WO2015142305A1

WO2015142305A1 - Monoclonal antibodies developed against h-fabp and the use thereof

Info

Publication number: WO2015142305A1
Application number: PCT/TR2015/000115
Authority: WO
Inventors: Mehmet Ozgun OZEN; Saime Ismet GURHAN
Original assignee: S.K Teknoloji Arastirma Gelistirme San. Ve Tic. Ltd. Sti.
Priority date: 2014-03-20
Filing date: 2015-03-19
Publication date: 2015-09-24

Abstract

With this invention, monoclonal antibodies that has affinity to heart-type fatty acid binding protein (h-FABP) have been determined. Monoclonal antibodies were developed against the h-FABP protein. The said monoclonal antibodies can be used in determining h-FABP levels in blood, serum or plasma of an individual in immunological and serological tests. As a result, this immune test kit may be used in the diagnosis of the heart attack and/or in the detection of therapeutics which have toxicity towards cardiac muscles.

Description

DESCRIPTION

MONOCLONAL ANTIBODIES DEVELOPED AGAINST H-FABP

TECHNICAL FIELD

The present invention encompasses obtaining hybridoma cell lines, named 1F114F6P3G11 and 1F114F6P3H8, which enable the detection of heart-type fatty acid binding protein (h-FABP) which reaches a detectable level in blood stream of an individual within the 15 minutes that follow a heart attack and hence, which enable the production of diagnostic kits which can be used in the detection of heart attacks and the production of monoclonal antibodies produced by these hybridoma lines. As the method used is in vitro immunization, the immunization period is shorter, and the resulting monoclonal antibodies belong to IgM isotypes.

PRIORT ART

With this invention, it has been shown that diagnostic, test kit or systems wherein h-FABP amounts above the reference values in the serum, plasma or blood can be qualitatively or quantitatively measured to manage the diagnosis of an individual who has undergone acute myocardial infarction within 1-3 hours of the infarction by means of in vitro immunization against h-FABP protein by improved IgM class antibodies.

Modem medicine, while offering personalized treatment models, the models include the patient's individual needs and risks. The most important of these risks is that cardiovascular problems are direct causes of morbidity and mortality (1). Therefore, reliable diagnosis of cardiovascular problems is highly important for success of the treatment. Due to high risk of cardiac death of individuals with acute coronary syndrome, they need to be distinguished from patients with non-traumatic chest symptoms and be directed by means of appropriate treatment (2). Clinical diagnosis of acute myocardial infarction (AMI);

• Admission symptoms and findings, i · Electrocardiogram (EKG),

• Carried out based on the amount of anaiyte to pass into the bloodstream of the individual resulting from damage to the cardiac muscle (3).

In 33% of individuals with complaints of AMI, it has been determined by the fact that no chest pain was observed is not a reliable indicator in terms of diagnosis (4). On the otherhand, diagnostic ECG helps provide a net diagnosis in %40 of patients in their first diagnosis (4). In addition to chest pain and ECG results, anaiytes passing to the bloodstream need to also be determined in AMI diagnosis (5).

The level of detection of the reagents, used in the literature and in clinical practice, after cardiac muscle damage and the period of time in which they can be detected in is presented in below table.

Table - 1. Reagents released from damaged cardiac muscle cells and durations in which they are clinically significant.

Unlike the above reagents, it has been determined that h-FABP contains certain clinical advantages due to its 15 kDa molecular weight and the fact that it is only present in cardiac muscle cells (5). It is released from damaged cardiac muscle cells within 1-3 hours, reaches its highest value within 6-8 hours and returns to its normal level within 24-30 hours (11, 12, 13). Moreover, it has also been reported that h-FABP released from damaged cardiac muscle cells can mix into the blood not only after a heart attack but also in other damages to peripheral blood myocytes and that h-FABP is a useful reagent also for therapeutics that exhibit toxicity above the cardiac muscle cells (16).

