GB2118300A - Method of immunoassay - Google Patents
Method of immunoassay Download PDFInfo
- Publication number
- GB2118300A GB2118300A GB08204236A GB8204236A GB2118300A GB 2118300 A GB2118300 A GB 2118300A GB 08204236 A GB08204236 A GB 08204236A GB 8204236 A GB8204236 A GB 8204236A GB 2118300 A GB2118300 A GB 2118300A
- Authority
- GB
- United Kingdom
- Prior art keywords
- antibody
- labelled
- complex
- prolactin
- immobilised
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 32
- 238000003018 immunoassay Methods 0.000 title claims abstract description 12
- 108010057464 Prolactin Proteins 0.000 claims abstract description 37
- 229940097325 prolactin Drugs 0.000 claims abstract description 37
- 230000000890 antigenic effect Effects 0.000 claims abstract description 10
- 230000000984 immunochemical effect Effects 0.000 claims abstract description 7
- 239000012876 carrier material Substances 0.000 claims abstract description 5
- 102000003946 Prolactin Human genes 0.000 claims abstract 15
- 101000687438 Homo sapiens Prolactin Proteins 0.000 claims description 23
- 239000012530 fluid Substances 0.000 claims description 14
- 238000011534 incubation Methods 0.000 claims description 14
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 239000002131 composite material Substances 0.000 claims 2
- 239000000700 radioactive tracer Substances 0.000 description 28
- 102100024819 Prolactin Human genes 0.000 description 22
- 210000004027 cell Anatomy 0.000 description 20
- 238000003556 assay Methods 0.000 description 19
- 239000003153 chemical reaction reagent Substances 0.000 description 17
- 239000002609 medium Substances 0.000 description 13
- 102000004169 proteins and genes Human genes 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 12
- 239000000427 antigen Substances 0.000 description 11
- 102000036639 antigens Human genes 0.000 description 11
- 108091007433 antigens Proteins 0.000 description 11
- 238000002360 preparation method Methods 0.000 description 9
- 238000005119 centrifugation Methods 0.000 description 8
- 206010003445 Ascites Diseases 0.000 description 7
- 229920002684 Sepharose Polymers 0.000 description 7
- 230000001419 dependent effect Effects 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 7
- 210000004408 hybridoma Anatomy 0.000 description 6
- 239000007790 solid phase Substances 0.000 description 6
- 238000002820 assay format Methods 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 229960001760 dimethyl sulfoxide Drugs 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 108060003951 Immunoglobulin Proteins 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 229940000425 combination drug Drugs 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 102000018358 immunoglobulin Human genes 0.000 description 3
- XOJVVFBFDXDTEG-UHFFFAOYSA-N pristane Chemical compound CC(C)CCCC(C)CCCC(C)CCCC(C)C XOJVVFBFDXDTEG-UHFFFAOYSA-N 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 150000004982 aromatic amines Chemical class 0.000 description 2
- 210000003567 ascitic fluid Anatomy 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 229940072221 immunoglobulins Drugs 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 230000010807 negative regulation of binding Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000007423 screening assay Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 1
- 108010032595 Antibody Binding Sites Proteins 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102000002265 Human Growth Hormone Human genes 0.000 description 1
- 108010000521 Human Growth Hormone Proteins 0.000 description 1
- 239000000854 Human Growth Hormone Substances 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000012979 RPMI medium Substances 0.000 description 1
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 1
- 241000425571 Trepanes Species 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 150000003973 alkyl amines Chemical class 0.000 description 1
- 229960003896 aminopterin Drugs 0.000 description 1
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000005289 controlled pore glass Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 238000000326 densiometry Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 239000008241 heterogeneous mixture Substances 0.000 description 1
- 210000004754 hybrid cell Anatomy 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000006192 iodination reaction Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 229910000077 silane Inorganic materials 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 238000007693 zone electrophoresis Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Endocrinology (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method of immunoassay for prolactin, characterised in that monoclonal IgG antibodies are used. The method preferably comprises the use of two different monoclonal antibodies which bind respectively at different antigenic sites on the prolactin molecule. One antibody is labelled and the other is immobilised on a water-insoluble carrier material, whereby an immunochemical complex comprising labelled antibody, prolactin and immobilised antibody is formed.
Description
SPECIFICATION
Method of immunoassay
This invention relates to a method of immunoassay, in particularforprolactin.
