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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

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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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Thank you

www.ubio.in