BRIEF DESCRIPTION OF THE INVENTION

With this invention, the production of a fast qualitative or quantitative diagnostic and/or test kit, device, ELISA system which can be applied in bedside of patient in places where experts need to be present (such as ambulances, aircraft, medical cabinets, infirmary and particularly hospitals and clinics) or patient suffering from heart disease or who has undergone cardio- vascular surgery can carry on himself, can purchase from pharmacies, can easily use in a home environment, which provides fast results and which can make use of monoclonal antibodies produced by 1F114F6P3G11 and 1FU4F6P3H8 hybridoma clones specified in the invention is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Diagram showing standard protein curve. By means of the formula obtained from the standard curve, the quantity of the purified monoclonal antibodies has been determined.

Figure 2: Graphic providing the formula wherein antibody titer values obtained via ELISA are converted to antibody quantity. Quantity of produced antibodies were determined via the titers obtained in the indirect ELISA controls performed prior to the purification of the antibodies.

Figure 3: The placement of pads in the test kit following lamination.

DETAILED DESCRIPTION OF THE INVENTION

With this invention, monoclonal antibodies, that have affinity to heart-type fatty acid binding protein (h-FABP) have been determined. Monoclonal antibodies were developed against h-FABP protein. The said monoclonal antibody can be used in determining the h-FABP levels in blood, serum or plasma of an individual in immunological and serological tests. As a result, this immune test kit may be used in the diagnosis of the heart attack.

The present invention enables the use of the antibodies produced by the hybridoma clones named as 1F114F6P3G11 and 1F114F6P3H8, against recombinant human heart-type fatty acid binding protein (h-FABP) specifically in immune tests. The said antibodies may be used in human clinics generally for research and diagnosis.

Production of Antibodies l) Immunogen: The h-FABP to be used as immunogen was procured in commercial form as having been purified from human heart tissue (Product code: P196-1, Genway Biotech. Inc. 6777 Nancy Ridge Drive San Diego, CA 92121, USA). 2) Immunization:

2.1) Isolation of Splenocytes: Three Ba!b/c mice were taken from 950 μΙ of 10% ketamine- HCI solution (Alfasan International BV, The Netherlands) and 500 μ! of 2% alfazyn A (Alfasan International BV, the Netherlands) and the volume was increase to 2 ml with 0.9% NaCI. From this mixture, the test animals were anesthetized by means of 1 ml/kg injection (14) after weight of the test animals was checked, and were euthanized by means of cervical dislocation after controlling for foot and eye reflex. Surface of the test animals was disinfected with EtOH 70% (Sigma Chemical Co., St. Louis, MO, USA) and they were then taken to the laminar flow cabinet and the process was continued therein. The spleens of the test animals were isolated under aseptic conditions and after their peripheral connective tissue and organ membrane were separated, they were passed through a sterile steel filter using a 5 ml syringe plunger, and the cell aggregates were obtained in a 12 ml serum-free RPMI 1640 nutrient medium (Biochrom AG, Leonorenstr. 2-6, 12247 Berlin , Germany). The cell suspension was washed 2 times with 12 ml HBSS buffer (Biochrom AG is Leonorenstr. 2-6, 12247 Berlin, Germany) and their aggregates were dissolved by means of pipetting. In washing process, the supernatant was removed by centrifugation, 1000 rpm, 4 °C, 6 minutes (Eppendorf, Germany).

The cells were then washed with a 5 ml, 0.83% NH₄CI sol. (Sigma Chemical Co., St. Louis, MO, USA) and cell lysis of erythrocytes was provided under a hypotonic environment. The centrifugation process was repeated (1000 rpm, 4 °C, 6 minutes). The resulting B-lymphocytes were resuspended with serum-free and phenol red-free PMI nutrient medium (Biochrom AG, is Leonorenstr. 2-6, 12247 Berlin, Germany). A 100 μΙ sample was taken and stained with 0.4% trypan blue (Sigma Chemical Co., St Louis, MO, USA) and the live/dead cell number and viability % were determined. According to the process of cell counting with Neubauer chamber, the cell concentration was adjusted as 1 x 106 cells/ml. 2.2) Thymocyte EL-4 Cell Cultures: EL-4 thymocyte cells were obtained commercially (CLS Cell Lines Service GmbH, Dr. Eckener-Strasse 8 69214 Eppelheim, Germany). Cells were produced with DMEM (Biochrom AG, Leonorenstr. 2-6, 12247 Berlin, Germany) (10% donor horse serum [(Sigma Chemical Co., St. Louis, MO, USA), 2 mM L-glutamine, 10 ug/ml gentamicin (Biochrom AG, Leonorenstr. 2-6, 12247 Berlin, Germany)]. Cells were stocked at - 196 °C using freeze medium consisting of 90% fetal bovine serum 10% DMSO. When cells reached 1 x 10⁶ cells/ml cell concentration in logarithmic phase, they were cultured again with fresh nutrient medium in the presence of 10 Mg/ml phorbol myristate-12-myristate-acetate (PMA). After 24 hours the culture supernatant was collected by centrifugation (3,000 rpm, at 4 °C for 10 minutes) and used in in vitro immunization procedure. Unused supernatant were stored in the freezer at - 20 °C until used.