Immunoassay methods are known in general for determining the presence or concentration of a substance in a fluid, based on the use of antibodies specific to that substance. Eitherthe antibody orthe antigen is usually labelled to enable itto be estimated.
Labelling systems which are commonly used employ fluorogenic materials, enzyme markers and radioisotopes.
Immunoassay procedures generally require a step involving the separation of the immunochemically complexed products from the surrounding incubation medium. This can be facilitated by providing one of the species involved in an immobilised, insoluble form. It is known that antigenic substances or antibodies can be bonded to various water-insoluble carrier materials without substantial loss of biological activity. At an appropriate stage in the immunoassay procedure, the solid phase immobilised reagent can be readily separated, e.g. by centrifugation, and the label estimated either in the separated solid phase or in the liquid phase. This technique is known as a solid phase immunoassay.
An improved solid phase immunoassaytechnique involvestaking advantage of polyvalentantigens,that is antigens having molecules which can bind antibody at two or more different sites. For example, the antigen is initially complexed with an immobilised antibody, followed by centrifugation. The solid phase product is then reacted with labelled antibodies which complex with the antigen already complexed on the solid phase antibodies. The uptake of labelled antibody is directly correlated to the amount of antigen in the test solution. Alternatively, the antigen mayfirst be reacted with labelled antibody, followed by the addition of immobilised antibody and subsequent centrifugation. In both cases, the eventual product is a "sandwich" of immobilised antibody, antigen and labelled antibody.These techniques are known as sandwich immanoassays and examples are described in our U.S. Patents Nos. 4,034,072 and 4,098,876.
In all immunoassaytechniques, it is desirableforthe antibody used to show as high an affinity and specificity as possible for the antigen to be estimated.
Conventionally produced antibodies are, of course, complex mixtures and even afterdifficult purification procedures they are by no means homogeneous in respect to their antigen binding properties. Recent techniques have made it possible to produce antibodies which are homogeneous in respect to their antigen binding properties. This is by the so-called hybridoma technique described by Köhler G, and
Mi Istein C., "Continuous cultures of fused cells secreting antibody of predefined specificity", Nature 256,495-497,1975. This procedure involves the
production of continuous cell lines of genetically
stable fused cell hybrids capable of producing large amounts of monoclonal antibodies against specific antigens.Such cell lines are produced by hybridisation between antibody-producing cells and myeloma cells. After the cell lines have been fused, clones are grown from individual hybrid cells and clones producing the desired antibodies are selected for antibody production. The cloned cells can then be propagated and maintained either in vitro or can, for example, be grown in the ascitesfluids of suitable animals. It is thereby possible to obtain large quantities of monoc lonal antibodies which offer particular advantages for immunoassay procedures.
The present invention is based on the development of monoclonal antibodies which are specificfortwo or more sites on the prolactin molecule. Thus, in one aspect, the invention provides a method of im munoassayfor prolactin, characterised in that monocional IgG antibodies are used.
Preferably, the invention relates to a "two-site" assay, i.e. one in which the labelled and immobilised antibodies are directed to different antigenic determinants ofthe prolactin molecule.
Thus, the invention also provides a method of immunoassayfor prolactin, involving the use oftwo different monoclonal IgG antibodies which bind respectively at two different antibody-binding sites, one ofthe antibodies being labelled and the other being immobilised on a water-insoluble carrier material whereby an immunochemical complex comprising labelled antibody, prolactin and immobilised antibody is formed.
Itwill be appreciated thatthe invention is particularly suitable for use in sandwich assays. These may be either forward assays, in which the fluid sample is incubated with immobilised antibody, followed by centrifugation and incubation with labelled antibody, or may be reverse sandwich immunoassays, in which the sample is first incubated with labelled antibody, then with immobilised antibody followed by centrifugation. In a particularly advantageous embodiment, a so-called "simultaneous addition" format is employed. The fluid sample is incubated simultaneously with labelled antibody and with immobilised antibody. This procedure is particularly useful as only one incubation and one centrifugation step are needed.
Two site assays em ideally suited to use in a simultaneous addition format because there is no competition forantigenic determinants bythesolid phase antibody and the tracer antibody which would occur if a heterogeneous mixtures of antibodies, as would be found in conventional antisera, were em pl oyed. The lack of competition intwo-siteassays gives advantages ofenhanced kinetics and sensitivity.
