WO1988007055A1

WO1988007055A1 - Diagnostic agents for systemic vasculitis

Info

Publication number: WO1988007055A1
Application number: PCT/GB1988/000190
Authority: WO
Inventors: Christopher Martin Lockwood; William Gordon Turnell
Original assignee: Royal Postgraduate Medical School
Priority date: 1987-03-11
Filing date: 1988-03-11
Publication date: 1988-09-22
Also published as: GB8705752D0; AU1428088A

Abstract

A peptide consisting of or comprising the amino acid sequence G - B - s/h - P - G - s/h - B', in which G represents glycine, P repesents proline, B represents K, R or N, K representing lysine, R representing arginine, N representing asparagine, B' represents V, A, T, I or L or B, V representing valine, A representing alanine, T representing threonine, I representing isoleucine, L representing leucine, s/h represents a small neutral residue, a hydrophobic residue, or tryosine may be used in an immunoassay for the diagnosis of systemic vasculitis.

Description

Diaqnostic Agents for Systemic Vasculitis

The present invention relates to an agent that can be used in an in vitro diagnostic test for systemic vasculitis and also in the treatment of that condition. The invention further relates to a kit for carrying out the diagnostic test and apparatus for the treatment.

Systemic vasculitis is a condition that is rarely diagnosed in its early stages as patients generally present initially with a variety of non-specific symptoms. Even in its later stages, when certain organs for example, the lungs and especially the kidneys are particularly involved, diagnosis remains difficult as there are still no specific symptoms. Indeed, diagnosis of the condition is often arrived at only by a process of elimination.

Tissue biopsy can give a positive diagnosis but it is not used in routine early screening because of its invasive nature and because of the difficulty in obtaining samples of informative material.

An example of the practical problems in diagnosing systemic vasculitis is shown in the diagnosis of rapidly progressive nephritis, which comprises a heterogeneous group of disorders. Only ten years ago the prognosis of this form of kidney failure was extremely poor and most patients lost all renal functions within two months. Recently, however, it has become clear that those forms of the disease associated with systemic vasculitis will respond to therapy with powerful immunosuppressive drugs and that recovery can be obtained even in patients who are dialysis-dependent at presentation.

Wegener's granulomatosis ( G) is a form of systemic vasculitis in which the pathoαenetic mechanisms are poorly understood. Recently an autoimmune aetiology has been implicated in G by the finding of circulating autoantibodies which were shown by indirect immunofluorescence techniques to bind to cytoplasmic components of alcohol-fixed normal human neutrophils (polymorphonuclear neutrophilic leucocytes) . Titres of these antibodies correlated with disease activity suggesting that their occurrence was not simply an epiphenomenon. The present invention provides an extract of acid- treated neutrophils, which extract is capable of interacting with antibodies characteristic of systemic vasculitis.

The present invention also provides a process for the production of a neutrophil extract of the invention which comprises treating neutrophils with an acidic medium, lysing the resulting acid-treated neutrophils and, preferably, obtaining the liquid phase of the lysed material. The neutrophils to be treated may be obtained from blood or an appropriate blood product. This is most conveniently obtained from normal donors but blood from other donors can be used. A number of ^'methods have been proposed for isolating and/or immobilising neutrophils, and any of these methods may be used in the present invention. The following is an example of such a procedure.

Whole blood (generally from normal donors) is layered over an appropriate density gradient, for example, a methyl cellulose/Hypaque gradient, and the cells allowed to settle at unit gravity. The erythrocytes agglutinate and sediment through the gradient. The supernatant plasma layer containing the buffy coat cells is diluted, centrifuged and resuspended on ice to lyse any remaining erythrocytes. The neutrophils to be extracted are preferably suspended in the acidic medium. The pH of the medium does not appear to be critical provided that the configuration of cell components is generally retained. The pH is, for example, within the range of from 2 to 6, preferably from 3.5 to 5, and especially from 4 to 4.5. The nature of the acid used also does not appear to be critical and an inorganic or organic acid for example, appropriately dilute hydrochloric or nitric acid, or acetic, propionic or butyric acid may be used. It is preferable to use a buffered acidic medium. Buffers generally in use are suitable, for example, an acetate_/ citrate or phosphate buffer, for example, a buffer comprising sodium acetate or sodium hydrogen phosphate.

The neutrophils are preferably allowed to stand in the acidic medium before lysis. The duration of acid treatment is not critical, but is preferably more than about 30 minutes, and the cells may be allowed to stand in the medium for several hours, for example, up to 18 hours. It is often convenient to allow the suspension to stand overnight.

The neutrophils are then lysed to release their contents. The lysis may be carried out on the reaction mixture, particularly when the acidic medium used is buffered or, if desired, the neutrophils may be isolated and suspended in another medium, especially a buffered medium, before lysis. Any method of lysis may be used, sonication being preferred. A particularly effective programme is sonication in one minute bursts to a total of 20 minutes.

The mixture resulting from lysis of the neutrophils is preferably separated into a solid phase and a liquid phase in a known manner, particularly by centrifugation, especially over 300 g. The resulting supernatant is retained and the cell debris discarded.