2.3) In vitro Immunization; Serum-free RPMI 1640 nutrient medium 1 x iO⁶ cells/ml cell suspension, 10 ug/ml MDP (N-acetyl-muramyl-L-alanyl-D-isoglutamine hydrate) (Sigma Chemical Co., St. Louis, MO, USA) and 200 ng/ml h-FABP (Genway Biotech. Inc. 6777 Nancy Ridge Drive San Diego, CA 92121, USA) were kept at room temperature for 20 minutes in serum-free and phenol red-free RPMI 1640 nutrient medium (Biochrom AG, Leonorenstr. 2-6, 12247 Berlin , Germany)

Then, the volume of said cell suspension was doubled, again using the same nutrient medium, and the following materials were added keeping in mind their concentration in the total volume: rabbit serum (2%) EL-4 thymocyte cell culture supernatant (25%), 2 mM L-glutamine, 10 ug/ml gentamicin, 10 IU penicillin, 10 μ/ml streptomycin (Biochrom AG, Leonorenstr. 2-6 , 12247 Berlin, Germany), 10 ng/ml IL-4, 2 ng/ml IL-6, 2 ng/ml IL-2, 10 pg/ml IL-la, 50 mM β- mercaptoethanol (Sigma Chemical Co., St. Louis, MO, USA); and the culture was cultured for 5 days at 37 °C in 5% C02 incubator. On 5^th day of the culture, 20 ng/ml IFN-γ and 10 ng/ml IL-10 (Sigma Chemical Co., St. Louis, MO, USA) was added. Incubation of the culture was continued under the same conditions. In supernatant samples taken from cultures on day 7, indirect EUSA determined the presence of anti-h-FABP antibody. The concentration of the cell suspension in culture dishes with positive antibody titer was determined by counting viable cells and the cells were collected for fusion. 3. Indirect ELISA: h-FABP protein was immobilized at 100 μΙ aliquots to 96 well ELISA plates at 50 ng/well. The plates were incubated overnight at 37 °C. After the predetermined period, 0.5% BSA (bovine serum albumin) (w/v) solution was added at 100 μΙ per well and the plate was covered to prevent evaporation and incubated at 37 °C for 1 hour. After 1 hour, the wells were washed with washing solution (200 μΙ/wel!) 3 times at 5 minute intervals [washing solution: PBS (Biochrom AG, Leonorenstr. 2-6, 12247 Berlin, Germany) containing 0.1% Tween 20 (Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany) and free of Ca⁺²-Mg⁺²]. After the washing process, 100 μΙ cell culture supernatant was added to the wells and the plates were covered to prevent evoporation and incubated at 37 °C for 1 hour. After 1 hour, the wells were washed with washing solution as specified above. 100 ml aliquots of conjugate solution were added to the washed wells [0.1% HRP-conjugated anti-mouse IgG antibody (Sigma Chemical Co., St. Louis, MO, USA), wash solution (v/v)]. The microplate were covered to prevent evaporation and incubated at 37 °C for 1 hour. At the end of incubation, the wells were washed as detailed and for the time specified above. 100 μΙ substrate solution was added to the wells (Substrate solution: 1 (OPD) tablet is dissolved in 60 ml ultrapure water, 0.05% of 30% H₂0₂ (Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany) is added to the part to be used.). The microplate was covered to prevent evaporation and incubated at 37 °C for 30 minutes. After 30 minutes, stopping solution, which stops the enzyme-substrate reaction, was added to the wells (Stopping solution: 4 M H2SO4, prepared with ultra-pure water). Reading at 492 nm was carried out at the spectrophotometer (VersaMax 190, Molecular Devices, USA). 4. Myeloma (Ag8) Cell Culture: Ag8 cell line obtained from HUKUK and used in the studies (ΗϋΚϋΚ, Sap Enstitusu, Ankara). The cells were cultured with 10% fetal bovine serum 2 mM L- glutamine, 10 IU penicillin, 10 ς/ΓηΙ streptomycin supplemented RPMI 1640 nutrient medium (Biochrom AG, Leonorenstr. 2-6, 12247 Berlin, Germany) at 5% C0₂ 37 °C. When cells reached to 1 x 10^s cells/ml concentration, they were passaged by dilution to 2 x 10^s cells/ml.

