WO1996033408A1

WO1996033408A1 - On-line coupling of biochemical detection to continuous-flow separation techniques

Info

Publication number: WO1996033408A1
Application number: PCT/EP1995/001523
Authority: WO
Inventors: Jan Van Der Greef; Hubertus Irth; Erik Van Der Vlics; Aaike Oosterkamp; Johan Haggblad
Original assignee: Rijksuniversiteit Leiden; Karo Bio Ab
Priority date: 1995-04-21
Filing date: 1995-04-21
Publication date: 1996-10-24
Also published as: AU2447595A

Abstract

The present invention relates to an on-line detection method comprising the on-line coupling of a fractionation and a biochemical detection technique, which method comprises the addition of a controlled amount of an affinity molecule to the effluent of the fractionation step to react with analytes in the effluent, the subsequent addition of a controlled amount of a detectable ligand capable of binding to the affinity molecule, and detection of the affinity molecule/detectable ligand complex. Further, the invention is directed to an on-line method for the screening of compounds on their binding capability to a known affinity molecule, which method comprises a fractionation step providing an effluent, the addition of a controlled amount of said affinity molecule to the effluent of the fractionation step and effecting a contact time sufficient to allow a reaction with or interaction between the compounds in the effluent, the subsequent addition of a controlled amount of a detectable ligand capable of binding to the affinity molecule, and detection of the affinity molecule/detectable ligand complex.

Description

ON-LINE COUPLING OF BIOCHEMICAL DETECTION TO CONTINUOUS-FLOW SEPARATION TECHNIQUES

The present invention relates to the on-line coupling of biochemical detection to continuous-flow separation techniques. Preferably, this on-line detection method is used as a screening method.

It is well-known to the person skilled in the art that immunoassays are highly sensitive detection techniques which combine the selectivity of biospecific interactions between antibodies and antigens with the sensitive detection of labels used a reporter molecules.

However, it is well known that immunoassays suffer from the problem of cross- reactivity in which reactions of antibodies with more than one analyte lead to erroneous results. For this reason, immunoassays are frequently combined with a fractionation step, e.g., a separation step using HPLC or another type of liquid chromatography.

In the field to which the present invention relates, there is a need for on-line coupling of a fractionation step and an immunoassay detection system. Several approaches have been proposed and described for performing continuous-flow immunoassays. Most of these immunoassays are in the form of a post-column reaction detection system based on a sequential additional type immunoassay.

Cassidy et al., Anal. Chem. 64 (1992), 1973-1977 disclose a kinetically controlled immunoassay based on the sequential addition of antibody, sample and label on a protein A column, performing immunoassays for albumin and transferrin in less than 1 minute.

Nilson et al. , J. Chromatography, 597 (1992) 383-389 describe a continuous-flow

competitive assay involving horse-radish peroxidase-labelled antibodies. The system

described was coupled to a size-exclusion column to allow the monitoring of enzyme

activity.

These prior art detection techniques, which are of a sequential nature, are not suitable

for on-line coupling to liquid chromatographic or other fractionation systems since these

techniques do not allow the continuous monitoring of the fractionation effluent.

In the article of Irth et al. in the Journal of Chromatography 633 (1992) 65072 and in

the articles of Oosterkamp et al. in the Journal of Chromatography 633 (1993) 65-72 and

in the Journal of Chromatography 653 (1994) 55-61, a method for the on-line detection

of digoxin and its metabolites is described. The on-line detection process comprises the

direct injection of a sample containing digoxin and its metabolites, a liquid

chromatographic (LC) fractional separation step, the mixing of the effluent of the LC column with fluorescein-labelled antibodies against digoxigenin, one of the metabolites,

the removal of free labelled antibodies from the mixture via passage through a small column packed with an antigen-bound support, and detection of the strongly fluorescent

irnmunocomplexes.

The digoxigenin system described is based on association reactions of antibodies and antigens eluting from the analytical column. By the use of fluorescein-labelled antibodies detection limits in the nanomolar-range are obtained.

The immunoreagent in this prior art assay consists of fluorescein-labelled fragments of

anti-digoxigenin antibodies which were immimopurified and are commercially available.

The commercial availability of purified, labelled antibodies is however exceptional. In

almost all cases, antibodies are only available in unlabelled state in crudely purified

antiserum. Although labelling and purification schemes for antibodies (or other affinity

proteins) are known to the person skilled in the art, it is preferred to use antisera, which

may be commercially available, without any pretreatment.

