WO2001013111A2

WO2001013111A2 - Allele detection of pgm1, gc, and aat

Info

Publication number: WO2001013111A2
Application number: PCT/GB2000/003038
Authority: WO
Inventors: David Albert Hopkinson; David Bertram Whitehouse
Original assignee: Medical Research Council
Priority date: 1999-08-13
Filing date: 2000-08-11
Publication date: 2001-02-22
Also published as: WO2001013111A3; AU6457000A; GB9919226D0

Abstract

Method of typing a biological sample comprising contacting the sample with one or more antibodies specific for any of the following polymorphisms: (i) R220 of PGM1, (ii) C220 of PGM1, (iii) Y419 of PGM1, (iv) H419 of PGM1, (v) D416 T420 of GC, (vi) E416 T420 of GC, (vii) D416 K420 of GC, (viii) H101 of AAT, (ix) R101 of AAT, (x) V264 of AAT, (xi) E264 of AAT, (xii) K342 of AAT, (xiii) E342 of AAT, (xiv) V213 of AAT and (xv) A213 of AAT, and determining whether the antibody binds a protein in the sample, binding of the antibody to the protein indicating that the sample contains a protein possessing the polymorphism for which the antibody is specific.

Description

ALLELE DETECTION The invention relates to a method of typing a biological sample, antibodies that can used in such a method, a method of diagnosis and to the treatment of a genetic disease. Forensic investigators often need to determine whether a biological sample is from a particular individual. This can be done by typing polymoφhisms in DNA or protein present in the sample and seeing whether they are the same as the polymoφhisms in a sample known to come from the individual. One system which has been used in forensic typing is Western blotting which assigns a protein phenotype based on the electrophoretic properties of the protein. Antibodies that bind all of the phenotypes are used to identify the position on the blot of the specific protein which is being typed. However, Western blotting systems require expensive reagents and equipment. Much of this method of typing has been replaced by DNA profiling based on restriction sites. Although this yields much more information which can be used to identify individuals very specifically it is a skilled, costly and time-consuming procedure.

The inventors have developed a typing system based on a panel of allele specific antibodies which is rapid, portable and can be carried out by a non-specialist user. The panel consists of antibodies specific for particular polymoφhisms in phosphoglucomutase-1 (PGMl), group specific component (GC) and α,-antitrypsin (AAT) protein. Two of the AAT polymoφhisms (V264 and K.342) which can be typed using the panel are associated with diseases caused by AAT deficiency and another AAT polymoφhism (A/V213) that affects the responsiveness of a patient to drug therapy. The invention therefore provides a method of typing a biological sample comprising contacting the sample with one or more antibodies specific for any of the following polymoφhisms: (i) R220 of PGMl, (ii) C220 of PGMl, (iii) Y419 of PGMl, (iv) H419 of PGMl, (v) D416 T420 of GC, (vi) E416 T420 of GC, (vii) D416 K420 of GC, (viii) HI 01 of AAT, (ix) R101 of AAT, (x) V264 of AAT, (xi) E264 of AAT, (xii) K342 of AAT, (xiii) E342 of AAT, (xiv) V213 of AAT and (xv) A213 of AAT, and determining whether the antibody binds a protein in the sample, binding of the antibody to the protein indicating that the sample contains a protein possessing the polymoφhism for which the antibody is specific.

The invention also provides a method of diagnosing (a) an AAT deficiency, (b) susceptibility to an AAT deficiency, (c) whether an individual is a carrier for a polymoφhism that contributes to an AAT deficiency, comprising contacting a sample from an individual with one or more antibodies specific for either of the following polymoφhisms: (x) V264 of AAT or (xii) K342 of AAT, and determining whether the antibody binds a protein in the sample, binding of the antibody to either polymoφhism indicating that the individual has an AAT deficiency or is susceptible to an AAT deficiency or is a carrier for a polymoφhism that contributes to an AAT deficiency.

The invention provides a method of diagnosing responsiveness to therapy, comprising contacting a sample from an individual with one or more antibodies specific for the polymoφhism (xv) A213 of AAT, and determining whether the antibody binds a protein in the sample, binding of the antibody to the protein indicating that the individual has a high responsiveness to therapy.

Typically each of the methods additionally comprises exposing the sample to denaturing conditions before or during the contacting.

The invention also provides antibodies which are specific for any of the polymoφhisms (v), (vi), (vii), (viii), (ix), (x), (xi), (xiv) or (xv).

The invention is illustrated by the accompanying drawings in which: Figure 1 shows the results of ELISAs testing the capacity of particular antibodies to discriminate between the target polymoφhism and the opposite polymoφhism. The horizontal axis shows 1/log dilution. Figure 2 shows immunoblots using the allele specific AAT antibodies to stain

AAT of known phenotype run on an IEF gel. The anode is at the top.

Figure 3 shows immunoblots using the allele specific AAT antibodies to stain AAT of known phenotype run on a SDS gel. The anode is at the bottom.

Figure 4 shows the effect of pH and NaCl concentrations on antibody binding. Figure 5 shows capture results with 2G2 anti-K342 antibody. The horizontal axis shows the phenotype of the test plasma sample. Figure 6a shows the effects of urea concentration on antibody binding in capture assays. The horizontal axis shows the starting urea concentration (M).

Figure 6b shows the effects of urea concentration on antibody binding in capture assays. Figure 7 shows urea-ELISAs with particular antibodies.

Figure 8 shows dot blot capture ELISAs using anti-AAT monoclonal antibodies to detect the phenotype in plasma samples.

In the nomenclature used herein to refer to the polymoφhisms (e.g. R220) the single letter amino acid code is used to specify the amino acid followed by the position of that amino acid in the protein. Some of the polymoφhisms are specified in terms of amino acids at two positions (e.g. E416 T420 of GC). The polymoφhisms (i) to (xiii) which are typed in the method of the invention are present in the corresponding peptides represented by SEQ ID NO's 1 to 13. In the description below the term 'method' is understood to refer to both the method of typing and the methods of diagnosis unless the context requires otherwise.

The sample which is typed in the method is typically one which is known or suspected of being a body sample from a human individual. In the case where a sample is suspected of containing proteins from the body of an individual the method of the invention may be carried out to simply determine whether or not it does contain such proteins.

