AU637701B2

AU637701B2 – Immunodiagnostic assay for rheumatoid arthritis
– Google Patents

AU637701B2 – Immunodiagnostic assay for rheumatoid arthritis
– Google Patents
Immunodiagnostic assay for rheumatoid arthritis

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Publication number
AU637701B2

AU637701B2
AU77370/91A
AU7737091A
AU637701B2
AU 637701 B2
AU637701 B2
AU 637701B2
AU 77370/91 A
AU77370/91 A
AU 77370/91A
AU 7737091 A
AU7737091 A
AU 7737091A
AU 637701 B2
AU637701 B2
AU 637701B2
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AU
Australia
Prior art keywords
iga
complex
antibody
val
ligand
Prior art date
1990-05-25
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AU77370/91A
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AU7737091A
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Inventor
Ian Victor Lewin
Sarita Nayyar
Denis Raymond Stanworth
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Sanofi Pasteur Holding Ltd

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British Technology Group Ltd
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1990-05-25
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1991-05-27
Publication date
1993-06-03

1990-05-25
Priority claimed from GB909011702A
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patent/GB9011702D0/en

1990-06-26
Priority claimed from GB909014227A
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patent/GB9014227D0/en

1991-05-27
Application filed by British Technology Group Ltd
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British Technology Group Ltd

1991-11-28
Publication of AU7737091A
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patent/AU7737091A/en

1992-09-10
Assigned to BRITISH TECHNOLOGY GROUP LIMITED
reassignment
BRITISH TECHNOLOGY GROUP LIMITED
Alteration of Name(s) of Applicant(s) under S113
Assignors: NATIONAL RESEARCH DEVELOPMENT CORPORATION

1993-06-03
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1993-06-03
Publication of AU637701B2
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patent/AU637701B2/en

1994-09-01
Assigned to PEPTIDE THERAPEUTICS LIMITED
reassignment
PEPTIDE THERAPEUTICS LIMITED
Alteration of Name(s) in Register under S187
Assignors: BRITISH TECHNOLOGY GROUP LIMITED

2011-05-27
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Classifications

C—CHEMISTRY; METALLURGY

C07—ORGANIC CHEMISTRY

C07K—PEPTIDES

C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins

C—CHEMISTRY; METALLURGY

C07—ORGANIC CHEMISTRY

C07K—PEPTIDES

C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

C07K16/38—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against protease inhibitors of peptide structure

Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC

Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

Y10S435/00—Chemistry: molecular biology and microbiology

Y10S435/81—Packaged device or kit

Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC

Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

Y10S435/00—Chemistry: molecular biology and microbiology

Y10S435/975—Kit

Abstract

The assay of rheumatoid arthritis by reference to IgA- alpha 1 antitrypsin complex present in analytes is facilitated by certain novel antibody reagents. These are ligands comprising an antibody domain specific for an antigenic determinant of a complex of human IgA and alpha 1-antitrypsin, this antibody domain being substantially non reactive with free human IgA and free human alpha 1-antitrypsin. Monoclonal antibodies to the naturally occurring IgA- alpha 1AT complex and monoclonal or polyclonal antibodies to a synthetic peptide are preferred. The synthetic peptide in itself part of the invention and preferably has an amino acid sequence: Val-Ser-Val-Val-Met-Ala-Glu-Val-Asp-Gly-Thr-Cys-Tyr (SEQ ID NO:

