WO1993007493A1 - Method for the determination of sulfidoleukotrienes in tissues and biological fluids and its application in diagnosis of allergies and other inflammatory diseases - Google Patents
Method for the determination of sulfidoleukotrienes in tissues and biological fluids and its application in diagnosis of allergies and other inflammatory diseases Download PDFInfo
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- WO1993007493A1 WO1993007493A1 PCT/EP1992/002238 EP9202238W WO9307493A1 WO 1993007493 A1 WO1993007493 A1 WO 1993007493A1 EP 9202238 W EP9202238 W EP 9202238W WO 9307493 A1 WO9307493 A1 WO 9307493A1
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- 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
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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- 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
- G01N33/88—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving prostaglandins or their receptors
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- 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
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
Definitions
- the present invention concerns a method for the determination of sul- fidoleukotrienes in tissues and biological fluids and its use in the diagnosis of allergies and other inflammatory diseases.
- the release of inflammatory mediators by various types of blood or tissue cells upon interaction with various stimulants is a common feature of inflammatory processes occurring in various acute or chronic diseases, such as rheumatic or kidney diseases.
- allergic reactions the release of histamine by blood basophils and/or tissue mast cells has long been considered a major feature.
- the determination of histamine in supernatants from suspensions of isolated blood leukocytes from allergic patients in vi ⁇ tro, following interaction with allergens to which they are sensitive, is a procedure which has been extensively used in allergy research.
- the only current diagnostic method in vitro widely used is the serologic determination of allergen-specific IgE antibodies, by various types of radio-immu- nological or immunoenzymatic assays (e.g. RAST assay).
- RAST assay radio-immu- nological or immunoenzymatic assays
- Such assays only detect the occurrence of antibodies, but do not reflect the most relevant pathophy- siological feature of the allergic reaction, namely the production of inflammatory mediators by the reactive cells upon interaction with the responsible allergen(s).
- practical and fiable cellular assays would be most desirable for the routine diagnosis of allergic and other inflammatory diseases.
- the object of the present inven ⁇ tion is to provide a series of novel cellular assays enabling to achieve that purpose.
- the sulfidoleukotrienes (sLT) LTC4, LTD4 and LTE4 are inflammatory mediators, which were previously collectively denominated Slow Reactive Substance of Anaphylaxis (SRS-A). They are synthesized in many cell types, such as tissue mast cells, blood basophils, macrophages, eosinophils and kidney mesangial cells. They play an important role in pathological events of inflammation and allergic reactions, particu ⁇ larly in IgE-mediated allergic reactions (Schleimer et al. J. Allergy clin. Immunol., 74, 473-481, 1984).
- SRS-A Slow Reactive Substance of Anaphylaxis
- a method which comprises a combina ⁇ tion of various, in part novel and original features, by which a routine in vitro cellular diagnostic assay for allergies and other inflammatory diseases can be established.
- the subject of the present invention is thus a method for detecting the sul ⁇ fidoleukotrienes sLT of the group LTC4, LTD4 and LT ⁇ 4 by a single immunoenzy- maticELIS assay, based on the interaction between one or several monoclonal anti- sLT antibodies and a sLT conjugated to a revealing enzyme, which method is charac- terized in that a biological sample, wherein a content of sLT is assumed, is contacted with a monoclonal anti sLT-antibody bound to a carrier, the sLT present in the sample are bound to the said antibody, subsequently a conjugate of a sLT with a revealing en ⁇ zyme is added to the charged carrier and eventually the amount of the conjugate bound to the carrier is evaluated, which amount is in an inverse correlation with the sLT bound on the carrier.
- a further subject of the present invention is an application of the above-de ⁇ fined method for diagnostic in vitro tests for the diagnosis of inflammatory diseases.
- a further subject of the present invention is a kit of reagents for carrying out the method, which kit comprises a microtiter plate having wells which are coated with monoclonal anti-sLT antibodies, and a conjugate of a leukotriene with a revealing enzyme.