In the method of diagnosis the AAT deficiency is one which is caused by possession of an AAT gene that expresses AAT with the (x) V264 of AAT (the S variant) or (xii) K342 of AAT (the Z variant) polymoφhism. The natural function of AAT is to inhibit neutrophil elastase. These polymoφhisms cause a reduction in plasma levels of AAT and/or cause a reduced AAT activity. This causes susceptibility to chest disease, such as emphysema (particularly in the ZZ and SZ genotypes). These polymoφhisms cause decreased secretion of AAT from hepatocytes causing AAT to accumulate in these cells. This causes susceptibility to liver disease (particularly in the ZZ genotype). The method may be used to diagnose (or aid or confirm diagnosis) in individuals suspected of having an AAT deficiency, such as those with any of the above symptoms. Diagnosis of the patient as having an AAT polymoφhism which causes or contributes to an AAT deficiency allows the physician to adjust therapeutic treatment accordingly.

The method may also be used to diagnose individuals who susceptible to AAT deficiency. Such individuals can then be advised on life style changes which may be required to decrease the likelihood of developing, or decrease the severity of, symptoms associated with AAT deficiency. The individuals may be treated prophylactically for the same puφose. AAT deficiency may modify the symptoms of cystic fibrosis. Therefore the method may be used to diagnose AAT deficiency in cystic fibrosis patients, for example so that appropriate changes may be made to the therapy given to the patient.

The method may additionally comprise contacting a sample from the individual with an antibody specific for the polymoφhism (xi) or (xiii) to determine whether the individuals are homozygous or heterozygous for the disease alleles. Individuals identified as having an AAT deficiency may treated therapeutically. This is discussed further below.

The method of diagnosis may also be used to determine whether an individual is a carrier of gene that expresses an AAT polymoφhism that may lead to an AAT deficiency. In the method of diagnosing responsiveness a sample from the individual may also be contacted with an antibody specific for polymoφhism (xiv) to determine whether the genotype of the individual at the locus encoding the polymoφhism. In one embodiment the sample is only contacted with antibody specific for polymoφhism (xiv), and not antibody specific for (xv). It will be understood that although the (xv) polymoφhism has been referred to causing high responsiveness this is in the context of a particular type of therapy of a disease in which AAT is implicated. For other therapies (xv) may confer low responsiveness (and (xiv) high responsiveness), and typing samples to determine such low responsiveness is included in the invention. The sample used in the method may be any material of human origin and in one embodiment the sample contains cells from the individual. The sample is typically a blood, saliva, hair root, seminal fluid, skin or foetal sample. The sample may be a product of conception, such as a placental sample. The sample may be processed before it is used in the method, for example it may be diluted, typically in water, saline or saline containing a buffer (any of these diluents may additionally comprise detergent).

An antibody used in the method of the invention may either be a whole antibody or a fragment thereof which is capable of binding the polymoφhism. Typically the antibody is monoclonal. Such a whole antibody is typically an antibody which is produced by any of the methods of producing an antibody which are discussed herein. Typically the antibody is a mammalian antibody, such as a primate, human, rodent (e.g. mouse or rat), rabbit, ovine, porcine, equine or camel antibody. The antibody can be any class or isotype of antibody, for example IgM, but is preferably IgG.

The fragment of whole antibody that can be used in the method comprises an antigen binding site, e.g. Fab or F(ab)₂ fragments. The whole antibody or fragment may be associated with other moieties, such as linkers which may be used to join together 2 or more fragments or antibodies. Such linkers may be chemical linkers or can be present in the form of a fusion protein with the fragment or whole antibody. The linkers may thus be used to join together whole antibodies or fragments which have the same or different binding specificities, e.g. that can bind the same or different polymoφhisms. The antibody may be a bispecific antibody which is able to bind to two different antigens, typically any two of the polymoφhisms mentioned herein. The antibody may be a 'diabody' formed by joining two variable domains back to back. In the case where the antibodies used in the method are present in any of the above forms which have different antigen binding sites of different specificities then these different specificities are typically to polymoφhisms at different positions or on different proteins. In one embodiment the antibody is a chimeric antibody comprising sequence from different natural antibodies, for example a humanised antibody. The antibodies used in the method are specific for the relevant polymoφhism, i.e. have antigen binding sites that do not bind the other polymoφhism mentioned herein which occurs at the same position in the protein (whilst it is understood that such antibodies can be joined to antibodies with different specificities in the manner discussed above). The antibodies may or may not bind the native form of the protein which contains the relevant polymoφhism, but generally do bind the relevant peptide represented by SEQ ID NO's 1 to 13. An antibody specific for the polymoφhism (xiv) or (xv) would generally bind a peptide of length 13 amino acids which has the same sequence as amino acids 207 to 219 of AAT containing the relevant polymoφhism at position 213. The antibodies may bind proteins that contain the relevant polymoφhism which have been denatured by any of the conditions discussed below.

In the method at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14 or all of the polymoφhisms are typed. Typically in the method the 2 or 3 polymoφhisms mentioned herein for the same position will all be typed. Preferably in the method the sample is contacted with at least one or more antibodies specific for any of the polymoφhisms (v), (vi), (vii), (viii), (ix), (x), or (xi). In one embodiment the sample is typed for the polymoφhisms (i), (iii), (iv), (vi), (vii), (viii), (ix), (xi), (xii) and (xiii). In another preferred embodiment all of the polymoφhisms are typed so that the sample is contacted with at least one antibody specific for each of the polymoφhisms (i), (ii), (iii), (iv), (v), (vi), (vii), (viii), (ix), (x), (xi), (xii), (xiii), (xiv) or (xv).

Typically each of the typings which are to be carried out in the method are performed in separate assays (such assays being physically separated from each other). In each assay the sample is contacted with a single antibody specific for the polymoφhism to be typed. For example the sample may be added to different wells of a microtitre plate, wherein each well contains a different antibody. However in one embodiment more than one of the typings is carried out in the same assay..

Generally the method is carried out in an aqueous solution. The sample and/or the antibody may be present is solution in the method. In particular embodiments (some of which are discussed below) the antibody or sample is immobilised on a solid support. Typically such a support is the surface of the container in which the method is being carried out, such as the surface of a well of a microtitre plate. In other embodiments the support may be a sheet (e.g. a nitrocellulose or nylon sheet) or a bead (e.g Sepharose or latex).

