SECTION 1 : IFA

Indirect Immunofluorescent Assays

The indirect immunofluorescence assay (IFA) is the most commonly used technique for the detection of antinuclear antibodies (ANA). Because IFA enables detection of a huge spectrum of auto-antibodies, it's generally used as a primary screening test for ANA. The technique is sensitive, can be reproduced and is relatively simple to perform.

The test involves incubating a dilution of the patient's sera with the substrate, followed by washing to remove unbound antibodies. A second incubation using a fluorescein labelled anti-human globulin conjugate is then performed, followed by a washing step to remove unbound conjugate. After mounting, the slide can be viewed under an appropriate fluorescent microscope for assessing and recording the intensity and patterns of fluorescence. The intensity can be expressed according to a scale of values compatible with the guidelines established by the reference centres (e.g. CDC. Atlanta) as + to ++++, or as negative. A semi-quantitative evaluation can be reached by performing serial dilutions of the sample.

At Bio-Diagnostics, we provide IFA products either as complete kits or individual components. Kits are compiled for convenience and economy, whilst individual components make our products flexible and user-friendly.

Individual slides are produced in a variety of formats, some of which are particularly suited to automation. Slides are available as single or multiple sections allowing laboratories to customise their testing methods. IFA kits are available as complete kits or individual components, providing a user-friendly and flexible solution.

Individual slides are produced in a variety of formats, some of which are particularly suited to automation. If you require a substrate slide in a non-standard format, we would be happy to try to accommodate your needs.

GASTRIC PARIETAL CELL (GPC)

SMOOTH MUSCLE

RODENT TISSUE SECTIONS

A variety of tissue sections can be used for IFA, but the primary screening substrates are liver, kidney and stomach sections (LKS). The traditional tissues are of rodent origin and comparative studies have shown that rat and mouse substrates are both equally reliable with respect to titre or pattern of fluorescence in ANA testing. The organ of choice for ANA is usually liver because of its simple structure and high density of nuclei. LKS sections are used for ANA, mitochondrial, smooth muscle and gastric parietal cell antibodies. We offer these tissue substrates either individually or in combinations to allow for identification of many auto-antibodies. Most ANA testing is now performed on HEp-2 substrate, which we also offer in a variety of slide and kit formats.

MONKEY OESOPHAGUS ENDOMYSIAL

MONKEY HEART

PRIMATE TISSUE SECTIONS

At Bio-Diagnostics, we produce a variety of monkey tissue substrate slides. Monkey tissues are very similar to human tissues both structurally and antigenically, and are used for the detection of many autoantibodies.

Most of our monkey substrate slides are produced in a 4 and 8 well format, with the exception of monkey oesophagus. Endomysial antibodies have become very important with a high specificity and sensitivity for coeliac disease. As use of this test is on the increase in the routine immunology laboratory, we offer monkey oesophagus in a 5 and 10 well format. These slides perform extremely well on automated IFA processors.

HEp-2 ANA CENTROMERE

HEp-2 - Jo1

HEp-2 SLIDES

EXPLANATION

The presence of antinuclear antibodies suggests the possibility of autoimmune disease. These antibodies were first associated with systemic lupus erythematosus (SLE). Now, ANA association includes rheumatoid arthritis (RA), scleroderma (progressive systemic sclerosis (PSS)), mixed connective tissue diseases (MCTD) and Sjögren's Syndrome (SS).

HEp-2 is human epitheloid cell line which is more sensitive for detection of ANA than animal tissue sections. The cells are cultured to maximise the number of mitotic figures which aid in pattern recognition and detection of previously unreported nuclear antigens. Bio-Diagnostics' highly sensitive HEp-2 cultures provide a reactive homogenous substrate not requiring artificial enhancement with genetic manipulation.

CRITHIDIA LUCILIAE

CRITHIDIA LUCILIAE SLIDES AND KITS

INTRODUCTION

Systemic Lupus Erythematosus (SLE) patients produce many different types of nuclear antibodies and those with the specificity for double stranded DNA (dsDNA) have a high correlation with SLE patients. Antibodies directed to native dsDNA cannot be detected by standard immunofluorescent antinuclear antibody (ANA) methods, which rely on different nuclear fluorescent patterns to determine the type of antibody. Antibodies to DNA that react with both double and single stranded DNA, produce the same rim and/or homogeneous patterns. Among the various ANA immunofluorescent patterns, the rim pattern confirms a clinical diagnosis of SLE and as many as 33% of these patients have some renal disease. Tests which can equivocally detect the presence of only native dsDNA antibodies should be performed to confirm the diagnosis of lupus nephritis. The DNA test kit using the substrate Crithidia luciliae which contains native dsDNA provides a simple technique for detection of antibodies to dsDNA.

PRINCIPLES

Antibodies directed against native dsDNA are not organ specific. The test for detection of antibodies to native dsDNA using immunofluorescent techniques utilises the giant mitochondrion kinetoplast of the non-pathogenic haemoflagellate Crithidia luciliae as a substrate for pure dsDNA. Good correlation of the results has been found between the immunofluorescence technique and the radioimmunoassay of Farr.

HUMAN GRANULOCYTE CYTOPLASMIC ANTIBODY

(C-ANCA)

HUMAN GRANULOCYTE ANTI-PERINUCLEAR ANTIBODY

(P-ANCA)

ANCA SLIDES

INTRODUCTION

Standard IFA methods allow the observation of several different patterns. Two patterns that have been well defined are C-ANCA (Cytoplasm) and P-ANCA (Perinuclear). The C-ANCA pattern shows an uneven granular staining of the cytoplasm. The P-ANCA has a perinuclear/nuclear staining pattern. During the 2nd International ANCA Workshop, it was agreed that these two different patterns should be used to subclassify the antibodies.

ANCA antibody detection by IFA methods has been a useful aid in the assessment of patient diagnosis, and to a certain extent their prognosis and response to therapy. Although there has been much work done with IFA ANCA, the availability of an EIA MPO and PR3 test has further enhanced the overall picture. The use of EIA and IFA together allows for the best possible patient assessment. At Bio-Diagnostics, we provide rapid and quantitative EIA kits for MPO and PR3, as well as our unique QuickCard systems.

IFA CONTROLS, CONJUGATES AND ACCESSORIES

To provide total flexibility, we offer a full range of autoimmune controls both individually or as ANA or ENA sets. All our positive controls are in liquid form and ready to use.

To complete our IFA range we provide conjugate, buffer, blotters, mounting media and coverslips to suit your requirements.