5 The cells were stocked at -196 °C using freezing medium consisting of 90% fetal bovine serum and 10% DMSO).

5. Fusion: Fusion process was carried out by modifying the method reported by IMalbantsoy et al. (15). The splenocytes in the culture determined, by means of indirect ELISA, as producing antibodies against h-FABP and Ag8 myeloma cells in logarithmic phase were collected by centrifugation in separate cultures (1000 rpm, 4 °C, 6 minutes). Cell counts were performed (with 0.4% trypan blue). Cell suspension was combined as 1 Ag8 cell per each splenocyte and homogenized by pipetting. The resulting cell suspension was washed in serum-free and phenol red-free RPMI 1640 nutrient and centrifuged (1000 rpm, 4 °C, 6 minutes). The resulting cell pellet was resuspended with the nutrient medium (100 - 300 μΙ) remaining in the tube using gentle strokes. 0.5 ml of PEG 1500 (Sigma Chemical Co., St. Louis, MO, USA) was added to the suspension drop wise over l min. 6 ml serum-free phenol red-free RPMI 1640 nutrient medium (Biochrom AG is Leonorenstr. 2-6, 12247 Berlin, Germany) was added drop wise over 3 minutes. Then, 6 ml of the same serum-free, phenol red-free nutrient medium was added for 3 minutes. Finally 4 ml of RPMI 1640 nutrient medium (phenol red-free) containing 20% FBS (Biochrom AG, is Leonorenstr. 2-6, 12247 Berlin, Germany) was added.

The cells were sedimented by centrifugation at 1000 rpm, 4 °C, for 6 minutes. The cell pellet was resuspended with 5 ml serum-free RPMI 1640 nutrient medium (phenol red-free) (Biochrom AG, is Leonorenstr. 2-6, 12247 Berlin, Germany) and mixed with pipetting. The cells were sedimented by centrifugation at 1000 rpm, 4 °C, for 6 minutes. The cell suspension was left to incubation at 37 °C in %5 C0₂ containing conditions with 50 ml HAT medium [phenol red-free 20% fetal bovine serum, 10% donor horse serum, 1 X HAT (10 μΜ Hypoxanthine, 0.4 μΜ aminopterin and 16 μΜ thymidine) (Sigma Chemical Co., St. Louis, MO, USA), 2mM L- glutamine, 10 μg/ml gentamycin (Biochrom AG, Leonorenstr. 2-6, 12247 Berlin, Germany) supplemented RPMI 1640 (Biochrom AG, Leonorenstr. 2-6, 12247 Berlin, Germany)]. The resulting 50 ml cell suspension was added in 96 well cell culture dishes at 100 μΙ/well (5 x 96 wells x 100 μΙ). 50 μΙ of the said nutrient medium was added to the wells twice at 72-hour intervals.

In this process, presence of colony like hybridoma growth was looked for. With 48-hour intervals, 50 μΙ of the 200 μΙ nutrient medium was replaced with fresh nutrient medium. After day 15, 100 μΙ nutrient medium was replaced. After day 15, 1 X HAT in the nutrient medium was replaced with 1 X HT (10 μΜ Hypoxanthine and 16 pM thymidine). 100 pL supernatant taken from the wells which has colony formation was tested with indirect ELISA explained before to determine whether or not the reproducing hybridomas produced antibodies against the h-FABP protein.

