ANCA Complete Kit

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Overview

ANCA IFA Complete Kit

Anti-Neutrophil Cytoplasmic Antibody (ANCA) Staining Examples

c-ANCA

Ethanol: Cytoplasmic
staining; lymphocytes negative
Formalin: Cytoplasmic
staining; lymphocytes negative

c-ANCA and ANA

Ethanol: Cytoplasmic
staining; lymphocytes positive
Formalin: Cytoplasmic
staining; lymphocytes positive

p-ANCA

Ethanol: Nuclear/perinuclear
staining; lymphocytes negative
Formalin: Cytoplasmic
staining; lymphocytes negative

p-ANCA and ANA

Ethanol: Nuclear/perinuclear
staining; lymphocytes positive
Formalin: Cytoplasmic
staining; lymphocytes positive

Negative

Ethanol: Negative staining Formalin: Negative staining

Atypical ANCA

Ethanol: Perinuclear staining Formalin: Negative staining

ANA Positive

Ethanol: Nuclear/perinuclear
staining; lymphocytes positive
Formalin: Negative staining;
lymphocytes positive
ANCA IFA Complete Kit

Anti-Neutrophil Cytoplasmic Antibody (ANCA) Testing

ANCA testing aids physicians in the diagnosis of necrotizing vasculitides and irritable bowel syndrome. As treatments will significantly vary, diagnosis and differentiation of antibody type is essential.

The Bio-Rad ANCA test system is made up of two types of immunofluorescent antibody tests (ethanol and formalin) for the detection and semi-quantitation of ANCA in human serum.


Ethanol Fixation

In ethanol fixed slides, MPO (p-ANCA) antigens migrate from the azurophilic granules and attach to the surface of the nucleus, whereas the PR3 (c-ANCA) antigens remain in the granules. ANCA positive samples will demonstrate either nuclear (p-ANCA) or cytoplasmic (c-ANCA) staining, dependent on antibody specificity.

 
MPO antigens migrate from the granules and attach to the surface of the nucleus   Cytoplasm contains azurophilic granules and the nucleus is lobular   PR3 antigens remain in the granules


Formalin Fixation

In formalin fixed slides, all ANCA antigens remain associated with the azurophilic granules, causing both c-ANCA and p-ANCA to produce cytoplasmic staining. Because antinuclear antibody positive samples may demonstrate a positive p-ANCA result, formalin fixed slides are typically used to confirm p-ANCA patterns.

MPO remains in the granules   Cytoplasm contains azurophilic granules and the nucleus is lobular   PR3 antigens remain in the granules

ANCA IFA Complete Kit

Anti-Neutrophil Cytoplasmic Antibody (ANCA) Test Method

The indirect immunofluorescence staining procedure is illustrated in the following figures.

Step 1. Bring all reagents to room temperature (18–26°C).

Step 2. Make 1:20 working solutions by diluting 50 µL of patient's serum in 1.0 mL diluent.

Step 3. Let pouch equilibrate to room temperature, then remove slide(s) from pouch.

Step 4. Remove slides from pouch. Place slide(s) into moisture chamber and add 50 µL of samples and controls. Cover and incubate 30 minutes.

Step 5. Rinse slide(s) by dipping into beaker with PBS. Transfer slide(s) into Coplin jar and wash 10 minutes.

Step 6. Blot edge of slide(s) on absorbent paper. Proceed immediately with next step.

Step 7. Apply ~50 µL conjugate to each well. Cover and incubate 30 minutes.

Step 8. Rinse slide(s) by dipping into beaker with PBS. Transfer slide(s) into Coplin jar and wash 10 minutes.

Step 9. Blot edge of slide(s) on absorbent paper. Proceed immediately with next step.

Step 10. Mount coverslip and read under fluorescent microscope.