- Such an assay involves the following steps :
- preincubation for a relatively short time e.g. incubation (5 - 10 minutes) of blood leukocytes with a special group of cytokines, namely EL-3 (interleukin 3), IL-5(interleukin 5), GM-CSF (granulocyte ⁇ nonocyte colony stimulating factor) or NGF (nerve growth factor) has the effect to considerably enhance the capacity of these cells to produce and release media ⁇ tors, when challenged with appropriate stimulants, such as allergens (for leukocytes of sensitized allergic patients) or non-specific mediator-releasing factors such as comple ⁇ ment components C5a and C3a, various basic peptides or bacterial-derived structures, such as formyl-methyl- peptide (fMLP). This phenomenon has been described as "priming".
- EL-3 interleukin 3
- GM-CSF granulocyte ⁇ nonocyte colony stimulating factor
- NGF nerve growth factor
- the inventor has now achieved similar results by pretreatment of whole leu ⁇ kocytes or diluted whole blood with purified human recombinant cytokines or with ap ⁇ basementtely prepared supernatants of cultures from activated lymphoid cells. Since maxi- mal priming is required for obtaining high sensitivity and release of sLT from the smallest possible number of blood cells and volume of blood, the preferred conditions for achiev ⁇ ing priming in whole diluted blood, as described in Example 3, are of utmost importance for the final performance of the test and for its applicability to routine diagnosis.
- the blood leukocytes either isolated by gradient centrifugation or other techniques known in the art, or as a suspension in whole diluted blood, are set up in the presence of specific allergens (such as pollen extracts, house dust mite extracts etc.) in a suitable medium for a period of several minutes to one hour.
- specific allergens such as pollen extracts, house dust mite extracts etc.
- the blood leu ⁇ kocytes of a putative allergic patient are set up with various doses of allergen extract in fluid form. After the required period of incubation, the cells are centrifuged and the su ⁇ pernatants harvested and analyzed separately for the amount of sLT produced in the sLT ELISA assay described below.
- This method is inconvenient for a routine diag ⁇ nostic technique, since it involves multiple manipulations, including the use of several doses per allergen and one centrifugation step.
- the allergens may also be pro ⁇ vided to the cells as bound to a solid phase. This may be achieved either by adsorption and binding of the allergen to the plastic or nitrocellulose bottom of a microtiter plate, or in the form of an allergen-coupled cellulose disk, similar to those used in the serological RAST assays for allergen-specific IgE antibodies.
- Appropriate binding conditions, in or ⁇ der to obtain high allergen concentrations per surface unit, such as made possible by pretreatment of the solid surface with glutaraldehyde or polylysine, by a biotin-avidin coupling system or by covalent coupling reagents, are to be preferred.
- a unique practical advantage is obtained, in that a single dose incubation with allergen is required in order to obtain optimal production and release of sLT to be measured in the cell supernatant.
- Similar procedures may be used for detecting the production of sLT under other circumstances and for the diagnosis of diseases other than IgE specific allergy.
- the capacity of blood leukocytes to produce sLT when challenged with a num ⁇ ber of non specific stimulants, such as component components C5a or C3a, as well as with bacterial structures like fMLP (see Example 5) may be used for evaluating the reac ⁇ tive capacity of the blood cells in various types of immunodeficiencies and of inflamma ⁇ tory diseases, like rheumatic diseases.
- the spontaneous production of sLT by blood or tissue cells, including biological fluid (e.g. synovia! fluid, urine) may be used for assess ⁇ ment of inflammatory disease activity. In that case, it may be necessary first to concen ⁇ trate the sLTs by extraction or passage on an adsorption chromatography column.
- the new tests may also be used for the diagnosis of so-called pseudo-allergies, in which inflammatory cells produce the same mediators than in IgE- mediated allergies but where the triggering mechanism is different.
- a classical example is the intolerance reactions to aspirin and other non steroidal anti-inflammatory drugs (NSAIDs).