Furthermore, it is difficult to selectively label only those antibodies in an antibody

preparation which are reactive with the analyte. In general, labelling techniques make

use of primary amine groups of antibodies resulting in the colabelling of all other

proteins present in the preparation. This leads to a drastic increase in the background

signal and, consequently, to an increase in detection limits.

The aim of the invention is to provide alternatives for the known technique, which uses

fluorescein-labelled antibodies. The present invention does not have the disadvantages

associated with the labelling of antibodies.

In more detail, the problem underlying the present invention is the provision of an on¬

line detection technique without requiring the use of labelled antibodies. This problem is overcome by the provision of an on-line detection comprising the on¬ line coupling of a fractionation step to a biochemical detection technique in which the

detection method comprises the addition of a controlled amount of an affinity molecule to an effluent of the fractionation step whereby the affinity molecules bind analytes in

the effluent and the subsequent addition of a controlled amount of a detectable ligand

capable of binding to the affinity molecule, and detection of the affinity

molecule/detectable ligand complex whereby reduction in the amount of detected affinity molecule/detectable ligand complex indicates the presence of an affinity molecule-

binding compound in the effluent.

In particular, in a first step, a suitable affinity molecule for analytes to be detected -

which affinity molecule may e.g be an affinity protein, such as an antibody or avidin - is

added to the effluent of a liquid chromatography or a capillary electrophoresis column to

react with ligands or analytes eluting from the column. Unbound affinity molecules

react in a second step with an excess of a detectable ligand, which may be the analyte to

be detected in labelled from, such as a fluorescein-labelled analyte to titrate the

remaining free binding sites; or as an alternative, a competition reaction occurs between

the affinity molecule, the analyte in the effluent and the detectable ligand. Normally, the

labelled ligand/affinity molecule complex is detected after a separation of free and bound

labelled ligand, preferably on the basis of the difference in molecular weight. Finally,

the labelled ligand/affinity molecule complex is detected.

The combination of the fractionation step with the biochemical detection step in accordance with the present invention greatly enhances the performance of both

techniques. The combined techniques provide an analytical method which is characterised by a high selectivity and a high sensitivity. Further, the problem

associated with cross-reactivity does no longer occur when using the method of the

present invention.

The method of the present invention uses bioaffinity molecules such as antibodies,

receptors, etc to detect any compounds showing high affinity for the ligand binding site of said affinity molecule. The compounds to be detected may be biological compounds

but are not in any way restricted thereto.

The method of the invention makes it possible to screen a mixture of different

compounds on their ability to bind to a certain affinity molecule, e.g., in order to find an inhibitor for said affinity molecule. Therefore, the present invention further relates to a

fast on-line method for the screening of compounds on their binding capability to a

known affinity molecule, which method comprises a fractionation strep providing an

effluent, the addition of a controlled amount of said affinity molecule to the effluent of the fractionation step and effecting a contact time sufficient to allow a reaction with or

an interaction between the compounds in the effluent, the subsequent addition of a

controlled amount of a detectable ligand capable of binding to the affinity molecule, and

detection of the affinity molecule/detectable ligand complex.

A comparison of the detection signal obtained when analytes are injected with the signal obtained when only the controlled amounts of affinity molecule and detectable ligand are introduced in the continuous-flow system, provides information in respect of the

percentage of the binding sites occupied by analytes present in the effluent of the

fractionation step. The person skilled in the art possesses the knowledge to process and

evaluate the data obtained from the detection method. The percentage binding sites

occupied by an analyte present in the effluent can be used to find new compounds which

show an interaction with a known affinity molecule. This information provides the

possibility to implement the on-line separation/affinity molecule detection process of the

present invention in e.g drug discovery.

A system of high-throughput screening to be used in drug discovery including the

method of the invention may, for instance, consist of the following steps. Complex

samples generated for example by an upstream combinational chemistry system, are

prefractionated in fractions containing compounds of similar polarity using, for example,

a solid-phase extraction technique or electrophoretic sample handling principles. Each

fraction may additionally be separated using, for example, either analytical or

preparative-scale liquid chromatographic separation columns. The compounds eluting

from said LC column are on-line detected using a suitable affinity molecule detection

technique. Where preparative-scale separation columns are applied, a post-column flow-

split will be made. One of the two flow streams is subjected to detection using the affinity molecule detection technique of the invention, the other stream is directed to a

fraction collector. Dependent on the signal obtained from the affinity molecule detector,

fractions containing compounds causing a positive response will be collected while fractions causing a negative response will be discarded. This complete screening method can be automated using valve-switching processes.