We have found that a neutrophil extract according to the invention comprises components having molecular weights of lOOkD, 6.2kD and 1.8kD as determined by gel fitration using molecular weight markers. (The range of molecular weights about the given values are in accordance with the method used, and as such are known to those skilled in the art, the range for the 100 kD components being about - 20, for the 6.2kD component about - 2kD, and for the 1.8kD component being about - IkD) .

The present invention accordingly provides a neutrophil extract comprising one or more of such components and provides, moreover, the individual components, that is to say, the present invention firstly provides a component obtained from a neutrophil extract according to the invention and having a

.. molecular weight of lOOkD - 20kD as determined by gel filtration using molecular weight markers. The present invention further provides a component obtained from a neutrophil extract according to the invention and having a molecular weight of 6.2kD - 2kD as determined by gel filtration using molecular weight ma ers. The present,invention further provides a component obtained from a neutrophil extract according to the invention the component having a molecular weight of 1.8kD - IkD as determined by gel filtration using molecular weight markers. Methods capable of separating components having the above molecular weights from a mixture comprising such components are well known and include separation methods based on charge, size, polarity and/or antibody/antigen interactions, for example, gel filtration and ion exchange chromatography, for example, in systems allowing the use of high pressure; affinity chro atography using polyclonal or monoclonal antisera; polyacrylamide gel electrophoresis; and density gradient chromatography. Of these, high performance liquid chromatography is particularly preferred.

Further embodiments of the invention are based on our surprising observations regarding the structure and inter-relationships of the components of the neutrophil extract of the invention.

The present invention further relates to a peptide consisting of or comprising the amino acid sequence of all or part of the loop portion of an enzyme having one or more loops the peptide being capable of interacting with antibodies characteristic of systemic vasculitis. Enzymes having one or more loops include lysozy e and the alkaline phosphatases, especially the human enzymes, and their precursors.

In particular the present invention provides a peptide consisting of or comprising the amino acid sequence I

G - B - s/h - p - G - s/h - B' (I) in which

G represents glycine P represents proline B represents K, R or

K representing lysine R representing arginine N representing asparagine B' represents V, A, T, I or L, or a residue B V representing valine A representing alanine T representing threonine I. representing isoleucine L representing leucine s/h represents a small neutral residue, a hydrophobic residue or tvrosine. (In some cases, the s/h residue adjacent to B' may be any L-amino acid residue.)

A small neutral residue is, for example, a glycine, alanine, serine or threonine residue, and a hydrophobic residue is, for example, a leucine, isoleucine, valine, phenylalanine, tryptopan or methionine residue.

An s/h residue is preferably a threonine or glycine residue (adjacent to 3) or an alanine or tryosine residue (adjacent to B"). The residue represented by B is preferably lysine or asparagine and the residue represented by B' is preferably valine or lysine.

Examples of peptides having amino acid sequences falling within the definition of peptide I are the following peptides II, III and IV

Gly - Lys - Thr - Pro - Gly - Ala - Val (II)

Gly - Asn - Gly - Pro - Gly - Tyr - Val (III)

Gly - Asn - Gly - Pro - Gly - Tyr - Lys (IV)

The present invention also provides a peptide consisting of or comprising the amino acid sequence II, III or IV, and further provides a peptide consisting of or comprising the amino acid sequence Ila, Ilia or IVa

Asp - Gly - Lys - Thr - Pro - Gly - Ala - Val - Asn - Ala Ila

Tyr - Gly - Asn - Gly - Pro - Gly - Tyr - Val - Leu - Lys Ilia

Tyr - Gly - Asn - Gly - Pro - Gly - Tyr - Lys - Val - Val IVa

or consisting of or comprising an amino acid sequence Ila, Ilia, or IVa, respectively, which has been modified in any one or more of the following ways: i) by the removal of one or two amino acid residues selected from the N-terminal residue and the two C-terminal residues, ii) by the replacement of one or more amino acid residues selected from the N-terminal residue and the two C-terminal residues by one or more other amino acid residues, respectively, iii) by the modification of one or more amino acid residues, with the proviso that a peptide consisting of or comprising a modified form of amino acid sequence Ila, Ilia or IVa respectively, must be capable of interacting with circulating antibodies characteristic of systemic vasculitis.

It is well known that it is possible in many cases to modify the structure of a peptide without causing loss of its function. Modifications include the addition or removal of an amino acid residue, the replacement of a residue by a different residue, in particular the replacement of a small or neutral residue by another small or neutral residue or of a hydrophobic residue by another hydrophobic residue, and the modification of a residue, for example, by protection or other alteration to a free, (reactive) group, for example, a free hydroxy or amino group.

In the present case, the replacement of any one or more of three replaceable residues may be by any other L-amino acid residue. In the case of the C-terminal residue, replacement by another small or neutral residue or tyrosine is preferred, for example in peptide Ilia the terminal tyrosine may be replaced by leucine.

Of these peptides, those consisting of or comprising amino acid sequence IV and IVa may be particularly useful in the diagnosis and/or treatment of systemic vasculitis.