The use oftwo monoclonal antibodies directed towards different antigenic sites on the same target
molecule allows the design of assays with a minimum cross reaction with other substances in sample fluids.
The following sections describe the procedures employedforthe production of monoclonal anti
bodies and assay reagents, the assay formats em
ployed and finally the evidence forthe two-site nature ofthe resultant human prolactin assay.
The drawing(s) originally filed were informal and the print here reproduced is taken
from a later filed formal copy.
1. PRODUCTION OFMONOCLONAL ANTIBODIES
TO HUMAN PROLACTIN
1.1 lmmunisationofmice: 5 g higly purified human prolactin (hPRL) in Freunds Complete Adjuvant were injected sub- cutaneously into female BABBLE mice on days 0 and 11.
5,ag hPRL in ammonium bicarbonate were injected on days 18 and 66.
1.2 Cell fusion and selection:
Spleen cell suspensions in RPMI medium containingfoetal calfserum (15%), penicillin (100 lU/ml), streptomycin (100,ag/ml) and glutamine (2 x 10-3M) were prepared on the sixth day from the final immunisation. Spleen white blood cells (SWBC) were fused with P3-X20 myeloma cells(ratio 4:1 respectively) in 42% (w/v) polyethylene glycol (m.wt. 1540) in RPMl 1640 medium containing 15% (v/v) dimethyl sulphoxide (DMSO) at 370C for one minute before dilution with medium over the next four minutes.The cells were then transferred into complete medium and addedtowells of 24well plates containing normal BALBIc SWBC as feeder cells in complete medium containing hypoxanthine (10-4M), aminopterin (4 x 10-7M), thymidine (1.6 x 1 0-5M) and 2 mercaptoethanol (4 x 10 5M), which is termed HAT medium.
On day 10 (afterfeeding with HAT medium on days 5 to7) asample of mediumwas removedforthe screening assay and replaced with fresh HAT medium.
The strongest growth wells on the basis of the
screening assay were grown to confluency, split in
half and transferred to a well of a fresh plate, termed
the secondary plate. From days 14to 28 cells were fed
with HAT medium and cells expanded to furtherwells of the secondary plate as necessary. When at least two
wells of each hybridoma cell colony are growing
strongly in the secondary plate rescreening was
instituted to determine which cells were to be cloned.
These hybridoma cells were either resuspended in
foetal calf serum (85%) DMSO (15%) and frozen at -709 or taken for cloning by limiting dilution.
1.3 Cloning of Hybridomas: Hybridomacellswere resuspended and viable cells were estimated by trepan blue exclusion. The cells were diluted in complete medium to give frequency of 0.5 cells/0.05 ml. and this volume was then added to each well of a 96 well tissue culture plate, containing normal mouse spleen feeder cells in complete medium. After7to 10 days at37 C in a 5% CO2, 95% air atmosphere each positive growth well was sampled for screening and the medium was replaced with fresh complete medium. A minimum of two specific anti
body-producing clones from each hybridoma cell colony were chosen for expansion to mass culture.
Several samples of all viable hybrids in mass culture werefrozen down in liquid N2 for permanent storage in 1 ml.foetal calfserum containing DMSO (15%).
1.4 Production of Ascitic Fluid
Mice were primed with 0.5to 1 ml. Pristane
(tetramethylpentadecane) approximately 14 days be
fore injecting cell suspension intraperitoneally. Ascitic
fluid was produced in approximately 10 to 14 days and
was collected by aspiration.
Ascites fluid containing monoclonal antibodies was
produced by this method. Immobilised antibody and tracer antibody reagents were prepared from antibodies in ascitesfluidsfrom two cloned hybridoma cell lines. These cell lines have been designated 79.4.A1. 1/18 and 77.4.D. 2/22.
2. PRODUCTION OFASSA YREAGENTS 2.1 Preparation ofantibodyfortracer: 2.1.1 Protein A-Sepharose isolation of specific mouse anti hPRL immunoglobulin.
IgG antibodies have been prepared from ascites fluid from both 79.4.A1.1/18 and 77.4.D4.2/22 cell lines by affinity chromatography on Protein A-Sepharose (Pharmacia Fine Chemicals). The method used was a modification of that described by Ey.P.L., Prowse, S.J., and Jenkin, S.R.; Isolation of pure IgG, lgG2a and IgG2b immunoglobulins from mouse serum using Protein
A-Sepharose. Immunochemistry 15,429-436, 1978.
