WO1998041871A1

WO1998041871A1 - Antibody fragment configurations for detecting analytes

Info

Publication number: WO1998041871A1
Application number: PCT/GB1998/000733
Authority: WO
Inventors: William Harris; Andrew Porter; Steven Grant
Original assignee: William Harris; Andrew Porter; Steven Grant
Priority date: 1997-03-14
Filing date: 1998-03-11
Publication date: 1998-09-24
Also published as: GB9705376D0; GB9805066D0

Abstract

The present invention relates to methods for the detection and measurement of concentration of small molecular weight analytes by exploiting the specificity's and higher sensitivities offered by recombinant antibody configurations. The method includes the substitution of recombinant antibody fragments for intact antibodies or Fab fragments in competitive immunoassays or biosensors. In a competitive mode the method includes preincubating a known quantity of recombinant antibody fragments with the test sample containing the analyte and a fixed amount of analyte conjugated to enzyme or signal generating moiety. The recombinant fragment is then trapped through a second, non-analyte binding motif. In non-competitive immunoassay, monomeric antibody fragment and test analyte are pre-incubated and all non-analyte containing antibody removed with immobilised analyte. Analyte-bound antibody is then detected using a second motif upon the monomeric fragment. Applications of the methods of the present invention to detection of atrazine are disclosed. Further kits useful for utilising the methods of the present invention are also disclosed.

Description

TITLE ANTIBODY FRAGMENT CONFIGURATIONS FOR DETECTING ANALYTES

TECHNICAL FIELD

The present invention relates to methods for detecting the presence or concentration of analytes in test samples by exploiting the chemical and biological properties of antibody fragment configurations In particular the present invention relates to the selection and use of particular antibody fragment configurations in immunoassay formats to provide improved sensitivity, as compared to the use of intact polyclonal or monoclonal antibodies, to detect or measure the concentration of small molecular weight analytes

BACKGROUND OF THE INVENTION

Major developments continue to be made in immunoassay technology using either labelled antigen (competitive approach) or labelled antibody for signal generation The non-competitive labelled antibody (immunometπc) approach using the two-site "sandwich assay" where the analyte is of sufficient size to be trapped between "capture" and"detector" antibody has led to benefits in diagnostic technology over the competitive assay in terms of more sensitive (through correct analyte-bound binding site measurement), rapid (through the use of excess reagent), specific and robust assays Such assays measure the fraction of capture antibody that has bound analyte and therefore the intensity of signal increases with increasing amount of analyte in the test sample By contrast competition immunoassays measure the "analyte-unbound" sites with the result that low concentrations of analyte are detected by a small reduction in the maximum signal Such assays are therefore inherently less sensitive

"Sandwich assay" cannot be applied to many small molecular weight analytes such as pollutants, drugs and metabolites since they do not have sufficient size to simultaneously bind a capture and detection antibody and major commercial use is currently made of competition assays using labelled antigen Methods are now being developed to improve the sensitivity of competition assays and bring the advantages of non-competitive immunoassays to this large group of key chemicals through using a variety of labels (e g enzymes, fluorescent, chemiluminescent compounds) to replace traditional radioisotope labelling of analyte or antibody, and devising homogenous assays which do not require the separation of bound and free moieties before a signal is measured (Self, C H & Cook, D B 1996, Curr Opin Biotechnol 7, 60-65)

Signal generation has been greatly improved using enzyme labels such as alkaline phosphatase and horseradish peroxidase with coloπmetπc, fluorescent and chemiluminescent substrates Biosensor detection methods include enzyme driven generation of signals for electrochemical detection, or direct measurements of antigen -bound antibody with optical, piezoelectric or electrochemical immunosensors (Gizeh, E & Lowe, C R 1996, Curr Opin Biotechnol 7, 66-71 ) One such system involved the immobilisation of a derivative of atrazine (a widely used organic herbicide) on the surface of a transparent film and its subsequent exposure to a monoclonal anti-atrazine antibody pre-incubated with the test sample The fraction of monoclonal antibody which had not bound atrazine was captured and detected using reflectrometπc interference (Brecht, A et al 1995 Anal Chim Acta, 31 1, 289-299) Despite this highly sensitive signal generation system, the detection limit was reported to be 0 25ppb still higher than that imposed by European Union regulations ( l e 0 lppb) In a continuation of their work( Lang, G et al , 1996 Fresemus J Analytical Chem 354 8570860) replaced monoclonal antibody with chemically derived monomeric Fab fragments but sensitivity was not improved