As indicated above, a peptide of the invention may consist of or comprise the specified amino acid sequence, that is to say, such an amino acid sequence may form part of a larger peptide even one large enough to be considered a protein. By way of example, peptide I may be part of the full amino acid sequence or a partial amino acid sequence of human lysozyme or a human alkaline phosphatase; peptide II or Ila may be part of the full or a partial amino acid sequence of human lysozyme, peptide III or Ilia may be part of the full or a partial amino acid sequence of human placental alkaline phosphatase or a precursor thereof, and peptide IV or IVa may be part of the full or a partial amino acid sequence of human liver/kidney/bone alkaline phosphatase or a precursor thereof.

It is to be understood that the term "peptide" as used in the present specification includes polypeptides and proteins.

Any modification to a peptide of the invention that does not result in loss of activity may be carried out. A peptide of the invention may be obtained from natural sources or may be produced synthetically. Any of the many known chemical methods of peptide synthesis may be used, especially those utilising automated apparatus. A peptide according to the invention may be produced using the techniques of recombinant DNA technology, for example, by construction of a gene, for example, by chemical synthesis or by reverse transcription from the corresponding m-RNA, insertion of the gene into an appropriate vector, for example, a plasmid, for example, pBR322, insertion of the vector into a host organism, for example, E. Coli, and expression of the gene in the host organism. Such procedures are now routine, particularly as vectors, for example, pBR 322 are available commercially. A peptide of the invention may be as defined above per se or as indicated, may form part of a larger molecule, for example, human lysozyme or a human alkaline phosphatase, for example, the present invention further provides a peptide consisting of or comprising the amino acid sequence of human lysozyme from the N-terminal residue to residue 76:

Lys - Val - Phe - Glu - Arg - Cys - Glu - Leu - Ala - - Arg

Thr - Leu - Lys - Arg - Leu - Gly - Met - Asp - Gly • - Tyr

Arg - Gly - He - Ser - Leu - Ala - Asn - Trp - Met - - Cys

Leu - Ala - Lys - Trp - Glu - Ser - Gly - Tyr - Asn - - Thr

Arg - Ala - Thr - Asn - Tyr - Asn - Ala - Gly - Asp - - Arg Ser - Thr - Asp - Tyr - Gly - He - Phe - Gin - He - - Asn

Ser - Arg - Tyr - Trp - Cys - Asn - Asp - Gly - Lys - - Thr

Pro - Gly - Ala - Val - Asn - Ala -

(The entire sequence of human lysozyme is given by Taiji I., Johnson N.L., North A.C.T., Philips D.C. and Ripley J.A. in Boyer P.D. The Enzymes, Academic Press 1982) .

As indicated above, a neutrophil extract according to the present invention interacts with antibodies characteristic of systemic vasculitides , as do the 100 kD, 6.2 kD and 1.8 kD components and also . the various peptides of the invention. The interaction in each case is of the antigen-antibody type. The present invention accordingly provides a method of qualitatively or quantitatively detecting antibodies characte istic of systemic vasculitis, which comprises carrying out an immunoassay using, as the antibody under investigation, a sample of body fluid and, as the antigenic reagent, any one or more of the following: a neutrophil extract of the invention, a lOOkD, 6kD or 1.8kD component of the invention, or a peptide of the invention.

The body fluid to be tested is generally serum, but other fluids may be used, for example, cerebrospinal fluid or fluid obtained from a joint. Im unoassay techniques, both qualitative and quantitative, are well known and include ELISA (enzyme linked immunosorbent assays) , Western blotting, fluid phase precipitation assays, coated particle assays and solid phase radio immunoassays (SPRIA) . Of these,

ELISA and solid phase radioimmunoassay are particularly convenient in the present case. Accordingly, aliquots of the antigenic reagent according to the present invention are adsorbed on to a solid phase support, for example, a plastics material, for example, the wells of plastics microtitre plates, samples of the body fluid under investigation are incubated in contact with the antigenic reagent and any resulting bound antibody is detected using a radio-labelled antibody that is capable of binding to the bound antibody under investigation. Anti-human i munoglobulin is preferred. Any appropriate radioisotope may be used as the label, for example, a 3-emitter or a γ-emitter, examples being ¹²⁵I, ¹³¹I, ³H, and ¹⁴C. The bound radioactivity may be measured conventionally.

An enzyme-linked immunoassay may be carried out conventionally and analogously to be SPRIA, using, for example, an enzyme which binds with or reacts with a covert coloured target.

A specific monoclonal antibody (see below) is especially useful as a control.

The present invention also provides a solid phase support having suitable for use in an immunoassay or in affinity chromategraphy and comprising an antigenic reagent as defined above.

The present invention further provides a kit for the investigation of systemic vasculitis, which comprises: . a) a solid phase support comprising an antigenic reagent as defined above, b) labelled antibody capable of reactinσ directly or indirectly with antibodies present in systemic vasculitis, in particular, a radiolabelled antibody, especially anti-human immunoglobulin and, preferably, c) a control reagent, especially a monoclonal antibody (see below) .

As the various embodiments of the invention are or comprise antigenic substances or haptens, it is possible to use them to produce corresponding polyclonal and monoclonal antibodies, and such polyclonal and monoclonal antibodies are themselves part of the present invention.

A neutrophil extract according to the invention is a heterogeneous mixture, and can be used to raise polyclonal antibodies. It is preferred, however, to use one of the isolated components or one of the peptides of the present invention as the antigen for monoclonal antibody production. Any of the known techniques may be used. (In the case of peptides that are too small to have antigenic properties themselves, it is necessary to conjugate such a peptide hapten to an appropriate larger molecule in the usual manner.)