- NSAIDs non steroidal anti-inflammatory drugs
- the drugs are inducing the production and release of histamine and sLT in intolerant patients by some other mechanism than IgE-E mediated basophil activation.
- the precise mechanism is, however, not yet known .
- the new sLT test also offers the possibility to explore for diagnostic purposes a number of other allergies and pseudo-allergies, hitherto difficult or impossible to detect in vitro, such as intolerance reactions to foods and food additives, drugs and other chemical agents, air pollutants, etc.
- a key element in the new assays is the method developed for assessing all sLTs quantitatively in a single easy-to-perform immunoenzymatic assay.
- HPLC High Pressure Liquid Chromatography
- RIA Radioimmunoassay
- the RIA however, has the disadvantages of using radioactive reagents, an increasingly objectionable procedure, encompassing high pro ⁇ duction costs. Furthermore, up to recently, only poiyclonal antibodies of sufficient af- finity and avidity for LT have been available for such assays. In particular, only a few poiyclonal antibodies of high specificity for LTC 4 have been described (Lindgren et al., (FEBS Lett. 152, 83-88, 1983; Wynalda et al. Anal. Chem. 56, 1862-1865, 1984).
- LTE4 is conjugated to bovine serum albumin, and coated to the wells of a microtiter plate.
- Biotinylated anti-sLT monoclonal antibody is reacted with the analytical sample con ⁇ taining sLT, followed by incubation in the microtiter plate coated with LTE4-BSA and revelation with avidin-coupled peroxidase.
- Figs.1 A and IB show diagrammatic drawings of a one-step and a two-step sLT-ELISA-Test, respectively, for its use in the diagnosis of allergies and in ⁇ flammatory diseases
- Fig. 2 shows in a diagram the capture of phosphatase from LTD4/AP reaction mixtures by solid phase monoclonal anti-sLT antibody.
- Fig. 3 shows in a set of two diagrams the LTD4 detection by inhibition ELISA using different combinations of (LTD)-AP and anti-sLT antibody.
- Fig. 4 shows in a diagram the correlation of sLT-measurement by ELISA and RIA
- Fig 5 shows in a diagram the allergen triggered sLT generation and histamine release of isolated human mononuclear cells (MNC).
- Fig 6 shows in a set of two diagrams the ELISA detection of allergen-trig- gered-sLT generation in diluted whole blood.
- Fig. 7 shows in a set of two diagrams the correlation between the amount of sLT or Mstamine generated by mononuclear cells isolated or in whole blood, in 12 individuals; the cells were challenged with anti-IgE antibody.
- Fig. 8 shows in a set of three diagrams the comparison of skin test, RAST and sLT production from 38 patients tested with 5 allergens.(6-Grasses , Birch, D.pt eronyssinus, Mugwort, Plantain).
- Fig. 9 shows in a diagram the sLT production of an aspirin-sensitive patient upon challenge with acetylsalicylic acid (ASA) in the sLT assay.
- ASA acetylsalicylic acid
- Fig 10 shows in a set of diagrams the comparison of the sLT release in IL3- primed and untreated (HACM alone) diluted whole blood of two donors after 2, 4 and 6 h; the cells were specifically challenged with fMLP or anti-IgE anti ⁇ bodies.
- the monoclonal anti-sLT antibody is coated onto microtiter plates, or other solid phases such as nitrocellulose membranes, without any modifica ⁇ tion, either directly, with the help of coating agents such as glutaraldehyde or polylysine, or of a biotin-avidin coupling system, or indirectly with the help of an anti- mouse IgG antibody.
- coating agents such as glutaraldehyde or polylysine, or of a biotin-avidin coupling system, or indirectly with the help of an anti- mouse IgG antibody.
- sLTs produced by the cells are directly bound.
- the revealing step of the assay is provided by mere addition of an LTD4- or LTE4- alkaline phosphatase conjugate, the binding of which is inhibited in direct correlation to the amount of sample sLT bound.