Ascites fluid from 79.4.A.1/18 yielded two distinct antibody perparations by this method. These antibodies have been designated; peak 2 and peak 3 antibodies, with respect to their elution position.
Ascites fluid from 77.4.D4.2/22 yielded one antibody preparation.
w.1.2 Radio-iodination
The method used was a modification of that described by Hunter & Greenwood; Preparation of lodine-131 labelled human growth hormone of high specific activity. Nature 194495-496, 1962.
Tracer reagents were prepared by radioiodination with 1251 ofthe antibody preparations obtained by the
Protein A-Sepharose procedure, ie. 79.4.A.1/1 8 peak 1, 79.4.A1.1/18 peak2 and77.4.D4.2/22 antibodypreparations.
2.2 Preparation of Immobilised Antibody Reagents
Immobilised antibody reagents (IMAs) have been prepared by chemically coupling ascitesfluid or protein A Sepharose purified immunoglobulins to control pore glass (approx. 1 FLm diameter; ssoA pores). This can be achieved by a number of procedurex to a protein loading of approx. 40 mg protein/ gm CPG; e.g. when CPG particles are reacted with a silane having an arylamine oralkylamine organofunctional portion, the resulting alkylamine or arylamine CPG can be modified for coupling to proteins, as described in U.S. Patent 3,975,511.
Immobilised antibody reagents were prepared by coupling ascites fluids from 77.7.D4.2/22 and 79.4.A1.1/18 cells lines, and the two Protein A
Sepharose prepared antibody preparations, 79.4.A1.1/18 peak2 and79.4.A1.1/18 peak3,to controlled pore glass particles.
2.3 Preparation of Standards:
Human prolactin was added to filtered human serum to concentrations between 2 and 100 ng/ml (equivalentto 65 to 3250 mlU/1 of the International
Reference Preparation 75/504).
3. ASSAYFORMATS
Two different assays format have been tested:
3.1 "Reverse Sandwich" Format:
This consists of an incubation ofsample or
standard with the tracer reagent followed by a second
incubation with the immobilised antibody reagent
(IMA) before the subsequent separation of bound and
free radioactivity by centrifugation and decanting.
The method employed consisted oftwo 2 hour
incubation periods.
50,us of serum is pipetted into tubes followed by the addition of 200 iLl radio-iodinated monoclonal lgG (approximately 5 ng in phosphate buffered saline containing Bovine Serum albumin pH 7.4). After mixing the reaction between the hPRL in the serum and the 1251-antibody is allowed to proceed for two hours at room temperature. 500 it1 of the immobilised antibody suspension (in the above buffer) is then added and after mixing the reaction ofthis antibody with the hPRL-1251-antibody complex formed in the first reaction is allowed to proceed for a further 2 hours.
Two millilitres of deionised water is then added as a 'dilution' wash, the mixture agitated and then centrifuged at 1500 xg for 10 minutes to separate the immobilised antibody from the supernatant. The supernantant is decanted to waste, the tube drained and the radioactivity associated with the pellet determined. The amount of radioactivity is directly related to the hPRL present in the standard or test serum.
The following monoclonal antibody reagents were evaluated in the reverse sandwich assay format.
Immobilised antibodies Tracers 77.4.D4.2/22 77.4.D4.2/22 79.4.A1.1/18 79.4.A1.1/18 (peak 2) 79.4.A1.1/18 (peak 2) 79.4.A1.1/18 (peak 3) 79.4.A1.1/18 (peak 3) The different combinations of immobilised antibody and tracer antibody were incubated with a series of prolactin standard solutions. The results were plotted as percentage of radioactivity in the bound fraction (%B) versus the concentration of the prolactin standards.
The results are shown in Figures 1-5. It is clear that the following combinations result in prolactin concentration dependent binding and thus have the potential to provide effective human prolactin assays:
77.4.D4.2/22 IMAwith 79.4.A1.1/18 peak 2 Tracer (fig.3) 79.4.A1 .1/18 IMAwith 79.4.A1.1/18 peak 3 tracer (fig.4)
or77.4.D4.2/22tracer (fig. 5) 79.4.A1 .1/18 (peak 2) IMA with 77.4.D4.2/22 tracer (fig.2) or79.4.A1.1/18(peak3)tracer(fig. 1) 79.4.A1.1/18 (peak3) IMAwith 79.4.A1.1/18 (peak 2) tracer (fig.1) 3.2 'Simultaneous Addition' Format:
In this procedure there was just one period of incubation, during which all reagents were present.