Description

637701
AUSTRALIA
PATENTS ACT 1990 COMPLETE SPECIFICATION NAME OF APPLICANT(S): -N a tio n a R s c a r h B e-l p m n C o r -po ra tio n 1 -o ADDRESS FOR SERVICE: DAVIES COLLISON Patent Attorneys 1 Little Collins Street, Melbourne, 3000.
INVENTION TITLE: Immunodiagnostic assay for rheumatoid arthritis The following statement is a full description of this in vention, including the best method of performing it known to me/us:-
V.
S
5*S 5.
*9 S 5 555 *555
S
S.
S S S5S S
S
500 S. S S S S5 la- Background of the invention Field of the invention The present invention is directed to a method of assay of rheumatoid arthritis More particularly, the present invention Is directed to the assay of human immunoglobulln Aal-antitrypsin complex (IgA-alAT) In patients who are suspected of having or are being treated for RA.
Description of the prior art Rheumatoid arthritis has been described as an unresolved systemic inflammation in which immune dysfunction and genetic susceptibility play roles. In earlier stages, it is often characterised by fluctuating remissions and exacerbations, and in later stages by a chronic granulatamous response (pannus formation) leading to tissue destruction notably of bone and 15 cartilage. The synovial membrane in RA has many of the characteristics of a hyperactive immunologically stimulated lymphoid organ and the ratio of T suppressor to T helper lymphocytes has been shown to be significantly reduced.
se*: Since there is no unambiguous test distinguishing RA from S 20 other acute or chronic inflammatory diseases, differentiating RA S* from other arthritides, such as systemic lupus erythematosus (SLE), ankylosing spondylitis polyarticular gout (PAG), or psoriatic arthritis (PsA) is often difficult. Diagnosis of RA Is usually made according to American Rheumatism Association (ARA) 25 criteria, i.e.: 0 morning stiffness; joint tenderness or pain on motion; soft-tissue swelling of the joint; soft-tissue swelling of a second joint (within three months); soft-tissue swelling of symmetrical joints (excludes distal interphalangeal joint); Subcutaneous nodules; X-ray changes; Se’um positive for rheumatoid factor 2wherein diagnosis of 3 or 4 of these factors is considered representative of probable RA and diagnosis of 5 or more of the factors is considered representative of definite RA.
The most widely used immunodiagnostic assay of RA, the so-called Waaler-Rose assay, is based upon an antibody (rheumatoid factor) to the Fc region of IgG. Rheumatoid factor (RF) is present in about 60% to 70% of those individuals afflicted with RA. The test is not satisfactory because it has been found to give unacceptably large numbers of false positives or negatives, and it does not assess the response to therapy or predict activation or reactivation of the disease process.
see* Moreover, based as it is on a haemagglutination or latex agglutination end point, it is difficult to standardize from one clinical laboratory to another. More seriously, it can provide a 15 positive result on only about 70% of chronic sufferers from this disease and, in any case, the immunopathogenic role of RF has never been established.
Recently, evidence has begun to point to the covalently linked complex between IgA and al-antitrypsin (clAT) as a 20 major immunopathological factor in RA. This complex is found at grossly elevated levels in the sera of patients with IgA m Alomatosis but has also been detected in abnormally high amounts in the circulation of RA patients. The following evidence suggests that the measurement of circulating levels of IgA-cIAT complex would provide a more relevant immunodiagnostic indicator of rheumatoid arthritis than those currently used: It is present in abnormally high levels in the circulation and joint fluids of virtually all patients with untreated chronic rheumatoid arthritis. (The currently utilized rheumatoid factor is detectable in the sera of only about 70% of such patients).
The serum level of IgA-alAT complex appears to fall In those patients who show a beneficial clinical response to treatment with second line anti-rheumatic drugs.
It is also detectable at abnormally high levels in those I I 3 AS patients who show erosive joint changes.
Unlike other so-called disease markers which are measured in RA, such as rheumatoid factor and acute phase proteins haptoglobin and C-reactive protein), both in vitro and in vivo studies have provided a plausible explanation of the immunopathogenicity of the IgA-clAT complex. Thus, not only can the formation of the IgA-oiAT complex lead to the consumption of as much as one-third of the total available alAT (one of the major anti-proteases) in rheumatoid patients’ serum or joint fluids, but the IgA-lAT complex itself is capable of eliciting release of degradative proteolytic enzymes from isolated macrophages (by a cytolytic process dependent on the activation of the alternative complement pathway). Furthermore, .4 injection of the isolated complex into normal rabbits’ knee 15 joints results in the rapid development of an acute arthritis, which shows the gross anatomical and histopathological features of the clinical condition. (See, Stanworth, “IgA dysfunction in rheumatoid arthritis”, Immunology Today, New Directions in Research, 6. pp. 43-45 (1985); Stanworth D.R. “The 20 role of IgA in the immunopathogenesis of rheumatoid arthritis” Chapter 7 in “Immunogenic Mechanisms of Arthritis” Eds.
3. Goodacre and D.W. Carson, pp 122-142 (19B7) and Dawes, P.T., Jackson Shadforth, Lewin, and Stanworth, D.R., “The relationship between the complex of immunoglobulin A and al-antitrypsin, its constituent components and the acute phase .response, as measured by C-reactive protein in rheumatoid arthritis treated with gold or D-penicillamine”, British Journal of Rheumatology, 26. pp. 351-353 (1987)).
The current method of measuring IgA-caAT complex relies on a two-dimensional immunoelectrophoresis comprising a first dimensional electrophoretic separation of complex from free iaAT in agarose, and its identification by second dimensional electrophoresis into agarose-containing antiserum directed specifically against alAT. The amount of complex is then quantitated (in arbitrary units) by determining the area under -4its precipitation peak (on the subsequently dried and stained plate) by planimetry. However, this is a laborious and time-consuming procedure.
Accordingly, it would be desirable to provide an easily performed assay for the IgA-aiAT complex. Initial attempts were made to use an ELISA in which either anti-IgA or anti-alAT antibody was first coated onto the wells of micro-titration plates followed by incubation with an IgA-alAT complex-containing test specimen, reaction with either anti-alAT IgG or anti-IgA IgG antibody, respectively and final development with an enzyme labelled anti-IgG antibody. However, it is a problem that such a complex cannot be detected reliably by an assay which depends on the binding of the IgA-aiAT complex to an antibody to IgA or aAT, since this approach would result in the binding of 15 uncomplexed IgA or alAT which are also present in unquantified amounts in the sample from the patient, thus interfering with the quantitative measurement of the IgA-alAT complex.
It has been necessary, therefore, to produce an antibody directed specifically against human IgA-L 1 AT complex. An initial 20 attempt to accomplish this by immunising rabbits with purified IgA-alAT complex failed, as the resultant antisera reacted also S with uncomplexed IgA and olAT.
Summary of the invention It has surprisingly been found that a monoclonal antibody can be produced which is virtually unreactive with free IgA and alAT, but is specific to the naturally occurring IgA-alAT complex.
Moreover, when attempting to prepare such antibodies from the fusion of mouse spleen cells with myeloma cells, it was found that yields of spleen cells were very low, making the production of the hybridomas impossible. It was necessary to find a solution to this problem, which, as it transpired, was caused by toxicity of the human IgA-alAT complex to the mouse macrophages.
It was demonstrated that incubation of isolated peritoneal mouse macrophages with human IgA-oIAT complex leads to a substantial release of the cytoplasmic enzyme LDH and a subsequent destruction of the macrophages. The problem was eventually overcome as described below.
Furthermore, an immunogenic peptide has been synthesised comprising a first peptide fragment having an amino acid sequence or an analogue thereof found in the Fc region of human IgA and a second peptide fragment having an amino acid sequence or an analogue thereof found in human alAT, said first and second fragments being covalently bound to one another, wherein an antibody raised against said peptide is substantially non-reactive with free human IgA, is substantially non-reactive with free human alAT, and binds to the naturally-occurring moo complex of human IgA and caAT (IgA-alAT). Surprisingly.
polyclonal antibodies raised against the said peptide were also found to be substantially non-reactive with free IgA and alAT.
15 Monoclonal antibodies of the same or better specificity will doubtless be raisable against it. Moreover, the recently developed chimeric antibodies (Reichman, Clark, M., Waldmann, H. and Winter Nature 332, pp 323-327, 1988)), single chain antibodies (PCT Patent Application Publication 20 Number WO 88/01649 Genex Corporation) and single domain antibodies (Ward, GUssow, Griffiths, Jones, P.T.
and Winter, G. Nature 341. pp 544-546, (19B9)) having elements anti to the said naturally occurring complex and peptide can be expected to be producible.
Accordingly the invention provides a ligand comprising an antibody domain specific for an antigenic determinant of a complex of human IgA and al-antitrypsin, said antibody domain being substantially non-reactive with free human IgA and free human al-antitrypsin.