In the method, determining whether the antibody binds a protein in the sample may be performed any method known in the art for detecting binding between two moieties. The binding may be determined by measurement of a characteristic in either the antibody or protein that changes when binding occurs, such as a spectroscopic change.

In a preferred embodiment the antibody is immobilised on a solid support

(such as the supports discussed above). When the sample is contacted with the antibody proteins in the sample containing the polymoφhism bind to the antibody.

Optionally the surface of the solid support is then washed to remove any protein from the sample which is not bound to antibody. The presence of the protein bound to the solid support (through the binding with the antibody) can then be determined, indicating that the protein is bound to the antibody. This can be done for example by contacting the solid support (which may or may not have protein bound it) with an agent that binds the protein specifically. This agent may be labelled either directly or indirectly by a detectable label.

Typically the agent is a second antibody which is capable of binding the protein in a specific manner whilst the protein is bound to the first immobilised antibody that binds the polymoφhism. This second antibody can be labelled indirectly by contacting with a third antibody specific for the Fc region of the second antibody, wherein the third antibody carries a detectable label.

Another system which can be used to determine the binding between the protein and the antibody is a competitive binding system. One embodiment of such a system determines whether protein in the sample is able to inhibit the binding of the antibody to a reference compound which is capable of binding the antibody. The reference compound may for example be a known amount of labelled PGMl, GC or

AAT protein containing the polymoφhism which the antibody recognises. If protein in the sample is able to inhibit the binding the between the antibody and reference compound then this indicates that such a protein contains the polymoφhism recognised by the antibody. Examples of detectable labels include enzymes, such as a peroxidase (e.g. of horseradish), phosphatase, radioactive elements, gold (or other colloid metal) or fluorescent labels. Enzyme labels may be detected using a chemiluminescence or chromogenic based system. The method may additionally comprise exposing the sample to denaturing conditions before or during the contacting. Thus in the method the antibody may be contacted with a protein which is denatured (such as by any of the conditions mentioned herein). The denaturing conditions alter the conformation of the protein so that parts of the protein which are not on the surface in the native conformation may become more accessible to the antibody. Thus exposing the sample to denaturing conditions generally increases the ability of the antibody to bind the relevant polymoφhism in the protein. Since the denaturing conditions generally make the polymoφhism more accessible they also generally increase the susceptibility of the polymoφhism to being labelled by a labelling agent, e.g. such as iodination by an iodinating agent.

The denaturing conditions may be increased temperature (such as a temperature of at least 45°C, such as at least 50, 60 or 70°C) or UV radiation The denaturing may comprise contacting the sample with an agent that causes denaturation. Such denaturing conditions or agents are typically able to disrupt the interactions between the protein in the sample and the solvent in which it is present and/or disrupt intramolecular interactions inside the protein. Typically the conditions or agents cause unwinding of the structure of the protein (typically rendering it nonfunctional). The conditions or agents may be such that they denature native AAT in normal human serum (in which AAT is present at about or at 2mg/ml) sufficiently to allow an antibody made using the peptide represented by SEQ ID NO:7 as immunogen in the process disclosed herein, to bind the AAT (such an antibody would not bind native AAT which had not been denatured).

The agent may be able enter and associate with hydrophobic regions inside the protein and/or may bind to the surface of the protein. The agent may bind the protein in a reversible or non-reversible manner.

In one embodiment the agent is a water soluble chemical, such as a chaotropic agent (e.g. urea or guanidinium compounds such as guanidinium HCl), an alkylating agent (e.g. iodoacetamide or iodoacetic acid) or a strong acid or alkali (e.g. hydrochloric or sulphuric acid, or sodium hydroxide) . In a preferred embodiment exposing the sample to denaturing conditions comprises contacting the sample with urea. Typically the sample is contacted with a concentration of urea of from 2 to 8 M, such as from 3 to 6 M or 4 to 5M.

The invention also provides antibodies specific for any of the polymoφhisms (v), (vi), (vii), (viii), (ix), (x), or (xi). The antibodies provided by the invention (and those which are used in the method of the invention) may be made by culturing a cell that expresses the antibody and optionally purifying the antibody from the cell.

The cell used in the process may be one which is obtainable by administering a peptide comprising any of the polymoφhisms mentioned herein to a mammal, extracting B cells from the mammal and selecting a cell from these based on the ability to express an antibody which binds the polymoφhism. Optionally the B cells are immortalised after extraction or selection, for example by fusing them with an immortal cell (to form a hybridoma) or by infection with EBV virus.

Cells that express the antibody comprise a polynucleotide that is capable of expressing the antibody, a polynucleotide that encodes the antibody.

The mammal to which the peptide is administered may be any of the mammals mentioned herein. Preferably the mammal is a mouse. The peptide is typically any of the peptides represented by SEQ ID NO's 1 to 13.

Another type of cell which can be used to make the antibody is one which is recombinant for a polynucleotide which expresses the antibody. Such a cell may be prokaryotic or eukaryotic (such as from any of the mammals mentioned herein). The invention also includes a dipstick which can be used to carry out the method of the invention. The dipstick comprises a porous material capable of chromatographically transporting a liquid and one or more of the antibodies mentioned herein. When the dipstick is contacted with the sample it draws up liquid from the sample towards a detection region on the dipstick. Proteins in the sample comprising the polymoφhisms mentioned herein are detected by their binding to detection region. In one embodiment the liquid is drawn through a region in the dipstick containing the antibodies of the invention. These antibodies bind to proteins containing the relevant polymoφhism forming an antibody/protein complex. This complex is drawn towards the detection region which contains an agent (immobilised on the dipstick) that binds and thus immobilises the complex in the detection region. The agent is typically a specific binding agent (e.g an antibody) that binds either the antibody or the protein of the complex. The antibody/protein complex is typically detected in the detection region by the use of a label which is attached to the polymoφhism-specific antibody. In another embodiment protein in the sample is labelled before it is drawn up the dipstick. The labelled protein is then drawn up the dipstick (which has been contacted with sample) and is detected by binding the polymoφhism specific antibody (which is bound to the detection region).

Typically the label used in the dipstick systems described above is a visually detectable label which becomes visually detectable (i.e. can be seen with the human eye) when enough antibody/protein complex becomes immobilised in the detection region. A suitable label is a gold (or other colloidal metal) particle or a fluorophore (e.g. fluoroscein).

