Intended Use The NOVA Lite IgG F-Actin kit is an indirect immunofluorescent assay (IFA) on rat intestine epithelial substrate for the screening and semi-quantitative determination of IgG anti-F-Actin antibodies in human serum. The presence of IgG anti-F-Actin antibodies can be used in conjunction with clinical findings and other laboratory tests to aid in the diagnosis of autoimmune liver diseases such as autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC).
Summary and Explanation of the test
IgG anti-F-Actin autoantibodies are the main component of what have been called anti-smooth muscle antibodies (ASMA).1-6 These antibodies are found in up to 80% of autoimmune hepatitis (AIH) patients and up to 30% of patients with primary biliary cirrhosis2,6, but may also be found, usually at low titers, in individuals without AIH. ASMA are heterogeneous and include antibodies to filamentous (F) and globular (G) forms of actin as well as non-actin components such as tubulin and intermediate filaments.3,4,7 Several autoantibodies are associated with AIH. However, IgG antibodies to F-Actin are the most specific autoantibody for AIH. 2,5 While IgG F-Actin ELISA tests, such as the QUANTA Lite Actin IgG ELISA, allow detection of IgG anti-F- Actin antibodies in an objective and quantifiable manner, some laboratories prefer IFA methodology to screen for IgG anti-F-Actin antibodies or to confirm their presence when detected by the conventional rodent kidney/stomach/liver (KSL) tissue sections. Differentiating anti-F-Actin from other antibodies to components of smooth muscle is often difficult using conventional IFA substrates such as KSL tissue sections.2,5 The identification of anti-F-actin is an important aid in the diagnosis of AIH. At the moment, a “gold standard” method for the discrimination of anti-F-actin from other reactivities is not available. 1,2 Slides made with rat intestine epithelial cells prepared using proprietary growth and fixation methods offer a new substrate for detection of IgG anti-F-Actin antibodies by IFA. This substrate overcomes some of the limitations of other IFA procedures since the IgG anti-F-Actin pattern is distinct and other antibodies that cause an interfering pattern on KSL slides do not interfere on the rat epithelial cell substrate.
Principles of the Procedure
In the indirect immunofluorescent technique, samples are incubated with the antigen substrate, and unbound antibodies are washed off. The substrate is then incubated with specific fluorescein labeled conjugate followed by a wash to remove the unbound reagent. When viewed through a fluorescence microscope, samples will exhibit an apple green fluorescence corresponding to areas where autoantibody has bound. A sample is considered positive if specific staining is observed in the F-Actin fibers surrounding most cells in a hexagonal pattern, and sometimes observed in the fibers crossing over cells.
Reagents 1. F-Actin Slide; 6 wells/slide, with desiccant 2. FITC IgG Conjugate (Goat), 1 vial of fluorescein labeled in buffer containing Evans Blue and 0.09% sodium azide 3. IgG F-Actin Positive Control, 1 vial of buffer containing 0.09% sodium azide and human serum antibodies to F-Actin, prediluted 4. IFA System Negative Control, 1 vial of buffer containing 0.09% sodium azide and no human serum antibodies to F-Actin, prediluted 5. PBS Concentrate (40x), 2 vials 6. Mounting Medium, 0.09% sodium azide, 1 vial 7. Coverslips
Warnings 1. All human source material used in the preparation of controls for this product has been tested and found negative for antibody to HIV, HBsAg, and HCV by FDA cleared methods. No test method, however, can offer complete assurance that HIV, HBV, HCV or other infectious agents are absent. Therefore, the IgG F-Actin Positive Control and IFA System Negative Control should be handled in the same manner as potentially infectious material.8
1 2. Sodium Azide is used as a preservative. Sodium Azide is a poison and may be toxic if ingested or absorbed through the skin or eyes. Sodium azide may react with lead or copper plumbing to form potentially explosive metal azides. Flush sinks, if used for reagent disposal, with large volumes of water to prevent azide build-up. 3. Use appropriate personal protective equipment while working with the reagents provided. 4. Spilled reagents should be cleaned up immediately. Observe all federal, state and local environmental regulations when disposing of wastes.
Precautions 1. This product is for in vitro diagnostic use. 2. Substitution of components other than those provided in this kit may lead to inconsistent results. 3. Incomplete or inefficient washing of IFA wells may cause high background. 4. Adaptation of this assay for use with automated sample processors and other liquid handling devices, in whole or in part, may yield differences in test results from those obtained using the manual procedure. It is the responsibility of each laboratory to validate that their automated procedure yields test results within acceptable limits. 5. A variety of factors influence the assay performance. These include the starting temperature of the reagents, the strength of the microscope bulb used, the accuracy and reproducibility of the pipetting technique, the thoroughness of washing and the length of the incubation times during the assay. Careful attention to consistency is required to obtain accurate and reproducible results. 6. Strict adherence to the protocol is recommended.