The invention also provides a method of assay of rheumatoid arthritis (RA) in an analyte suspected to contain a complex of human IgA and al-antitrypsin (IgA-alAT) as an indicator of RA, which comprises detecting or measuring immunological binding between the aid complex and the above said ligand.
-6- This invention further provides an assay kit for carrying out a method of assay of human RA in an analyte suspected to contain a complex of human IgA and cl-antitrypsin (IgA-alAT) as an indicator of RA, the kit comprising an IgA-olAT complex and a ligand whose antibody domain is specific for an antigenic determinant of IgA-alAT, but substantially non-reactive with free human IgA and free human alAT.
Description or the preferred embodiments The following definitions are used throughout the present specification: “Assay” means a method of detection or measurement.
“IgA-aiAT complex” means the complex as found in human I patients serum, unless otherwise stated or the context otherwise requires.
15 Fab’ fragment represents one “arm” of the two “arms” of the shaped antibody configuration; the fragment retains antigen-binding ability.
F(ab’)2 fragment represents two Fab’ “arms” linked by disulphide bonds; the fragment retains antigen-binding ability.
20 Fc fragment represents the single “tail” or central axis of the shaped antibody.
The ligands of the invention comprise an antibody domain specific for an antigenic determinant of a complex of human IgA and human c 1 AT (IgA-alAT). The said antibody domain is relatively non-reactive with free human IgA and free alAT. The complex of IgA and alAT (IgA-aAT) is the naturally occurring complex found in analytes taken from patients suffering from rheumatoid arthritis. Most preferably, but as exemplified below, not necessarily, the ligand comprises a monoclonal antibody raised against such a complex. The most preferred monoclonal antibodies are obtainable from hybridomas which are the subject of patent deposits described herein below. Polyclonal antibodies raised against the purified, naturally occurring, complex were not found to be specific for the IgA-alAT complex, the resulting antisera reacting also with uncomplexed IgA and alAT.
-7- Alternatively the ligand can be an antibody raised against a synthetic peptide of the invention. This synthetic peptide is a covalently linked conjugate of short chain peptides representative of those parts of the IgA heavy chain and c.lAT chain sequences which comprise an IgA-cIAT complex-specific immunogenic determinant. In a particular embodiment, the present invention provides and utilizes a first peptide fragment having an amino acid sequence found in the Fc region of human IgA or an analogue of said sequence, and a second peptide fragment having an amino acid sequence found in human alAT or an analogue of said sequence, which are covalently bound to one another. The preferred form of covalent bonding is an S-S linkage which preserves the immunogenic three dimensional conformation of the linkage of the penultimate cysteine residue, relative to the r* 15 C-terminal end of human IgA, in the Fc region of human IgA to human alAT. The structure of the preferred IgA-caAT complex has not yet been completely elucidated. However, it is likely that the covalent S-S bridging occurs between the only cysteine residue (No. 232) of human acAT and the cysteine residue 20 (No. 495) occupying the penultimate cysteine position in the -chain of human IgA, to which 3-chain is known to conjugate in the formation of polymeric IgA.
SQ The first peptide fragment preferably corresponds to an amino acid sequence of the Fc region of human IgA containing the penultimate cystelne residue, relative to the C-terminal end of :6 human IgA, comprising 5 to 20 amino acid residues, preferably to 15 amino acid residues, or analogues thereof. The first peptide fragment preferably contains at least the amino acid sequence Val-Met-Ala-Glu-Val-Asp-Gly-Thr-Cys-Tyr which corresponds to residues 487-496 of the human IgA a-chain, or an analogue thereof. This is the minimum sequence length which will lead to the formation of a stable conjugate. Most preferably the amino acid sequence comprises Val-Ser-Val-Val-Met-Ala-Glu-Val-Asp-Gly-Thr-Cys-Tyr -8which corresponds to residues 484-496 of the human IgA a-chain or an analogue thereof.
The second peptide fragment preferably corresponds to an amino acid sequence of human alAT, including the cysteine residue which bonds covalently to the IgA e-chain, comprising 5 to amino acid residues, preferably 10 to 15 amino acid residues, or analogues thereof. The second peptide fragment preferably comprises at least the amino acid sequence His-Cys-Lys-Lys which corresponds to residues 231-234 of human al-AT, or an analogue thereof. This is considered to be the minimum sequence
*O@
length which will lead to the formation of a stable conjugate.
Most preferably the amino acid sequence comprises Gly-Met-Phe-Asn-Ile-Gln-His-Cys-Lys-Lys-Leu-Ser-Ser 15 which corresponds to the residues 225-237 of the human al-AT or f e an analogue thereof.
Accordingly, a particularly preferred immunogenic peptide of this invention comprises at least the following amino acid sequence: Val-Met-Ala-Glu-Val-Asp-Gly-Thr-Cys-Tyr **S4 His-Cys-Lys-Lys 20 or an analogue thereof.
Most preferably, the immunogenic peptide comprises the peptide conjugate of the following amino acid sequence: Val-Ser-Val-Val-Met-Ala-Glu-Val-Asp-Gly-Thr-Cys-Tyr Gly-Met-Phe-Asn-Ile-G1n-His-Cys-Lys-Lys-Leu-Ser-Ser or an analogue thereof (hereinafter designated peptide F017-F018).
Antibodies to the IgA-caAT complex (whether the natural complex or synthetic peptide) may be readily prepared by techniques well-known in the art. Thus, polyclonal antibodies can be obtained from immunised rabbits by exsanguination.
Monoclonal antibodies can be prepared by the Kohler-Milsteln method in mice provided that spleen cells are always used in excess in the fusion and that cytolysis of the resultant hybridomas is combatted as necessary by addition of fresh spleen cells. The hybridomas have then to be screened for specificity -9to the complex. Fab’ and F(ab’)2 fragments of such monoclonal or polyclonal antibodies may be prepared in well-known ways. Any of these molecules providing antibody domains to the complex can be used.
For diagnostic purposes, the antibody will react with the naturally occurring IgA- 1 AT complex from the individual under test to produce a detectable product. An antibody composition used in any test designed to quantitate the presence of IgA-alAT must contain sufficient antibody to react with all of the naturally occurring IgA- 1AT complex. Such diagnostically effective amounts o-f antibody will vary appreciably with a number of factors well known to those skilled in the art. These include, for example, the sensitivity and specificity of the test employed, the instrumentation available and the amount of analyte under test. The most preferred analyte is a serum sample since this gives a better indication of RA than joint fluids.
Detection and measurement of levels of the IgA-caAT complex, preferably in serum or joint fluid, may also be used as a prognostic indicator of RA in order to facilitate the better 20 management of patients with “early” (pre-erosion stage) RA, where diagnosis is normally difficult.
While enzyme-linked immunosorbent assay (ELISA) is preferred in this invention, other assays, e.g. radio-immunoassay, precipitation, agglutination, direct and indirect immunofluorescence and complement fixation can be used. These assays may employ any protocol such as competitive, inhibition or sandwich type.
i* The assays generally require a detectable label. The anti-IgA-alAT antibody, an anti-antibody goat anti-rabbit serum), an anti-IgA antibody or an anti-mAT antibody may be labelled. Useful labels include fluorescent labels such as fluorescein, rhodamine or auramine and radioisotopes such as 14C, 1311, 1251 and 35 S. The preferred enzyme labels include horseradish peroxidase, B-D-glucosidase, (-D-galactosidase, urease, glucose oxidase plus peroxidase, and acid phosphatase.
10 Currently available procedures for detecting the arorementioned labels are well-known and include colorimetric, luminometric and fluorometric techniques, as well as various instrumental methods of detecting radio isotopes.
The assays will normally be carried out so that the detectable product becomes bound to a support, so as to ensure ready separation from the unbound serum sample.
Usable supports include glass or plastic surfaces, especially the inner surface of test tubes or a surface of a test plate.
Typical examples of flat surfaces useful in the enzyme-linked immunoassay procedure (ELISA) or the radioimmunoassay procedure (RIA) include glass, nitrocellulose paper or plastics such as polystyrene, polycarbonate or various polyvinyls. Particles which can be “,ed for macroscopic procedures wherein the reaction 15 product can be detected visually, e.g. the hemagglutination procedure, include biological particles such as sheep red blood cells or human group 0 red blood cells, and biologically inert particles such as charcoal, bentonite or latex beads. Such beads can be formed of polystyrene, polyvinylpyrrolidone or various 20 polyvinyls.
Attachment to the support surfa.ce may be by direct adsorption, forced adsorption or chemical coupling in accordance with known procedures.
Preferred binding schemes are as follows labelled substance): S Sandwich Assays support/anti-IgA-alAT/IgA-alAT analyte/anti-IgA*; support/anti-IgA-oAT/IgA-olAT analyte/anti-ccAT*; support/anti-IgA-alAT/IgA-alAT analyte/anti-IgA-alAT* (the 2nd antibody having a different specificity from the first) Inhibition Assays Support/IgA-olAT/anti IgA-AI analyte (pre-incubated before addition to support/IgA-alAT) 11 Competition Assays IgA-ctAT* Support/anti-IgA- AT IgA-alAT analyte.