Polyclonal antibodies or, especially, a monoclonal antibody of the invention may be used in affinity chromatography as an im unosorbent in the purification of antigens according to the present invention, and the present invention provides both an immunosorbent comprising polyclonal antibodies or, especially, a monoclonal antibody of the present invention, generally adsorbed on or otherwise carried by a solid support in a conventional manner (see below) , and a method of purifying antigens of the present invention using such supported antibodies ( immunosorbents) . An antigenic reagent of the invention, as defined above, and especially a peptide of the invention, preferably in as pure a form as possible, in particular, when purified by affinity chromatography using a monoclonal antibody of the invention and especially when produced synthetically, may be used for the treatment of blood of patients with systemic vasculitis e.g. by affinity chromatography. Such a method is a further embodiment of the present invention.

Such treatment may be carried out continuously, using apparatus analogous to dialysis apparatus but utilising a method of affinity chromatography, or batch-wise. In the former case, a column comprising an antigenic reagent, especially a peptide, of the invention adsorbed or otherwise bound on to a solid support, for example, Sepharose (Trade Mark), silica gel, or a membrane, for example, of vinyl acetate, may be interposed in an extracorporeal circuit. In the latter case, samples of blood may be removed from a patient, and treated with suitable immobilised peptide, for example, adsorbed or bound to a support as described above, then returned to the patient's body. Large peptides of the invention, even human lysozyme or a human alkaline phosphatase, can be used, but are generally not preferred because of possible antigenic effects and also, in the case of enzymes or partial sequences of enzymes that retain an active centre, the requirement in most cases to inhibit the enzyme activity.

An advantage of the use of a peptide of amino acid sequence I, II, III, IV, Ila, Ilia and IVa in affinity chromatography is that the peptide is too small to be antigenic per se, so if any peptide is washed off during the treatment of a patient's serum, there will be no undesirable immunogenic response in the patient on the return of the plasma to the patient. These small peptides are also easier to synthesise chemically.

This method of treatment of systemic vasculitis provides an alternative to the use of powerful immunosuppressive agents with their attendant risks.

The relationship between the various embodiments of the invention appears to be the presence of at least one common antigenic determinant, which appears to be or to be related to the loop sequence of certain loop sequence-bearing enzymes, for example, lysozyme, and the various forms of alkaline phophatase. Amino acid sequence I is a template for recognition of such loop sequences and amino acid sequences Ila, Ilia and IVa are loop sequences of human lysozyme (residues 67 to 76 inclusive) , of human placental alkaline phosphatase and its precursor (residues 394 to 403 inclusive of the enzyme, residues 415 to 424 inclusive of its precursor) , and of human liver/bone/ kidney alkaline phosphatase and its precursor (residues 415 to 424 inclusive of the precursor) , respectively. Amino acid sequence Ila was obtained by sequencing the 1.8kD component according to the invention and amino acid sequences Ilia and IVa by template matching. Amino acid sequencing of the 6.2kD component indicates a peptide having at least the first 20 N- terminal amino acids of lysozyme. Reduction and alkylation of the 6.2 kD component gives the 1.8kD component, which retains its antgenicity. The 6.2kD component is considered to consist of or comprise the amino acid sequence IV given above.

A monoclonal antibody raised against the 6.2 kD component (monoclonal antibody W8) bound to all three components of a neutrophil extract according to the invention, (100 kD, 6.2 kD and 1.8 kD) , indicating that common determinants are present in the three substances. It is believed that, as indicated above, it is the loop sequence that is or comprises this common antigenic determinant. Peptides Ila and Ilia have been produced by chemical synthesis and tested with the monoclonal antibody W8. A positive result was obtained in each case, which further confirms the role of the loop sequence.

When sera of patients suffering from different types of systemic vasculitis were tested using the neutrophil extract, the isolated 100 kD, 6.2 kD and 1.8 kD components and also peptides Ila and Ilia produced by chemical synthesis, it was found that sera from patients with WG bound to each of the three components (100 kD, 6.2 kD, and 1.8 kD) and to synthetic peptides Ila and Ilia, whereas sera from patients with microscopic polyarteritis (MP) bound only to the 100 kD component. This result enables differential diagnosis to be made. However, as the treatment for both WG and MP is the same, it is not essential to determine which form of the disease is present.

The immunoassay of the invention may be used to diagnose systemic vasculitides, in particular in the case of patients presenting with renal failure, and also subsequently to monitor the course of the disease and the effect of treatment. This is particularly useful in the present case since the drugs of choice are powerful immunosuppressive agents, and the ability to tailor the drug therapy to disease activity should lessen the risk of opportunistic infection attendent on continuous high dose immunosuppression.

The terms "systemic vasculitis" and "systemic vasculitides" are used herein to mean any one or more of the forms of this condition. The term "antibodies characteristic of systemic vasculitis" denotes antibodies that are characteristic either of any one particular form of systemic vasculitis or of all forms of systemic vasculitis.

