WO2014121802A1 - Innovative nano-elisa single test for the detection of hcv, hiv antibodies and hbsag - Google Patents

Abstract

The invention represents a novel economic and reliable technique to extend the utilization of conventional elisa protocol for detection of single pathogen to accommodate the capability to detect multiple pathogens (such as HIV, HCV and HBSAG) in a single test with enhanced sensitivity. This was achieved via incorporating a large number of a specific type of anybody or multiple types of antibodies into distinctively designed swellable self-assembled and/or hydrogel nanoparticles followed by binding the antibodies-loaded nanoparticles to the elisa plate.

Description

INNOVATIVE NANO-ELISA SINGLE TEST FOR THE DETECTION OFHCV. HIV ANTIBODIES AND HBSAG

TECHNICAL FIELD

THIS INVENTION COULD BE APPLIED IN MEDICINE AS IT AIMS TO PERFORM EASY SCREENING OF BLOOD FOR MULTIPLE PATHOGENS; E.G. HCV ANTIBODIES, HIV ANTIBODIES AND HBSAG IN A SINGLE TEST.

BACKGROUND ART

ELISA IS THE ABBREVIATION OF "ENZYME-LINKED IMMUNOSORBENT ASSAY". IT IS A USEFUL AND POWERFUL METHOD IN ESTIMATING NG/ML TO PG/ML ORDERED MATERIALS IN THE SOLUTION, SUCH AS SERUM, URINE, SPERM AND CULTURE SUPERNATANT. ELISA HAS BEEN WIDEL Y USED IN THE LIFE SCIENCE RESEARCHES.

THE BASIC PRINCIPLE OF AN ELISA IS TO USE AN ENZYME TO DETECT THE BINDING OF AN ANTIGEN (AG) OR AN ANTIBODY (AB). THE ENZYME CONVERTS A COLORLESS SUBSTRATE (CHROMOGEN) TO A COLORED PRODUCT, INDICATING THE PRESENCE OF AG-AB BINDING. AN ELISA CAN BE USED TO DETECT EITHER THE PRESENCE OF AGS OR ABS IN A SAMPLE, DEPENDING ON HOW THE TEST IS DESIGNED.

PREPARATION OF ELISA MICROTITRE PLATES INVOLVES THREE MAJOR STEPS: (1) BINDING ANTIGEN OR ANTIBODY TO THE PLATE; (2) BLOCKING NON-SPECIFIC BINDING SITES ON THE PLATE; AND (3) COATING THE PLATE WITH A STABILIZER TO ALLOW DRY STORAGE OF THE PLATES FOR LONG PERIODS OF TIME.

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THE NANOP ARTICLES TTECNOLOGY ARE BASED ON A WIDE RANGE OF POLYMERS THAT INCLUDE, BUT NOT LIMITED TO, MODIFIED CHITOSAN, MODIFIED PEG, PI GA, AND CARRAGENAN. THE DEVELOPMENT OF THE NANOP ARTICLES HAS BEEN ACHIEVED THROUGH SEVERAL APPROACHES THAT INCLUDES, BUT NOT LIMITED TO, (1) SELF- ASSEMBLING OF THE MODIFIED POLYMERS INTO NANOP ARTICLES DEPENDING ON THEIR AMPHIPHILIC CHARACTERISTICS, AND (2) PHYSICAL CROSSLINKING OF THE MODIFIED POLYMERS INTO HYDROGEL NANOP ARTICLES. IN BOTH CASES, THE NANOP ARTICLES PREPARATION WAS CARRIED OUT IN MILD AQUEOUS MEDIA TO ENSURE THE STABILITY OF THE (PRE- OR POST-LOADED) ANTIBODIES/ ANTIGENS.