A wide variety of kits are possible for carrying out assays of the present invention. They comprise a ligand of the invention and an IgA-alAT complex. Preferably th. assay kit will provide a means of assaying the complex either by a sandwich assay wherein the kit provides in addition to the above, a second detection ligand which comprises an antibody domain capable of o detecting an IgA-alAT complex when bound to the first ligand, or ‘m a competitive or inhibition assay in which the said IgA-alAT **o complex component of the assay kit is an immunogenic analogue, 10 and is more preferably an immunogenic synthetic peptide, of the naturally occurring complex. The ligands are preferably polyclonal or monoclonal antibodies as set forth above and Fab’ or F(ab’)2 fragments thereof or single domain or single chain antibodies as will be apparent to one skilled in the art.
15 The detection ligand in a sandwich assay need not be an antibody which has specificity to the whole complex. Any such ligand which provides a means of attaching label to the analyte IgA-lAT (without interfering with the binding of the analyte to the capture antibody) is usable. Thus it could conveniently be an antibody raised against IgA or a 1
AT.
The detection ligand in the sandwich assay, the antibody which competes with the analyte in a competition assay and the antibody which is pre-reacted with the analyte in a, inhibition assay have to be labelled at some stage. While these reagents can be provided as ready labelled conjugates it is normally more convenient merely to label them by providing a further antibody thereto which is labelled as a separate component. Typically the second antibody is an immunoglobulin and the further antibody provides anti-immunoglobulin by being raised in a different host animal.
12 Normally, all components of the kit will be provided in separate containers.
Appropriate washing, enzyme substrate and buffer solutions would be provided with the assay kit, together with a detailed instruction sheet, including advice on the calculation and interpretation of the results.
Although the synthetic peptide or purified naturally occurring IgA-oIAT complex (being covalently linked) is relatively stable, it could become dissociated if test samples were mishandled exposed to reducing conditions).
It is important to keep analytes such as specimens of sera and joint fluids at 4 0 C over the short term, awaiting assay. If, Showever, they cannot be tested within a day or two they should be stored in the frozen state (at or preferably below -20 0 after 6* 15 having had cellular and non-cellular debris removed from them by gentle centrifugation.
The following Examples illustrate the invention. “Tween” is a Registered Trade Mark.
Example 1 20 Formation of mixed disulphide between peptides F017 and F018 Both peptides F017 and F018 were synthesized using the 9-fluorenylmethoxycarbonyl (Fmoc) solid phase peptide synthesis chemistry in an LKB Biolynx 4170 peptide synthesizer. The cysteine (Cys) residues in both peptides F017 and F018 had 25 side-chain protection of S-triphenylmethyl (TRT).
S*
15 mMol Iodine in acetic acid: water was added to a mixture of 5 rnMol F018 and 5 mMol F017 in acetic acid-water The mixture was gently mixed on addition and then left at 4°C for 16 hours.
Peptides F017-FOL8 were also treated in a similar way separately to act as controls. Each peptide preparation was then run on a Necleosil 5 C18 reverse-phase HPLC column with a methanol gradient (A 5% methanol in water, B 95% methanol in water). The HPLC traces were compared and the fractions comprising the extra peak obtained from the F017 and F018 mixture 13 were collected and used as F017-F018 peptide complex.
In a similar manner, the peptides Val-Met-Ala-Glu-Val-Asp-Gly-Thr-Cys-Tyr and His-Cys-Lys-Lys can be prepared.
Likewise, the peptide conjugate Val-Met-Ala-Glu-Val-Asp-Gly-Thr-Cys-Tyr
I
His-Cys-Lys-Lys can also be prepared in the s..e manner.
Example 2 Production of rabbit anti IqA-c 1
AAT
New Zealand White rabbits were injected subcutaneously with 10 200 pg of purified human IgA- 1 AT complex or 200 pg of peptide conjugate (F017-F018) emulsified in complete Freund’s adjuvant, followed by further injections of the same amount of complex or pept’de conjugate emulsified with incomplete Freund’s adjuvant at 14 to 28 days. About a month later the animals were bled.
Example 3 Isolation of IqG from rabbit antiserum and the preparation of various cleavage fragments One volume of saturated (NH 4 2 S0 4 solution pH 6.5 was added to one volume of rabbit serum (to give a final salt concentration 20 of 50% saturated), drop wise with stirring at 4 0
C.
After being left to stand for 6 hours, the precipitate was separated by centrifugation (3000g for 30 minutes) and the supernatant was discarded. The precipitate was redissolved in
S..
S0.3 volumes of 0.01M phosphate buffer pH 8.0 and dialysed against S 25 3 changes of the same bUffer. This final dialysed solution 5ml) was placed on a DEAE-Sephadex column 12.0 x 1cm) which was pre-equilibrated with 0.01M phosphate buffer pH and eluted with the same buffer. 2.0ml fractions were collected. The fractions corresponding to the protein (i.e.
IgG) peak were pooled and concentrated by ultrafiltration.
Further IgG containing fractions were retrieved from the column by application of a salt gradient 0.O1M 0.10M P0 4 using 3 column volumes of each buffer in a gradient maker.
14 The composition of all fractions was recovered and checked by immunoelectrophoresis against anti-whole human serum; and those fractions containing only IgG were pooled, concentrated by ultrafiltration and stored below -20 0
C.
Preparation of proteolytic cleavage fragments Preparation of Fab’ and Fc fragments Native IgG is hydrolysed in the hinge region by papain to yield two antigen-binding fragments, Fab’ and one dimer of the C-terminal half of the heavy chain, Fc’ (Porter 1959). These are all of similar size (50,000 molecular weight) but they can be separated by ion-exchange chromatography. In general, proteolytic fragments of immunoglobulins can be separated under non-denaturing conditions because they are not held by non-covalent bonds.
15 Procedure: S1. Dissolve 1 mg of papain in 100 pl of 0.1M sodium phosphate buffer and quickly add 50 pl of this to the IgG, Mix gently and incubate at 37 0 C overnight (16 hours).
2. Dialyze against water and then 3 x 500 ml of 0.01M sodium 20 acetate pH 3. Equilibrate the ion exchanger with the 0.01M acetate buffer and pack into the column, wash with the same buffer at “room temperature.
4. When both sample and exchanger are fully equilibrated, apply the sample to the column and elute with at least 60 ml of So. starting buffer until the absorbance at 280 nm has returned to ;baseline. Then apply a linear gradient, total volume 200 ml, from 0.01M to 1M acetate all at room temperature. Collect 5 ml fractions and monitor the absorbance at 280 nm.
5. Protein eluted with the starting buffer and the first peak in the gradient consists mostly of Fab’. The third peak is Fc. The protein yield in the three peaks should be about 90% of the original IgG.
15 Preparation of F(ab’)2. Fab’ and pFc’ fragments Native IgG is also hydrolysed by pepsin. However, this enzyme cleaves on the C-terminal side c~ at least one a-a-chain disulphide bond to give a divalent antigen-binding fragment, F(ab’)2. It also degrades part of the Fc portion to small peptides to leave a dimer of the C-terminal quarter of the a-chain, pFc’. The F(ab’)2 fragment can be reduced to the monovalent Fab’ fragment.
Procedure: 1.Dissolve 2 mg of protein in 200 pl of the acetate buffer and add 100 pl of this to the IgG solution. Mix gently and incubate at 37 0 C overnight (16 hours).
2. Neutralizt with 2M tris (approximately 300 pl this 9* Irreversibly inactivates the enzyme) and centrifuge at 2000 g for 15 10 minutes to remove any precipitate.
3. Apply the supernatant to the G-200 column and elute with TBS. Collect 2.5 ml fractions and monitor the absorbance at 280 nm.
4. The first major peak is F(ab’)2. In front of this is 20 undigested material and just behind it any Fab’ or intact Fc formed. These minor products are sometimes not completely resolved from F(ab’)2 and form shoulders on the main peak. pFc’ in the next peak and small peptides are eluted in the total column volume Fab’. F(ab’)2 can be directly reduced to Fab’, if required, by the following procedure: A. Pool the fractions containing F(ab’)2 and concentrate to 5 ml (thi: should give a protein concentration of about 6 mg/ml). Add 0.5 ml of the IM tris buffer and 50 pl of EDTA solution.
B. Add 50 pl of dithiothreitol solution (0.1M dithiothreitol in IM tris buffer, freshly prepared) and incubate in a sealed tube at room temperature for 1 hour with stirring.
C. Cool on ice, cover with foil and add 50 pl of lodoacetamide solution. Incubate in an ice bath for 30 minutes with stirring.
16 D. Add 5 iI of dithiothreitol solution, incubate at room temperature for 15 minutes and apply the mixture to the G-200 column. Elute as for the peptic digest. There will be a small peak of undissociated F(ab’)2 in its original position followed by a major peak of Fab’.
Example 4 Assessment of specificity of rabbit (polyclonal) anti-complex antisera 96-well flexible assay plates (Falcon 3912) were coated with antigen, by overnight incubation at 4″C with 120 pl aliquots of one of the following: i) IgA-alAT (5 pg/ml) ii) IgA (5 pg/ml) (iii) clAT (5 pg/ml) 15 made up in 0.05 M carbonate/bicarbonate buffer (pH 9.6).
The plates were then washed 3 times for 1 minute each with phosphate buffered saline (PBS), pH 7.2 containing 0.05% Tween (PBS/Tween).
Normal (NRS) and test (anti-complex) rabbit sera (100 pl), as 20 prepared in Example 2, were titrated in PBS/Tween (neat to 1 in 2 dilutions or neat to 1 in 5 dilutions) and added to the antigen coated plates. The plates were then incubated for 1 hour at 37″C. (Negative Control: PBS/Tween used alone or blank plate, incubated with normal rabbit serum). The plates were washed after incubation as before.