This formatwas tested used a 2 hour and 4hour incubation period.
50,at of serum is pipetted into tubes followed by 200 iL1 of the tracer as described above and immediately followed by 500 it1 of the scurry described above. All tubes are mixed thoroughly and both reactions described for the reverse sandwich assay allowed to proceed simultaneously for eithertwo or four hours and the I MA pellet separated as described above. Again the amount of radioactivity bound is
directly related to the hPRL present in the standard or
sample.
To evaluate the simultaneous assay format the
following combinations of antibody reagents were
incubated in the presence of human prolactin stan
dardsfortwo orfour hours.
77.4.D4.2/22 IMA with 79.4.A1 .1/18 Peak 2 tracer 79.4.A1 .1/18 IMAwith 79.4.A1 .1/18 Peak 3 tracer
79.4.A1.1/18lMAwith 77.4.D4.2/22tracer The results are given in Table 1. All the combina- tions resulted in prolactin dependent binding, thus
proving the feasibility of a simultaneous sandwich
assay for human prolactin utilising monoclonal
antibodies.
4. EVIDENCE FORA 'TWO-SITE' MONOCLONAL
HUMAN PROLACTINASSA Y
The two site nature ofthe previously described
human prolactin assays can be established from the
reverse sandwich assay studies, the results of which
are shown in Figures 1 - 5.
Figure 1 shows the results of the combination of
each of 79.4.A1 .1/18 Peak 2 and Peak 3 antibodies
with each ofthe immobilised antibody reagents of
these antibody preparations, in the presence of
prolactin standard concentrations. It is clear that the
homologous combinations, i.e. Peak 2 Tracer with
Peak2 IMA, and Peak3tracerwith Peak31MA, do not
result in prolactin concentration dependent binding.
In contrast the heterologous combinations of Peak 2 tracerwith Peak 3 IMA and Peak 3 tracer with Peak 2
IMA do result in prolactin concentration dependent
binding.
These results can be explained by proposing that
the use of the same antibody as tracer and immobil
ised antibody leads to inhibition of binding due to
competition between these antibodiesforthe same
antigenic site on the prolactin molecule. It follow
that Peak 2 and Peak 3 antibodies react with different
antigenic sites on the prolactin molecule, as the
heterologous combinations result in prolactin depen
dent binding.
Figure 2 shows the results obtained when an antibodytracer derived from Protein-ASepharose pu rified antibody of 77.4.D4 22/22 ascites flu id was
combined with immobilised antibodies of Peak 2 and Peak3, 79.4.A1.1/18 ascites fluid (Notethat Protein-A Sepharosefractionation of 77.4.D4.2/22 ascitesfluid
yielded only one antibody population). The combina
tion of 77.4.D4.2/22 tracer with 79.4.A1.1/18 Peak 2
immobilised antibody resulted in prolactin depen
dent binding, butthe combination of this tracer with
Peak3 IMAshowed almosttotal inhibition of binding.
These resu Its imply that 77.4.D4.2/22 anti body reacts
with the same antigenic site as 79.4.A1.1/18 Peak 3 antibody but to a different site than 79.4.A1 .1/18 Peak
2 antibody. This hypothesis is borne out by the results
of an experimentwhich tested the combination of 79.4.A1 .1/18 Peak or Peak3tracerwith an immobil
ised antibody from 77.4.D4.2/22 ascitesfluid, (figure
3).
Figures65 show results obtained by testing the
immobilised antibody prepared from unfractionated 79.4.A1.1/18 ascitesfluid. This immobilised antibody,
79.4.A1.1/18 IMA must therefore contain both consti
tuent Peak2 and Peak3 antibodies.
Figure 4 presents the results of the combination of 79.4.A1.1/18 IMAwith 79.4.A1 .1/18 Peak 2 or 79.4.A1.1/18 Peak 3 tracers. It is clearthatthe combination with Peak 3 tracer results in much higher binding than the combination with Peak 2 tracer.