THE EFFECT OF RELATIVE NANOPARTICLES COMPOSITIONS IN ADDITIO TO THE VARIOUS PREPARATION PARAMETERS ONTO THE PHYSICOCHEMICAL CHARACTERISTICS OF THE OBTAINED ANTIBODIES-LOADED NANOPARTICLES HAS ALSO BEEN EXTENSIVELY INVESTIGATED. DISCLOSURE OF THE INVENTION

THE COATING OF ELISA PLATE WITH THE TARGETS- LOADED NANOPARTICLES INSTEAD OF TARGETS ONLY WOULD ALLOW BINDING SEVERAL TYPES OF TARGETS (ANTIBODIES OR ANTIGENS) INSTEAD OF ONLY ONE TYPE WHICH ENABLES DETECTION OF SEVERAL PATHOGENS IN A SINGLE TEST. ALSO, THE HIGH SURFACE AREA OF THE NANOPARTICLES CARRIERS ENHANCES THE ENTIRE DIAGNOSIS SENSITIVITY AS COMPARED TO THE STANDARD ELISA.

ADVANTAGES

1- THE INVENTED TECHNIQUE ENHANCES THE DIAGNOSTIC SCREENING FOR COMMON PATHOGENS; HIV, HCV AND HBSAG, AND OTHERS.

2- THE SENSITIVITY OF THE WAS CONSIDERABLY ENHANCED THROUGH THE USE OF NANOPARTICLES.

3- THE ASSAY PROCEDURE OF THE MODIFIED ELISA IS THE SAME AS THAT OF THE STANDARD ELISA TECHNIQUE, SO THE TEST

PROCEDURES ARE SIMPLE AND REQUIRE NO EXTRA TRAINING.

4- IT SPARES THE TIME NEEDED TO PERFORM THREE OR MORE SEPARATE ELISA TESTS TO DETECT VARIOUS PATHOGENS. 5- THE COST OF PERFORMING THE NOVEL TECHNIQUE WILL BE ECONOMIC WITH SIGNIFICANTLY COMPARABLE COST TO A CONVENTIONAL ELISA TEST. 6- THIS NOVEL SINGLE NANO-ELISA TEST MAY BE THE METHOD OF CHOICE AMONG POPULATIONS WITH LOW INCOME AND WHEREAS A RAPID DECISION SHOULD BE TAKEN ABOUT THE SAFE BLOOD TRANSFUSION. BREIF DESCRIPTION OF DRAWINGS

FIGURE (1) SHOWS THE INVENTED STRATEGY IN ITS COMPLETE FORM; A: ANTIBODY ,B: LABELING REAGENTS ,C: PATIENT'S SAMPLE, D: SWELLABLE NANOP ARTICLES LOADED WITH LARGE NUMBER OF A SPECIFIC ANTIBODY OR MULTI ANTIBODIES ,1 : BINDING SITE ,2: TARGET ANALYTE (ANTIGEN) ,3: SECOND , ANTIBODY OR TARGET ANALYTE ,4: ENZYME ,5: SUBSTRATE ,6: PRODUCT ,7: INCUBATION ,S: WASHING STEP ,9: ADDITION OF LABELING AGENT 10: INCUBATION ,1 1 : WASHING STEP , 12: ADDITION OF SUBSTRATE ,13: INCUBATION 14: READING.

AS ILLUSTRATED IN THE DRAWINGS , SPECIFICALLY DESIGNED NANOP ARTICLES LOADED WITH VARIOUS TYPES OF ANTIBODIES AND / OR ANTIGENS ARE BOUND TO THE POLYSTYRENE WELL PLATE. THEN, UPON ADDITION OF THE PATIENT'S SERUM OR PLASMA SAMPLE FOLLOWED BY INCUBATION, THE TARGET ANALYTES (ANTIGENS AND / OR ANTIBODIES) ARE LINKED TO THE BINDING SITES IN THEIR CORRESPONDING SITES. WASHING STEP IS THEN CARRIED OUT TO REMOVE ANY UNBOUND ANTIGENS AND / OR ANTIBODIES. AFTERWARDS, ENZYME LABELED REAGENTS (SECOND ANTIBODY CONJUGATED TO ENZYME OR TARGET ANALYTE) IS ADDED AND THE WELL PLATE IS INCUBATED AGAIN. WASHING STEP IS REPEATED FOR REMOVAL OF UNBOUND LABELING REAGENTS. IF THE PATIENT'S SAMPLE CONTAINS THE ANTIGEN AND / OR ANTIBODIES SPECIFIC FOR THE TEST, THE ENZYME CONJUGATED TO THE SECOND ANTIBODY WILL BREAK AND OPEN THE CHROMOGENIC SUBSTRATE LEADING TO A COLOR APPEARANCE (USUALLY BLUE, GREEN OR YELLOW). IF THERE IS NO SAMPLE TARGET, THE WELL REMAINS COLORLESS