- Such a simplified sLT inhibition ELISA assay requires substantially less synthetic leukotriene for conjugate production, shows improved sen ⁇ sitivity and reagent stability, and allows simple and effective determination of sLT gen ⁇ erated by small numbers of basophils in whole blood.
- Synthetic LTD4 was conjugated to glutaraldehyde-activated alkaline phos ⁇ phatase (AP) (0.2 % glutaraldehyde and 175 nM in phosphate buffer saline pH 7.4) for 2 hr at room temperature. The conjugate was then dialyzed overnight at 4°C or puri- fied by FPLC. After dialysis or FPLC purification, 1 % BSA (w/v) was added and the preparation of LTD4-AP was stored in aliquots.' Variations in the amount of LTD4 used for the coupling reaction lead to different LTD4 / AP ratios, and lead to differences in the amount of phosphatase activ ⁇ ity picked up by anti-sLT antibody (Fig. 2).
- AP glutaraldehyde-activated alkaline phos ⁇ phatase
- Fig. 2 Diagram of Fig. 2: AP solution was reacted with increasing amounts of LTD4 in the presence of glutaraldehyde.
- reaction mixtures were incubated with anti-sLT moAb 1 A-LDR1 (final dilutions 1/25 and 1/500, respectively) in GaM-coated micro wells (1 ⁇ m/well) for 4 hours at 4°C (solid circles).
- Non-specific binding was determined in a similar way but with omission of anti-sLT antibody (open circles).
- the phosphatase substrate pNPP was added and optical density at 405 nm was measured after 4 hours at 37°C. Data are duplicate means (variability range ⁇ 15%).
- BG indicates the spontaneous hydrolysis of pNPP alone.
- conjugate ratios of LTD4 /AP ratios of 4:1 to 20:1 are optimal for performance of the assay in terms of sensitivity.
- Such conjugates show practically no unspecific binding to microtiterplate wells not coated with anti-sLT antibody and therefore a very low background in the sLT assay.
- Such conjugates have also been found to be stable for over 6 months under appropriate stor ⁇ age conditions.
- microtiterplate wells or other solid phase recipients known in the art may be coated directly with anti- sLT antibody, possibly with the help of binding agents such as glutaraldehyde or polylysine or of a biotin-avidin coupling system.
- a preferred method is to coat the well with a mixture of anti-mouse IgG antibody and anti-sLT antibody since this considerably increases sensitivity and reduces the amount of anti-sLT antibody re ⁇ quired per well.
- the analytical sample and a fixed amount of LTD4-AP conjugate are added and incubated for periods which may vary from 30 minutes to 4 hours at 4°C.
- phosphatase is re-vealed by incubation with para-nitrophenyl phosphate (pNPP) (1 mg ml) added and incubated for 1 to 4 h at 37 °C in phosphatase detection buffer (diethanolamine 97 ml, MgCl2.6H 2 O : 100 mg; sodium azide 0.2 g per 1000 ml H O; pH 9.8).
- phosphatase detection buffer diethanolamine 97 ml, MgCl2.6H 2 O : 100 mg; sodium azide 0.2 g per 1000 ml H O; pH 9.8.
- the optical density of the solution at 405 nm is then measured by an appropriate densitometer.
- optimal conditions for the sLT ELISA test may be found by varying the amounts of anti-sLT antibody coated and the amount of LTD4- AP conjugate used for revelation.
- FIG. 3 Diagrams of Fig. 3: The upper panel shows the effect of varying concentrations of anti- sLT moAb 1A-LDR1 at a constant amount of (LTD4)4-AP (final dilution 1/25), The lower panel shows inhibition curves obtained with different amounts of (LTD4)4-AP at a fixed dilution of anti-sLT moAb 1A-LDR1 (1/500).
- NSB indicates non-specific binding of (LTD4)-AP, i.e. in the absence of anti-sLT antibody. Data are means of duplicates (variability range ⁇ 15%)
- the lower detection limit was 6 pg LTD4/100 ⁇ l sample and 50 % inhibition of the standard LTD4 curve was obtained around 65 pg LTD4.