The dipstick may comprise a denaturing agent that causes denaturation of the protein which is drawn up the dipstick. In one embodiment the sample is exposed to denaturing conditions (e.g. contacted with a denaturing agent) before it is contacted with the dipstick.

The invention also provides a kit comprising an antibody of the invention or a dipstick of the invention. The kit may additionally comprise other reagents which assist in carrying out the method of the invention. Thus it may additionally comprise one or more of the following: a denaturing agent, an antibody that binds PGMl, GC or AAT when they bound to any of the allele specific antibodies mentioned herein, an antibody that binds the Fc portion of an allele specific antibody mentioned herein, a means of labelling protein in a sample, or a means of detecting a labelled antibody or labelled PGMl, GC or AAT. The kit may comprise buffer solutions or positive and negative controls (such as PGMl, GC or AAT protein, or peptides which are able to bind the allele specific antibodies).

In one embodiment the kit may comprise the agent discussed below which is to be administered to an individual who is diagnosed as having or being at risk of getting an AAT deficiency. Any of the antibodies, polynucleotides or cells (such as those polynucleotides or cells capable of expressing the allele specific antibodies e.g. those antibodies provided by the invention) may be present in substantially isolated form, or may be in substantially purified form, in which case they will generally comprise at least 90%, e.g. at least 95%, 98% or 99% of the protein, polynucleotide, cells or dry mass in the preparation.

The method (of typing and diagnosis) of the invention is generally performed in vitro but in one embodiment is performed in vivo (e.g. on the skin surface). Thus the allele specific antibodies may be present in the form of a diagnostic composition comprising the antibody and a pharmaceutically acceptable carrier or diluent. The invention provides the antibody for use in a method of diagnosis practised on the human body.

As discussed above individuals identified as having an AAT deficiency (or being susceptible to such a deficiency) by the method of diagnosis of the invention may then be treated therapeutically (including prophylactically). Thus the invention provides a method of treating an individual with an AAT deficiency, or who is susceptible to an AAT deficiency, comprising diagnosing whether the individual has an AAT deficiency or is susceptible to an AAT deficiency by the method of diagnosis of the invention and then administering an agent therapeutic for the AAT deficiency to the individuals who have been diagnosed as having AAT deficiency or being susceptible to an AAT deficiency.

The invention provides use of an agent therapeutic for an AAT deficiency in the manufacture of a medicament for treating an AAT deficiency in an individual diagnosed as having, or being susceptible to, an AAT deficiency, wherein said diagnosis is by a method of the invention. The agent may complement the activity of AAT, and thus in one embodiment has at least some AAT activity. Typically the agent is AAT or a polynucleotide that is capable of being expressed to provide AAT (i.e. expressed in vivo inside cells of the patient). The AAT may be native human AAT, or a homologue or fragment of native AAT or homologue (typically with a length of at least 5, such as at least 10, 20 or 50 amino acids). The homologues are typically at least 70% homologous to the native human

AAT, preferably at least 80 or 90% and more preferably at least 95%, 97% or 99% homologous thereto. Such homology may exist over a region of at least 15, preferably at least 30, for instance at least 40, 60 or 100 or more contiguous amino acids. Methods of measuring polynucleotide or polypeptide homology are well known in the art. For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (Devereux et al (1984) Nucleic Acids Research 12, p387- 395) typically on its default setting. The homologues may differ from the native AAT by at least 2, 5, 10, 20, 50 or more mutations (each of which may be an insertion, substitution or deletion). The AAT is typically capable of binding one or more of the antibodies mentioned herein when it is denatured (e.g. as described by conditions herein). The polynucleotide that is capable of expressing AAT may be administered as 'naked DNA' or within a vector, such as a cellular or viral vector.

The condition of a patient suffering from an AAT deficiency can therefore be improved by administration of the agent. A therapeutically effective non-toxic amount of the agent may be given to a human patient in need thereof.

The formulation of the agent for use in preventing or treating the AAT deficiency will depend upon factors such as the nature of the AAT deficiency. Typically the agent is formulated for use with a pharmaceutically acceptable carrier or diluent. For example it may be formulated for aerosol (via nose or mouth), intravenous, intramuscular or subcutaneous administration. A physician will be able to determine the required route of administration for each particular patient. The pharmaceutical carrier or diluent may be, for example, an isotonic solution.

The dose of product may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient. A suitable dose may however be from 0.01 to 100 mg/kg body weight such as 1 to 40 mg/kg body weight.

The following Examples illustrate the invention: Examples

In order to streamline the detection of AAT, PGMl and GC variants for forensic and medical applications, we have investigated strategies for generating allele-specific monoclonal antibodies that would discriminate between allelic variants of these proteins (in the case of AAT between Ml -like (Ml and M3), M2-like (M2 and a less common variant M4), S and Z). Such antibodies could be adapted for use in rapid immunoassay formats. Our aim was to produce high affinity antibodies that specifically recognised allelic variants of the intact native AAT, PGMl and GC proteins.

Single or double amino acid substitutions underlie all the variants we selected and we opted to use small synthetic peptides as immunogens. In the case of AAT the amino acid sequence changes for Ml, M2, M3, M4, S and Z have been determined from DNA sequence analysis. The most common variant, Ml, is characterised by Arg 101, Glu 264, Glu 342 and Glu 376. We have used two polymoφhic residues at lOland 342 as target epitopes. Residue 101 determines the difference between Ml and M3 (Arg 101) versus M2 and M4 (His 101). Residue 342 determines the Z variant (Lys 342) versus the M and S variants (Glu 342). We did not use residue 376 which determines Ml and M4 (Glu 376) versus M2 and M3 (Asp 376) since it was found to be somewhat inaccessible in the 3D model of intact native AAT.

For each of the two polymoφhic sites a pair of peptides, that was analogous to the corresponding region of the AAT protein and that differed only at the polymoφhic residue, was synthesised. All four peptides produced anti-peptide responses in mice and allele-specific monoclonal antibodies that recognised intact protein were generated against all four epitopes. SEQ ID NO's 1 to 13 show the peptide immunogens which can be used to generate antibodies specific for polymoφhisms (i) to (xiii). Here we describe the production and validation for the mAbs, and in particular of 3A2 (anti M2-like), 10a (anti non M2-like), 2G2 (anti Z) and 6G5 (anti non Z). We report the development of simple immunoassays that enable a genetic profile be determined rapidly and without the need for isoelectric focusing. The use of a rapid immunogold membrane panel test for the presence of the relevant polymoφhism in protein in serum or whole blood has also been demonstrated.