Storage Conditions 1. Store all the kit reagents at 2-8°C. Do not freeze. Reagents are stable until the expiration date when stored and handled as directed. 2. Diluted PBS buffer is stable for 4 weeks at 2-8°C.
Specimen Collection This procedure should be performed with a serum specimen. Addition of azide or other preservatives to the test samples may adversely affect the results. Microbially contaminated, heat-treated, grossly hemolyzed, or lipemic specimens containing visible particulates should not be used.
Following collection, the serum should be separated from the clot. CLSI (NCCLS) Document H18-A3 recommends the following storage conditions for samples: 1) Store samples at room temperature no longer than 8 hours. 2) If the assay will not be completed within 8 hours, refrigerate the sample at 2-8°C. 3) If the assay will not be completed within 48 hours, or for shipment of the sample, freeze at -20°C or lower. Frozen specimens must be mixed well after thawing and prior to testing.
Procedure Materials provided 5 F-Actin Slide (6 well) 1 7mL FITC IgG Conjugate 1 0.8mL IgG F-Actin Positive Control 1 0.5mL IFA System Negative Control 2 25mL PBS Concentrate (40x) 1 7mL Mounting Medium 1 Package of 10 Coverslips
Additional materials required but not provided
Micropipets to deliver 15-1000μL volume Distilled or deionized water Squeeze bottles or Pasteur pipets Moist chamber 1L container (for diluting PBS) Coplin jar Fluorescent microscope with 495nm exciter and 515nm barrier filter
2 Method Before you start 1. Bring all reagents and samples to room temperature (20-26oC). 2. Dilute PBS Concentrate: IMPORTANT: Dilute the PBS Concentrate 1:40 by adding the contents of the PBS Concentrate bottle to 975mL of distilled or deionized water and mix thoroughly. The PBS buffer is used for diluting patient samples and as a wash buffer. The diluted buffer can be stored for up to 4 weeks at 2-8°C. 3. Dilute Patient Samples: a. Initial Screening: Dilute patient samples 1:40 with diluted PBS buffer (i.e., add 50μL of serum to 1.95mL of PBS buffer). b. Titration: Make serial 2-fold dilutions from the initial screening dilution for all positive samples with diluted PBS buffer (i.e. 1:80, 1:160, 1:320... to endpoint).
Assay procedure 1. Prepare Substrate Slides: Allow the substrate slide to reach room temperature prior to removal from its pouch. Label it with pencil and place it in a suitable moist chamber. Add 1 drop (20-25μL) of the undiluted positive and the negative control to wells 1 and 2 respectively. Add 1 drop (20- 25μL) of diluted patient sample to the remaining wells. 2. Slide Incubation: Incubate the slide for 30 ± 5 minutes in a moist chamber (a dampened paper towel placed flat on the bottom of a closed plastic or glass container will maintain the proper humidity conditions). Do not allow the substrate to dry out during the assay procedure. 3. Wash Slides: After incubation, use a plastic squeeze bottle or pipet to gently wash off the serum with diluted PBS buffer. Orient the slide and stream of PBS buffer so as to minimize wash-over of samples between wells. Avoid directing the stream directly onto the wells to prevent substrate damage. If desired, place the slides in a Coplin jar of diluted PBS buffer for up to 5 minutes. 4. Addition of Fluorescent Conjugate: Shake off the excess PBS buffer. Place the slide back in the moist chamber and immediately cover each well with a drop of fluorescent conjugate. Incubate the slides for an additional 30 ± 5 minutes. 5. Wash Slides: Repeat Step 3. 6. Coverslip: Coverslip procedures vary from lab to lab; however, the following procedure is recommended: a. Place a coverslip on a paper towel. b. Apply mounting medium in a continuous line to the bottom edge of the coverslip. c. Shake off the excess PBS buffer and touch the lower edge of the slide to the edge of the coverslip. Gently lower the slide onto the coverslip in such a way that the mounting medium flows to the top edge of the slide without air bubble formation or entrapment.
Quality Control The IgG F-Actin Positive Control and IFA System Negative Control should be run on every slide to ensure that all reagents and procedures perform properly. Additional suitable control sera may be prepared by aliquoting pooled human serum specimens and storing at < -70oC. In order for the test results to be considered valid, all of the criteria listed below must be met. If any of these are not met, the test results should be considered invalid and the assay repeated. 1. The undiluted IgG F-Actin Antibody Positive Control must be > 3+. 2. The IFA System Negative Control must be negative.