The following Examples illustrate the-invention. Material and Methods

(a) Patient sera

Samples of sera were obtained from 8 patients with WG (characterised clinically by predominant upper respiratory tract involvement with cavitating lesions on chest X-ray and/or histologically by granuloma formation in biopsy material), and 4 patients with MP (characterised by clinical evidence of small vessel vasculitis - small gut disease, mononeuritis multiplex or cutaneous leucocytoclastic vasculitis) . All patients had renal biopsy evidence of necrotising glomerulitis. The sera were stored at -20°C until used.

Control sera were obtained from patients with systemic lupus erythematosus (10) , tuberculosis (5) , sarcoidosis (8) , asthma (6) , bronchitis (6) , pneumococcal pneumonia (5) , fibrosing alveolitis (8) and patients with glomerulonephritis, either of the primary idiopathic variety (17) or developing in association with autoantibodies to the glomerular basement membrane (31) . Normal sera (10) were obtained from healthy, non-laboratory, personnel.

(b) Indicator sera

Fluorescein conjugated (FITC) rabbit anti-human light chain sera (Dako) were used for indirect immunofluorescence studies. 1251-labelled goat human IgG was used for radioim unoassays. (c) Separation of neutrophils

A standard separation procedure was used to isolate and/or immobilise the neutrophils. Whole blood (from normal donors) was layered over a methyl cellulose/hypaque gradient (1.25% methyl cellulose and 13% hypaque) and the cells allowed to settle at unit gravity. The erythrocytes agglutinated and sedimented through the gradient. The supernatant plasma layer containing the buffy coat cells was diluted 1/2 in phosphate buffered saline (PBS, Oxid) pH 7.2 and centrifuged at 700 g at 4°C to deposit the neutrophils.

(d) Indirect immunofluorescence studies on normal neutrophils

Neutrophils were obtained as described in c) above, the centrifugation at 700 g being carried out for 10 minutes. After centrifugation the supernatant was examined microscopically to confirm that all the neutrophils had sedimented and the supernatant was discarded. The separated neutrophils (and monocytes) were washed in PBS and attached to plain glass microscope slides (Chance) in a cytocentrifuge or multi- well glass slides (Henley-Essex) suitable for titration studies. The cells were then fixed with 100% ethyl alcohol. All procedures, including washing between stages, were carried out at 4°C.

Sera from patients with WG, MP or from controls were diluted to 1/16 in PBS for cytoprep qualitative work and from 1/16 to 1/512 for quantitative titrations before overlay on the immobilised white cells. After incubation for one hour at 4°C the slides were washed three times in PBS. Specific IgG antibody binding was recognised using FITC rabbit anti-human IgG diluted 1/32 and incubation for a further hour at 4°C. Slides were then examined by UV microscopy. Sera were considered positive for neutrophil antibodies when the majority of the cells examined showed bright cytoplas ic fluorescence.

We were able to confirm that sera from patients with active WG contained an autoantibodv which bound to neutrophil (and monocyte) cytoplasmic constituents in a granular pattern. Fluorescence positivity could be detected up to a dilution of 1/256 in sera from patients with active vasculitis. Sera from normals or controls did not bind to the cell preparations at the screening dilution of 1/16 used.

Example 1:

Acid extraction of neutrophil antigens

Neutrophils were separated as described in c) above, the centrifugation being carried out at 700 g for 5 minutes at 4°C. The neutrophils were resuspended in NH_*C1 for 5 minutes on ice to lyse any . remaining erythrocytes. After washing with PBS the cells were resuspended in sodium acetate (200 mmol/1, pH 4.2) to a volume of 10ml. The suspension was transferred to a thick glass tube on ice and sonicated in bursts of 1 minute to a total of 20 minutes.

Following centrifugation at 8000 g for 30 minutes at 4°C, the supernatant was removed, and the optical density at 280nm (OD230) ^was determined before storage in one ml aliquots in glass tubes at -70°C. Example 2 :

Solid phase radioimmunoassay (SPRIA) using neutrophil acid extract

The OD₂8o °f ^he ^aci<3 extract was adjusted to 0.3 in complement fixation diluent (CFD, Oxoid) and then diluted 1/50 in the same buffer. One hundred il aliquots of the neutrophil acid extract were then coated to the wells of plastics microtitre plates (Dynatech) by incubation overnight at 4°C. The plates were washed three times in CFD containing 0.05% Tween

(CFDT). Test or control sera, diluted 1/4 in CFDT, were incubated for one hour at 37°C. After washing, the binding of specific antibody was recognised by addition of 100!l ¹²⁵I-goat anti-human IgG 200,000cpm (Sp Ac 3^ClΛg) added to each well, for one hour at 37°C. After washing, the wells were counted in an 1KB multichannel gamma counter.

Binding of sera from patients with WG or MP was readily identifiable. For screening sera for antibody activity and for quantitative studies the binding of test sera was expressed as a percentage of the binding of a reference strong positive serum (100%) previously standardised to the binding of a pool of normal sera. Figure 1 shows the range of binding of normals and of patients with systemic vasculitis (SV) pre and post treatment, primary glomerulonephritis (1°GN) and systemic lupus erythematosus (SLE) . Patients diagnosed clinically pretreatment as WG are shown as « those with MP as Θ . Binding of normal sera was 8.1% + 3.9% (+ 2 SD) that of a reference strong positive serum. Control sera gave values of binding similar to those obtained with normals except for 2/10 patients with SLE, see Figure 1. The coefficient of variation between assays was 16.5% .