- the important practical consequence of such an increase in sensitivity is that multiple assays, for example the detection of reactions of the patient's blood cells to several different allergens, may be performed with much smaller blood samples (e.g. 25 ⁇ l blood per assay, see Example 4). With less sensitive assays, the amounts of blood required for routine diagnosis become prohibitive.
- the absolute sensitivity of the assay can still be increased by modifying the conditions of conjugation of LTD4 to AP, for example by using polymerized AP and/or by selecting optimal conjugate fractions through FPLC purification.
- the signal generation step may also be enhanced , for example by using a luminogenic phos ⁇ phatase detection reagent (e.g. AMPPD) in conjunction with microtiter luminescence imaging (Maly et al. Anal. Biochem. 168, 462-469, 1988) or by employing enzyme amplification with the NADP/alcohol dehydrogenase-diaphorase p- iodonitrotetra- zolium violet system (Self, J.Immunol.
- a luminogenic phos ⁇ phatase detection reagent e.g. AMPPD
- microtiter luminescence imaging Mely et al. Anal. Biochem. 168, 462-469, 1988
- the sensitivity of the sLT- ELISA assay is of the same order as that of the best RIA available, and the results cor ⁇ relate very well with those obtained with an RIA, at a fraction of the cost and without the inconveniences caused by the RIA (environmental hazards, short reagent shelf life).
- basophil-containing mononuclear cell sus- pensions, leukocytes or diluted whole blood are distributed in aliquots in a first series of containers (test tubes or microtiterplate wells), pre-treated with appropriate cytoki ⁇ nes such as IL3, IL5 or GM-CSF and then left to interact with allergens provided either in a fluid phase or as bound to some solid phase.
- cytoki ⁇ nes such as IL3, IL5 or GM-CSF
- allergens provided either in a fluid phase or as bound to some solid phase.
- the containers are centrifuged and the supernatants harvested. These supernatants are then analyzed for sLT content in a second series of containers, usually microtiterplate wells coated with anti-sLT antibody and treated for an sLT-ELISA as described in Example 2.
- peripheral blood mononuclear cell suspensions blood anticoagulated with 10 mM EDTA is mixed with 0.25 vol ⁇ umes of 6 % dextran and the erythrocytes allowed to sediment for 90 minutes at room temperature.
- Leukocytes are pelleted from the supernatant by centrifugation (150 g, 20 - min at room temperature) and resuspended in HA buffer (20 mmol Hepes, 125 mmol Nad, 5 mmol KC1, 0.5 mmol glucose and 0.025 % BSA) .
- the cells are then further fractionated by Ficoil-Hypaque density gradient (specific gravity 1.077) centrifugation (600 g, 40 min at room temperature) .
- MNC are harvested from the interphase, washed 3 times (150 g, 10 min at 4°C) in HA buffer and are finally resuspended in HACM buffer (HA buffer supplemented with 1 mmol CaC-2 and 1 mmol MgCl 2 ) at a cell density of 10 7 or 5 x 10 ⁇ cells / ml.
- HACM buffer HA buffer supplemented with 1 mmol CaC-2 and 1 mmol MgCl 2
- dextran-sedimented leukocytes may be used directly, without washing, following centrifugation and resuspension in HACM buffer.
- sLT For generation of sLT by leukocytes or mononuclear cells, 100 ⁇ l/well of cell suspension in HACM buffer are heated at 37°C for 10 min in flat-bottomed mi ⁇ crotiter plates. IL 3 or GM-CSF (50 ⁇ l, final concentration 10 ng/ml) or HACM buffer are added either simultaneously or for 10 min before adding the stimulant (50 ⁇ l, aller- gen extract or non-specific stimulant such as C5a or fMLP). The reaction is stopped 30 - 40 min after the addition of stimulants by cooling the microtiterplate on ice. After centrifugation (600 g, 5 min at 4°C), 100 ⁇ l of supernatant are transferred into the ELISA assay microtiter plate and assayed for sLT as described in Example 2.