Interpretation of Results Negative Reaction: A sample is considered negative if specific staining as described below in the section “Positive Reaction” is less than or equal to the IFA System Negative Control. Samples can exhibit various degrees of specific or background staining to other cellular components, but be negative for IgG anti- F-Actin antibodies. Positive Reaction: A sample is considered positive if specific staining at a greater intensity than the IFA System Negative Control is observed in the F-Actin fibers surrounding most cells in a hexagonal pattern, and sometimes observed in the fibers crossing over cells. Determine the fluorescence grade or intensity using these criteria: 4+ Brilliant apple green fluorescence 3+ Bright apple green fluorescence 2+ Clearly distinguishable positive fluorescence 1+ Lowest specific fluorescence that enables the F-Actin staining to be clearly differentiated from the background fluorescence.
3 Pattern Interpretation: A variety of patterns of nuclear and/or cytoplasmic staining can be exhibited depending on the types and relative amounts of autoantibodies present in the sample. Only the pattern described above in the heading “positive reaction” should be considered positive for IgG anti-F-Actin antibodies. All other patterns should be considered negative.
Limitations of the Procedure
1. High-titered IgG anti-F-Actin pattern is suggestive of AIH but should not be considered diagnostic. The IgG anti-F-Actin result should be considered in combination with other laboratory results as well as the overall clinical history of the patient. 2. A variety of external factors influence the test sensitivity including the type of fluorescence microscope used, the bulb strength and age, the magnification used, the filter system and the observer. 3. If a band pass filter is used instead of a 515nm barrier filter, increased artifactual staining may be observed. 4. Only pencil should be used to label the slides. Use of any other writing material may cause artifactual staining. 5. All coplin jars used for slide washing should be free from all dye residues. Use of coplin jars containing dye residue may cause artifactual staining. 6. The assay performance characteristics have been established for serum, but not for plasma or other specimen types.
Expected Values The ability of the NOVA Lite IgG F-Actin Kit to detect IgG F-Actin antibodies was evaluated by comparison to commercially available QUANTA Lite Actin IgG ELISA (Inova Diagnostics, Inc). ELISA results were determined to be positive if the patient sample was 20 units or greater and negative if less than 20 units.
Normal Range Four hundred ninety three samples from normal blood donors were run using the NOVA Lite IgG F- Actin kit. All but 4 of the 493 normal samples (99.2% specificity) were negative on NOVA Lite IgG F- Actin.
Comparison between NOVA Lite IgG F-Actin IFA and QUANTA Lite Actin IgG ELISA To determine the positive and negative percent agreement of the assays, 992 samples containing antibodies to a wide variety of antigens were tested with the NOVA Lite IgG F-Actin IFA kit and the QUANTA Lite Actin IgG ELISA. These samples included 493 normal blood donors, 60 patients with clinically defined diseases (rheumatoid arthritis, SLE, and scleroderma), 40 samples with defined antibodies (infectious diseases and autoantibodies) and 399 samples from patients suspected of having liver disease.
QUANTA Lite Actin IgG QUANTA Lite Actin IgG
N=992 ELISA ELISA Total Positive Negative NOVA Lite IgG F-Actin Positive 269 21** 290 NOVA Lite IgG F-Actin Negative 69* 633 702
5 References 1. Chretien-Leprince P, Ballot E, Andre C, et al. Diagnostic value of anti-F-actin antibodies in a French multicenter study. Ann NY Acad Sci 1050: 266-273, 2005. 2. Villalta D, Bizzaro N, Da Re M, et al. Diagnostic accuracy of four different immunological methods for the detection of anti-F-actin autoantibodies in type 1 autoimmune hepatitis and other liver related disorders. Autoimmunity 41: 105-110, 2008. 3. Czaja A, Norman G: Autoantibodies in the diagnosis and management of liver disease. J Clin Gastroenterol 37: 315-329, 2003. 4. Toh BH. Smooth muscle autoantibodies and autoantigens. Clin exp Immunol 38: 621-628, 1979. 5. Fusconi M, Cassani F, Zauli D, et al. Anti-actin antibodies: a new test for an old problem. Journal of Immunological Methods 130: 1-8, 1990. 6. Granito A, Muratori L, Muratori P, et al. Antibodies to filamentous actin (F-actin) in type 1 autoimmune hepatitis. J Clin Path 59: 280-284, 2006. 7. Dighiero G, Lymberi P, Monot C. Sera with high levels of anti-smooth muscle and anti- mitochondrial antibodies frequently bind to cytoskeleton proteins. Clin exp Immunolol 82: 52-56, 1990. 8. Biosafety in Microbiological and Biomedical Laboratories. Centers for Disease Control/National Institute of Health, 2007, Fifth Edition.
6 Manufactured By: :
Inova Diagnostics, Inc.
9900 Old Grove Road San Diego, CA 92131 United States of America Technical Service (U.S. & Canada Only) : 877-829-4745 Technical Service (Outside the U.S.) : 00+ 1 858-805-7950 support@inovadx.com