Example 3:

HPLC f actionation of neutrophil acid extract

Gel filtration was carried out on a Toyosoda TSK- 2000 column using a Waters 6000-A delivery system and the optical density of the fractions at 280nm was measured by a Waters model 440 absorbance detector and recorded on a Data Module. One ml fractions were collected by an LKB 2211 fraction collector. Separate studies had confirmed that the TSK 2000 column was capable of resolving molecules having molecular weights within the range of from 100,000 to 1,000 Daltons.

The column was equilibrated with acetate buffer (200 mmol/1, pH 4.2) at a flow rate of lml/min. The acid extract of the neutrophil antigen (OD 0.3 at 280nm) was passed through a 0.45^-m filter and 100/^.1 applied to the column. The' one ml fractions were collected in glass and 100

volumes were aliquotted in duplicate, in sequence, into microtitre plates which were incubated for 18 hours at 4°C, and then washed three times in CFDT. A printed record of each filtration as a chromatogram was also produced.

Sera from patients with well characterised WG or MP were then diluted 1/4 CFDT and tested for their binding to the HPLC fractionated antigen by SPRIA as described in Example 2 using the fractionated antigens instead of the neutrophil acid extract. Control sera were tested in the same way.

The elution profile (as determined by optical density at 280 nm) of the neutrophil acid extract fractionated on the TSK 2000 gel filtration column showed up to 12 peaks of absorbance see Figure 2(a) . Generally only four of these peaks could readily be detected and the binding of WG or MP sera to such a fractionated extract is shown in Figure 2(b). Sera from patients with WG bound to three separate OD280 P^{eal s} of approximately 100 (peak 2) 6.2 (peak 6) and 1.8

(peak 10) kD. Sera from patients with MP bound only to the 100 kD peak. Such contrasting patterns of binding were seen with six patients with WG and four patients with MP. Occasionally only one peak, corresponding to peak 6 was obtained after fractionation of the neutrophil extract. Other studies (not shown) suggested that peak 6 was a constitutive component of resting neutrophils and that the other peaks represented the products of activation of these cells.

Example 4:

Production of a monoclonal antibody to an acid extractable neutrophil antigen

A standard hybridoma technique (cf. Galfre G, Howe SC, Milstein C, Butcher GW and Howard JC. Antibodies to major histocompatibility antigens produced by hybird cell lines. Nature (1977) 266, 550-552) were used to raise monoclonal antibodies in Balb/C mice to the single protein peak P6, isolated from non-activated neutrophils. The antigen (200/ .; OD₂go 0.3) was given in an equal volume of complete Freund's adjuvant intramuscularly and this immunisation was followed by a boost with an equal dose at day 14. Spleens were harvested 5 days later. Fusions were carried out as described by Pressey A. Pusey CD, Dash A, Peters DK and Lockwood CM. Production of a monoclonal antibody to autoantigenic components of human glomerular basement membrane. Clin. Exp. Immunol (1983) 54, 178 - 184. In detail, 4 x 10 mouse myeloma cells (mouse strain NF1) were fused with spleen ^'cells obtained from 2 mice. After fusion, the cells were plated into 480 wells (20 plates, 24 wells per plate) . Hybridoma supernatants were screened by solid phase radioimmunoassay using neutrophil acid extract as solid phase ligand as described in Example 2.

Seventeen positive clones were found on testing at day 9 and day 22. After cloning out five times by limiting dilution two strongly positive clones, W8 and W18 , were obtained. The mouse monoclonal antibody W8 was tested on the fractionated neutrophil acid extract as ligand on tne SPRIA using a cross reacting I-labelled anti-rat IgG (gift of C. Barrett) to detect specific binding. Three peaks of binding activity were obtained over the same fractions recognised by WG sera (as shown in Figure 3) .

Example 5:

Gel filtration of acid extractable neutrophil antigen

i) Mobile phase : barbital buffered saline pH 7.6 0 ii) Stationary phase : TSK 2000 column iii) Flow rate : 1 l/min iv) Temperature : Ambient

100^1 aliquots of acid extractable neutrophil antigen in 0.2M sodium acetate were run on a TSK 2000 column as described in Example 3 except that borbital buffered saline pH 7.6 0.15 M was used as the mobile phase. Peaks 6 and 10 now at neutral pH were collected and their antigenicity confirmed by coating to microtitre plates and retesting with positive sera and monoclonal antibody W8.

Example 6:

Reverse phase chromatography of purified peaks 6 and 10

i) Mobile phase:

A = H₂0, 0.02% (C₂H₅)₃N, triethylamine, acetic acid to pH 4.5

B = 50:50 H₂0 : acetonitrile, 0.02% triethylamine, acetic acid to pH 4.5 ii) Stationary phase : Novapak C18 iϋ) Flow rate : 1 ml/min iv) Temperature r 35°C

100^1 aliquots of peak 6 or peak 10 obtained according to Example 5 were run in gradient A - B. The column was equilibrated with 99% A, 1% B at 1 ml/min at 35°C. The gradient operation was as follows:

Time (Mins) Flow (ml/min) %A %B Slope

Initial 1.0 99 X -

2.0 1.0 99 1 Linear

3.5 1.0 87 13 Curvilinear

8.0 1.0 50 50 Linear

12.0 1.0 50 50 Linear

13.0 1.0 90 1 Linear

99.0 1.0 99 1 Linear

100.0 0.1 99 1 Linear

The column was allowed to equilibrate for 10 minutes between runs.