MATERIALS AND METHODS

Peptides. The choice of the peptide immunogens was constrained by the position of the polymoφhic substitution sites. In all cases the polymoφhic residue(s) was positioned towards the centre of the peptide. Wherever possible, a mix of charged, polar and non-polar residues was included. Peptides were synthesised with an exotic N or C terminal cysteine to facilitate coupling to the carrier proteins via sulphur chemistry using malsac Aldwin et al (1987). The sequences of the immunogens are shown in SEQ ID NO's 1 to 13. In the case of AAT the four peptide immunogens were: M2-like/non M2-like, 12mer, 97 cQELL[H R]TLNQPDS 108; Z/non-Z, 1 lmer, 337 cVLTID[K/E]KGTEA 347. Note that the exotic amino terminus cysteine is shown in lower case, the alternative allelic amino acids in bold face and the flanking numbers represent the start and finish positions of the peptides in the mature AAT protein. In the case of polymoφhisms (xiv) and (xv) a peptide of length 9 to 13 with AAT sequence from around the polymoφhic site will be used, for example the sequence of amino acids 207 to 219 of AAT (with the exotic cysteine on the N or C terminus). The peptides were synthesised on an Abimed continuous flow peptide synthesiser as free acids with protected side groups on Wang resins using solid phase f-moc chemistry. Following the final cleaning steps after cleavage of peptides from the resin and deprotection, the peptides were checked for purity by HPLC and mass spectrometry.

Preparation of immunogens. The peptides were coupled to pure protein derivative of M. tuberculin (PPD) using standard sulphur chemistry (Aldwin et al (1987). Prior to coupling to the carrier protein, the peptides were cleaned by washing twice in ether. Immunisation. For each peptide conjugate, six 6-week-old female Balb c mice were primed with 100 μl BCG vaccine by subcutaneous injection two weeks before 1st peptide-PPD immunisation. The mice were immunised by subcutaneous injection with 300 μL of emulsion of peptide conjugate (100 μg of peptide per mouse) and Incomplete Freund's Adjuvant (IF A). After 21 to 28 days, the mice were boosted using the immunogen prepared and delivered exactly as for the 1st peptide injection. Four days after the second boost, 50 μl blood was collected from the tail vein for gel based and simple two step ELISA tests (see screening ELISA) to monitor antibody levels. If test bleeds were negative, the mice were rested for 14 days and re-boosted as above. Following a further test bleed, the best responder for each immunogen was selected for fusion. Four days prior to fusion the mouse was boosted as follows. Three dilutions of immunogen peptide conjugate were prepared in sterile isotonic saline (1/50 = 2 μg peptide per 100 μl; 1/10 = 10 μg peptide per 100 μl; Vi = 50 μg peptide per 100 μl). Over a course of 5 hours the mouse was injected (intraperitoneal) six times at hourly intervals with increasing concentrations of immunogen as follows: T₀, lOOμl at 1/50; T,. lOOμl at 1/50; T₂, lOOμl at 1/10; T₃, lOOμl at 1/10; T₄, lOOμl at 'Λ; T₅, lOOμl at - . The next day, 100 μl of undiluted immunogen was injected into a tail vein. The mouse was left for four days whereupon it was killed and the spleen removed aseptically.

Cell fusion. Fresh immunised spleen cells were fused with Sp2/0-Ag 14 mouse myeloma cells in the ratio of 5 to 1 essentially as described by Paton (1995). Following fusion in the presence of polyethylene glycol (PEG1500, Boehringer, 500g per litre), the cells were suspended in selective medium (Iscoves modified Dulbeccos medium, Gibco BRL) containing 17% fetal calf serum (Gibco BRL hybridoma grade), single strength HAT (hypoxanthine, aminopterin, thymine, Sigma), 0.1% gentamycin (Sigma) and 10% Domadrive (ImmuneSystems). Three dilutions of cells, lin 2, lin 5 and lin 9, were plated at 100 μl per well onto 18 (i.e. 6 plates per dilution) polystyrene 96 well culture plates (Gibco BRL) and cultured without further medium supplementation at 37 C in 5% CO2 for 10 days.

Purification of AAT. alpha- 1 Antitrypsin from plasma of individuals of known phenotype (determined by IEF pH 4.2-4.9) was purified on immunoaffinity columns using goat anti-human AAT polyclonal antibodies (IncStar) conjugated to CNBr Sepharose 4B (Amersham Pharmacia Biotech). AAT proteins of phenotype

Ml, M1M4, M2, and M2Z were prepared. Note that the M4 protein has His at residue 101 and is thus identical to M2 at this position.

Screening for positive hybridomas. The antibody response of the hybridomas was assayed using conventional ELISA on plates coated with AAT, PGMl or GC of known phenotype. Allele-specific antibodies were identified by their capacity to bind the intended allelic variant protein but not the alternative allele. For each fusion experiment, 36 PVC ELISA plates (Greiner) were coated with partly purified AAT protein or PGMl or GC protien containing the intended allele (18 plates) or the alternative allele (18 plates) by incubation of 100 μl of antigen (100 μg/ml in pH 9.6 bicarbonate coating buffer) overnight at room temperature. A two step screening strategy was employed. In the first round the highest dilution culture plates were tested. If several positive allele-specific clones were identified from this round the remaining culture plates were discarded, if not further plates were analysed. A standard two step ELISA method was used to identify antibody positive culture wells. Following incubation with cell culture supernatant and appropriate washing steps, bound antibody was detected using rabbit immunoglobulins to mouse Ig conjugated to horseradish peroxidase (Dako) diluted 1 in 1000 and orthophenyl diamine (OPD) plus H₂O₂as substrate (Sigma). The ELISA reactions were stopped with 10% H₂SO₄ and recorded using an Anthos micro-plate reader at 492 nm.