HAT medium, HT medium, production environment, PBS, FBS diluted at 1/200, DHS diluted at 1/200 and serum-free nutrient medium was used as negative control. Of the wells in which anti- h-FABP antibodies were produced, the ones with the highest antibody titer were cloned.

6. Cloning by Limiting Dilution Method: The hybridomas that formed colonies and which provided the highest antibody titers were cloned. They were taken from the wells in which they formed colonies were diluted with HT medium [20% fetal bovine serum, 1 x HT (10 pM hypoxanthine and 16 pm thymidine) (Sigma Chemical Co., St. Louis, MO, USA), 2mM L- glutamine, 10 pg/ml gentamycin containing P I 1640 (Biochrom AG, Leonorenstr. 2-6, 12247 Berlin, Germany)], counted with 0.4% trypan blue solution and dispersed in 96-wel! culture plates at 1-2 cells per well and incubated at 37 °C at 5% C0₂. 50 pi of the above nutrient medium was added to wells in which colony formation was observed (within 4-6 days). When hybridoma colonies expanded in wells, exchanged nutrient medium volume increased to 100 pl/well. Wells with high titers of antibodies that has more than one colony was cloned again according to the procedure given above. Antibody titers of the wells were determined with indirect ELISA explained above, in which colony type hybridoma growth is observed after cloning. HAT medium, HT medium, production environment, PBS, FBS diluted at 1/200, DHS diluted at 1/200 and serum-free nutrient medium was used as negative control. Wells with high antibody titers were scaled up or stored at -196 °C.

7. Scale-up and Storage: The clones determined as producing the desired monoclonal antibody were transferred to 2 wells of a 24-well plate with Production Medium [20% fetal bovine serum (Sigma Chemical Co., St. Louis, MO, USA), 2mM L-glutamine, 10 pg/ml gentamycin (Biochrom AG Leonorenstr. 2-6, 12247 Berlin, Germany) containing RPMI 1640 (Biochrom AG, Leonorenstr. 2-6, 12247 Berlin, Germany)] and frozen using the previously defined freezing medium and stored at - 196 °C liquid nitrogen. Two clones with the highest titer, 1F114F6P3G11 and 1F114F6P3H8, were subjected to further characterization. For the scale up processes, the 1F114F6P3G11 and 1F114F6P3H8 clones were produced in 24-well microplates at 2 ml/well and in 6-well microplates at 5 ml/well. While the same nutrient medium is used in the production in suspended culture flasks, the fetal bovine serum rate was reduced to 10%.

8. h-FABP Monoclonal Antibody Isotyping: Isotyping of monoclonal antibodies was performed according to ISO-2® Isotyping Kit (Sigma Chemical Co., St Louis, Mo, USA) and according to manufacturer's instructions. Briefly, isotype specific antibodies (IgGl, IgG2a, IgG2b, IgG3, IgA, IgM) were diluted with ultrapure water at 1/1000. From each diluted isotype specific antibodies, 100 μΙ aliquot was added to two wells of 96-weil microplates. Isotype specific antibody coated microplate was incubated at 37 °C for 1 hour. After that, coating solution was poured and the wells were washed 3 times for 5 minutes with 200 μΙ washing solution. 100 μΙ culture supernatant was added to each well (without any dilution). Microplate was incubated for 1 hour at room temperature, supernatants were poured and the wells were washed 3 times for 5 minutes with washing solution. 100 μΙ of conjugate solution (0.1% HRP- labeled anti-mouse IgG antibody) was added to each well and incubated for 30 minutes at room temperature. The washing process was repeated. After the washing process, 100 pL of substrate solution [prepared by adding 1 OPD tablet (Sigma Chemical Co., St. Louis, MO, USA) dissolved in 60 ml of ultrapure water at 0.05% of 30% H202 (Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany) to the part to be used] was added to each well, After incubation for 30 minutes at room temperature, stopping solution (Stopping solution: 4 M H₂S0₄, prepared with ultrapure water) was added to each well and absorbance was measured with a spectrophotometer (VersaMax 190, Molecular Devices, USA) at 492 nm.

Monoclonal antibodies produced by 1F114F6P3G11 and 1F114F6P3H8 clones were determined as IgM subtype.