Thus the immobilised parental antibody behaves as if it contains mostly Peak 2 antibody. This was substantiated by a zone electrophoresis study. Electrophoresis of 79.4.A1 .1/18 ascitesfluid separated two proteins corresponding to Peak 2 and Peak 3 antibodies, and subsequent densitometry showed thattherewas a greater mass of protein associated with Peak 2 than Peak 3.
Figure 5 presents the results of combining 77.4.D4.2/22 tracer with 79.4.A1 .1/18 IMA or 77.4.D4.2/22 IMA. This again shows that having the same antibody present as tracer and IMA, (the combination of 77.4.D4.2/22 IMA and 77.4.D4.2/22 tracer), results in lack of binding due to competition of the antibodies for the same antigenic site on the prolactin molecule. In contrast the combination of 77.4.D4.2/22 tracer and 79.4.A1.1/18 IMA gives prolactin concentration dependent binding.This supports the previous deductions firstly that 77.4.D4.2/22 antibody is directed tothe same antigenic site as 79.4.A1.1/18 peak 3 antibody and secondly the 79.4.A1.1/18 ascitestluid contains a higher proportion of 79.4.A1.1/18 peak 2 antibodythan peak 3 antibody.
Thusthe results of testing the various combina- tions of antibody tracers and immobilised antibodies in human prolactin 'reverse-sandwich' assay format, have shown that antibody 79.4.A.1/1 8 Peak 2 reacts with one site on the prolactin molecule and that antibodies 79.4.A. 1118 peak 3 and 77.4. D4.2/22 are directed to a separate site on the prolactin molecule. It therefore follows that assays formed by the following combinations of reagents are indeed 'two-site' immunoassays.
79.4.A1 .1/18 Peak 2 IMA with 77.4.D4.2/22 tracer (fig. 2) 79.4.A1.1118 Peak IMAwith 79.4.A1.1/18 peak 3 tracer (fig. 1) 79.4.A1.1/18 Peak3 IMAwith 79.4.A1.1/18 peak 2 tracer (fig.1) 77.4.D4.2/22 IMA with 79.4.A1 .1/18 peak 2 tracer (fig. 3) Various combinations of monoclonal reagents at the two different assayformats were evaluated for useash.PRLassays byconstructing standard curves of the binding of radioactivity at a range of h.PRL concentrations, as shown in Figures 1-5 and Table 1.
The standard curves achieved by combinations of monoclonal reagents in the 'reverse sandwich' format are presented in Figures 1-5. The combinations which have the potential to provide effective human prolactin assaywereidentified, (Section 3.1).
The standard curves achieved by selected combinations of monoclonal reagents in the 'simultaneous addition' format are presented in Table 1.
Each of these combinations has the potential to achieve an effective human prolactin assay, with the advantages of single incubation and centrifugation steps.
Additionallythetwo-site nature of the resultant assay systems has been deduced from the reverse
sandwich assay results.
We have thus described a number of monoclonal two-site immunoradiometric assay systems for hu
man prolactin.
Thesuccessforthese assays depends on the availability of monoclonal antibodies specific to different portions ofthe prolactin molecule and the use of the Protein A-Sepharose procedure fortheir purification.
Claims (7)
1. A method of immunoassay for prolactin, characterised in that monoclonal IgG antibodies are used.
2. A method according to claim 1, comprising the.
use of two different monoclonal antibodies which bind respectively at different antigenic sites on the prolactin molecule, one antibody being labelled and the other being immobilised on a water-insoluble carrier material, whereby an immunochemical complex comprising labelled antibody, prolactin and immobilised antibody is formed.
3. A method according to claim 1, which comprises the steps of:
a) incubating a fluid sample containing human prolactin with labelled anti-human prolactin antibody under conditions sufficient to form a first labelled immunochemical complex;
b) incubating that complex with a composite comprising anti-human prolactin antibody immobilised on a water-insoluble carrier material, the incubation being underconditionssufficienttoform a second labelled complex comprising the first complex complexedtothe composite;
c) separating the second labelled complex from the incubation medium;
d) determining the amount of label associated with the second complex; and
e) relating the determination of step (d)to a standard to determine the concentration of human prolactin inthefluid,
characterised in thatthe anti-human prolactin antibody instep a) and/orthe anti-human prolactin antibody in step b) is a monoclonal antibody.