THE INTERACTION BETWEEN THE DEVELOPED PLAIN AND ANTIBODY-LOADED POLYMERIC NANOP ARTICLES AND THE POLYSTYRENE OF THE 96-WELL PLATE HAS BEEN INVESTIGATED. THE PRELIMINARY RESULTS SHOWED A GOOD ATTACHMENT BETWEEN THE NANOP ARTICLES ESPECIALLY THAT MADE OF CHITOSAN AND CHITOSAN DERIVATIVES, WITH THE WELL PLATE SURFACE. BESIDES, THE ANTIBODY BINDING TO THE DEVELOPED POLYMERIC NANOP ARTICLE S MADE OF VARIOUS POLYMERS INCLUDING BUT NOT LIMITED TO CHITOSAN WAS ASSESSED BY USING A STANDARD ELISA TECHNIQUE. PLATES (96 WELLS) WERE COATED WITH ABOUT 5 μβ OF POLYMERIC NANOP ARTICLES SUSPENSION AND ALLOWED TO DRY. PLATES WERE WASHED 3 TIMES WITH 0.05% TWEEN/PBS, AND BLOCKED OVERNIGHT AT 4°C BY THE ADDITION OF 5% BOVINE SERUM ALBUMIN (BSA)/PBS. PLATES WERE THEN WASHED 3 TIMES AGAIN, AND THE DESIRED MONOCLONAL ANTIBODIES AND ANTIGENS OF HCV, HIV AND HBV WERE SERIALLY DILUTED IN 5% BSA/PBS AND DISPENSED IN TRIPLICATE. PLATES WERE INCUBATED OVERNIGHT AT 4°C, AND WASHED WITH TWEEN/PBS. ANTIBODY BINDING WAS DETECTED BY THE ADDITION OF 100 ML GOAT ANTI-MOUSE IMMUNOGLOBULIN G CONJUGATED WITH HRP DILUTED 1 : 1000 IN PBS/BSA.

PLATES WERE INCUBATED FOR 2 H AT ROOM TEMPERATURE, AND THEN WASHED IN TWEEN/PBS. REACTIONS WERE DEVELOPED BY THE ADDITION OF TMB. THE OPTICAL DENSITY (OD) AT 450 NM WAS MEASURED BY USING A MICROPLATE SPECTROPHOTOMETER READER, AND THE BINDING WAS EXPRESSED AS OD UNITS. THE PRELIMINARY DATA HAS DEMONSTRATED A DESIRABLE BINDING BETWEEN THE DEVELOPED NANOP ARTICLES MADE OF ONE OR MORE POLYMERS, INCLUDING BUT NOT LIMITED TO CHITOSAN, AND CHITOSAN DERIVATIVES, WITH THE VARIOUS INVESTIGATED MONOCLONAL ANTIBODIES. MODES FOR CARRYING OUT THE INVENTION