Non-competitive methods for small molecular weight analytes are also being developed, such as anti-immune complex assays and selective antibody methodology (Self, C H 1994, In Rapid Methods and Automation in Microbiology and Immunology, Eds Spencer, RC et al , A_ndover,UK pp 185-190) In the selective antibody technology (also termed "ldiometπc" immunoassay) the amount of analyte- bound capture antibody is measured by the addition of a reagent that binds to residual analyte unbound sites (e g analyte analogue or anti-idiotypic antibody) that prevents these molecules binding a secondary "reporter" antibody The "reporter" antibody then only binds to analyte-bound antibodies (Mares, A et al , 1995, Immunol Methods, 181 83-90) This assay method described for oestradiol is considered appropriate for a dip-stick format for "point of need" testing

The advantages inherent in non-competitive lmmunometπc methods and the active research in this area make it likely that these lmmunodiagnostic methods for small molecular weight analytes could provide the "point of need" immunochemical testing that is becoming increasingly important, particularly for the general public with applications such as pregnancy testing, fertility monitoring, and monitonng of chemical pollutants in the environment, particularly water and soil

There is extensive pollution of our environment through the widespread use of synthetic chemicals which are extremely recalcitrant to natural degradation and which move with the mass flow of water in the environment to accumulate in soil, waters and move up through the food chain Concern over these events has prompted most countries to issue regulations and legislation to control such pollution and to lay down "acceptable' levels of contamination The European Community through the European Drinking Water Act, 1980 set an upper limit of 0 lug 1 ' for any on one of the organic pollutants on the 'red alert' list of compounds This necessitates routine testing and monitoring for these chemicals using methodologies with ? sensitivity to meet the above limits Current standard methodologies include high performance liquid chromatography, gas chromatography and mass spectrometry, methods which are technically demanding and expensive In contrast immunotechnologies are quick simple and inexpensive

A small number of immunoassay based kits for detection of organic pollutants are commercially available through companies such as Guildhay Ltd, Mil pore Ltd Transia, though in most cases improvements in sensitivity are required to meet regulatory demands and expand the market for such products Improvement technologies based upon novel signal generations, assay formats and biosensors have been alluded to above One object of the invention is to provide immunoassay systems of improved sensitivity through modification by recombinant DNA technology or otherwise of the monoclonal antibodies used as capture or "detector" molecules in such assay systems and improvements in immunoassay format The invention can be used independently or in combination with any of the improvements in technologies of signal generation or assay format described above

Recombinant DNA technology is being exploited in a number of ways to improve immunoassay technology including production in E coli of antibody fragment-enzyme fusion products In one such system a fusion product consisting of a dimer of bacterial alkaline phosphatase and fragments of a monoclonal antibody against human IgG was produced in the peπplasm of E coli and was effective in an ELISA assay (Ezan, E et al , 1994, J Immunol Methods 169, 205-211) In another system a genetically engineered hpid tagged antibody has been produced (Laukkanen,M-J et al , 1995, J Immunol Methods, 185, 95-102) and used to construct a fluorescence assay with europium chelate loaded hposomes

It is well known to those in the field that expression in E coli of antibody fragments such as Fab fragments or single chain Fv fragments (Figure 1 ) results in a mixture of monomeric and dimeπc fragments (McGregor,D et al , 1994, Molec Immunol, 31,219-226 ), and methods of genetic engineering can be employed to ensure that only dimeπc antibody fragments are produced (Holhger, P et al , 1993, Proc Nat Acad Sci USA,90, 6444-6448)

Published work from the laboratones of Kramer & Hock (Food & Agricultural Immunology,! 996 8, 97-109) and Ward et al (Ward, V K et al , 1993 Protein Engineering 6, 981-988) found that recombinant antibody fragments derived against s- tπazines were much less sensitive in immunoassays compared with the parental monoclonal antibody In contrast, previous work from our laboratories have demonstrated that monomeric antibody fragments against atrazine (Byrne et al, 1996 Food and Agricultural Immunology 8, 1996) and paraquat (Graham, B et al, 1995 J Chem Tech Biotechnol 63, 279-289) offer increased sensitivity in a heterogenous competition immunoassay compared with intact monoclonal antibody though in the described format still some way from the current European legislative limit

One aspect of the invention is the use of configurations of recombinant antibody fragments (in place of monoclonal or polyclonal antibodies or Fab antibody fragments) to increase the sensitivity of homogenous competition immunoassays