Peak 6 ran as two components, retention times 26 and 32 minutes. Peak 10 ran as major single peak, retention time 10.6 - 10.8 minutes. The antigenicity was tested and confirmed using monoclonal antibody W8.

Example 7:

5 Sequencing data

Peak 6 fractions a and b, and peak 10 after reverse phase chromatography as described in Example 6 were collected, lyophilised and sequenced in an Automated Biosystems Sequencer according to the 10 manufacturer's instructions.

Peak 6 (a) = sequencing was carried out as far as the N terminal 20 amino acids. The sequence obtained is shown below and corresponds to the first 20 15 N-terminal amino acids of human lysozyme:

Lys - Val - Phe - Glu - Arg - Cys - Glu - Leu - Ala - Arg - Thr - Leu - Lys - Arg - Leu - Gly - Met - Asp - Gly - Tyr -

(b) = not sequenceable Peak 10 = residues (65,66) 67-76 (77,78) of human lysozyme 67 76

Asp - Gly - Lys - Thr - Pro - Gly - Ala - Val - Asn - Ala A s trong sequence homology wi th res idues 394-403 of human placental alkaline phosphatase and of res idues 415 to 424 of the precur sor of human liver/bone/kidney alkaline phosphatase was shown by template match ing : 394 403

Tyr - Gly - Asn - Gly - Pro - Gly - Tyr - Val - Leu - Lys

415 424

Tyr - Gly - Asn - Gly - Pro - Gly - Tyr - Lys - Val - Val

Example 8 :

Synthetic peptides

A peptide consisting of residues 67-76 of human lysozyme and a peptide consisting of residues 394-403 of human placental alkaline phosphatase, the amino acid sequences of which peptides are shown in Example 7, were synthesised chemically by Cambridge Research Biochemicals, using the F oc method (fluorenyl methoxycarbonyl protected amino acids) in automated sequencer apparatus (Applied Biosystems Ltd) cf Ashton C. and Hardfield D. An Approach to Automated Peptide Synthesis, also Sheppard R.C. and Williams B.J. and Williams T.H.T., J.C.S. Chem. Comm., 587, 1982 and Sheppard R.C, and Williams B.J., Int. J. Peptide and Protein Research, 20, 451, 1982. Both peptides were tested with monoclonal antibody W8 and human sera in the solid phase assay described in Example 2 and both were shown to be active, that is to say, both bound to the antibody.

Claims

Cla ims :

1. A peptide consisting of or comprising the amino acid sequence I

G - B - s/h - P - G - s/h - B' (I)

in which

G represents glycine P represents proline B represents K, R or N

K representing lysine

R representing arginine

N representing asparagine B¹ represents V, A, T, I or L or B

V representing valine

A representing alanine

T representing threonine

I representing isoleucine

L representing leucine s/h represents a small neutral residue, a hydrophobic residue, or tryosine.

2. A peptide as claimed in claim 1 , consisting of or comprising an amino acid sequence II, III or IV

Gly - Lys - Thr - Pro - Gly - Ala - Val (II)

Gly - Asn - Gly - Pro - Gly -^■ Tyr - Val (III)

Gly - Asn - Gly - Pro - Gly - Tyr - Lys (IV)

3. A peptide as claimed in claim 1, consisting of or comprising the amino acid sequence Ila, Ilia or IVa -26-

sp - Gly - Lys - Thr - Pro - Gly - Ala - Val - Asn - ^Ala I^la

Tyr - _Gly - Asn - Gly - Pro - Gly - Tyr - Val - Leu - Lys I^lia

Tyr - _Gly - Asn - Gly - Pro - Gly - Tyr - Lys - Val - ^Val IVa

or consisting of or comprising an amino acid sequence Ila, Ilia or IVa which has been modified in any one or more of the following ways: i) by the removal of one or two amino acid residues selected from the N-terminal residue and the two C-terminal residues, 0 ϋ) by the replacement of' one or more amino acid residues selected from the N-terminal residue and the two C-terminal residues by one or more other amino acid residues, respectively, iii) by the modification of one or more amino acid 5 residues; with the proviso that a peptide consisting of or comprising a modified form of amino acid sequence Ila, Ilia or IVa must be capable of interacting with circulating antibodies characteristic of systemic 0 vasculitis.

4. A peptide as claimed in claim 1, wherein the amino acid sequence is part of the full or a partial amino acid sequence of human lysozyme or a human alkaline phosphatase.

5 5. A peptide as claimed in claim 3, wherein the amino acid sequence II is part of the full or a partial amino acid sequence of human lysozyme; the amino acid sequence III is part of the full or a partial amino acid sequence of human placental alkaline phosphatase or a precursor thereof; and the amino acid sequence IV is part of the full or a partial amino acid sequence of human liver/kidney/bone alkaline phosphatase or a precursor thereof.