THIS ELISA IS A TWO STEP INCUBATION "SANDWICH TYPE" ENZYME IMMUNOASSAY, WHICH USES POLYSTYRENE MICROWELL STRIPS PRE- COATED WITH RECOMBINANT HEPATITIS C VIRUS (HCV) ANTIGEN (C22-3, C200 AND NS5), ANTIGEN REPRESENTING IMMUNO-DOMINANT EPITOPES OF HIV- 1 GP41 AND HIV-2 GP36 AS WELL AS ANTIBODIES AGAINST THE ANTIGEN P24 &(HBSAG). PATIENT'S SERUM OR PLASMA SAMPLE IS ADDED, AND DURING THE FIRST INCUBATION STEP, THE SPECIFIC HCV AND/OR HIV 1/2 ANTIBODIES, AS WELL AS HBSAG AND OR ANTIGEN P24 WILL BE CAPTURED INSIDE THE WELLS IF PRESENT. THE MICRO WELLS ARE THEN WASHED TO REMOVE UNBOUND SERUM PROTEINS. A COCKTAIL OF RECOMBINANT ANTIGENS AND ANTIBODIES CONJUGATED TO THE ENZYME HORSERADISH PEROXIDASE (HRPCONJUGATE) AND EXPRESSING THE SAME EPITOPES AS THE PRE-COATED ANTIGENS AND ANTIBODIES IS ADDED, AND DURING THE SECOND INCUBATION, THEY WILL BIND TO THE CAPTURED ANTIGENS AND OR ANTIBODIES. THE MICROWELLS ARE WASHED TO REMOVE UNBOUND CONJUGATE, AND CHROMOGEN SOLUTIONS ARE ADDED INTO THE WELLS. IN WELLS CONTAINING THE ANTIGEN- ANTIBODY- ANTIGEN (HRP) "SANDWICH" IMMUNOCOMPLEX, THE COLORLESS CHROMOGEN (TMB) IS HYDROLYZED BY THE BOUND HRP CONJUGATE TO A BLUE COLORED PRODUCT. THE BLUE COLOR TURNS YELLOW AFTER THE REACTION IS STOPPED WITH SULFURIC ACID. THE AMOUNT OF COLOR INTENSITY CAN BE MEASURED AND IT IS PROPORTIONAL TO THE AMOUNT OF ANTIBODIES AND/OR ANTIGENS CAPTURED IN THE WELLS. WELLS CONTAINING SAMPLES NEGATIVE FOR HCV, HB SAG AND HIV REMAIN COLORLESS.

INTERPRETATIONS OF RESULTS : POSITIVE RESULT IS INDICATED BY THE DEVELOPMENT OF COLOR AFTER THE ADDITION OF THE STOPPING REAGENT. OPTICAL DENSITY IS MEASURED FOR THE WELLS ON AN ELISA READER WITH A WAVE LENGTH OF 450 NM. ANY SAMPLE OD EXCEEDS THE CUTOFF VALUE IS CONSIDERED REACTIVE FOR ONE OR MORE OF THE TESTED ANTIBODIES OR ANTIGENS SENSITIVITY AND SPECIFICITY OF THE TEST THE INNOVATIVE DESIGN OF THIS ELISA PROVED TO BE MORE SENSITIVE AND SPECIFIC OVER THE CONVENTIONAL ELISA TECHNIQUES.

INDUSTRIAL APPLICABILITY

SUCH INVENTION COULD BE USED IN PRODUCING A STANDARD KIT TO BE APPLIED FOR BLOOD SCREENING FOR HCV, HIV AND HBSAG.THE DESIGN OF THIS METHOD RESEMBLES ORDINARY ELISA, THE ONLY DIFFERENCE IS THE INNOVATIVE DESIGN OF COATING THE ELISA PLATE.SO THERE IS NO DIFFICULTY IN PRODUCING SUCH KITS THAT ARE CAPABLE OF DETECTING THREE PATHOGENS IN ONLY ONE ELISA FORMAT.

Claims

1- A NOVEL ECONOMIC AND RELIABLE TECHNIQUE TO EXTEND THE UTILIZATION OF CONVENTIONAL ELISA PROTOCOL FOR DETECTION OF SINGLE PATHOGEN TO ACCOMMODATE THE CAPABILITY TO DETECT MULTIPLE PATHOGENS IN A SINGLE TEST WITH ENHANCED SENSITIVITY.

2- THE NOVEL TECHNIQUE OF CLAIM 1, WHEREIN THE DESIGNED ELISA IS ABLE TO -DETECT MULTIPLE PATHOGENS THAT INCLUDES BUT NOT LIMITED TO; HIV-IGM, HCV-IGM, HBSAG, TOXOPLASMA IGM, 10 RUBELLA-IGM, CMV-IGM, AND HSV-IGM.

3- THE NOVEL TECHNIQUE OF CLAIM 1, WHEREIN THE DESIGNED ELISA IS ABLE TO DETECT ALSO OTHER MULTIPLE PATHOGENS THAT INCLUDES BUT NOT LIMITED TO; HIV-IGG, HCV-IGG, TOXOPLASMAS IGG, RUBELLA-IGG, CMV-IGG, AND HSV-IGG.

