lateral flow assay in animal disease diagnosis
Post on 15-Nov-2014
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ubio
Bishor V. I
Lateral Flow Immuno Assay in Disease DiagnosisLateral Flow Immuno Assay in Disease Diagnosis
Director, ubio Biotechnology Systems Pvt. Ltd, Kinfra Hi-tech Park, Kochi
www.ubio.in
ubio
Lateral flow assays
• Rapid, Immunochromatography based.• Easy to do in the field.
– Quick results in just a few minutes.– Require no instrumentation, – Allows decentralized implementation (should not require
serum transport)– No, or low, capital expenditure.– Able to execute the test and read result in the field itself.– Room temperature storage, no refrigeration required.
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TYPICAL TEST STRUCTURETYPICAL TEST STRUCTURE
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• The signal results from the formation of an immunocomplex on the membrane
Y
Detector Reagent
Analyte
Capture Reagent
Membrane
Chromogen
Immunocomplex
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Assay Principle example
• Eg Leptospira Antibody test in dogs– Test line: Pooled Leptospira antigen– Control line: Mouse IgG– Conjugate pad: Protein A colloidal gold– Sample pad: Buffered with Tris buffer.
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Test methodology
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Lateral flow assays in companion animal diagnosis• Canine distemper: antigen and antibody tests
• Specimen: nasal or conjunctival swabs• Canine Parvovirus: Antigen test and antibody test
• Specimen: fecal sample/serum• Canine Adeno virus: Antigen tests: Nasal swabs• Corona virus: antigen: Fecel swabs• Canine brucellosis: antibody tests • Ehrlichia canis: antibody test• Giardia : antigen in feacal sample• Heartworm : antigen: blood• Leptospira test: antigen and Ab tests• Feline tests: FeLV/FIV combo test
Brucella/E.canis/Ab tests
CP/CD/Giardia test
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Vaccination titer check
• Antibodies for CDV, CPV and Rabies can be assessed.
• Color intensity of test line is assessed for titer
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Lateral flow assays in Farm animal practice
Common tests are• Bovine Brucella Ab• Bovine TB Ab• FMD NSP Ab• Swine fever Ab test.• Rota virus test.
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Avian Tests
AIV Antigen test NDV Antigen test IBD Antigen test
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EXTARCTION• A rapid screening test which can detect
Aflatoxin B1 in feed is very of critical importance in farms and feed manufacturing plants.
• Lateral flow assays : using a competitive immunoassay format.
• an easy and rapid extraction procedure using ethyl acetate.
• Quantification from 2 to 100ppb. • This test is easier and cheaper compared to
HPLC or TLC methods.• No need to handle toxic Aflatoxin standards
and solvents.
POSITIVE
NEGATIVE
Food testing and feed testing
SEPARATION
DILUTION
TESTING
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Residue testing
• Antibiotic residue testing in milk and meat.
• Chloramphenecol in aquatic foods.
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Reagent Application
Drying Process
Lamination of Components
Inspection/Marking
Cutting
Quality Control
Assembly/Packaging
SUMMARY OF MANUFACTURING PROCESSES
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Reagent Application
Dispense: Test, control, conjugate
Coat: Conjugates, blocking, other pad treatmentsInspection/Marking: Dispensing defectsDrying Process
Objective is to reduce water content to less than 4 % level.
Used after reagent application processes.Lamination of Components
Individual layers are bonded, using thin layers of adhesive.
Adhesive layers are usually prelaminated to plastic backing and cover layers.Inspection/Marking
Lamination alignment defects
SUMMARY OF MANUFACTURING PROCESSES
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Cutting
Separate into individual test strips: 3-10 mm
Remove bad parts marked in inspection process
Quality Control
Sample testing of manufacturing lot for functionality
Assembly/Packaging
Seal test strip in foil pouch with desiccant, pipette, etc.
SUMMARY OF MANUFACTURING PROCESSES
Manufacturing facility• Temperature and humidity controlled facility.• Relative Humidity below 25%
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Batch Manufacturing EquipmentBatch Manufacturing Equipment
Line dispenser laminatorManual Dip TrayGuillotine Cutter
Reel-to-reel web handling and dispensing platform Auto Laminator
In-line manufacturing equipmentIn-line manufacturing equipment
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Batch Process Line Layout
T/C
XYZ XYZ
Conjugate Inspect/Mark
Dip Tank
Block
Oven
Dry
Cutter Clamshell Clamshell
Cut/Sort Inspect/Mark Laminate cover Laminate Master Strip
Assemble
Assembly Roller
Assemble Assemble
SealSealSeal
Typ. 1.5 million tests limited by assembly & pouching
12-15 + operators
Increase to 4-6 millionby adding pouchingand/or assemblymachine
Final QC
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New generation Lateral flow assay• Nucleic acid lateral flow assays: PCR
amplified products can be detected.
• Biochips: Multiplex microarray lateral flow format
• Microfluidic chips: Lab on a chip: from sample extraction to detection without human intervention
• Biosensors: electrochemical detection of immunoassays.
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