The Kallestad™ Autoimmune ANCA IFA Complete Kits contain all necessary reagents for performing tests: human neutrophil substrate slides, FITC conjugate, PBS, mounting medium, negative and positive controls, Evans blue counterstain, cover slips and blotters. Kallestad™ is the most trusted name in autoimmune testing for more than 25 years. Customers can test for p-ANCA and c-ANCA on a single slide with large cells for clear reactivity. All controls are pre-diluted and ready to use. Wells are spaced for use with a multichannel pipette. Kits are packaged for flexible volume ordering and automation options are available.

Human anti-neutrophil cytoplasmic antibodies (ANCA) are directed against antigenic components mainly present in primary granules of neutrophils. They are serological markers of primary necrotizing systemic vasculitis, particularly in Wegener’s granulomatosis. In addition, they have a prognostic interest because, in most cases, their titer is usually correlated to clinical activity during the disease. The IFA method is considered the gold standard for detecting ANCA. When fixed in ethanol, two types of ANCA staining reaction patterns occur: cytoplasmic (c-ANCA) and perinuclear (p-ANCA). The p-ANCA reactions can be confirmed by repeat testing on formalin-fixed slides where p-ANCA reactions convert to c-ANCA.

Complete ANCA Kit With Ethanol Fixed Slides

29403
Ethanol fixed slides, reagents and controls for immunofluorescent anti-neutrophil cytoplasmic antibody assays (10 x 6 wells)

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ANCA Combination Ethanol/Formalin Fixed Slides

29419
Combination of ethanol and formalin fixed slides for anti-neutrophil cytoplasmic antibody immunofluorescence assays (5 x 6+6 wells)

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ANCA Formalin Fixed Slides

29418
Formalin fixed slides for anti-neutrophil cytoplasmic antibody immunofluorescence assays (5 x 6 wells)

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ANCA Ethanol Fixed Slides

29417
Ethanol fixed slides for anti-neutrophil cytoplasmic antibody immunofluorescence assays (5 x 12 wells)

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ANCA Ethanol Fixed Slides

29416
Ethanol fixed slides for use in anti-neutrophil cytoplasmic antibody immunofluorescence assays (5 x 6 wells)

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ANCA Anti-Human IgG FITC Conjugate

29411
Fluorescein conjugated to IgG for anti-neutrophil cytoplasmic antibody immunofluorescence assays (5 mL)

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Sample Diluent

29407
Solution to dilute samples for immunofluorescent anti-neutrophil cytoplasmic antibody assays (60 mL)

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Mounting Medium

29410
Solution to adhere cover slips to slides for anti-neutrophil cytoplasmic antibody immunofluorescence assays (5 mL)

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ANCA 6-Well Blotting Paper

29414
6-well blotters for anti-neutrophil cytoplasmic antibody immunofluorescence assays (box of 12)

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ANCA 12-Well Blotting Paper

29415
12-well blotters for anti-neutrophil cytoplasmic antibody immunofluorescence assays (box of 12)

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c-ANCA Positive Control

29400
Positive control for cytoplasmic staining pattern in immunofluorescent anti-neutrophil cytoplasmic antibody assays (0.5 mL)

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ANCA Negative Control

29408
Negative control for anti-neutrophil cytoplasmic antibody immunofluorescence assays (0.5 mL)

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ANCA Formalin IFA (10 x 6w formalin fixed slides)
ANCA Formalin IFA (10 x 6w formalin fixed slides)

29420
Complete test kit for ANCA Formalin immunofluorescence assays; includes 10 x 6 wells formalin-fixed slides (60 tests)

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ANCA Combi IFA (5 x 12w 6 + 6w) ethanol/formalin fixed slides)
ANCA Combi IFA (5 x 12w 6 + 6w) ethanol/formalin fixed slides)

29421
Complete test kit for ANCA combination immunofluorescence assays; includes 5 x 12 wells ethanol-fixed slides, 6 x 6 wells formalin-fixed slides (60 tests)

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p-ANCA Positive Control

29401
Positive control for nuclear and perinuclear staining pattern in immunofluorescent anti-neutrophil cytoplasmic antibody assays (0.5 mL)

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