4. A method according to claim 1,which compris es the steps of: a) incubating a fluid sample containing human prolactin with immobilised antibody thereto, under conditions sufficientto form a first immunochemical complex;
b) separating the said first complex from the incubation medium;
c) incubating the said first complex with labelled antibody under conditions sufficient to form a second labelled complex comprising the first complex complexed to the labelled antibody;
d) separating the second labelled complex from the incubation medium;
e) determining the amount of label associated with the second complex; and
f) relating the determination of step (e) to a standard to determine the concentration of human prolactin inthefluid.
characterised in that the anti-human prolactin antibody in step a) and/orthe anti-human prolactin antibody in step c) is a monoclonal IgG antibody.
5. A method according to claim 1, which compris es the steps of: a) incubating a fluid sample containing human prolactin simultanteously with labelled anti-human prolactin antibody and with an immobilised antihuman prolactin antibody under conditions sufficient to form a labelled and immobilised immunochemical complex of labelled antibody, prolactin and immobilised antibody;
b) separating the immunochemical complexfrom the incubation medium;
c) determining the amount of label associated with the complex; and
d) relating the determination of step c) to a standard to determinethe concentration of human prolactin in the fluid sample,
characterised in that the immobilised antibody and/orthe labelled antibody is a monoclonal IgG antibody.
6. A method according to any of claims 1 to 5, wherein the antibody is labelled with -7251.
7. A method according to any of claims 1 to 5, wherein the antibody is labelled with materials other than radioisotopes, such as enzymes, and fluorescent or chemiluminescent molecules.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB08204236A GB2118300B (en) | 1982-02-12 | 1982-02-12 | Method of immunoassay |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB08204236A GB2118300B (en) | 1982-02-12 | 1982-02-12 | Method of immunoassay |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| GB2118300A true GB2118300A (en) | 1983-10-26 |
| GB2118300B GB2118300B (en) | 1985-06-19 |
Family
ID=10528309
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB08204236A Expired GB2118300B (en) | 1982-02-12 | 1982-02-12 | Method of immunoassay |
Country Status (1)
| Country | Link |
|---|---|
| GB (1) | GB2118300B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61124868A (en) * | 1984-08-21 | 1986-06-12 | ポ−ル・コ−ポレ−シヨン | Ligand concentration method and active membrane used for said method |
| EP0343932A1 (en) * | 1988-05-24 | 1989-11-29 | Applied Research Systems Ars Holding N.V. | Method of Assay |
| WO1992003738A1 (en) * | 1990-08-23 | 1992-03-05 | Enfer Technology Limited | Hormone detection methods |
| DE19532655A1 (en) * | 1995-08-24 | 1997-02-27 | Pavel Dr Strohner | Method for the two-dimensional determination of samples in immunoassays |
| WO1999053324A1 (en) * | 1998-04-16 | 1999-10-21 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Salivary prolactin test for serotonergic activity |
| EP1010980A1 (en) * | 1998-12-17 | 2000-06-21 | Socolab S.A. | Immunoassay of proteins |
| CN104098699A (en) * | 2006-08-18 | 2014-10-15 | 诺华有限公司 | PRLR-specific antibody and uses thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2074727A (en) * | 1980-04-25 | 1981-11-04 | Hoffmann La Roche | Immunological diagnostic method |
| EP0044219A1 (en) * | 1980-07-16 | 1982-01-20 | Unilever Plc | Methods of immuno analysis using monoclonal antibodies |
| EP0045103A2 (en) * | 1980-07-28 | 1982-02-03 | Akzo Nobel N.V. | Method for the determination of antigens with the aid of two or more monoclonal antibodies |
| GB2086041A (en) * | 1980-08-04 | 1982-05-06 | Hybritech Inc | Immunassay utilising monoclonal antibodies |
| GB2095831A (en) * | 1981-02-18 | 1982-10-06 | Mochida Pharm Co Ltd | Monoclonal antibody reagent and method for immunological assay |
-
1982
- 1982-02-12 GB GB08204236A patent/GB2118300B/en not_active Expired
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2074727A (en) * | 1980-04-25 | 1981-11-04 | Hoffmann La Roche | Immunological diagnostic method |
