HUMAN ANNEXIN A1 ELISA Product Data Sheet Cat. No.: RD191371200R For Research Use Only Page 1 of 24 ENG.001.A CONTENTS 1. INTENDED USE 3 2. STORAGE, EXPIRATION 3 3. INTRODUCTION 4 4. TEST PRINCIPLE 6 5. PRECAUTIONS 6 6. TECHNICAL HINTS 7 7. REAGENT SUPPLIED 7 8. MATERIAL REQUIRED BUT NOT SUPPLIED 8 9. PREPARATION OF REAGENTS 8 10. PREPARATION OF SAMPLES 10 11. ASSAY PROCEDURE 11 12. CALCULATIONS 13 13. PERFORMANCE CHARACTERISTICS 14 14. DEFINITION OF THE STANDARD 17 15. PRELIMINARY POPULATION DATA 17 16. METHOD COMPARISON 18 17. TROUBLESHOOTING AND FAQS 19 18. REFERENCES 20 19. EXPLANATION OF SYMBOLS 21 This kit is manufactured by: BioVendor – Laboratorní medicína a.s. Use only the current version of Product Data Sheet enclosed with the kit! Page 2 of 24 ENG.001.A 1. INTENDED USE The RD191371200R Human Annexin A1 ELISA is a sandwich enzyme immunoassay for the quantitative measurement of human annexin A1. Features It is intended for research use only The total assay time is less than 3.5 hours The kit measures annexin A1 in human serum, plasma (EDTA, citrate, heparin), urine and saliva samples Assay format is 96 wells Standard is recombinant protein based Components of the kit are provided ready to use, concentrated or lyophilized 2. STORAGE, EXPIRATION Store the complete kit at 2 – 8 °C. Under these conditions, the kit is stable until the expiration date (see label on the box). For stability of opened reagents see Chapter 9. Page 3 of 24 ENG.001.A 3. INTRODUCTION The annexin superfamily is composed of 12 members in humans [1], characterized by the unique Ca2+-binding-site architecture, which enables them to peripherally attach to negatively charged membrane surfaces [2,3]. Annexin A1 (AnxA1), also known as annexin I or lipocortin I, was characterized as a glucocorticoid-regulated anti-inflammatory protein [4,5]. This 37-kDa protein is comprised of a homologous core region of 310 amino acid residues, representing almost 90% of the structure, attached to a unique N-terminal region [3]. AnxA1 is a Ca2+-regulated phospholipid-binding protein [6]. To mediate membrane binding, Ca2+ ions can induce a conformational change that leads to the exposure of the bioactive N-terminal domain [3,7,8]. The different functions of AnxA1 lie within the unique N-terminus [9]. AnxA1 is expressed in several tissues and involved in different cell processes including cell survival, proliferation, apoptosis, differentiation and migration [10]. AnxA1 can be nuclear, cytoplasmic and/or membrane-associated [2,11,12]. Depending on the human tissue/cell line, AnxA1 can be cleaved at different positions by different proteases [6]. The membrane- associated AnxA1 can be proteolytically cleaved to become accessible to its cognate partners, the formyl-peptide receptors (FPRs) [6]. The AnxA1/FPR complex has been found to be involved in inflammation [5], neuroendocrine system regulation [13], skeletal muscle differentiation [14] and cancer progression [15]. To date, FPRs are the only known receptors of externalized AnxA1 [6]. As these receptors can be activated or silenced by a variety of synthetic ligands, they represent an attractive family of pharmacological targets [16]. AnxA1 and inflammation: AnxA1 is able to counterregulate inflammatory events [4]. AnxA1 and its mimetic peptides inhibit neutrophil tissue accumulation by reducing leukocyte infiltration and activating neutrophil apoptosis [4]. Some evidence has suggested the ability of AnxA1 to induce macrophage reprogramming towards a resolving phenotype, resulting in reduced production of proinflammatory cytokines and increased release of immunosuppressive and proresolving molecules [4]. Thus, AnxA1 could be a promising tool for development of new therapeutic strategies for treating inflammatory diseases [4]. AnxA1 and cancer: Deregulation of AnxA1 often correlates with cancer, and its role in tumour initiation, proliferation and metastases has been established [15,17]. AnxA1 could be a tumorigenic mediator between cancer cells and their microenvironment [6]. Efficient inhibitors of AnxA1 could potentiate cell response to chemotherapy [6]. The overexpression of AnxA1 in the cytoplasm of tumour cells has been observed in several cancers [18]. AnxA1 is specifically expressed in tumour vasculature (at least in breast, kidney, liver, lung, brain and prostate cancers) and could be a promising target for human tumour imaging, drug delivery and internal radiotherapy [6]. In some cancers, AnxA1 is found at the cell surface, where it stimulates FPRs to trigger oncogenic pathways [6]. Page 4 of 24 ENG.001.A Areas of investigation: Immune Response, Infection and Inflammation Autoimmunity Oncology Apoptosis Page 5 of 24 ENG.001.A 4. TEST PRINCIPLE In the BioVendor Human Annexin A1 ELISA, standards and samples are incubated in microplate wells pre-coated with polyclonal anti-human annexin A1 antibody. After 60 minutes incubation followed by washing, biotin-labelled polyclonal anti-human annexin A1 antibody is added and incubated with the captured annexin A1 for 60 minutes. After another washing, streptavidin-HRP conjugate is added. After 30 minutes incubation and the last washing step, the remaining conjugate is allowed to react with the substrate solution (TMB). The reaction is stopped by addition of acidic solution and absorbance of the resulting yellow product is measured. The absorbance is proportional to the concentration of annexin A1. A standard curve is constructed by plotting absorbance values against concentrations of standards and concentrations of unknown samples are determined using this standard curve. 