00 pl aliquots of goat-anti-rabbit/IgG/horseradishperoxiease were added at a dilution of 1/1000 PBS/Tween and the plates were then incubated for 1 hour at 37″C. After incubation, the plates were washed as before.
100 pl aliquots of substrate were added, the substrate comprising: mg o-phenylenediamine; 250 pl H 2 0 2 and ml 0.15 M citrate phosphate buffer (pH The colour was allowed to develop for 5-15 minutes and then 17 the enzymatic colour reaction was stopped by addition of 25 pl of
H
2 S0 4 to all wells.
The optical density of the contents of each well was read at 492 nm (00D492) in a Titertek automated plate reader. The results set forth in Table 1 indicate that the antisera had a considerable specificity for the complex, giving a high 0D492 at high antibody dilutions, some reaction towards alAT and no significant difference over controls towards IgA.
:0.
UO
a 4 e a S
S
S
S
S S S S S.
*5 S S St. S S S S S S*.
555 S55 SSS 5.5 4 S S S S S S S S S S S S S S S S S S S S S Table I Assessment of specificity of polyclonal rabbit anti-IgA-a 1 AT complex antiserum by ELISA.
Mean zeroed values of optical density measured at 492 nm.
ANTIGEN COATING ON ELISA PLATE IgA (5pg/ml) IgA-alAT Antibody NRS anti- NRS anti- NRS antidilution complex complex complex antibody antibody antibody Neat 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 1/1024 112048 0.761 0.665 0.452 0.254 0.117 0.046 0.644 0.703 0.496 0.250 0.172 0.083 0.035 0.005 0.652 0.585 0.353 0.235 0.176 0.094 1.519 1.526 1.536 1.558 1.573 1.514 1 .425 1.425 1.04 0.799 0.489 0.276 0.364 0.754 0.551 0.362 0.253 0.143 0.082 0.052 0.016 0.001 1 .388 1 .447 1 .455 1.457 1.456 1 .452 1 .445 1 .436 1.435 1 .412 1.284 1.191 19 Example Production of monoclonal antibodies Immunisation BALB/c mice were Injected intraperitoneally with IgA-aAT complex emulsified in equal volumes of Freund’s complete adjuvant. Injections were repeated on day 14 and 28 with IgA-x(AT complex emulsified in Freund’s incomplete adjuvant.
Test tail bleeds taken on day 28 or later were assayed for the presence of anti-peptide antibodies by indirect ELISA. Three days prior to fusion, mice showing raised serum antibody titres received a further booster injection of 50pg IgA-alAT complex in PBS.
s ees Fusion Hyperimmunized mice were sacrificed by cervical-dislocation, 15 the spleen removed and cells isolated and washed. The spleen cells were fused with a mouse myeloma cell line (Ag. 8.653 or NSO or NSI) from a culture in logarithmic growth. By modification of the Kohler and Milstein method (Kohler, G. and Milstein, C., Nature (London) 256, pp. 495 (1975)), spleen and myeloma cells 20 were fused at a ratio of 2:1, respectively, using 40% PEG (polyethylene glycol mol. weight 1450). Iml PEG was added dropwise over a 1 minute time period to the pellet of mixed cells (spleen and myeloma) and diluted with serum-free medium. The fusion suspension was distributed into 96-well plates and cultured in medium containing HAT (Hypoxanthine, aminopterin and thymidine). The poor growth of hybridoma cells was rectified by the addition of normal mouse spleen cells (immediately after fusion), as a source of fresh macrophages to replace those cytolysed by the injected IgA-x 1 AT complex.
After 10 days, plates were examined for growth of hybridomas. Supernatant removed from these cells was screened for the presence of anti-IgA-alAT complex antibodies by indirect ELISA. The following binding scheme was employed labelled substance): Support/IgA-alAT/Anti-IgA-caAT/goat anti-mouse IgG* 20 Cloning When positive wells were identified as producing the desired antibody, the hybrid cells were cloned by limiting dilution and clones assayed again. Hybridomas were cultured in flasks or grown in mice. Ascitic fluid was raised in BALB/c mice primed with pristane (0.5 ml injected a few days prior to injecting with 105 hybrid cells. Tumour formation should result after some 2-4 weeks and accumulated ascitic fluid removed by sacrificing the mouse and removing the contents of the abdominal cavity with a pipette. The concentration of monoclonal antibody in ascitic fluid was determined at every tumour passage; this ranged from 5-15 mg/ml.
Screening The procedure employed in screening the monoclonal antibodies was as follows. Plates were prepared comprising the following o layouts:o ff plate coated with human IgA-zlAT complex (by incubation with a solution containing 5pg protein/ml) cell supernatant goat anti-mouse-peroxidase labelled antibody; S 20 plate coated with free human IgA (5pg/ml solution) subsequent steps as above; plate coated with free human 1 AT (5pg/ml solution) subsequent steps as above.
off a Cells producing those supernatants which reacted positively only in system above, were selected as hybridomas which were producing monoclonal antibody directed specifically against the IgA-alAT complex (whilst being unreactive with free IgA or free c* 1
AT).
Two such hybridoma cell lines, secreting monoclonal antibodies to the naturally occurring IgA-cl-AT complex, have been deposited at the European Collection of Animal ‘ell Cultures, PHLS Centre for Applied Microbiology and Research, Porton Down. Salisbury, Wiltshire SP4 OJG, England. The first, designated NLW.54, was deposited on 6th February 1990 under the accession number ECACC 90020611, under the provisions of the 21 a
S
i 0 a 00 0 0
S.
a a @0 Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The most preferred antibody, designated NLW.50, was deposited on 13th December 1990 under the accession number ECACC 90121302.
Example 6 Measurement of IgA-cLAT complex by 2-Dimensional Immunoelectrophoresis (2D-IEP) A solution of 1% agarose (Sea kem HGT Agaose ICN Biomedical Ltd.) in 0.05M barbitone buffer pH 8.6 was prepared. 4ml of this melted agarose solution was poured onto a 7.6 x 5.0cm glass plate and allowed to set, whereupon 1.1 x 5.0cm strips were cut and transferred onto clean 7.6 x 5.0cm glass plates (1 strip of agarose per plate). A 2mm diameter well was cut into the agarose, 15mm from the left edge and 7mm from the bottom of the 15 plate. 3pl of the test serum was applied to the well and a small spot of bromophenol blue was added to the sample. The plates were placed onto the electrophoresis apparatus with the sample well Pearest the cathode and the agarose strip running length-ways to the anode. Filter paper wicks were placed on the agarose, 1cm in from each end. The plates were then electrophoresed at 20mA/6 plates with cooling, until the slower moving (albumin bound) bromophenol blue marker spot reached the wick on the anode side of the apparatus (approximately 2 hours).
50pl of sheep anti-human alAT was added to 4ml of melted agarose 25 at 56°C and poured onto the glass plates into the space above the 1.1 x 7.6cm strip. The plates were then electrophoresed at to the first electrophoresis at 20mA/6 plates overnight. The plates were removed from the electrophoresis apparatus and placed between weighted filter paper for 30 minutes. The plates were then transferred to an incubator until fully dried. The plates were stained with 0.05% Coomassie Brilliant Blue (Coomassie is a Registered Trade Mark) for 10 minutes and then destained with methanol/acetic acid/water (40:4:56) until precipitin lines could clearly be seen. The area of the ‘slower’ moving peak (IgA-alAT complex) was measured using a planimeter, and the ccmplex
S
S.
S
5
S
5. 5 5 is 5 00 22 concentration quoted as area in cm 2 The concentration of IgA-alAT complex in a panel of test pathological specimens was measured by this method. The results are set forth in Table 2 below.
Table 2 Patient Diagnosis Specimen IgA-#1AT Complex 2D-IEP Concentration Rank (2D-IEP value) (Arbitrary units) 1 RA Serum 4.05 4 2 RA Serum 2.80 3 Swollen Knee Serum 1.00 9 4 RA Serum 2.40 7 5 AS Serum 0.45 6 IgA myelomatosis Serum 26.00 1 7 IgA myelomatosis Serum 12.00 2 8 RA Joint Fluid 2.80 9 RA Serum 6.85 3 Polymyalgia Serum 0.70 11 11 RA Serum 1.20 8 12 RA* Joint Fluid 0.00 12 RA AS Rheumatoid Arthritis Ankylosing Spondylitis Steroid treated patient Example 7 Measurement of IqA-alAT complex by sandwich ELISA assays General method A 96-well flexible assay plate (Falcon 3912) was coated with a first or capture antibody of optimal concentration made up in a 23 coating buffer (0.05M carbonate/bicarbonate buffer pH 9.6).
120pl aliquots of this antibody 1/16000 dilution) were adsorbed onto the plate by incubation at 37 0 C for 1 hour, room temperature for 1 hour or overnight at 4*C. The plate was then washed 3 times for 1 minute each time with phosphate buffered saline (pH 7.2) containing 0.05% Tween 100 pl aliquots of IgA-alAT complex or test serum samples (diluted 2 fold or 5 fold) were added to the plate and incubated fNr 1 hour at 37*C. The plates were then washed as before.
100 pI aliquots of a second antibody of optimal concentration 1/6000 Rabbit-anti-IgA-aAT complex) were added to the plate and then incubated at 37″C for 1 hour. The plate was again washed as before. Then, 100 pl aliquots of a third antibody of optimal dilution were added. This antibody Goat-anti- 15 Rabbit IgG) was labelled with the enzyme horseradish peroxidase.
The plates were incubated at 37″C for 1 hour and then washed as before.
100 pl aliquots of the substrate for the enzyme horseradish peroxidase was added. The substrate comprised 20 o-phenylenediamine, 2 50pl H 2 0 2 and 50ml 0.15M citrate-phosphate bufrer, pH 5.0. The colour was allowed to develop for 5-15 minutes and then the enzymatic colour reaction was stopped by the addition of 25pl of 25% H 2
SO
4 to all the wells. The optical density of the contents cf each well was read at 492 nm (00492) in a Titertek automated plate reader.