- stimulant 50 ⁇ l, aller- gen extract or non-specific stimulant such as C5a or fMLP
- the diagram of Fig 5 shows the allergen triggered sLT generation and hista ⁇ mine release of isolated human mononuclear cells (MNC).
- MNC 2.5 x lO ⁇ /well
- A.U. mite allergic donor
- DF Dermatophagoides farinae allergen
- Supernatants were harvested after 40 minutes, and generation of sLT and release of hista- mine were determined by ELISA and RIA, respectively, as detailed in this specification.
- Spontaneous histamine release was 1.2 %. Data are duplicates means (variability range >15%)
- the isolated MNC (250O00 / microwell) of an individ ⁇ ual allergic to the house dust mite Dermatophagoides pteronyssinus generate sLT in re- sponse to increasing doses of the relevant allergen.
- These sLT were easily detected by sLT-ELISA and their release was accompanied by a correlated release of histamine.
- MNC contain about 1% basophils and as only 100 ⁇ l of the total incubate (250 ⁇ 1) are used for sLT measurements, the sLT-ELISA detects generation of sLT from about 1000 basophils. Changing incubation volume and supernatant volume used for sLT-ELISA may allow working with even less mononuclear cells.
- the practicability " of the sLT as- say would be much improved if isolation of mononuclear cells could be bypassed and the test performed directly in whole blood.
- the inclusion of blood plasma proteins in the sLT -ELISA assay does not markedly affect sLT recovery in the sLT- ELISA assay and varies from 70 - 90 % depending upon the plasma concentration. Therefore, detection of sLT generated in diluted whole blood upon stimulation of al ⁇ lergen appeared feasible.
- venous whole blood is drawn into suitable closed containers with heparin (final concentration 12 U/ml).
- 100 ⁇ l per well of heparinized blood diluted 1 :4 with HACM buffer are pipetted into microtiter plates, after which the procedure is exactly as described above for isolated mononuclear cells.
- the allergen Dermato ⁇ phagoides pteronyssinus stimulates release of sLT in diluted whole blood from a mite allergic individual.
- Phleum pratense to which this individual is not allergic, does not cause significant release of sLT. even in cells prein- cubated with 1L3.
- the specificity of the mite allergen-induced sLT generation is further demonstrated by the fact that non allergic individuals show no sLT generation when challenged with either allergen, with or without IL 3 "priming".
- Fig 6 shows the ELISA detection of allergen-trig- gered-sLT generation in whole blood.
- Heparinized blood 25 ⁇ l/well
- DF dermatophagoides farinae
- PP phleum pratense
- sLT are given with reference to total J__*a_r_ine present in the sample, a measure of blood basophil content, i.e. as pg sLT per total amount of histamine (ng)
- Fig. 6 The results shown in Fig. 6 reflect sLT generation by approximately 100 - 300 basophils and are in the range expected from experiments with isolated MNC. As shown in Fig. 7, there is an excellent correlation between the results obtained with di ⁇ luted whole blood and with isolated MNC.
- Fig. 7 shows in a set of two diagrams the correlation between the amount of sLT (upper panel) or histamine (lower panel) generated by mononuclear cells isolated (PBL) or in full blood, in 12 individuals; the cells were challenged with anti-IgE antibody 100 ⁇ g/ml.
- Fig. 8 shows in a set of three diagrams the comparison of skin-test, RAST and sLT production from 38 patients tested with 5 allergens.(6-Grasses , Birch, D.pt. eronyssinus, Mugwort, Plantain).
- the results obtained in parallel with skin tests and with the classical serological RAST assay for allergen-specific IgE show that there is a better correlation between the sLT assay and the skin test, and hence with the clinical status of the patient, than between the RAST assay and the skin test. This shows that the theoretical advantage of using a cellular assay for detecting allergic hypersensitivity is for the first time confirmed in practice.