4- THE INNOVATIVE ELISA DESCRIBED IN CLAIM 1, WHEREIN THE DIAGNOSTIC SCREENING PROCESS IS ACHIEVED IN A SINGLE TEST WITH ENHANCED SENSITIVITY.

5- THE NOVEL TECHNIQUE OF CLAIM 1, WHEREIN THE DESIGNED ELISA OBTAINED VIA INCORPORATING A LARGE NUMBER OF A SPECIFIC TYPE OF ANIBODY (OR ANTIGEN) OR MULTIPLE TYPES OF ANTIBODIES (OR ANTIGENS) LNTO DISTINCTIVELY DESIGNED l o

SWELLABLE SELF-ASSEMBLED AND/OR HYDROGEL NANOP ARTICLES.

6- THE NOVEL TECHNIQUE OF CLAIM 4, WHEREIN THE INCORPORATION PROCESS OF ANIBODY (OR ANTIGEN) OR THE MULTIPLE TYPES OF ANTIBODIES (OR ANTIGENS) INTO THE NANOP ARTICLES IS ACCOMPANIED BY IN SITU OR FOLLOWED BY BINDING THE ANTIBODIES/ANTIGEN S-LOADED NANOP ARTICLES TO THE PLATE.

7- THE NANO-ELISA OF CLAIM 4, WHEREIN SAID POLYMERS ARE SELECTED FROM THE GROUP CONSISTING OF SODIUM ALGINATE, CHITOSAN, HYDROXYPROPYL CHITOSAN, GLYCOL CHITOSAN, QUATERNARY AMMONIUM CHITOSAN, CARRAGEENAN, CARBOXYMETHYL CELLULOSE, SODIUM HYALURONATE, POLY(LACTIC-CO-GLYCOLIC ACID), MUCINS, CARBOXYMETHYL CHITOSAN AND MDCTURES THEREOF.

8- THE NANO-ELISA OF CLAIM 6, WHEREIN SAID POLYMERS ARE MODIFIED WITH VARIOUS FUNCTIONALITIES THAT INCLUDE BUT NOT LIMITED TO OH, COOH, CHO, NH2, NHAC, C=O, AND MIXTURES THEREOF.

9- THE NANO-ELISA OF CLAIM 6, WHEREIN SAID POLYMERS ARE MODIFIED WITH HYDROPHILIC MOIETIES SUCH AS BUT NOT I I

LIMITED TO AN ^* END-CAPPED POLYETHYLENEGLYCOL POLYMER.

10- THE NANO-ELISA OF CLAIM 6, WHEREIN SAID POLYMERS ARE ALSO MODIFIED WITH SMALL HYDROPHOBIC MOIETIES SUCH AS STEARIC, CHOLANIC AND OLEIC ACIDS.

1 1- THE NANO-ELISA OF CLAIM 4, WHEREIN THE DEVELOPMENT OF THE NANOPARTICLES CAN BE ACHIEVED THROUGH SEVERAL APPROACHES THAT INCLUDE BUT NOT LIMITED TO; (1) SELF- ASSEMBLING OF THE MODIFIED POLYMERS INTO NANOPARTICLES DEPENDING ON THEIR AMPHIPHILIC CHARACTERISTICS, (2) PHYSICAL CROSSLINKING OF THE MODIFIED POLYMERS INTO HYDROGEL NANOP ARTICT ,ES

12- THE NANO-ELISA OF CLAIM 10, WHEREIN THE NANOPARTICLES PREPARATION IS CARRIED OUT IN MILD AQUEOUS MEDIA TO ENSURE THE STABILITY OF THE LOADED ANTIBODIES/ ANTIGENS.

13- THE NANO-ELISA OF CLAIM 4, WHEREIN THE INCORPORATION OF THE ANTIBODIES/ ANTIGENS IN THE NANOPARTICLES CAN BE ACHIEVED VIA CHEMICAL CONJUGATION AND/OR PHYSICAL ENTRAPMENT (PRE- OR POST- LOADING).