Cloning: Monoclonal hybridomas from antibody positive wells were obtained by limiting dilution. A series of doubling dilutions were prepared such that the highest had an expected density 1 cell per 6 wells; all hybridomas reported here were picked from the highest dilution plates. For long term storage, the cells were pelleted and resuspended in 2 ml of 90% fetal calf serum and 10% dimethyl sulphoxide (Sigma) and divided between three 1ml cryo- vials (Nunc). The vials were cooled at 1 degree C per minute in a cell freezing device in a -80°C freezer. The following day the cells were transferred to a liquid N2 biological freezer.

SDS electrophoresis. SDS electrophoresis was based on the method of Laemmli (1970). The separation gel consisted of 11% polyacrylamide (37.5:1, acrylamide to bis-acrylamide) and 0.14% SDS; the stacking gel consisted of 3% total monomer and SDS. Electrophoresis was carried out using a Hoefer "Mighty Small" gel apparatus at 150 V until the bromophenol blue from the sample dissolving buffer had reached the anodal end of the gel. For immunodetection, proteins were electroblotted onto nitrocellulose sheets (Schleicher and Schuell, 0.45 μm) using a BioRad Transfor apparatus at 60 mA for 1 hour. The blots were incubated in a suitable dilution of monoclonal antibody, either Ig fraction or culture supernatant, or goat anti-human AAT polyclonal antibodies IgG fraction (1 in 1000), followed by incubation with either horseradish peroxidase labelled rabbit anti-goat globulins or rabbit anti -mouse globulins (both at 1 in 1000 dilution) and the result recorded by chemiluminescence using the ECL system (Amersham Pharmacia Biotech).

Isoelectric focusing. Polyacrylamide gel isoelectric focusing (IEF; gradient pH 4.2 - 4.9 with ACES), pre-treatment of plasma samples with dithiothreitol and iodoacetic acid, immunodetection and protein detection was carried out as described (Whitehouse et al (1989)). To prevent overstaining, plasma samples to be used for immunodetection were diluted in water. In the case of AAT this was as follows: Z plasma, 1 in 5; Ml, MIS and M1Z plasma, 1 in 12. AAT phenotypes were detected non-specifically by Coomassie blue R250 staining or specifically by immunodetection. For immunodetection, proteins were passive blotted from IEF gels onto nitrocellulose sheets (Schleicher and Schuell, 0.45 μm) for 2 h at room temperature. The blots were developed as described for SDS electrophoresis.

Capture ELISA assays. ELISA plates were coated with 501/well, Protein G affinity purified monoclonal antibodies diluted 1 in 10 in 0.1 M bicarbonate coating buffer (pH 9.6) for 2 hours at room temperature or overnight at 4°C. The plates were washed three times in PBS containing 0.1% Tween 20 (PBST) and then blocked with 100 1/well 1% BSA in PBST for 1 hour. The plates were washed twice in PBST. The plasma test sample was diluted with an equal volume of 8M urea and incubated at room temperature for 20 minutes. Treatment with urea is essential and serves to increase the accessibility of the epitope to the paratope. Following incubation the plasma/urea mixture was diluted to 1 in 50 final plasma/serum concentration, 0.16 M urea, with 1% BSA-PBST and 501 added in duplicate to the test wells. The final urea concentration of 0.16 M did not interfere with antibody binding. The plates were incubated for 2 - 3 hours at room temperature after which they were washed four times in PBST. Biotinylated anti-human AAT polyclonal antibodies, diluted 1 : 1000 in 1% BSA-PBST were added to each well (1001/well) and the plate incubated for lhour at room temperature. The plate was washed four times followed by the addition of streptavidin - HRP, diluted 1 :500 in 1% BSA- PBST (1001/well) and incubated for 30 - 60 minutes at room temperature. The plate was then washed six times in PBST and a further three times in pure water before adding the substrate (orthophenyl diamine plus H₂O₂) at 1001/well. The reaction was stopped with 10% H₂SO4 and the plate read at 492 nm. Pre absoφtion of second antibodies was as follows. For biotinylated anti-human AAT polyclonal antibodies, 1 part normal mouse serum was mixed with 5 parts of antibodies and incubated at 4°C overnight before use.

Nitrocellulose rapid ELISA panel tests: A set of 5 x 1cm panels from BA85 (Schleicher & Schuell) nitrocellulose sheets were prepared as required. 4ul diluted capture antibody (1 :5 in 0.1M Bicarbonate coating buffer pH 9.6) was spotted onto the strips and left to dry at 37°C for 20 mins. The membranes were blocked with 5% skimmed dried milk (Marvel) in PBST for 1 hour. The test serum/plasma samples were treated with urea as before and incubated with the capture membrane for 15 mins at room temperature. After washing four times in PBST, the membrane was incubated with biotinylated anti-human AAT polyclonal diluted 1 :500 in 2.5% Marvel-PBST for 10 minutes at room temperature. Following an identical washing step the membrane panel was incubated for 5 minutes in streptavidin - HRP diluted 1 :250 in 2.5% Marvel-PBST for 5 minutes at room temperature and then washed 6 times in PBST and once in PBS. The membrane panel was then incubated in 3 '3' diaminobenzidine plus H₂O₂in PBS until the colour on the positive control strip developed. The reaction was stopped by washing panel with tap water.

RESULTS

We present the results of fusion experiments using the AAT peptide immunogens described. The immunisation and fusion details and the results of cell characterisation are shown in Table 1. All of the immunogens elicited antipeptide responses and produced antibodies that cross-reacted specifically with the intended protein variant but not the opposite isoform. Following cloning to produce Mabs, four monoclonal cell lines were selected for further analysis: 2G2, anti-K342 (Z); 6G5, anti-E342 (non-Z); 3A2, anti-HlOl (M2); 10A, anti-RlOl (non-M2). Isotyping revealed that all were IgGls except 2G2 which was IgG2. Antibody characterisation The mono allelic specificity of each Mab against undenatured AAT of known phenotype was assessed using a simple two step ELISA (based on the hybridoma screening ELISA) and western blot analysis of IEF gels. SDS gel immunoblots were used to assess the specificity of the Mabs for denatured AAT.