Results: 1. Sandwich ELISAs incorporating polyclonal antibodies directed against IgA or axlAT. The capture antibody is anti-IgA The procedure for carrying out the sandwich EL1SA was as described above. 4 assays were carried out employing 2 sheep and 2 rabbit polyclonal anti-IgA antibodies as the capture or first antibody. The binding schemes for these ELISA assays are shown below:- Assay 1: Support/Sh.(1)anti-IgA/IgA-clAT complex/Sh.anti-c 1 AT labelled anti-sheep antibody.
24 a 4a
V
.a a. a 4
S.
Assay 2: Support/Sh.(2)anti-IgA/IgA- AT complex/Sh.anti-AT labelled anti-sheep antibody.
Assay 3: Support/Rb.(1)anti-IgA/IgA-alAT complex/Sh.anti-alAT labelled anti-sheep antibody.
Assay 4: Support/Rb.(2)anti-IgA/IgA-mlAT complex/Sh.anti-clAT labelled anti-sheep antibody.
Sh. Sheep Rb. Rabbit The results of the above assays 1-4 are set forth in Table 3 below. The samples 1-12 are the same as those samples in Table 2 of Example 6, where the concentrations of IgA-alAT complex were measured by 2D-IEP. Their rankings in the 2D-IEP are reproduced in Table 3 for ease of comparison.
Table 3 Sample No. Assay 1 Assay 2 Assay 3 Assay 4 Rank in 00492 Rank 00492 Rank 00492 Rank OD492 Rank 2D-IEP 1. 0.828 2 0.355 2 0.900 2 0.355 2 4 2. 0.924 1 0.578 1 1.157 1 0.578 1 6 3. 0.657 7 0.173 4 0.176 5 0.173 4 9 4. 0.607 9 0.153 9 0.144 11 0.153 9 7 0.580 10 0,164 7 0.141 12 0.164 7 6. 0.702 4 0.160 8 0.155 8 0.160 8 1 7. 0.664 6 0.167 6 0.147 10 0.167 6 2 8. 0.653 8 0.158 10 0.191 4 0.148 10 9. 0.406 12 0.116 12 0.157 7 0.116 12 3 0.762 3 0.170 5 0.566 3 0.170 5 11 11. 0.663 5 0.178 3 0.154 9 0.178 3 8 12. 0.466 11 0.132 11 0.159 6 0.132 11 12 a La aa a. a a *S pd a As can be seen qualitatively can be confirmed by statistical confirm the results of the 2D-IEP.
by comparing analysis, the For example, the rankings and ELISA failed to in assays 1 and 25 3, samples 6, 7 and 9 all gave low values despite their high 2D-IEP values, the concentration of complex in sample 10 appeared low by 2D-IEP but high by ELISA value.
In assays 2 and 4, samples 6, 7 and 9 all gave low ELISA values, despite their high 2D-IEP values. These results show that this approach cannot be used to measure IgA-x 1 AT complex.
The discrepancies in results obtained by the two methods (ELISA and 2D-IEP) are probably due to the free IgA binding preferentially to the anti-IgA antibody coating the ELISA plate, thereby preventing tne binding of IgA-cIAT complex.
abt, 2. Sandwich ELISA systems incorporating polyclonal antibodies directed against IgA or ~alAT. The capture antibody is anti-iaAT A sandwich ELISA procedure was carried out as described above. The following binding scheme was used:- 15 fupport/Sh.anti-aiAT/IgA-a1AT complex/polyclonal anti IgA labelled antibody S”In this Example, different pathological specimens were measured and compared to measurements of the same samples by 2D-IEP. The results are shown in Table 4 below:d S J 93.
Sa Oem S U S U 0 0 e eec 0 0 C Ce ee C
S.
S 0 S Ce e.g 0 6 04 CO Table 4 Patient’s Name Di agnos is ELISA results+ at serum duln, of: 1/10 1/20 1/40 ComplIe x level determ.
by 2 Dimen. I.E.* We aver Whyte Re id Barton Wright Snape Al bandol Jones 0.98 1 .03 0.71 0.11 0.22 0.12 0.07 0.13 0.94 0.91 0.45 0.10 0.12 0.10 0.09 0.12 0.85 0.72 0.21 0.08 0.09 0.09 0.08 0.09 0.77 0.52 0.08 0.07 0.06 0.06 0.06 0.07 4.05 2.80 1 .00 2 0.45 1 .00 26.00 12.00 MyelIoma 11 OD (optical density) arbitrary units units 27 The wide differences in the results obtained by the two methods, as seen e.g. in the rankings, are probably attributable to the binding of free alAT to the coating antibody preferentially, thereby inhibiting binding of the IgA-olAT complex.
3. ELISA sandwich assays employing antibody (monoclonal or polvclonal) to the naturally produced complex as capture or first antibodies Carrying out a sandwich ELISA procedure as described, the following binding schemes were used: 1. Support/Mc.anti-IgA-alAT/Iga-liAT complex/Sh.Pc.anti-IgA labelled anti-sheep antibody.
2. Support/Mc.anti-IgA-(lAT/Iga-(lAT complex/Sh. Pc.anti-acAT labelled anti-sheep antibody.
15 3. Support/Mc.anti-IgA-lAT/IgA-xiAT complex/Rb. anti IgA-alAT labelled anti-rabbit antibody.
go 4. Support/Rb.Pc.anti-IgA-alAT/IgA-caAT complex/Sh.Pc.
A anti-IgA labelled anti-sheep antibody.
Support/Rb.Pc.anti-IgA-iAT/IgA-caAT complex/Sh.Pc.
anti-alAT labelled anti-sheep antibody.
6. Support/Rb.Pc.anti-IgA-LAT/IgA-caAT complex/Mc.anti-IgAalAT labelled antibody.
Pc. Polyclonal antibody raised against the purified complex, IgA or alAT as indicated in the above binding schemes.
Mc. Monoclonal antibody secreted by hybridoma NLW54 in accordance with the invention.
Sh. Sheep.
.oO. Rb. Rabbit.
So The results of a sandwich ELISA incorporating binding scheme above are shown in Table 5 below. This table shows a comparison of OD492 values obtained from the ELISA with results from 2D-IEP.
28 Table Serum No. 2D-IEP ELISA (0D492) Values Rank Values Rank 19 0.9 10 0.65 0.5 11 0.64 9 21 1.5 9 0.63 6 22 2.3 8 0.63 6 23 2.5 7 0.63 6 24 2.9 6 0.60 3.2 5 0.77 3 26 3.5 4 0.60 27 3.9 3 0.76 4 28 5.0 2 0.81 2 purified isolated IgA-IAT complex (2mg/ml) 1 0.99 1 eese s o 00 0 419 0 4
C
See.
4 R
C
C CR
C.
As seen in Table 5 above, three of the four samples from binding scheme giving high 2D-1EP values units) also show higher 0D492 values. Although in serum samples containing lower levels of IgA-alAT complex, there is little difference in the ELISA values measured, this result may be remediable by increasing the sensitivity of the assay.
The resulr- from ELISA assays incorporating binding schemes 1 and 2 (data not shown) reveal a similar trend to that for scheme These results show that in a sandwich assay the detection antibody can be anti- to the whole complex or to either component thereof.
The results obtained from assays incorporating binding schemes 4, 5 and 6 reveal that it is not possible to measure the 29 IgA-alAT complex by using plates coated with polyclonal rabbit anti-complex antibody (data not shown).
Example 8 Measurement of IgA-tAT complex by inhibition ELISA A 96-well rigid plate (Falcon 3040) was coated with 500pg ml1 bovine serum albumin (BSA). Aliquots of 200 pl were added to each well and the plate was incubated at 37 0 C for 1 hour. The plate was washed three times for 1 minute each time with phosphate buffered saline (PBS) pH 7.2 containing 0.05% Tween (PBS/Tween 20). 100pl aliquots of IgA-oaAT complex or test serum samples were titrated (2 fold or 5 fold dilutions in PBS/Tween then 100pl aliquots of monoclonal antibody to the IgA- 1
AT
complex from the NLW54 cell line were added to an optimal concentration 1/20000 to give a final concentration of 15 1/40000) and the plate was incubated overnight at 4°C.
9 After the overnight incubation described above, the plate was S, ‘centrifuged at 3000 rpm for 15 minutes, and 90pl aliquots were transferred from each well onto another plate pre-coated with IgA-cAT complex. This plate was precoated as follows. A 96-well flexible assay plate (Falcon 3912) was coated with 100p aliquots of IgA-caAT complex of 5pg ml-1 concentration, made up Sin coating buffer (0.05M carbonate/bicarbonate buffer pH 9.6).
This plate was then incubated at 37″C for 1 hour, room temperature for 2 hours or 4°C overnight and then washed as S 25 described above.
100pl aliquots of antibody labelled with the enzyme horseradish peroxidase, of optimal dilution labelled goat anti-mouse IgG at 1/1000 dilution) were added and the plate was incubated at 37°C for 1 hour. The plate was washed as before.
100pl aliquots of substrate solution were added. The substrate solution comprised 20 mg o-phenylenediamine, 250pl 11202 and 50 ml 0.15M citrate phosphate buffer, pH 5.0. The colour was allowed to develop for 5-15 minutes before the enzymatic eaction was terminated by adding 2 5pl of 25% H 2 S0 4 to all the wells.
The optical density of the contents of each well was read at *949 9 0@*S 9
S
SO
9.
.3 3 p S 0 ‘j 9 rt 0e 30 492nm (0D492) in a Titertek automated plate reader. The calculation of percentage inhibition was as follows:inhibition 1- 0D492 sample 0D492 blank x 100 00D492 uninhibited sample OD492 blank The results of this assay are shown in Table 6. The results were compared to measurement of the same sera by a conventional 2D-lEP procedure. The ELISA results are expressed in terms of the reciprocal of the dilution of serum (the titre) required to be added to give 50% inhibition of the labelled antibody.
Table 6 Rheumatoid Reciprocal of Rank 2D-IEP 2D-IEP Serum Serum titre giving (Arbitrary Rank 50% inhibition Area Units) 29 423 3 0.75 3 1405 5 1.15 4 31 1553 7 1.90 32 2051 8 2.45 6 33 1494 6 2.50 7 34 1310 4 2.75 8 35 52 1 0.60 1 36 344 2 0.65 2 As will be seen from the rankings in Table 6, there was a good agreement between the ELISA and the 2D-IEP. This has been calculated statistically as about 69%. This percentage is even more impressive if the results for serum 32 (the sample giving the highest ELISA inhibition value) are ignored.
Example 9 Measurement of IqA-ocAT complex by a double antibody capture ELISA 96-well flexible assay plates (Falcon 3912) were coated with capture antibody of optimal concentration made up in coating 31 buffer (0.05M carbonate/bicarbonate; pH 9.6).
Aliquots (120 pi) of antibody at 1/1000 dilution were absorbed onto a plate by incubation at 37°C for 1 hour, at room temperature for 2 hours or at 4°C overnight. The plate was then washed (3 x 1 minute) with phosphate-buffered saline (PBS), pH 7.2, containing 0.05% Tween To the above pre-coated plate, monoclonal antibody to IgA-lAT complex as secreted by hybridoma NLW54 in accordance with the invention, was added and the assay carried out according to the protocol previously described in Example 7. The following binding scheme was employed:- Support/Mc. rat anti-mouse IgG/Mc mouse anti-IgA-clAT complex/ IgA-mlAT complex/ Pc.Rb. anti-IgA.c 1 AT/labelled anti-rabbit antibody Mc. Monoclonal antibody Pc. Polyclonal antibody o Rb. Rabbit 15 The results of determining the level of Ig-o 1 AT complex in a panel of test sera (rheumatoid arthritis and normal controls) by the above double antibody capture method were compared with the levels of IgA-lAT complex measured by 2D-immunoelectrophoresis, single antibody capture ELISA (as described in Example 7(3) 20 following binding scheme 3) and inhibition ELISA techniques (as described in Example These results are shown in Table 7.