A suitable fractionation method to be used in the method of the present invention

comprises a liquid chromatography separation or a capillary electrophoresis step. Other

separation or fractionation techniques which are known to the person skilled in the art

and which allow a relatively continuous output stream can, however, be used as well.

In a preferred embodiment, the liquid chromatography separation step is a reversed

phase HPLC step.

The detection method which forms an integral part of the methods of the present

invention can be chosen dependent on a suitable detectable ligand. Examples of suitable

detection methods comprise methods based on all types of luminescence detection

principles, such as fluorescence, time-delayed fluorescence and chemiluminescence

spectroscopy; methods based on electrochemical detection and on radiometric detection.

In the methods of the present invention all molecules capable of interaction with or

binding to other molecules can be used as affinity molecule. In preferred embodiments,

the affinity molecule is selected from the group of cytosolic receptors, e.g. , the

estrogen, glucocorticoid receptors; solubilized membrane bound receptor, e.g. , a β-

receptor; affinity proteins, such as antibodies, enzymes, avidin; polynucleotides and

polysaccharides. Furthermore, the affinity molecule may be an "orphan receptor". Such molecules are thought to be nuclear receptors, as they are structurally related to characterised

receptors, but for which no ligand has been identified. The determination of ligands for orphan receptors is relatively slow. By using the method of the present invention, this determination can be accelerated.

The term "detectable ligand" as used in the present description and claims refers to a

ligand which is capable of interacting or reacting with the afore-defmed affinity

molecule, and which can be detected in a biochemical detection method. An example of

a detectable ligand is a labelled analyte.

If labelled antigens are used instead of the labelled antibodies used in the prior art

techniques described above, two major advantages are obtained. First, it is easier to

prepare and, particularly, purify low-molecular weight labelled antigens. Second, the

background noise signal problem obtained in the preparation step of labelled antibodies

does not occur when labelled antigens are used for immunodetection.

On-line coupling as used in the methods of the present invention requires fast reaction

times in order to minimize extra-column band broadening. This means that affinity

molecule-ligand interactions having reaction times in the order of minutes rather than

hours should be considered.

In one embodiment of the method of the invention, biotinylated DNA-fragments are determined by the continuous addition of avidin to the eluate of a HPLC column. The

avidin is allowed to react with the biotinylated DNA-fragments in a so-called reaction

coil. Subsequently, a continuous flow of a solution comprising fluorescein-labelled biotin is added in the flow leaving the reaction coil. In a following step in the

continuous flow process, free and avidin-bound fluorescein-labelled biotin are separated

from each other, e.g., by the use of a separation procedure making use of the difference

in molecular weight between free and bound biotin, such as passing a column packed

with C_lg-silica restricted access support. The high-molecular weight labelled

ligan/protein complex passes the C₁₈-column unretained and is subsequently detected by

means of fluorescence detection. For the biotin-avidin system a detection limit in the

order of 100-500 fmol was obtained for biotin.

The methods of the present invention can be applied as well to systems wherein a DNA

or RNA probe is used as a receptor for DNA and/or RNA derived analytes. In this

case, the detectable ligand may be at least part of complementary strand which can be,

e.g., labelled with a radioactive isotope.

In a very advantageous embodiment within the scope of the present invention the affinity

molecule used is the estrogen receptor and the detectable ligand is coumestrol or another

natively fluorescent estrogenic ligand. In this embodiment it is also possible to use the

steroid binding domain of the human estrogen receptor, which can, e.g., be prepared by

gene technology, instead of the complete estrogen receptor. This embodiment does not require a separation step wherein bound and free coumestrol are separated. Coumestrol O 96/33408 PC17EP95/01523

is a compound which provides a strong fluorescent signal when bound to the estrogen receptor, and provides a weak fluorescent signal in free state. Since coumestrol and the

estrogen receptor are introduced in the continuous-flow system of the present invention

in controlled amounts, a comparison of the fluorescence signals obtained will give

information as to how many binding sites of the estrogen receptor are occupied by

coumestrol and how many binding sites are occupied by competitive analytes. Although

this embodiment using the steroid binding domain of the estrogen receptor and

coumestrol or another natively fluorescent estrogenic ligand does not require a

separation step wherein bound and free coumestrol are separated, the fluorescence data

will provide more detailed qualitative and quantitative information if such a separation

step is carried out.