For the ELI S As each anti-peptide Mab was tested for its capacity to discriminate between the target isoform and the opposite isoform (Figure 1 ) using partly-purified AAT protein as antigen, except for 6G5 where we used the peptides since homozygous Z protein was not available. For each Mab there was clear discrimination of the target from the opposite isoform across a range of dilutions. Note that IgG fractions of the Mabs were used for these experiments. For the IEF experiments, EDTA plasmas from individuals of known AAT phenotype were focused on narrow gradient pH 4.2-4.9 gels. The results of the immunoblots, which were probed with diluted (1 in 10) Mab hybridoma supematants, showed complete correlation with the ELISAs for each Mab (Figure 2). In all cases only those isoforms containing the target epitope were seen, thus in heterozygous samples the intensity of the pattern was approximately half that of the corresponding homozygous sample. The full range of AAT components was recognised by each Mab suggesting that the epitopes were not obscured by carbohydrate side-chains. To check for spurious crossreactivities of the Mabs with proteins with different pl values, blots of plasma samples were prepared from broad gradient pH 3.5-10 gels and probed with the Mabs 2G2 and 6G5. No further bands were observed indicating the monospecificity of these reagents for AAT.

SDS gel analysis revealed the expected pattern of allele specific antibody recognition in all cases. Intensely stained AAT bands of the expected mobility were seen when the target epitope was present and no AAT bands when it was absent (Figure 3). When whole plasma was used, two additional very weakly staining components were sometimes seen migrating either side of AAT. These were most evident in samples where the target variant was absent. These minor proteins are unlikely to be related to AAT since they were not visible on blots of purified AAT, and they were not detected on blots of plasma samples when polyclonal goat anti- human AAT was used. The avidity of the four Mabs was tested using simple ELISAs and a range of pH values and salt concentrations based on the method of Hoffmann et al (1990). For each antibody, optimal binding was observed between pH 5.0 and pH 9.0. Outside this optimal range there was slight decrease in binding between pH 9.0 and pH 10.0 and considerable decrease from pH 5.0 to pH2.0 (Figure 4a). The effect of NaCl concentration differed between the Mabs. Three, 2G2, 10A and 3A2, showed optimal binding at all NaCl concentrations (between 0.14M and 3.5M) whereas the binding of 6G5 decreased steadily with increasing salt concentration over the same range (Figure 4b). Capture ELISA In order to utilise these four reagents in simple immunoassays for genetic phenotyping various capture ELISA formats were investigated. The principle of these tests was to attach an allele specific Mab antibody to the solid phase and use this to capture the target AAT protein from a complex mixture such as a plasma sample. The presence of the captured AAT target protein was detected by its reactivity towards polyclonal goat anti-human AAT which was linked to an HRP system.

For the microtitre plate ELISAs we compared three methods for attaching the Mabs to the solid phase. Two, hydrazide and streptavidin coated plates required pretreatment of the Mab by oxidation and biotinylation respectively. The third approach was passive adsoφtion of the Mab to the PVC surface of the plate. Since there was no evidence for increased binding when using coated plates (data not shown) whereas the expense is much greater, passive adsoφtion was adopted for general use. In the first capture experiments, polyclonal rabbit anti-goat globulins conjugated to HRP were used to identify the presence of polyclonal goat anti-human AAT. This approach was of limited value since there appeared to be marked crossreactivity between the rabbit anti-goat globulins and the Mab attached to the solid phase. Absoφtion of the conjugate with mouse serum did not improve this problem (data not shown). To circumvent the problems of antibody-antibody interactions, the polyclonal goat anti-human AAT was biotinylated. This scheme allowed direct detection of goat anti-human AAT using a strepavidin-HRP complex.

2G2 anti-AAT Z

The results for 2G2 (anti K342) which is specific for the AAT Z protein, demonstrate absolute discrimination between the target epitope on the Z protein and the complementary non-Z protein (Figure 5). Whilst the OD readings suggested a small degree of crossreactivity with the non-Z protein, this was not the case since similar readings were obtained from the no-plasma control wells. This illustrates the feasibility of producing a Mab against a small synthetic peptide, which is capable of recognising the native target isoform in solution, but not the opposite isoform which differs by a single amino acid. The assay was tested in duplicate using 248 plasmas of known AAT type including 139 that were non-Z and 109 that contained the Z protein. The test was carried out blind to the operator and in all cases the expected result was observed, there were no false positive or false negatives. Urea-ELISA for 6G5, 10A and 3A2

The initial results for the other three Mabs 6G5, 10A and 3A2, were not satisfactory as in each case there were ambiguities of antibody binding. With some plasma samples the expected recognition of the target isoform did not occur, whereas with others crossreaction with the opposite isoform was observed. To overcome these difficulties with recognising the epitope in the native antigen protein in solution, the AAT protein was partially denatured with urea prior to dilution for the capture ELISA. This treatment led to an increased accessibility of the epitopes to the paratopes. The optimal range of urea concentration was found to lie between 3M and 4M (Figure 6a). In practice, incubation of test plasmas with 4M urea led to the complete suppression of spurious reactivities with the opposite isoform and enhancement of binding with the target isoform, irrespective of which Mab was used for antigen capture (Figure 6b). The results of urea-ELISA were entirely conclusive (Figure 7), in each case the expected reactivity was observed. In the case of 3A2, 107 plasma samples were tested in duplicate, without the tester knowing the AAT phenotype and the expected result was obtained in each case when the experiment was decoded. Smaller numbers were tested for 10A and 6G5 but again the results were satisfactory without exception. No false positives or false negatives have yet been recorded for any of the antibodies.

The effect of heat denaturation treatment on plasma samples was also investigated. The results were similar to urea treatment although the difference between positive and negative samples was less marked (data not shown). Rapid nitrocellulose ELISA tests To circumvent the time-demanding pipetting and washing steps, rapid capture ELISAs were formatted using nitrocellulose panels as the solid phase instead of microtitre plates. Exactly the same protocols including urea concentration for sample preparation when using 6G5, 10A and 3A2, were used as before except that the washing steps and incubation times were compressed. Minor modifications to the blocking solution and higher concentrations of secondary antibody and HRP- streptavidin were necessary to produce clean backgrounds and adequate staining discrimination between positive and negative plasmas. The results of the optimised nitrocellulose panels tests, that were be scored by eye, were obtained in less than one hour from sample application (Figure 8).

Other allele specific antibodies

Allele specific antibodies to the polymoφhisms (i), (iii), (iv), (vi), (vii) and (xi) were tested in the same IEF and ELISA systems as described above and were found to bind the relevant polymoφhism.