Table 7 Sample 2D-iEP Double Ab Single Ab Inhibition (cm ELISA (serum ELISA (serum ELISA dil. 1/40) dil. 1/40) Inhibition O* 492 nm O.D. 492 nm Titre 29 0.5 0.298 0.674 196 1.2 0.564 0.877 309 31 3.3 0.342 0.654 221 32 3.9 0.538 0.748 356 Normal sera 1.1 0.310 0.644 202 Complex 5.2 0.571 0.79 443 containing sera 32 This sample is anomalous in that it consistently shows high ELISA values and low 2D-IEP values. It is probably giving false low 2D-IEP readings.
Example Comparison of results from employing murine monoclonal NLW.50 and NLN.54 in ELISA system A double antibody capture assay was also carried out in which the monoclonal antibody to IgA-oxAT complex was secreted by hybridoma NLW.50 in accordance with the invention. In this assay, polystyrene plates were employed (Dynatech-Immulon 4).
The assay was carried out as described above in Example 9 except that the test serum was diluted 1/100. The use of a better S* monoclonal antibody coupled with the use of polystyrene plates contributed to the increased sensitivity of this assay. The 15 following binding scheme was employed: support/Rat Mc Anti-mouse IgG/Mc anti-IgA-alAT/IgA–alAT complex/ Rb Pc anti-IgA-lAT/labelled goat anti-rabbit antibody.
Mc Monoclonal antibody Pc Polyclonal antibody G Goat Rb Rabbit The results are shown in Table 8 below.
Table 8 SERUM 20-IEP RESULTS ELISA RESULTS (OD 492) SAMPLE (arbitrary units) NLW.50 NLN.54 Value Rank Value Rank Value Rank
OS
37 0.75 10 0.572 10 0.634 8 38 1.20 9 0.628 9 0.596 39 1.40 8 0.685 7 0.635 7 2.00 7 0.724 6 0.634 8 41 2.50 6 0.882 1 0.663 42 2.75 5 0.851 3 0.813 2 43 3.30 3 0.651 8 0.637 6 44 3.60 2 0.868 2 0.897 1 4.40 1 0.756 5 0.679 4 46 3.00 4 0.781 4 0.712 3 47 0.60 11 0.386 11 0.374 11 33 When the results are plotted as ELISA results for NLN.54 and vs the 2D-IEP measurement, then the correlation coefficient are 0.56 and 0.72 respectively confirming that is slightly more sensitive than NLW.54 and the reason why it is thus the preferred antibody.
Example 11 Effect of having one or two detection antibodies in a double antibody capture ELISA assay A double antibody capture ELISA assay was carried out as previously described in Example 9 except that polystyrene plates (Dynatech-Immulon 4) were employed and the test sera was diluted 1/100. The effect of using one or two detection antibodies in the ELISA assay was investigated. The following binding schemes were employed: S 15 support/Rat Mc anti-mouse IgG/Mouse Mc anti-IgA-alAT/IgA–caAT complex/Sh Pc anti-IgA/D anti Sheep labelled antibody.
support/Rat Mc anti-mouse IgG/Mouse Mc anti-IgA-ajAT/IgA-alAT complex/Sh Pc anti-IgA labelled.
Mc Monoclonal antibody Pc Polyclonal antibody Sh Sheep D Donkey S* The monoclonal anti-IgA-alAT antibody was that secreted by S hybridoma NLW.50 in accordance with the invention.
The results are shown in Table 9 below.
*0 34 Table 9 IgA-aAT SERUM COMPLEX CONCENTRATION (arbitrary units) Binding Binding Scheme Scheme 1 2 IgA-a AT SERUM COMPLEX CONCENTRATION (arbitrary units) Binding Binding Scheme Scheme 1 2 0 0 0 9 0 00 0 0.3 0.43 0.8 0.4 0.55 0.36 0.52 0.41 0.72 0.35 0.41 0.6 0.49 0.45 <0.2 0.39 0.75 5.0 0.39 0.9 1.9 0.52 0.8 1.1 0.66 0.67 0.8 0.53 0.67 1.45 1.1 0.63 <0.2 0.48 1.7 0.32 0.58 0.27 0.36 0.33 0.54 <0.2 0.3 0.45 0.34 0.32 0.325 0.53 0.35 0.275 0.44 <0.2 0.52 0.46 0.38 1.05 0.47 0.84 0.39 1.05 0.29 0.40 0.57 0.61 0.45 0.34 0.69 0.41 0.35 0.74 0.3 0.85 0.69 The results of this assay show that there was no loss of sensitivity when employing one detection antibody instead of two as is the tradition in ELISA assays. This has the advantage of reducing the time needed for carrying out the assay by one hour, and the cost of carrying out such an assay is reduced also. When comparing the results of the two assays by plotting the results gained for binding scheme 1 against binding scheme 2, the correlation coefficient was 0.88 and the standard deviation <0.001 which is highly significant. The point representing serum sample number 2 has been eliminated from this statistical analysis (if included the correlation coefficient is 0.97). 35 Example 12 Comparison of ELISA sandwich assay emploving one detection antibody only with 2D-IEP measurements A double antibody capture ELISA assay was carried out in accordance with the method described in Example 9. Polystyrene plates (Dynatech-Immulon 4) were employed and the test sera was diluted 1/100. The monoclonal capture antibody was that secreted by hybridoma NLW.50 in accordance with the inventiun. The following binding scheme was employed: support/Rat Mc anti-Mouse IgG/Mc anti-IgA-ccAT/IgA-alAT complex/ Sh Pc anti-IgA/labelled D anti-sheep IgA antibody. Mc Monoclonal antibody Pc Polyclonal antibody D Donkey 15 Sh Sheep Measurement of IgA-caAT complex by 2D-IEP was carried out according to Example 6. The results are shown in Table 10 below. Table SERUM 2D-IEP (cm 2 ELISA (arbitrary units) Value Rank Value Rank 38 1.5 9 0.77 8 6 1.3 11 0.87 7 8 3.0 3 2.3 3 54 3.2 2 1.28 4 03 1.3 11 0.54 12 29 1.1 13 0.56 11 32 2.3 4 2.35 2 1.4 10 0.96 65 1.8 7 0.94 6 S24 1.7 8 0.58 9 7 0.6 15 0.47 13 44 3.3 1 2.5 1 58 2.3 4 0.57 2.2 6 0.4 1 0.9 14 0.44 14 When these samples were plotted on a graph comparing 2D-IEP and ELISA, the correlation coefficient was 0.73, which is significant. Claims (22) 1. A ligand comprising an antibody domain specific for an antigenic determinant of a complex of human IgA and al-antitrypsin, said antibody domain being substantially non reactive with free human IgA and free human al-antitrypsin. 2. A ligand according to claim 1 wherein said domain is specific for the naturally occurring complex of IgA and al-antitrypsin (IgA-alAT). 3. A ligand according to claim 1 wherein said domain is specific for a synthetic peptide comprising a first peptide fragment having an amino acid sequence or an analogue thereof found in the Fc region of human IgA and a second peptide fragment covalently bonded to the first, and having an amino acid sequence or an analogue thereof found in human acAT. 4. A ligand according to claim 1, 2 or 3 in the form of a S monoclonal antibody. 5. A ligand according to claim 1, 2, 3 or 4 In the form of an Fab' fragment of said antibody. 6. A ligand according to claim 1, 2, 3 or 4 in the form of an F(ab')2 fragment of said antibody. 7. An immunogenic peptide comprising a first peptide fragment $**see I a having an amino acid sequence or an analogue thereof found in the Fc region of human IgA and a second peptide fragment having an amino acid sequence or an analogue thereof found in human oaAT, said first and second peptide fragments each containing a cysteine residue and being covalently bound to one another through said cysteine residues, wherein an antibody raised against said peptide Is substantially non-reactive with free human IgA, is substantially non-reactive with free human alAT, and binds to the naturally-occurring complex of human IgA and alAT. 8. An Immunogenic peptide according to Claim 7, wherein said first peptide fragment comprises an amino acid sequence Including the penultimate cysteine residue of the Fc region of human IgA or an analogue thereof, relative to the C-termlnal end of human IgA. ft 37 9. An immunogenic peptide according to Claim 7 or 8, wherein said first peptide fragment comprises a Val-Met-Ala-Glu-Val-Asp-Gly-Thr-Cys-Tyr sequence or thereof. 10. The immunogenic peptide according to Claim 9, first peptide fragment comprises a Val-Ser-Val-Val-Met-Ala-Glu-Val-Asp-Gly-Thr-Cys-Tyr an analogue thereof. 11. An immunogenic peptide according to claim 7, wherein second peptide fragment comprises a His-Cys-Lys-Lys sequence or an analogue thereof. 12. An immunogenic peptide according to claim 7, wherein said second peptide comprises a Gly-Met-Phe-Asn-Ile-Gln-His-Cys-Lys-Lys-Leu-Ser-Ser 15 an analogue thereof. an analogue wherein said sequence or 8, 9 or 8, 9 or sequence or b 0 a. S. Y 0. 13. An immunogenic peptide according to claim 7 wherein the first and second fragments comprise a Val-Ser-Val-Val-Met-Ala-Glu-Val-Asp-Gly-Thr-Cys-Tyr Gly-Met-Phe-Asn-Ile-Gln-His-Cys-Lys-Lys-Leu-Ser-Ser sequence or an analogue thereof. too#, $6 -N 1 14. A\==ef-t 49dy according to claim 3 wherein the synthetic 20 peptide is a peptide claimed in claim 7, 8, 9, 10, 11, 12 or 13. An antibody produced by the hybridoma cell line designated deposited on 13th December 1990 at the European Collection of Animal Cell Cultures PHLS' Centre for Applied Microbiology and Research, Porton Down, Salisbury, Niltshire SP4 03G, England SO** 25 under the accession number ECACC 90121302. 16. A method of assay of human rheumatoid arthritis (RA) in an analyte suspected to contain a complex of human IgA and al-trypsin (IgA-lAT) as an indicator of RA, which comprises detecting or measuring immunological binding between the said complex and a ligand claimed in claim 1, 2, 3, 4, 5, 6, 14 or 17. A method according to claim 16 whcrein the assay is of a sandwich type and comprises capturing said IgA-c l AT by causing it to bind to said ligand, as a first or capture ligand, and assaying said binding by causing said IgA-cIAT to bind to a second, labelled ordetection ligand which comprises an antibody domain capable of detecting said IgA-a..AT, and detecting or measuring the amount of label thus captured. 