Methods in accordance with the present invention will be described in further detail by

way of example only with reference to the following Examples.

Example 1

Biotin was applied to a liquid chromatography (LC) column. The LC separation was

carried out on a 100 x 3.0 mm internal diameter stainless-steel column packed with

Nucleosil C₁₈ (5μm particles, Macherey-Nagel, Dϋren, Germany) using methanol/aqueous triethylammonium acetate (10 mmol/1; pH 7.0) 19:90 (v/v) as a

mobile phase, which mobile phase was delivered by means of a Kratos-ABI (Ransey,

U.S.A.) Spectroflow 400 pump. In the effluent of the LC separation step, avidin was delivered in binding buffer consisting of sodium phosphate (10 mmol/1, pH 8.0; Merck, Darmstadt, Germany) containing 0.5 mol/1 sodium chloride (analytical grade; Merck,

Darmstadt, Germany), the avidin concentration being 34 nmol/1, by means of a

Pharmacia (Uppsala, Sweden) P3500 pump (flow rate 0.4 ml/min). The combined

avidin-biotin flow was introduced into a knitted 0.5 mm interanl diameter poly -

tetrafluoroefhylene reaction coil with an internal volume of 800 μl. Subsequently,

fluorescein-labelled biotin (Sigma, St. Louis, U.S. A) was delivered in the mobile phase

at a flow rate of 0.8 ml/min using a Kratos-ABI Spectroflow 400 pump. The flow

obtained was introduced into a knitted 0.5 mm internal diameter polytetrafluoroethylene

reaction coil with an internal volume of 400 μl. The reactions were performed at 20°C.

Separation of free and avidin-bound fluorescein-biotin was performed using a 10 x 40

mm interanl diameter column packed with C_rg alkanediol silica (described in DE-A-41

30 475). After this column, the flow was introduced into a fluorescence detector (Merck

1080 fluorescence detector; excitation wavelength 486 ran; emission wavelength 520

nm).

The absolute detection limit obtained for biotin was 160 fmol. The detection system

provided a linear signal ranging from 8 to 200 nmol/1 (20 μl injections).

Example 2 A mixture consisting of T₆-biotin (0.5 μM) and T₃-biotin-T₃ (0.5 μM) representing

biotinylated DNA-fragments was injected onto an LC column. The LC separation was

carried out on a 100 x 3.0 mm internal diameter stainless-steel column packed with Nucleosil C_g (5 μm particles, Macherey-Nagel, Dϋren, Germany) using

acetonitrile/aqueous triethylammonium acetate (10 mM, pH 7.0) 7:93 (v/v) as a mobile phase, which mobile phase was delivered by means of a Kratos-ABI (Ramsey, U.S. A) Spectroflow 400 pump. In the effluent of the LC separation step, avidin was delivered

in binding buffer consisting of sodium phosphate (10 nmol/1, pH 8.0; Merck,

Darmstadt, Germany) containing 0.5 mol/1 sodium chloride (analytical grade; Merck,

Darmstadt, Germany), the avidin concentration being 34 nmol/1, by means of a

Pharmacia (Uppsala, Sweden) P3500 pump (flow rate 0.4 ml/min). The combined

avidin-biotinylated compounds flow was introduced in a knitted 0.5 mm internal

diameter polytetrafluoroethylene reaction coil with an internal volume of 800 μl.

Subsequently, fluorescein-labelled biotin (Sigma, St. Louis, U.S. A) was delivered in the

mobile phase at a flow rate of 0.8 ml/min using a Kratos-ABI Spectroflow 400 pump.

The flow obtained was introduced into a knitted 0.5 mm internal diameter

polytetrafluoroethylene reaction coil with an internal volume of 400 μl. The reactions

were performed at 20°C.

Separation of free and avidin-bound fluorescein-biotin was performed using a 10 x 4.0

mm internal diameter column packed with C₁₈ alkanediol silica (described in DE-A-41 30 475). After passing this column, the flow was introduced in a fluorescence detector

(Merck 1080 fluorescence detector; excitation wavelength 486 nm; emission wavelength

520 nm).

It appeared that the retention times of T₃-biotin-T₃ and T₆ were 6.2 and 6.6 minutes, respectively.

