5% Triton 
(30 min., 
(30 min., 
loading 
material 
RT) (30 min., RT) 
20.degree. C.) 
30.degree. C.) 
__________________________________________________________________________ 
Streptavidin 
Luran 13.9 42 17.8 28 
(comparison) 
polystyrene 
11 25 9.6 11.6 
thermo-BSA- 
Luran 2.1 8 3.1 11.6 
streptavidin 
polystyrene 
1.5 2.1 1.6 1.7 
(according 
to the 
present 
invention) 
__________________________________________________________________________ 
RT = ambient temperature 
The per cent dissolving off is determined according to the methods known to 
the skilled worker, for example, by .sup.125 F-labelling of streptavidin 
and streptavidin-thermo-BSA or by an enzymatic determination. 
For the enzymatic determination of the per cent dissolving off, the coated 
test tubes are incubated with 50 mMole/liter potassium phosphate buffer, 
pH 7.0, to which a detergent according to Table II was added, under the 
conditions according to Table II. 
Subsequently, incubation is carried out at ambient for one hour, washing 
takes place with the above-mentioned buffer, and incubation is carried out 
with a conjugate of biotin POD (200 mU/ml. POD activity) for one hour at 
ambient, washing takes place, and 2 ml ABTS.RTM. solution is added thereto 
(Example 7). After substrate reaction for one hour, the extinction is 
measured at 405 nm, and from this the per cent dissolving off of 
streptavidin and streptavidin-thermo-BSA is determined via a standard 
calibration curve. 
EXAMPLE 6 
Determination of the Hydrophobicity of Proteins with Hydrophobic 
Interaction Chromatoqraphy (HIC) 
The hydrophobicity of various compounds is investigated with a liquid 
chromatograph (Hewlett Packard 1090 LUSI). The pre-column is a BioRad 
Biogel TSK-phenyl-5PW column (length 5 mm..times.internal diameter 4.6 
mm.). Column: BioRad-Biogel TSK-phenyl-5PW (length 75 mm..times. internal 
diameter 7.5 mm., 10 .mu.m. 1000 .ANG.). as detector, there is used a 
Hitachi F 1000 fluorimeter. Eluents/gradient (see Table 3). 
a. 1.5 mol/liter ammonium sulphate solution in 1/100 mole/liter 
monopotassium dihydrogen phosphate buffer (pH 6.8) 
b 1/100 mole/liter monopotassium dihydrogen phosphate buffer (ph 6.8). 
TABLE 3 
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a (%) b (%) minutes retention time 
______________________________________ 
100 0 0 
100 0 5 
0 100 30 flow - 0.5 min. 
0 100 5 
100 0 5 
100 0 5 
______________________________________ 
Working temperature: cold chamber +7.degree. C. 
Sample preparation: 
The samples are used undiluted. The sample volume is 10 .mu.l. 
The compounds to be investigated are dissolved at a concentration of 0.2 to 
1.4 mg./ml. in 10 mMole/liter potassium phosphate buffer (pH 6.8). 
The following Table 4 summarises the retention times for various proteins 
and specifically bindable substances. The longer is the retention time, 
the greater is the hydrophobicity. 
TABLE 4 
______________________________________ 
retention time t.sub.p 
protein (min.) 
______________________________________ 
Fab TSH 41.5 
BSA 23.2 
.gamma.-globulin 32.0 
.beta.-galactosidase 
39.6 
crosslinked .beta.-galactosidase 
40.2 
streptavidin 38.3 
thermo-BSA (Example 1e) 
not elutable 
.gamma.-globulin, crosslinked 
not elutable 
(Example 1a) 
______________________________________ 
The proteins which are not elutable under there conditions are especially 
suitable and are preferably used, thermo-BSA being quite especially 
preferred. 
EXAMPLE 7 
Adsorption of Thermo-BSA-Streptavidin on Glass Fibre Fleece 
A glass fibre fleece (6.times.6 mm.) is soaked in its absorption volume 
(ca. 30 .mu.l.), with a solution of 30 .mu.g/ml. thermo-BSA-streptavidin 
(prepared according to Example 2c) in 50 nMole/liter potassium phosphate 
buffer, pH 7.0, and dried at 50.degree. C. in circulating drier. 
For determining the biotin binding capacity, the strip is soaked with 30 
.mu.l. of a reagent consisting of a conjugate of peroxidase (POD) and 
biotin and having a POD activity of 50 mU/ml., biotin standard with 
concentrations of 0.5, 10, 20, 30, 40, 50, 100, 200, 1000 ng/ml. biotin, 
and incubated for 2 minutes. Thereafter, 2 % o-Tween-20 washing is carried 
out once with a surplus of 50 mMole/liter potassium phosphate buffer, pH 
7.0, and subsequently 100 mMole/liter citrate buffer, pH 4.4; 3.2 
mMole/liter perborate; 1.9 mole/liter ABTS 
(2,2'-azino-di[3-ethyl-benzthiazolinesulphonic acid(6)] -diammonium salt 
are introduced in 2 ml. ABTS solution and agitated for 15 minutes. After 
substrate reaction for 1 hour, the extinction is measured at 405 nm., and 
from this the biotin binding capacity is determined via the semimaximum 
signal drop.