18. A method according to claim 17 wherein the assay Is carried out in solution, the capture ligand is bound to an insoluble support, after said binding the support is separated from solution and the presence or amount of label on the support is detected or measured. 19. A method according to claim 16, 17 or 18 wherein the detection ligand is an antibody to IgA, aoAT or a complex thereof. An assay kit for carrying out a method of assay of human "rheumatoid arthritis in an analyte suspected to contain a complex of human IgA and al-trypsin (IgA-alAT) as an indicator of RA, the 15 kit comprising: a ligand claimed in claim 1, 2, 3, 4, 5, 6 or 14 and an IgA-aIAT complex. 21. An assay kit according to claim 20 for use in a sandwich assay claimed in claim 17 wherein said ligand is intended for use as a capture ligand, the complex is provided for testing the kit and which further comprises: S* a second ligand intended for use as a labelled or detection ligand, which comprises an antibody domain capable of detecting an IgA-alAT complex when bound to said ligand 22. An assay kit according to claim 20 for use in a competitive or inhibition assay in which the said IgA-cLAT complex component of the kit is an immunogenic analogue of the naturally occurring complex which binds competitively therewith. 23. A kit according to claim 22 wherein the said immunogenic analogue is a peptide according to any one of claims 7-13. 24. A ligand according to claim 1, an immunogenic peptide according to claim 7, a method according to claim 17, or an assay kit according to claim 20, substantially as hereinbefore described with reference to the Examples. disclosed herein or referred to or in din the specification and/or cl shis application, individu collectively, and any and all combinations _n tw mere of said a- r; eE s ee 0 SL DATED this TWENTY SEVENTH day of MAY 1991 by DAVIES COLLISON SEC. Patent Attorneys for the applicant(s)13 LU U ABSTRACT IMMUNODIAGNOSTIC ASSAY FOR RHEUMATOID ARTHRITIS The assay of rheumatoid arthritis by reference to IgA-al antitrypsin complex present in analytes is facilitated by certain novel antibody reagents. These are ligands comprising an antibody domain specific for an antigenic determinant of a complex of human IgA and al-antitrypsin, said antibody domain being substantially non reactive with free human IgA and free human al-antitrypsin. Monoclonal antibodies to the naturally occurring IgA-caAT complex and monoclonal or polyclonal antibodies to a synthetic peptide are preferred. The synthetic peptide in itself part of the invention and preferably has an amino acid sequence: Val-Ser-Val-Val-Met-Ala-Glu-.Val-Asp-Gly-Thr-Cvs-Tvr d S e* 40. o *t S. d S. S t B AU77370/91A 1990-05-25 1991-05-27 Immunodiagnostic assay for rheumatoid arthritis Ceased AU637701B2 (en) Applications Claiming Priority (4) Application Number Priority Date Filing Date Title GB909011702A GB9011702D0 (en) 1990-05-25 1990-05-25 Immunodiagnostic assay for rheumatoid arthritis GB9011702 1990-05-25 GB9014227 1990-06-26 GB909014227A GB9014227D0 (en) 1990-06-26 1990-06-26 Immunodiagnostic assay for rheumatoid arthritis Publications (2) Publication Number Publication Date AU7737091A AU7737091A (en) 1991-11-28 AU637701B2 true AU637701B2 (en) 1993-06-03 Family ID=26297113 Family Applications (1) Application Number Title Priority Date Filing Date AU77370/91A Ceased AU637701B2 (en) 1990-05-25 1991-05-27 Immunodiagnostic assay for rheumatoid arthritis Country Status (13) Country Link US (1) US5827668A (en) EP (1) EP0605410B1 (en) JP (1) JPH05508835A (en) AT (1) ATE126540T1 (en) AU (1) AU637701B2 (en) CA (1) CA2082529A1 (en) DE (1) DE69112225T2 (en) DK (1) DK0605410T3 (en) ES (1) ES2077857T3 (en) FI (1) FI103975B1 (en) GB (1) GB2246780B (en) NO (1) NO304237B1 (en) WO (1) WO1991019001A1 (en) Families Citing this family (12) * Cited by examiner, † Cited by third party Publication number Priority date Publication date Assignee Title US5955076A (en) * 1989-06-15 1999-09-21 Peptide Therapeutics Limited Immunoactive peptides and antibodies and their use in anti-allergy treatment GB8913737D0 (en) * 1989-06-15 1989-08-02 Univ Birmingham A novel anti-allergy treatment GB9125024D0 (en) * 1991-11-25 1992-01-22 Kirby Julian Rheumatoid arthritus treatment GB9416865D0 (en) * 1994-08-19 1994-10-12 Peptide Therapeutrics Limited Treatment of inflammatory diseases of the gastro-intestinal tract GB9416864D0 (en) * 1994-08-19 1994-10-12 Peptide Therapeutrics Limited Treatment of IgA nephropathy SK284856B6 (en) 1996-03-01 2006-01-05 Novartis Ag Immunogenic molecule, pharmaceutical composition, ligand, method for the preparation of an immunogenic molecule and its use ATE255228T1 (en) * 1997-10-16 2003-12-15 Scil Technology Holding Gmbh DETECTION OF CARTIAL DISEASES WITH MIA US6913749B2 (en) * 1998-11-02 2005-07-05 Resistentia Pharmaceuticals Ab Immunogenic polypeptides for inducing anti-self IgE responses US20140147441A1 (en) * 2006-09-12 2014-05-29 The General Hospital Corporation Compositions containing alpha-1-antitrypsin and methods for use US8574848B2 (en) * 2006-09-13 2013-11-05 Oncimmune Ltd. Immunoassay methods US20080118524A1 (en) * 2006-10-20 2008-05-22 Stefan Persson Anti-IgE Vaccines US8093018B2 (en) * 2008-05-20 2012-01-10 Otsuka Pharmaceutical Co., Ltd. Antibody identifying an antigen-bound antibody and an antigen-unbound antibody, and method for preparing the same Family Cites Families (9) * Cited by examiner, † Cited by third party Publication number Priority date Publication date Assignee Title GB1179435A (en) * 1966-06-17 1970-01-28 Ortho Pharma Corp Stable Reagent for the Detection of Rheumatoid Arthritis ES471585A1 (en) * 1977-07-15 1979-01-16 Behringwerke Ag Carrier-bound immunoglobulin fission product and its use in immunologic analyses DE3364347D1 (en) * 1982-04-09 1986-08-07 Fujirebio Kk Anti immune complex antibody and preparation thereof US4499186A (en) * 1982-12-30 1985-02-12 University Of Illinois, Board Of Trustees Diagnosing autoimmune rheumatoid arthritis by measuring proteolytic activity of α2 -macroglobulin GB8408193D0 (en) * 1984-03-30 1984-05-10 Cambridge Patent Dev Antibodies US4645748A (en) * 1985-02-01 1987-02-24 Charles Hurwitz Protein which is characteristic of rheumatoid arthritis GB2171999A (en) * 1985-03-04 1986-09-10 Iq Antibodies GB8616174D0 (en) * 1986-07-02 1986-08-06 Antibody Technology Ltd Monoclonal antibodies US5223441A (en) * 1986-10-09 1993-06-29 Syntex (U.S.A.) Inc. Receptors for immune complexes 1991 1991-05-24 GB GB9111215A patent/GB2246780B/en not_active Expired - Fee Related 1991-05-24 ES ES91909926T patent/ES2077857T3/en not_active Expired - Lifetime 1991-05-24 AT AT91909926T patent/ATE126540T1/en not_active IP Right Cessation 1991-05-24 DK DK91909926.7T patent/DK0605410T3/en active 1991-05-24 DE DE69112225T patent/DE69112225T2/en not_active Expired - Fee Related 1991-05-24 CA CA002082529A patent/CA2082529A1/en not_active Abandoned 1991-05-24 WO PCT/GB1991/000821 patent/WO1991019001A1/en active IP Right Grant 1991-05-24 JP JP91509764A patent/JPH05508835A/en active Pending 1991-05-24 EP EP91909926A patent/EP0605410B1/en not_active Expired - Lifetime 1991-05-27 AU AU77370/91A patent/AU637701B2/en not_active Ceased 1992 1992-11-24 FI FI925321A patent/FI103975B1/en active 1992-11-25 NO NO924555A patent/NO304237B1/en not_active IP Right Cessation 1995 1995-08-10 US US08/513,514 patent/US5827668A/en not_active Expired - Fee Related Also Published As Publication number Publication date JPH05508835A (en) 1993-12-09 GB2246780A (en) 1992-02-12 EP0605410B1 (en) 1995-08-16 FI103975B (en) 1999-10-29 FI103975B1 (en) 1999-10-29 GB2246780B (en) 1994-04-27 DK0605410T3 (en) 1995-12-04 US5827668A (en) 1998-10-27 NO924555D0 (en) 1992-11-25 EP0605410A1 (en) 1994-07-13 DE69112225T2 (en) 1996-01-04 CA2082529A1 (en) 1991-11-26 ATE126540T1 (en) 1995-09-15 FI925321A0 (en) 1992-11-24 WO1991019001A1 (en) 1991-12-12 FI925321A (en) 1992-11-24 ES2077857T3 (en) 1995-12-01 NO304237B1 (en) 1998-11-16 DE69112225D1 (en) 1995-09-21 NO924555L (en) 1992-11-25 AU7737091A (en) 1991-11-28 GB9111215D0 (en) 1991-07-17 Similar Documents Publication Publication Date Title EP2292662B1 (en) 2014-04-23 Assay and reagents for quantifying hBNP JP4834136B2 (en) 2011-12-14 Method for detecting proBNP using a monoclonal antibody that binds to amino acids 41 to 46 WO1997032900A9 (en) 1997-11-06 ASSAY AND REAGENTS FOR QUANTIFYING hBNP AU637701B2 (en) 1993-06-03 Immunodiagnostic assay for rheumatoid arthritis EP1007570B1 (en) 2009-06-10 Monospecific antibody reactive with fibrinogen and fibrinopeptide b JP4913034B2 (en) 2012-04-11 ADAMTS13 activity assay antibody and activity assay method US5712157A (en) 1998-01-27 Methods and materials for the immunoassay of apolipoprotein(a) and lipoprotein(a) EP0506523A2 (en) 1992-09-30 Monoclonal antibodies Aulikki Santavuori et al. 1991 Application of a new monoclonal antibody for time-resolved fluoroimmunoassay of human pancreatic phospholipase A2 KR960008672B1 (en) 1996-06-28 Monoclonal antibody recognizing atrial natriuretic polypeptide KR940009281B1 (en) 1994-10-06 Alpha-hanp recognizing monoclonal antibody and immunologically measuring reagent for alpha-hanp US5679583A (en) 1997-10-21 Monoclonal antibodies for the selective immunological determination of intact procollagen peptide (type III) and procollagen (type III) in body fluids US5124248A (en) 1992-06-23 Monoclonal antibody recognizing gamma atrial natriuretic polypeptide EP0485525B1 (en) 1994-09-14 Monoclonal antibody to acetylated lysine residues in recombinant bovine somatotropin JPH0677017B2 (en) 1994-09-28 A method for the immunoassay of human dehydrogenase type IV collagen EP2495564A1 (en) 2012-09-05 5.9 kDa PEPTIDE IMMUNOASSAY METHOD JPH11286500A (en) 1999-10-19 Measurement of laminin fragment IE930440L (en) 1986-04-29 Monoclonal antibodies special for Hb Alc Legal Events Date Code Title Description 2002-01-03 MK14 Patent ceased section 143(a) (annual fees not paid) or expired
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