Another aspect of the invention is a method which uses monomeric antibody fragments in a non-competitive immunoassay to capture analyte from solution, then remove analyte-unbound antibody fragments and detect analyte-bound antibody fragments with a recognition motif on the monomeric fragment

The invention also comprises a kit performing the invention SUMMARY OF THE INVENTION

The present invention relates to the use of configurations of recombinant antibody fragments in immunoassays to detect the presence or concentration of small molecular weight analytes Small molecular weight analytes are defined as those whose size is insufficient for detection using the well established immunometric two-site "sandwich assay" Monomeric antibody fragments possess only 1 analyte binding domain per molecule in contrast to intact antibodies or dimeric antibody fragments (Figure 1) which are bivalent with two analyte binding domains per molecule Established immunoassay methodologies use intact antibodies or in some cases monomeric fab fragments The present invention provides higher sensitivity in competition immunoassays compared with immunoassays based upon intact antibodies or Fab fragments by replacing these molecules with configurations of recombinant antibody fragments

The present invention is also embodied in novel assay formats and kits The format and kit includes a monomeric antibody fragment specific to a particular analyte desired to be tested for The antibody fragment and test sample are mixed in solution Non- analyte bound monomeric antibody fragment is then removed by absortion to analyte immobilised upon a solid surface The analyte-bound monomeric antibody fragment is then detected using a signal generator which recognises a separate domain upon the antibody fragment. The kits may further include necessary reagents to carry out the methods of the present invention.

DETAILED DESCRIPTION OF THE EMBODIMENT OF THE INVENTION

The immunoassay format described in the accompanying examples are ELISA assays based upon detection with the enzyme horseradish peroxidase, using wells upon plastic multiwell plates as solid surface to capture antibody It will be appreciated by those skilled in the art that alternate detection methodologies using fluorescence, chemiluminescence, biosensors or alternate enzymes are equally applicable as are alternate solid matrices such as polystyrene, nylon, inorganic beads, and flat surfaces such as dipsticks and membranes

The illustrations refer to detection of the pesticide atrazine, an organic chemical of molecular weight 216 daltons, as a typical example of small molecular weight analytes which are too small in size to allow simultaneous binding of capture and detection antibodies as used in well known immunometric sandwich assays It will be appreciated by those skilled in the art that many insecticides, pesticides, herbicides drugs, oestrogen mimetics and industrial chemicals such as petroleum products, organochlorines, alkylphenolics, phthalates could equally well be presented as example

The invention will be described by way of illustration only, with reference to the accompanying drawings and in the following examples

In the Figures -

Figure 1 illustrates the structure of an antibody and various antibody fragment configurations, Figure 2 illustrates the composition of a typical gene vector for expression of antibody fragments in E coli The protein product (scAb) consists of a single chain antibody Fv fragment with a human CK protein motif and a 6-hιstιdιne tail fused to the VK of the antibody Either the human CK or polyhistidine tail can be used to purify the ScAb

Figure 3 shows the HPLC analysis of antibody fragments purified either by (A) metal chelate chromatography (which generates monomeric antibody fragments of retention time 16 8 mm ) or (B) anti-human CK immunoaffinity purification (which generates mainly dimeric antibody fragments of retention time 15 4mιn)

Figure 4 shows ELISA assays for detection of atrazine in (A) a solid-liquid phase heterogeneous competition assay and (B) a liquid-liquid homogenous competition assay

Figure 5 illustrates the increased sensitivity in ELISA assay for detection of atrazine in a liquid-liquid homogenous competition assay using recombinant scAb (single-chain antibody) compared to intact monoclonal and Fab fragments

Figure 6 illustrates the increased signal which can be generated using monomeric antibody fragments in non-competitive ELISA assays