5. PRECAUTIONS For professional use only Notice: Wear gloves, face mask (or another mouth covering), hair cover and laboratory coat when handling ELISA components and during ELISA assay. Skin flakes and saliva can contain annexin A1 protein and contamination in any ELISA step could cause false positive results Do not drink, eat or smoke in the areas where immunodiagnostic materials are being handled This kit contains components of human or animal origin. These materials should be handled as potentially infectious Avoid contact with the acidic Stop Solution and Substrate Solution, which contains hydrogen peroxide and tetramethylbenzidine (TMB). Wear gloves and eye and clothing protection when handling these reagents. Stop and/or Substrate Solutions may cause skin/eyes irritation. In case of contact with the Stop Solution and the Substrate Solution wash skin/eyes thoroughly with water and seek medical attention, when necessary The materials must not be pipetted by mouth Page 6 of 24 ENG.001.A 6. TECHNICAL HINTS Reagents with different lot numbers should not be mixed Use thoroughly clean glassware Use deionized (distilled) water, stored in clean containers Avoid any contamination among samples and reagents. For this purpose, disposable tips should be used for each sample and reagent Substrate Solution should remain colourless until added to the plate. Keep Substrate Solution protected from light Stop Solution should remain colourless until added to the plate. The colour developed in the wells will turn from blue to yellow immediately after the addition of the Stop Solution. Wells that are green in colour indicate that the Stop Solution has not been mixed thoroughly with the Substrate Solution Dispose consumable materials and unused contents in accordance with applicable national regulatory requirements 7. REAGENT SUPPLIED Kit Components State Quantity Antibody Coated Microtiter Strips ready to use 96 wells Biotin Labelled Antibody Conc. (100x) concentrated 0.13 ml Streptavidin-HRP Conjugate ready to use 13 ml Master Standard lyophilized 2 vials Dilution Buffer ready to use 50 ml Wash Solution Conc. (10x) concentrated 100 ml Substrate Solution ready to use 13 ml Stop Solution ready to use 13 ml Product Data Sheet + Certificate of Analysis - 1 pc Page 7 of 24 ENG.001.A 8. MATERIAL REQUIRED BUT NOT SUPPLIED Deionized (distilled) water Test tubes for diluting samples Glassware (graduated cylinder and bottle) for Wash Solution (Dilution Buffer) Precise pipettes to deliver 5 – 1000 l with disposable tips Multichannel pipette to deliver 100 l with disposable tips Plate cover Absorbent material (e.g. paper towels) for blotting the microtiter plate after washing Vortex mixer Incubator adjustable to 37 °C Microplate washer (optional). [Manual washing is possible but not preferred] Microplate reader with 450 10 nm filter, preferably with reference wavelength 630 nm (alternatively another one from the interval 550 – 650 nm) Software package facilitating data generation and analysis (optional) 9. PREPARATION OF REAGENTS All reagents need to be brought to room temperature prior to use Always prepare only the appropriate quantity of reagents for your test Do not use components after the expiration date marked on their label Assay reagents supplied ready to use: Antibody Coated Microtiter Strips Stability and storage: Return the unused strips to the provided aluminium zip-sealed bag with desiccant and seal carefully. Remaining Microtiter Strips are stable 3 months stored at 2 - 8 °C and protected from the moisture. Streptavidin-HRP Conjugate Dilution Buffer Substrate Solution Stop Solution Stability and storage: Opened reagents are stable 3 months when stored at 2 - 8 °C. Page 8 of 24 ENG.001.A Assay reagents supplied concentrated or lyophilized: Human Annexin A1 Master Standard Refer to the Certificate of Analysis for current volume of Dilution Buffer needed for reconstitution of standard!!! Reconstitute the lyophilized Master Standard with Dilution Buffer just prior to the assay. Let it dissolve at least 15 minutes with occasional gentle shaking (not to foam). Vortex is recommended. The resulting concentration of human annexin A1 in the stock solution is 3.2 ng/ml. Prepare set of standards using Dilution Buffer as follows: Volume of Standard Dilution Buffer Concentration Stock - 3.2 ng/ml 250 l of stock 250 l 1.6 ng/ml 250 l of 1.6 ng/ml 250 l 0.8 ng/ml 250 l of 0.8 ng/ml 250 l 0.4 ng/ml 250 l of 0.4 ng/ml 250 l 0.2 ng/ml 250 l of 0.2 ng/ml 250 l 0.1 ng/ml Prepared Standards are ready to use, do not dilute them.