| EP0044219A1 (en) * | 1980-07-16 | 1982-01-20 | Unilever Plc | Methods of immuno analysis using monoclonal antibodies |
| EP0045103A2 (en) * | 1980-07-28 | 1982-02-03 | Akzo Nobel N.V. | Method for the determination of antigens with the aid of two or more monoclonal antibodies |
| GB2086041A (en) * | 1980-08-04 | 1982-05-06 | Hybritech Inc | Immunassay utilising monoclonal antibodies |
| GB2095831A (en) * | 1981-02-18 | 1982-10-06 | Mochida Pharm Co Ltd | Monoclonal antibody reagent and method for immunological assay |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61124868A (en) * | 1984-08-21 | 1986-06-12 | ポ−ル・コ−ポレ−シヨン | Ligand concentration method and active membrane used for said method |
| EP0280840A1 (en) * | 1984-08-21 | 1988-09-07 | Pall Corporation | Methods of preparing biologically active membranes and uses thereof |
| JPH0689169B2 (en) | 1984-08-21 | 1994-11-09 | ポ−ル・コ−ポレ−シヨン | Ligand concentration method and active membrane used therefor |
| EP0343932A1 (en) * | 1988-05-24 | 1989-11-29 | Applied Research Systems Ars Holding N.V. | Method of Assay |
| WO1989011655A1 (en) * | 1988-05-24 | 1989-11-30 | Ares-Serono Research & Development Limited Partner | Method of assay |
| WO1992003738A1 (en) * | 1990-08-23 | 1992-03-05 | Enfer Technology Limited | Hormone detection methods |
| US5460976A (en) * | 1990-08-23 | 1995-10-24 | Enfer Technology Limited | Detection of reproductive hormone levels in equines |
| DE19532655A1 (en) * | 1995-08-24 | 1997-02-27 | Pavel Dr Strohner | Method for the two-dimensional determination of samples in immunoassays |
| WO1999053324A1 (en) * | 1998-04-16 | 1999-10-21 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Salivary prolactin test for serotonergic activity |
| EP1010980A1 (en) * | 1998-12-17 | 2000-06-21 | Socolab S.A. | Immunoassay of proteins |
| CN104098699A (en) * | 2006-08-18 | 2014-10-15 | 诺华有限公司 | PRLR-specific antibody and uses thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| GB2118300B (en) | 1985-06-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4703003A (en) | Monoclonal antibody with a high affinity for digoxin | |
| US5262296A (en) | Freeze-dried composition containing enzyme-labeled anti-human interferon-β antibody and enzyme immunoassay kit containing the composition | |
| US4536479A (en) | Use of anti-idiotype antibodies in immunoassays | |
| EP0119736A2 (en) | Two-site immunoassays using monoclonal antibodies of different classes or subclasses and test kits for performing same | |
| US4720455A (en) | Progesterone assay method for mammals and monoclonal antibody therefor | |
| US4713325A (en) | Hybridomas producing monoclonal antibodies specific for FeLV p27 | |
| JPH0367678B2 (en) | ||
| EP0048357A1 (en) | Method for the determination of an antigen in solution | |
| US4892824A (en) | Fast track method for producing monoclonal bi-specific immunoglobulins | |
| JP2527706B2 (en) | Lewis blood group phenotyping method | |
| Lewis et al. | Conformation-specific monoclonal antibodies directed against the calcium-stabilized structure of human prothrombin | |
| US4841025A (en) | Antibody preparations | |
| US5316914A (en) | Method for determining human collagen peptides by way of enzyme immunoassay | |
| GB2118300A (en) | Method of immunoassay | |
| US4692330A (en) | Process for accelerating antigen-antibody reaction | |
| FI83669C (en) | Method for Specific Determination of Pancreatic Amylase | |
| EP0274198B1 (en) | Immunoassay kit | |
| US4610960A (en) | Monoclonal antibody to thrombospondin and method for assaying for and isolating thrombospondin | |
| US5856182A (en) | Monoclonal antibodies specific for the PSA-ACT complex | |
| EP0401370A1 (en) | Enzyme immunoassay according to sandwich method of human iv-type collagen | |
| CA2281262C (en) | Anti-human medullasin monoclonal antibody, process for producing the same and immunoassay using the same | |
| EP0314338A1 (en) | Method for measuring human insulin | |
| EP0133540A1 (en) | Receptor assays using labeled monoclonal anti-idiotypic antibodies | |
| US5650324A (en) | Inhibitor and anti-inhibitor monoclonal antibodies specific for horseradish peroxidase | |
| EP0131415A2 (en) | Monoclonal antibodies, processes for their preparation and methods for their use |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PCNP | Patent ceased through non-payment of renewal fee |