6. A peptide as claimed in any one of claims 1 to 5, which has been produced by chemical synthesis or by recombinant DNA technology.

7. The use of a peptide as claimed in claim 1 in an immunoassay for systemic vasculitis or in the treatment of blood or a blood product to remove antibodies characte istic of systemic vasculitis.

8. An extract of acid-treated neutrophils, the extract being capable of interacting with antibodies characteristic of systemic vasculitis.

9. A neutrophil extract as claimed in claim 8, comprising the fluid phase of lysed, acid-treated neutrophils.

10. A process for producing a neutrophil extract capable of interacting .with antibodies characteristic of systemic vasculitis which comprises treating neutrophils with an acidic medium, lysing the resulting acid-treated neutrophils, and obtaining the liquid phase of the lysed material.

11. A process as claimed in claim 10, wherein the pH of the acidic medium is within the range of from 2 to 6.

12. A process as claimed in claim 11 , wherein the pH is within the range of from 3.5 to 5.

13. A process as claimed in claim 12, wherein the pH is within the range of from 4 to 4.5.

14. A neutrophil extract as claimed in claim 8 or claim 9, which comprises a component having a molecular weight of 100 kD - 20 kD, a component having a molecular weight of 6.2 kD - 2 kD, and a component having a molecular weight of 1.8 kD - 1 kD, the molecular weights being determined by gel filtration using molecular weight markers.

15. A component isolated from a neutrophil extract as claimed in any one of claims 8, 9, and 14 and having a molecular weight of 100 kD - 20 kD as determined by ge. filtration using molecular weight markers.

16. A component isolated from a neutrophil extract as claimed in any one of claims 8, 9, and 14 and having a molecular weight of 6.2 kD - 2 kD as determined by gel filtration using molecular weight markers.

A component isolated from a neutroohil extract as claimed in any one of claims 8, 9, and 14 having a molecular weight of 1.8 kD - 1 kD as determined by filtration using molecular weight markers.

18. A peptide as claimed in claim 1, when present in an extract as claimed in claim 8 or when obtained from an extract as claimed in claim 8.

19. A method of determining qualitatively or quantitatively antibodies characteristic of systemic vasculitis, which comprises carrying out an immunoassay using, as the antibody under investigation, a sample of body fluid and, as the antigenic reagent, a neutrophil extract as claimed in any one of claims 8, 9, and 14, a component as claimed in any one of claims 15 to 17, or a peptide as claimed in any one of claims 1 to 6 or claim 18.

20. A method as claimed in claim 19, wherein the body fluid is serum, cerebrospinal fluid or joint fluid.

21. A method as claimed in claim 19 or claim 20, wherein the immunoassay is a solid phase radio¬ immunoassay or an enzyme-linked immunoassay.

22. A solid phase support suitable for use in an immunoassay or in affinity chromotography and comprising an antigenic reagent as defined in claim 19.

23. A kit for investigation of systemic vasculitis, which comprises: a) a solid phase support comprising an antigenic reagent as defined in claim 19, and b) a labelled antibody capable of reacting directly or indirectly with antibodies present in systemic vasculitis.

24. A kit as claimed in claim 23, wherein the labelled antibodies are labelled anti-human immunogloblin.

25. A kit as claimed in claim 23 or claim 24, which also comprises one or more control reagents.

26. Polyclonal antibodies directed against any one or more of the antigenic reagents defined in claim 19.

27. A monoclonal antibody directed against a component as claimed in any one of claims 15 to 17 or a peptide as defined in any one of claims 1 to 6 or claim 18.

28. A solid phase support suitable for use in an immunoassay or in affinity chromatography which comprises polyclonal antibodies as claimed in claim 26.

29. A solid phase support suitable for use in an immunoassay or in affinity chromatography which comprises a monoclonal antibody as claimed in claim 27.

30. A method of purifying an antigenic reagent as defined in claim 19, which comprises subjecting a liquid comprising the antigenic reagent to affinity chromatography using polyclonal antibodies as claimed in claim 26 or one or more monoclonal antibodies as claimed in claim 27.

31. A method as claimed in claim 30 for purifying a peptide as claimed in any one of claims 1 to 6 or claim 18, which comprises subjecting a mixture comprising the peptide to affinity chromatography using the corresponding monoclonal antibody as claimed in claim 27.

32. A method of treating blood or a blood product to remove antibodies characte istic of systemic vasculitis, which comprises contacting the blood or blood product with an antigenic reagent as defined in claim 19.

33. A method as claimed in claim 32, wherein the antigenic reagent is a peptide as claimed in any one of claims 1 to 6 or claim 18.

34. A method as claimed in claim 32 or claim 33, wherein the antigenic reagent is carried on a solid phase support as claimed in claim 22.

35. A solid phase support suitable for use in the method as claimed in claim 32, which comprises a peptide as claimed in any one of claims 1 to 6 or claim 18.

36. The use of an antiqenic reagent as defined in claim 19 in an immunoassay for systemic vasculitis.

37. The use of an antigenic reagent as defined in claim 19, in the treatment of blood or a blood product to remove antibodies characteristic of systemic vasculitis.