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Overview

Rodent Tissues IFA

Kallestad™ Mouse Kidney and Mouse Stomach/Kidney Autoimmune IFA

Clinical Utility

The detection and semi-quantitation of autoantibodies aid in the diagnosis of autoimmune diseases. Laboratories have used indirect fluorescent antibody (IFA) procedures to detect autoantibodies since 1957. In these procedures, a fluorescent antibody serves as a marker for an antigen-antibody binding reaction that occurs on the surface of the substrate. Among the substrates commonly used in the IFA procedure are mouse kidney and stomach, human epithelial (HEp-2) cells, and Crithidia luciliae, a hemoflagellate. Observation of a specific pattern of fluorescence on the substrate indicates the presence of autoantibodies in the patient's serum.

A high anti-mitochondrial antibody (AMA) titer supports the diagnosis of primary biliary cirrhosis. Low titers of AMA may be detected in other liver disorders, which include chronic active hepatitis and cryptogenic cirrhosis. Anti-smooth muscle antibodies (ASMA) are present in high titers in the serum of 70% of patients with chronic active hepatitis. In addition, 50% of these patients are positive for anti-nuclear antibodies (ANA), while 25% demonstrate low AMA titers. Low ASMA titers may be present in viral infections, malignancies, and good health. ASMA usually does not appear in systemic lupus erythematosus (SLE).

Anti-parietal cell antibodies (APCA) occur in the serum of 90% of patients with autoimmune pernicious anemia. With other clinical and laboratory data, a positive APCA result helps to distinguish autoimmune pernicious anemia from other megaloblastic anemias.

References

  1. Peter JB, Dawkins RL. Evaluating autoimmune disease. Diagn Med. Sept–Oct 1979:68–76.
  2. Doniach D, Roitt IM, Walker JG, Sherlock S. Tissue antibodies in primary biliary cirrhosis, active chronic (lupoid) hepatitis, cryptogenic cirrhosis and other liver diseases and their clinical implications.
    Clin Exp Immunol. 1966 1:237–262.
  3. Toh BH. Smooth muscle autoantibodies and autoantigens. Clin Exp Immunol. 1979 38:621–628.
  4. Lange A, Smolik R, Chmielarczyk W, Garncarek D, Gielgier Z. Diagnostic specificity of autoantibodies. II. Clustering of autoantibodies—role in diagnosis and in comparison to E—and EAC-RFC peripheral blood profiles and immunoglobulin levels. Arch Immunol Therap Exp. 1978 26:881–885.
Rodent Tissues IFA

Kallestad™ Mouse Stomach/Kidney Autoimmune IFA

Autoimmune IFA Procedure


Step 1. Reconstitute PBS with distilled water.

Step 2. Prepare screening dilutions of the test sera as specified in the instruction manual.

Step 3. Remove appropriate number of slides from freezer or refrigerator and equilibrate to room temperature in foil bags (20 minutes). Bring all reagents to room temperature.

Step 4. Remove slide from foil bag and place in moisture chamber. Immediately add 25 µL of controls or diluted test sera to appropriate wells.


Step 5. Cover moisture chamber/slide holder. Incubate substrate slides at room temperature for 20 minutes.

Step 6. Gently rinse each slide with PBS buffer and remove excess buffer.

Step 7. Wash slides for 10 minutes in a staining dish filled with PBS. Gently agitate several times or use a magnetic stirrer during the wash period.

Step 8. Remove each slide or slide holder from staining dish and blot excess PBS from around wells using blotting strips. Do not touch strip to substrate.


Step 9. Place slide(s) in moisture chamber and immediately dispense 25 µL FITC conjugate to each well.


Step 10. Cover moisture chamber/slide holder. Incubate 20 minutes at room temperature.

Step 11. Rinse each slide briefly with a stream of PBS. Wash slides for 10 minutes in a staining dish filled with PBS or perform an optional counterstaining procedure (using Evans Blue counterstain) as specified in the instruction manual.


Step 12. Remove each slide or slide holder from PBS and drain briefly on a paper towel. Apply 4 ±1 drops mounting media per slide, making sure to cover all wells and add coverslip.

Step 13. Examine the reactions on the slide under a fluorescent microscope.


 

Rodent Tissues IFA

Kallestad™ Mouse Stomach/Kidney Autoimmune IFA

Result Interpretation

Result Interpretation
Negative No specific pattern of fluorescence on the substrate, such as ANA (Homogeneous—diffuse, Rim—Peripheral, Speckled, Nucleolar), AMA (Mitochondrial), ASMA (Smooth Muscle), APCA (Parietal Cell), Brush Border or Reticulin or < +1 fluorescence
Positive When substrate shows a specific pattern of fluorescence with ≥ +1 fluorescence

Kallestad™ Autoimmune IFA Rodent Tissue Kits are indirect fluorescent antibody (IFA) assays for the detection and semi-quantitation of human autoantibodies. Each kit includes a universal procedure for HEp-2, MSK and Crithidia luciliae. High-quality rodent tissue sections ensure high sensitivity and specification; manufacturing is standardized and rigidly controlled. All controls are ready to use; wells are spaced for use with a multichannel pipette and automation options are available.

Bio-Rad’s rodent tissue substrates can be used to detect anti-mitochondrial antibodies (AMA), anti-smooth muscle antibodies (ASMA), anti-parietal cell antibodies (APCA) and other organ-specific antibodies. The use of this tissue section minimizes interference from blood group isoantibodies while providing easily discernible fluorescent patterns.

Kallestad™ Mouse Stomach/Kidney Kit

30443
Reagents, controls, and supplies for antinuclear antibody immunofluorescence assays on mouse stomach and kidney substrate slides (6 x 8 wells)

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