9. Dialysis of Antibodies: The monoclonal antibodies are dialyzed for ion exchange prior to purification process. 30 ml culture supernatants are concentrated at 3000 rpm for 25 minutes, at 25 °C with concentration tubes (Vivaspin 20, GE Healthcare, USA). 1 ml concentrate was dialysed against 1.25 M NaCI, 5 mM Tris with Spectra / Por® 1 (Spectrum, USA). The process is continued for 12 - 18 hours with the liquid phase being changed every 6 hours. Antibody solution in the membrane was used in the purification step afterwards, if not the solution was stored at - 20 °C. 10. Purification of Antibodies: The mannan-binding protein containing column which is working with the principle of affinity chromatography is conditioned with column preparation fluid (5 ml). The supernatant obtained after dialysis, is diluted at 1:1 with binding buffer prior to delivery to column. The column is reconditioned via 30 ml of binding buffer. After it is ensured that the diluted antibody solution adsorbed by the column packing material, 0.5 ml of additional binding buffer is added to column and the column caps were closed (first lower, then upper cap), and the column is left for incubation. These processes are performed at 4 °C and incubation time was 30 minutes. 42 ml of binding buffer was runned from the column and 3 ml fractions were collected. This process is carried out at 4 °C. After that column brought to room temperature. 30-42 ml of elution buffer is runned from the column and 3 ml fractions are collected. The fractions collected from both with binding and elution buffers are read using a spectrophotometer at 280 nm (with quartz cuvettes). The results obtained indicate the presence of protein in the sample.

Protein Determination: Additionally, protein determination was performed in order to determine the antibody amount quantity in the fractions obtained (BCA ™ Assay Kit QquantiPpro, Sigma, USA). In the analyses carried our according to the information provided by the manufacturer, a 150 μΙ sample was mixed with the prepared 150 μΙ Working Solution and incubated at 37 °C for 2 hours. Following the incubation, the microplate was read using a spectrophotometer at 562 nm. The graphics obtained under the presence of the standards prepared as described by the manufacturer is given in Figures as follows: 11. Labeling of Monoclonal Antibodies with Colloidal Gold Nanoparticles: 40 nm diameter colloidal gold nanoparticles (Arista Biologicals, USA) were centrifuged (10,000 rpm, 4 °C, 30 min) and then resuspended with 0.1 M boric acid buffer. Of 500 ng/ml antibody solution, 10 μΙ was added to the solution and incubated for 1 hour at room temperature (16). After 1 hour, 50 μΙ from the 10 mg/ml BSA solution was added and incubated for 30 minutes at room temperature. After 30 minutes, conjugated antibodies were centrifuged at 5.000 rpm, 4 °C for 20 minutes. Then, the pellet was resuspended with 250 μΙ ultrapure water.

12. Labelled Antibodies Being Turned Into Test Kits: Antibodies labelled and antibodies not labelled may be used in different tasks, in different formats; in lateral flow, dot-ELISA, dry ELISA and other methods. The use of the monoclonal antibodies produced according to the principle of the lateral flow test and test kit is as follows; 12.1. Capture Zone: Monoclonal antibodies produced by 1F114F6P3G11 clone are used in™T Zone" where the target protein h-FABP in the serum is captured. 0.033 μΙ/cm of antibody from 1 mg/ml monoclonal antibody solution is immobilized in the nitrocellulose membrane dried at room temperature. 12.2. Control Zone: Biotin - BSA solution (Arista, U5A) at 1 μΙ/cm is immobilized in the X Zone" prepared to check whether the test works from 1 mg/ml stock solution. It is dried at room temperature for 20 minutes or through incubation at 37 °C for 10 minutes. Nitrocellulose membrane that has the T and C zones was blocked with 1% BSA, Blocked membranes were allowed to pre-dry at room temperature for 15 minutes and then 5 x 5 minutes of washing was performed with 5 mM Na₂HP0₄ (pH 7.0) (Merck AG, Germany) buffer. After washing, membranes were dried at 37 °C.