- the microtiterplate needed for the sLT-ELISA and precoated with anti-sLT antibody are also simultaneously precoated with the intended stimulating agent, for example by allergens or allergen mixtures.
- the num- ber and succession of operations needed are :
- this one step assay may be due to interference between the allergens coated in the wells and some of the reagents in ⁇ volved, in particular with serum antibodies present in excess and which may neutralize the allergens. In that case, the use of allergen-coated disks and/or a centrifugation step to remove plasma proteins may prove necessary.
- the sLT -ELISA assay in the forms described represents the first type of cellular allergy diagnostic assay clearly suitable for economic and efficient in vitro allergy diagnosis.
- Fig. 10 shows in a set of diagrams the comparison of the sLT release in IL3 -primed and untreated (HACM alone) diluted whole blood of two donors after 2, 4 and 6 h; the cells were specifically challenged with fMLP or anti-IgE antibodies.
- Fig. 10 shows the reactivity of various donors to the fMLP stimulant as well as the priming effect of pretreatment of the MNC with IL 3. Since the evolution and stage of activity of inflammatory diseases as well as some states of immune defi- ciency are reflected by cellular hyper- or hyporeactivity, respectively, the sLT-ELISA test may be expected to play a useful role in the diagnosis and monitoring of such dis ⁇ eases.
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Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
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DE69217038T DE69217038T2 (en) | 1991-10-01 | 1992-09-25 | IN VITRO PROCEDURE FOR DETERMINATION IN ALLERGY DIAGNOSES AND OTHER INFLAMMATORY DISEASES |
JP5506593A JPH06503427A (en) | 1991-10-01 | 1992-09-25 | Methods for determining sulfide leukotrienes in tissues and biological fluids and their application in the diagnosis of allergies and other inflammatory diseases |
EP92920592A EP0559871B1 (en) | 1991-10-01 | 1992-09-25 | Method for an in vitro diagnosis of allergies and other inflammatory diseases |
US08/070,326 US5487977A (en) | 1991-10-01 | 1992-09-25 | Method for the determination of sulfidoleukotrienes in tissues and biological fluids and its application in diagnosis of allergies and other inflammatory diseases |
AU26525/92A AU651249B2 (en) | 1991-10-01 | 1992-09-25 | Method for the determination of sulfidoleukotrienes in tissues and biological fluids and its application in diagnosis of allergies and other inflammatory diseases |
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CH290491 | 1991-10-01 |
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PCT/EP1992/002238 WO1993007493A1 (en) | 1991-10-01 | 1992-09-25 | Method for the determination of sulfidoleukotrienes in tissues and biological fluids and its application in diagnosis of allergies and other inflammatory diseases |
Country Status (9)
Country | Link |
---|---|
US (1) | US5487977A (en) |
EP (1) | EP0559871B1 (en) |
JP (1) | JPH06503427A (en) |
AT (1) | ATE148234T1 (en) |
AU (1) | AU651249B2 (en) |
CA (1) | CA2097417A1 (en) |
DE (1) | DE69217038T2 (en) |
ES (1) | ES2097355T3 (en) |
WO (1) | WO1993007493A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998019163A1 (en) * | 1996-10-29 | 1998-05-07 | Nen Life Science Products, Inc. | A solid phase cell-based assay |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FI951778A (en) * | 1995-04-12 | 1996-10-13 | Aboatech Ab Oy | Method for diagnosing allergies |