EXAMPLE 1

EXPRESSION AND PURIFICATION OF MONOMERIC ANTIBODY FRAGMENTS

The antibody heavy and light chain genes from a hybπdoma cell line expressing murine anti- atrazine monoclonal antibody were cloned into the £ coli foreign gene expression vector construct pPMl-His described in Figure 2 The resulting protein product, referrred to as scAb (Figure 1 ), consisting of a single chain Fv anti-atrazine antibody fragment with a human CK motif and a polyhistidine tail fused to the murine VK is secreted into the peπplasm of £ coli on induction of gene expression with EPTG (isopropyl-B-D-thiogalactoside) £ coh XL1 -Blue cells transformed with vector were grown overnight in 5 ml of LB media containing 1% glucose, 12 5μg/ml tetracychne,50μg/ml ampicil n at 37°C Cells were pelleted by centπfugation at 3500 rpm, and resuspended in 7 5ml of fresh medium containing 50μg/ml ampicilhn After incubation at 25°C for lh, IPTG was added to a final concentration of 0 lmM Expression was continued at 25°C for 3-4h Cells were the pelleted, supernatant discarded and cells resuspended in 1ml of ice-cold osmotic solution I (30mM TRIS- HC1, 20%W/V sucrose, pH8 0) 1 OOmM EDTA pH8 0 was then added dropwise to a final concentration of lmM Cells were pelleted at 13000 rpm for 30 s and the supernatant recovered (T/S fraction) The cell pellet was resuspended in 1ml of ice- cold osmotic shock solution II (5mM MgS04) and mixed in ice for 5 mm Cells were pelleted as before and the supernatant recovered (M/S fraction) T/S and M/S were filtered through 0 45 μm filters, pooled to provide peπplasmic extracts, and used in the ELISA described in Figure 4

Previous studies in our laboratories have shown that crude extracts contain a mixture of monomeric and dimeric scAb (McGregor, D et al , 1993, Molec Immunol 31, 219-226). Periplasmic extract was purified by either anti-human CK or nickel chelate chromatography by methods well known to those skilled in the art and purified fragments analysed by HPLC analysis using Hydropore sec83-SO3-C5 size exclusion column (250 x 4.6mm) eluted with 0.2M Na₂PO₄ (pH 7.0) at a flow rate of 0.2ml/min. It can be seen that ScAb purified by Nickel Chelate Chromatography, which preferentially binds the polyhistidine tail (Figure 3 A) is predominantly monomeric (retention time 16.8 min.), while that purified by anti-CK is predominantly dimeric (retention time 15.4 min.). These separate preparations are hereafter referred to as monomeric and dimeric scAbs respectively.

EXAMPLE 2

ANTI-ATRAZINE COMPETITION ELISA ASSAYS USING MONOMERIC SCABS

(i) Heterogeneous Competition Assay. 96-well flat bottomed Immulon 4 ELISA microtitre plates (Dynatech Laboratories Ltd) were coated with lOOμl per well of atrazine-BSA conjugate (Dunbar conjugate) diluted 1 :2000 in PBS resulting in approximately 2ng conjugate per well. Plates were incubated for lh at room temperature and then washed three times with PBST (phosphate buffered saline containing 0.05% Tween-20). Plates were blocked with 200μl of 1% BSA in PBS for 1 h at 4°C, then washed three times with PBST. A decreasing range of different triazine concentrations were pre-mixed with 50μl of purified monoclonal antibody (mAb) or scAb and incubated for 1 h at 4°C before addition to the ELISA plate and incubated for a further 1 h at 4°C. Plates were washed three times with PBST then lOOμl per well of goat anti-mouse IgG (Fc specific) peroxidase conjugate diluted 1 : 1000 in PBS (to detect mAb), or goat anti-human IgG (Fab specific) to detect scAb, was added and plates incubated for 1 h at 4°C. Plates were washed four times with PBST and the ELISA developed using TMB (3, 3', 5, 5', tetramethylbenzidine dihydrochloride) and absorbance measured at 405nm.

The results in Figure 4(A) demonstrate that the IC50 (concentration of antibody required to achieve 50% inhibition of signal) is (a)10nM for monoclonal; (b) 7nM for dimeric scAb; (c)^%0.7nM for monomeric scAb, demonstrating the superior sensitivity of monomeric scAb in this assay. Figure 4 also demonstrates that scAbs retain the same specificity with respect to relative binding to the atrazine chemical analogues simazine and propazine, as compared to the monoclonal antibody.

(ii) Homogenous Competition Assay. 96-well flat bottomed Immulon 4 ELISA plates (Dynatech Laboratories Ltd) were coated with lOOμl per well of goat anti-mouse IgG (whole molecule) diluted 1 : 1000 in PBS, for 1 h at room temperature. Plates were washed three tines with PBST and blocked with 200μl of 1% BSA in PBS. .After lh at 4°C they were washed three times with PBST. Duplicate lOμl samples consisting of a range of decreasing concentrations of 25 μl triazine concentrations in H₂0, 25 μl of a 1 : 1250 dilution of atrazine-alkaline phosphatase tracer in PBS and 50μl purified MAB or ScAb were mixed and incubated at 4°C for lh, then added to the ELISA plates. Plates were incubated at 4°C for 1 h then washed four times with PBST. The ELISA was developed by adding lOOμl per well of a pNPP solution prepared by dissolving one Tris buffer tablet in 20ml H₂0 followed by one tablet (Sigma Chemical Co. Ltd) FAST pNPP(N-270). Development was allowed to proceed for lh and the absorbance read at 405nm.