12.3. Conjugate Pad Preparation: Monoclonal antibodies produced by 1F114F6P3H8 hybridoma clone, are labelled by colloidal gold nanoparticies as described in step 11. The conjugate pad blocked with 2 mM boric acid (Merck AG, Germany) solution (pH 7.4) containing 2.5% sucrose (Sigma, USA) was dried at 37 °C. Monoclonal antibody - colloidal gold nanoparticle conjugates were placed to dried pad by means of dipping, soaking or spraying methods. Pad was dried again at 37 °C. For C Zone, streptavidin conjugated with colloidal gold nanoparticies was soaked to the conjugate pad and dried at 37 °C.

12.4. Lamination: Pads dried or kept in appropriate conditions were laminated. Resulting layers after lamination were cut 4 mm wide and the test strips taken was schematized as follows (Figure 3). Strip components;

1. Sample pad: Filters the sample dropped onto the test strip, organizes the flow regime and transfers it to the conjugate pad.

2. Conjugate pad: Temporarily hosts the monoclonal antibodies conjugated with gold nanoparticies and streptavidin; when passing over the sample, if present in the sample content, it binds to h-FABP protein (anti-h-FABP antibody labelled with gold nanoparticies), transmits complex to nitrocellulose membrane containing test and control zones. 3. Nitrocellulose membrane: The membrane in which anti-h-FABP monoclonal antibodies (Test Zone, 3.a) and biotin-BSA (Control Zone, 3,b) were immobilized by means of it's protein retention capability.

4. Adsorbent Pad: It is included in the test to adsorb excessive sample after interaction of sample, mobile and immobilized antibodies in conjugate pad, Test Zone and Control zone respectively.

5. The support material that enables all components to remain together, as designed.

ABBREVIATIONS h-FABP : Heart-type fatty acid binding protein AMI : Acute myocardial infarction

EKG : Electrocardiogram kDa : Kilo Dalton

R PI : Roswell Park Memorial Institute

DMEM : Dulbecco's Modified Eagle's medium FBS : Fetal bovine serum

DHS : Donor horse serum

HBSS : Hank's Balanced Salt Solution

DMSO : Dimethyl sulfoxide

PMA : Phorbol- myristate-12-myristate-acetate MDP : N-acetyl-muramyl-L-alanyl-D-isoglutamine hydrate

IL - 1 o : Interleukin - 1 a

IL - 2 : Interleukin 2 IL - 4 : Interleukin - 4

IL - 6 : Interleukin - 6

IL - 10 : Interleukin 10

IFN-γ : Interferon gamma

ELISA : Enzyme-linked immunosorbent assay

BSA : Bovine serum albumin

PBS : Phosphate buffered saline

HRP : Horseradish peroxidase

IgM : Immunoglobulin M antibody

IgG : Immunoglobulin G antibody

OPD : o- phenylenediamine

PEG : Polyethylene glycol

HAT : Hypoxanthine - aminopterin - thymidine

HT : Hypoxanthine - thymidine

ISO-2® : Mouse Monoclonal Antibody Isotyping kit, Sigma

REFERENCES

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Claims

CLAIM

1. Monoclonal antibody which has affinity to heart-type fatty acid binding protein (h-FABP) characterized in that it is produced by the hybridoma clone coded as 1F114F6P3G11.

2. Monoclonal antibody which has affinity to heart-type fatty acid binding protein (h-FABP) characterized in that it is produced by the hybridoma clone coded as 1F114F6P3H8.

3. The antibody according to Claim 1 or 2, characterized in that it is used in the diagnostic kit,

4. Use according to Claim 3, characterized in that it includes lateral flow, dot-ELISA or dry ELISA methods.

5. Use according to Claim 4, characterized in that the said lateral flow test kit comprises: a) Capture zone obtained by means of the immobilization of an antibody according to Claim 1 to the nitrocellulose membrane,

b) BSA immobilized control zone,

c) Conjugate pad containing an antibody according to Claim 2 labelled with colloidal gold nanoparticles and streptavidin.

6. A method used in the detection of acute myocardial infarction, characterized in that it comprises:

a) Obtaining the sample,

b) Determination of the amount of heart-type fatty acid binding protein in the sample using antibodies produced by hybridoma clones coded as 1F114F6P3G11 or 1FU4F6P3H8,

c) Comparison of the h-FABP reference amount with the amount determined in step (b),

d) Detection of the presence of acute myocardial infarction.