US6958148B1 (en) | 1998-01-20 | 2005-10-25 | Pericor Science, Inc. | Linkage of agents to body tissue using microparticles and transglutaminase |
US6919076B1 (en) | 1998-01-20 | 2005-07-19 | Pericor Science, Inc. | Conjugates of agents and transglutaminase substrate linking molecules |
DE50100654D1 (en) * | 2001-06-05 | 2003-10-23 | Hanns-Wolf Baenkler | Diagnostic procedure based on lipid measurement parameter modulation / effector quotient profiles |
US8029454B2 (en) | 2003-11-05 | 2011-10-04 | Baxter International Inc. | High convection home hemodialysis/hemofiltration and sorbent system |
US8114276B2 (en) | 2007-10-24 | 2012-02-14 | Baxter International Inc. | Personal hemodialysis system |
EP3060244A2 (en) * | 2013-10-25 | 2016-08-31 | Lpath, Inc. | COMPOSITIONS AND METHODS FOR BINDING CYSTEINYL LEUKOTRIENES (cysLTs) FOR TREATMENT OF DISEASE |
CN112415191A (en) * | 2020-11-06 | 2021-02-26 | 郑州人福博赛生物技术有限责任公司 | Leukotriene detection kit |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE3218270A1 (en) * | 1982-05-14 | 1983-11-17 | Bayer Ag, 5090 Leverkusen | Method for determining metabolic products of arachidonic acid by means of a binding inhibition assay on an immunosorbent with monoclonal antibodies |
US4559310A (en) * | 1983-05-20 | 1985-12-17 | Dana Farber Cancer Institute | Assay methods and systems utilizing mast cell clones |
JPS6111663A (en) * | 1984-06-28 | 1986-01-20 | Ono Pharmaceut Co Ltd | Leukotriene labeled by enzyme used in enzyme immunomeasurement method |
ATE108560T1 (en) * | 1988-03-31 | 1994-07-15 | Sankyo Co | DETERMINATION OF ARACHIDONIC ACID DERIVATIVES AND EQUIPMENT THEREFOR. |
-
1992
- 1992-09-25 DE DE69217038T patent/DE69217038T2/en not_active Expired - Fee Related
- 1992-09-25 US US08/070,326 patent/US5487977A/en not_active Expired - Fee Related
- 1992-09-25 AU AU26525/92A patent/AU651249B2/en not_active Ceased
- 1992-09-25 EP EP92920592A patent/EP0559871B1/en not_active Expired - Lifetime
- 1992-09-25 WO PCT/EP1992/002238 patent/WO1993007493A1/en active IP Right Grant
- 1992-09-25 JP JP5506593A patent/JPH06503427A/en active Pending
- 1992-09-25 ES ES92920592T patent/ES2097355T3/en not_active Expired - Lifetime
- 1992-09-25 CA CA002097417A patent/CA2097417A1/en not_active Abandoned
- 1992-09-25 AT AT92920592T patent/ATE148234T1/en not_active IP Right Cessation
Non-Patent Citations (2)
Title |
---|
BIOCHIMICA ET BIOPHYSICA ACTA vol. 1081, no. 3, 1 March 1991, AMSTERDAM NL pages 274 - 278 M. REINKE ET AL. 'A monoclonal antibody against the sulfigopeptide leukotrienes ltc4, ltd4 and lte4.' cited in the application * |
PATENT ABSTRACTS OF JAPAN vol. 10, no. 157 (P-464)(2213) 1 June 1986 & JP,A,61 11 663 ( ONO YAKUHIN KOGYO ) 20 January 1986 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998019163A1 (en) * | 1996-10-29 | 1998-05-07 | Nen Life Science Products, Inc. | A solid phase cell-based assay |
Also Published As
Publication number | Publication date |
---|---|
AU2652592A (en) | 1993-05-03 |
ATE148234T1 (en) | 1997-02-15 |
US5487977A (en) | 1996-01-30 |
DE69217038D1 (en) | 1997-03-06 |
EP0559871B1 (en) | 1997-01-22 |
EP0559871A1 (en) | 1993-09-15 |
AU651249B2 (en) | 1994-07-14 |
ES2097355T3 (en) | 1997-04-01 |
JPH06503427A (en) | 1994-04-14 |
DE69217038T2 (en) | 1997-10-30 |
CA2097417A1 (en) | 1993-04-01 |
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