EXAMPLE 3

A mixture of 17β-estradiol, estriol, diethylstilbestrol, zeranol, estrone, progestrone,

testosterone and dexamethasone (concentration: 100 nmol/1; injection 20 μl) was

applied to a LC column. The LC separation was carried out on a stainless-steel column

packed with Nucleosil C₂ (5 μm particles, Macherey-Nagel, Dϋren, Germany) using

acetonitrile:methanol: potassium phosphate buffer (10 mM, pH 7.4) 20:20:60 (v/v) as a

mobile phase, which mobile phase was delivered by means of a Kratos-ABI (Ramsey,

U.S. A) Spectroflow 400 pump at a flow rate of 0.5 ml/min. In the effluent of the LC

separation step, estrogen receptor (human estrogen receptor steroid binding domain;

Karo-Bio, Huddinge, Sweden) was delivered in a potassium phosphate buffer (200

nmol/1, pH 7.4; Merck, Darmstadt, Germany), the estrogen receptor concentration being

5 nmol/1, by means of a Pharmacia (Uppsala, Sweden) P3500 pump (flow rate 0.5

ml/min). The combined estrogen-analytes flow was introduced into a knitted 0.5 mm

internal diameter polytetrafluoroethylene reaction coil with an internal volume of 900 μl.

Subsequently, coumestrol (Eastman-Kodak, Rochester, New York, U.S. A) in a

concentration of 111 nmol/1 in the potassium phosphate buffer was delivered in the

mobile phase at a flow rate of 0.5 ml/min using a Kratosd-ABI Spectroflow 400 pump.

The flow obtained was introduced into a knitted 0.5 mm internal diameter

polytetrafluoroethylene reaction coil with an internal volume of 570 μl. The reactions

were performed at 20°C. Subsequently, the flow was introduced into a fluorescence detector (Merck 1080 fluorescence detector; excitation wavelength 340 nm; emission wavelength 410 nm). It

was found that 17β-estradiol, estriol, diethylstilbestrol, zeranol and estrone could be detected in a concentration of 50 nmol/1, whereas progestrone, testosterone and

dexamethasone did not provide any response at concentrations of 100 nmol/1.

Claims

1. An on-line detection method comprising the on-line coupling of a

fractionation step to a biochemical detection technique in which the detection method

comprises the addition of a controlled amount of an affinity molecule to an effluent of

the fractionation step whereby the affinity molecules bind analytes in the effluent and the

subsequent addition of a controlled amount of a detectable ligand capable of binding to the affinity molecule, and detection of the affinity molecule/detectable ligand complex

whereby reduction in the amount of detected affinity molecule/detectable ligand complex

indicates the presence of an affinity molecule-binding compound in the effluent.

2. An on-line method for the screening of compounds for their binding

capability to a known affinity molecule, which method comprises adding a controlled

amount of said affinity molecule to the effluent of a fractionation step and effecting a

contact time sufficient to allow a reaction with or interaction between the compounds

and the affinity molecule in the effluent, the subsequent addition of a controlled amount

of a detectable ligand capable of binding to the known affinity molecule, and detection of

the affinity molecule/detectable ligand complex whereby reduction in the amount of

detected affinity molecule/detectable ligand complex indicates the presence of an affinity

molecule-binding compound in the effluent.

3. The method of claim 1 or 2, wherein the fractionation step is a liquid

chromatography separation or a capillary electrophoresis step.

4. The method of claim 3, wherein the liquid chromatography separation step is a HPLC or a reversed phase HPLC step.

5. The method of any one of the preceding claims, wherein the detection is

effected by fluorescence, time-delayed fluorescence or chemiluminescence spectroscopy,

or by a method based on electrochemical detection or radiometric detection.

6. The method of any one of the preceding claims, wherein the affinity

molecule is a cytosolic receptor, a solubilized membrane bound receptor, an antibody,

an enzyme, avidin, a polynucleotide or a polysaccharide.

7. The method of any one of the preceding claims, wherein the detectable

ligand is a labelled ligand or a ligand possessing native detection properties such as

fluorescence or electrochemical activity.

8. A method according to any preceding claim in which the affinity molecule is

an estrogen receptor or a portion thereof.

9. The method of claim 8, wherein the affinity molecule is an estrogen receptor

or the steroid binding domain thereof and the detectable ligand is coumestrol or another

natively fluorescent estrogenic ligand.

10. A method according to any preceding claim, in which the method is arranged downstream of a combinatorial chemistry system whereby the combinatorial chemistry system provides the fractionation step providing the effluent.

11. A method according to any preceding claim in which the affinity molecule is

an orphan receptor.

12. Compounds detected by the method according to any preceding claim.

13. The use of compounds detected by their method according to any one of

claims 1-11 as a ligand for the affinity molecule.