TABLE 1

REFERENCES

Aldwin L, Nitecki DE (1987) A water-soluble, monitorable peptide and protein crosslinking agent. Anal. Biochem. 164: 494-501

Hoffmann R, Braun A, Cleve H (1990) A monoclonal antibody against human vitamin-D-binding protein for the analysis of genetic variation in the group-specific component system (Gc). Hum Genet 84:137 - 146

Laemmli EK (1970) Cleavage of the structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685

Lovegrove JU, Jeremiah S, Gillett GT, Temple IK, Povey S, Whitehouse DB (1997) A new alpha 1-antitrypsin mutation, Thr - Met 85, (PI Z bristol) associated with novel electrophoretic properties. Ann. Hum. Genet. 61: 385 - 391

Paton A (1990) Preparation and characterisationof isoform- specific anti-peptide antibodies to protein kinase C. M Phil thesis, Oxford Brookes University.

Phillips C, Whitehouse D (1990) A rapid high resolution separator IEF technique for phenotyping alpha- 1 antitrypsin. Advances in Forensic Haemogenetics,

Springer- Verlag, Berlin, Heidelberg. pp274 - 276

Wallmark A, Aim R, Eriksson S (1984) Monoclonal antibody specific for the mutant PiZ alpha 1 -antitrypsin and its application in an ELISA procedure for identification of PiZ gene carriers. Proc Natl Acad Sci USA S1: 5690 - 5693 Whitehouse DB, Lovegrove JU, Hopkinson DA (1989) Variation in alpha-1- antitrypsin phenotypes associated with penicillamine therapy. Clin. Chim. Acta.

179:109-115

Zegers ND, Claassen E, Gerritse K, Deen C, Boersma WJ (1991) Detection of genetic variants of alpha 1-antitrypsin with site-specific monoclonal antibodies. Clin Chem 37: 1606 - 1611

Claims

1. Method of typing a biological sample comprising contacting the sample with one or more antibodies specific for any of the following polymoφhisms: (i) R220 of PGMl, (ii) C220 of PGMl, (iii) Y419 of

PGMl, (iv) H419 of PGMl, (v) D416 T420 of GC, (vi) E416 T420 of GC, (vii) D416 K420 of GC, (viii) HlOl of AAT, (ix) RlOl of AAT, (x) V264 of AAT, (xi) E264 of AAT, (xii) K342 of AAT, (xiii) E342 of AAT, (xiv) V213 of AAT and (xv) A213 of AAT, and determining whether the antibody binds a protein in the sample, binding of the antibody to the protein indicating that the sample contains a protein possessing the polymoφhism for which the antibody is specific.

2. Method according to claim 1 wherein the sample is contacted with at least one or more antibodies specific for any of the polymoφhisms (v), (vi), (vii),

(viii), (ix), (x), (xi), (xiv) or (xv).

3. Method according to claim 1 wherein the sample is contacted with at least one antibody specific for each of the polymoφhisms (i), (ii), (iii), (iv), (v), (vi), (vii), (viii), (ix), (x), (xi), (xii), (xiii), (xiv) or (xv).

4. Method of diagnosing (a) an AAT deficiency, (b) susceptibility to an AAT deficiency, (c) whether an individual is a carrier for a polymoφhism that contributes to an AAT deficiency, comprising contacting a sample from an individual with one or more antibodies specific for either of the following polymoφhisms: (x) V264 of AAT or (xii) K342 of AAT, and determining whether the antibody binds a protein in the sample, binding of the antibody to either polymoφhism indicating that the individual has an AAT deficiency or is susceptible to an AAT deficiency or is a carrier for a polymoφhism that contributes to an AAT deficiency.

5. Method of diagnosing responsiveness to therapy, comprising contacting a sample from an individual with one or more antibodies specific for the polymoφhism (xv) A213 of AAT, and determining whether the antibody binds a protein in the sample, binding of the antibody to the protein indicating that the individual has a high responsiveness to therapy.

6. Method according to any one of the preceding claims in which determining whether the antibody binds a protein in the sample is done by contacting the sample to an antibody as defined in any one of the preceding claims, which antibody is immobilised on a solid support, and then detecting whether any protein is bound to the antibody by contacting the support with a labelled agent that binds specifically to the protein, binding of the labelled agent to the support indicating that the antibody binds protein in the sample.

7. Method according to any one of the preceding claims which additionally comprises exposing the sample to denaturing conditions before or during the contacting.

8. Method according to claim 7 in which the said exposing comprises contacting the sample with urea.

9. An antibody as defined in claim 2.

10. A process of making an antibody as defined in claim 1 or 2 comprising culturing a cell that expresses the antibody and optionally purifying the antibody from the cell.

11. A process according to claim 10 in which the cell is one which is obtainable by administering a peptide comprising a polymoφhism as defined in claim 1 or 2 to a mammal, extracting B cells from the mammal and selecting a cell from these based on the ability to express an antibody which binds the polymoφhism.

12. A process according to claim 10 in which the cell is recombinant for a polynucleotide which expresses the antibody.

13. A dip stick comprising an antibody as defined in claim 1 or 2.

14. A kit comprising an antibody as defined in claim 1 or 2 or a dipstick as defined in claim 13.

15. A kit according to claim 14 that additionally comprises one or more of the following: a denaturing agent, an antibody that binds PGMl, GC or AAT when they bound to an antibody as defined in claim 1 or 2, an antibody that binds the Fc portion of an antibody as defined in claim 1 or 2, a means of labelling protein in a sample, or a means of detecting a labelled antibody or labelled PGMl, GC or AAT.

16. Use of a denaturing agent to increase the binding between an antibody as defined in claim 1 or 2 and a protein comprising a polymoφhism as defined in claim 1 or 2.

17. Method of treating an individual with an AAT deficiency, or who is susceptible to an AAT deficiency, comprising diagnosing whether the individual has an AAT deficiency or is susceptible to an AAT deficiency by the method of claim 4 and then administering an agent therapeutic for the

AAT deficiency to the individuals who have been diagnosed as having AAT deficiency or being susceptible to an AAT deficiency.

18. Method according to claim 17 wherein the agent is AAT or a polynucleotide that is capable of being expressed to provide AAT.

9. A kit according to claim 14 or 15 that additionally comprises an agent as defined in claim 17 or 18.