The results in Figure 4(B) demonstrate that the IC_5u (concentration of antibody required to achieve 50% inhibition of signal) is 5nM for monoclonal; 0.4nM for dimeric scAb; 0.2nM for monomeric scAb demonstrating the superior sensitivity of monomeric scAb in this assay. It can also be seen that monomeric scAb is more sensitive in the homogenous competition assay than in the heterogeneous competition assay .

Fab was prepared from 1 mg of Mab using Immunopure Fab preparation kit (Pierce,UK). Purity was confirmed by showing that the final preparation gave a strong positive ELISA signal using an Fab-specific antisera but no signal using Fc-specific antisera. A single band of approximate MW 45KD was obtained by SDS-PAGE gel electophoresis. Figure 5 demonstrates that recombinant single chain antibody fragments (scAb) have improved sensitivity over monomeric Fab fragments prepared by proteolytic digestion of monoclonal antibody

EXAMPLE 3

NON-COMPETITIVE ELISA ASSAY USING MONOMERIC ScAb

In competition assays antibody and test analyte are incubated and the proportion of analyte-free antibody detected either by addition of labelled analyte (Figure 4B) and capture of all antibody; or capture of analyte-free antibody on a solid surface coated with analyte and then measurement of this captured antibody (Figure 4A). The disadvantage of this assay format is that it is the fraction of antibody with unbound analyte which is measured and hence low concentrations of analyte are detected by a small reduction in the maximum signal.

Figure 5 illustrates one example of a non-competitive immunoassay format that can be adopted to detect the fraction of antibody that has bound analyte and therefore the intensity of signal increases with the amount of analyte. The higher sensitivity is achieved with monomeric scAb. In this illustrative example a low concentration of the analyte to be measured (e.g. atrazine) is mixed with either monomeric and dimeric anti- atrazine scAb, prepared as described in Example 1. Equimolar amounts of monomeric and dimeric scAb are included in this illustration. Samples are then added into wells of microtitre plates coated with immobilised atrazine-BSA. ScAbs containing unoccupied atrazine binding sites adsorb to the wells. Supernatant (now containing only scAb with bound atrazine) can be removed and its concentration measured by detecting a second motif upon the scAb (in this examples either human CK or polyhistidine tail). Figure 5 (a) illustrates monomeric scAb and (b) dimeric scab It can be seen that (a) provides a higher concentration of scAb for detection and hence a more sensitive assay. Dimeric scAb is included for purpose of comparison and does not represent an example of the invention.

Claims

1. A method for detection and measuring the concentration of low molecular weight analytes by incorporating recombinant antibody fragments in homogenous competition immunoassays.

2. A method according to claim 1 where recombinant antibody fragments are selected from the configurations Fv, single chain Fv, disulphide bonded Fv, scAb or protein fusion products which include one of these configurations.

3. A method for detection and measuring the concentration of low molecular weight analytes in non-competitive immunoassays comprising the steps of a) pre-incubating antibody fragments with test analyte

b) removing all antibody fragments without bound analyte c) detection of remaining analyte-bound antibody fragments

4. A method according to claim 3 whereby the antibody fragments consist of monomeric fragments.

5. A method according to claims 1 and 3 which incorporates enzymes, fluorescence , chemiluminescense, or biosensor methodologies into immunoassays.

6. A method according to claims 1 and 3 which incorporates matrices to remove analyte or antibody fragment which are not associated with each other such matrices to include, but not limited to plastic, polystyrene, nylon, inorganic beads and flat surfaces, dipsticks and membranes.

7. A method according to claims 1,3 and 6 which incorporates matrices or reagents to preferentially remove analyte-antibody complexes

8. A method according to any preceding claim which detects or measures the concentration of analytes of such a low molecular weight that they are of insufficient size to be trapped between "capture" and "detector" antibodies, such analytes to include, but not limited to, pesticides, herbicides, insecticides drugs, oestrogenic mimetics, petroleum products, alkyphenolics,phthalates,organochlorines.

6. Assay kits for the detection of analytes when used in any one of claims 1,2 and 3 disposed in, or on, a contact surface and means of performing competitive or non- competitive ELISA assay or biosensor measurements therewith.