Rabbit anti-Human CTLA-4 Monoclonal Antibody | anti-CTLA-4 antibody

CTLA-4 Antibody

Cat. Num. AAA58783

• Reactivity: Human
• Synonyms: CTLA-4, Antibody; CTLA-4 Antibody; anti-CTLA-4 antibody

• Host: Rabbit
• Reactivity: Human
• Clonality: Monoclonal

• Localization: Membranous, Cytoplasmic
• Positive Control: Tonsil
• Principles and Procedures: Visualization of the antigen present in tissue sections is accomplished in a multi-step immunohistochemical staining process, in conjunction with a horseradish peroxidase (HRP) or alkaline phosphatase (AP) linked detection system. The process involves the addition of the stated antibody (primary antibody) to a tissue slide, followed by a secondary antibody (linked to an enzyme complex) which specifically binds to the primary antibody. A chromogenic substrate is then added which reacts with the enzyme complex, resulting in a colorimetric reaction at the site of the antigen. Results are interpreted using a light microscope.
• Reconstitution, Mixing, Dilution, and Titration: The prediluted antibody does not require any mixing, dilution, reconstitution, or titration; the antibody is ready-to-use and optimized for staining. Any further dilution may affect the quality of the staining signal or antibody-antigen interaction. The concentrated antibody requires dilution using an Antibody Diluent Buffer, to the recommended working dilution range listed in the table above, prior to use.
• Specimen Collection and Preparation: Each tissue section should be fixed with 10% neutral buffered formalin, cut to the applicable thickness (4um), and placed on a glass slide that is positively charged. The prepared slide should then be baked for a minimum of 30 minutes in a 53-65°C oven (do not exceed 24 hours).; ; Note: Performance eva luation has been shown on human tissues only. Variable results may occur due to extended fixation time or variations in tissue preparation. Do not use alcohol containing fixatives as those may result in a loss of staining activity.
• Material Required but not Provided: The following materials are required but are not provided:; a) Detection system (ie. BOND Polymer Refine Detection Kit or UltraView/OptiView Universal DAB Detection Kit); b) Chromogen (ie. DAB Substrate Kit); c) IHC wash buffer and blocking solution; d) Hematoxylin or other counterstaining reagents; e) Ethanol or reagent alcohol, xylene or xylene substitute and mounting medium; f) Antibody diluents; g) Positive and negative control tissue
• Quality Control Procedures and Interpretation of Results: The immunohistochemical staining process results in a colorimetric reaction at the site of the antigen, localized by the primary antibody. The tissue specimen result should be interpreted only after the positive and negative control tissues have been analyzed. It is recommended to include a set of tissue controls with each staining run to monitor for antibody, tissue, and reagent performance. Tissue sections may contain both positive and negative staining elements. In these cases and where applicable, these sections may serve as both the positive and negative tissue control.
• Positive Control Tissue: A positive control tissue should be processed in the same manner as the specimen and run with each test condition to provide control for variables such as tissue processing, fixation, and staining. It should function to provide validity to the specimen results obtained and can consist of fresh autopsy, biopsy, or surg ical tissue. Once stained, the positive control tissue should analyzed first to ensure that the antibody and all reagents are performing as intended. Counterstaining will result in a blue coloration, which may range from pale to dark depending on the length of the incubation time and potency of the hematoxylin. If positive staining is not observed, the positive control tissue must be deemed invalid and the results obtained with the tissue specimen must also be treated as such.
• Negative Control Tissue: Some tissue sections can also function as an internal negative control due to the diversity of staining elements present. This, however, should first be confirmed by the user. Tissue components that do not stain should demonstrate an absence of specific staining. If specific staining is observed, the negative control tissue must be deemed invalid and the results obtained with the tissue specimen must also be treated as such.
• Tissue Specimens: Tissue specimens should only be analyzed after the positive and negative control tissues have been deemed valid. Negative staining indicates that the antigen was not detected in the tissue while positive staining represents the presence of the antigen. A tissue section stained with hematoxylin and eosin should be used to analyze the morphology of the tissue specimen and verified by a qualified pathologist.
• Peformance Characteristics: This antibody has been validated by immunohistochemistry using a FFPE human ti ssue microarray comprised of different types of normal and cancerous tissues. Positive staining was observed in tonsil, lymph nod and thymus tissues. No staining was seen in skeletal muscle and brain tissues. A representative positive staining image (see figure 1).
• Analytical Performance: Trueness of Measure was analyzed using tissue samples from peer-reviewed, published literature known to be either positive or negative, and this study found no unexpected results. Precision of Measure analysis involved known positive tissue samples performed to assess the repeatability and reproducibility of the antibody product over time and in different lots; standard acceptance criteria were met in all studies. Analytical Specificity testing fou nd the only known interference comes from alcohol containing fixatives, which demonstrated a loss of stain ing activity and thus should not be used in conjunction with the processing of samples to be stained using this product. Analytica l Sensitivity was tested using randomly chosen tissues representing different levels of antigen expression, a range of staining intensities was observed illustrating the product is sensitive to staining a diverse range of expression levels. Limits of Detection, Measurement Range and Linearity of Measure are all unable to be defined for qualitative and non-linear products such as this.
• Troubleshooting: 1. If tissue sections wash off the slide, this may be caused by:; a) Slides are not positively charged.; b) Inadequate neutral-buffering of the formalin used for the fixation process.; c) A thick t issue section.; d) Inadequate drying of the tissue section prior to staining.; ; 2. If the positive control tissue exhibits negative staining, this may be due to:; a) An issue with the primary antibody or one of the secondary reagents.; b) Improper collection, fixation or deparaffinization of the tissue section.; c) Errors in the IHC staining process.; ; 3. If the positive control tissue exhibits weaker staining than expected, this may be due to sub-optimal lHC conditions, partial degradation of the primary antibody or improper storage of secondary reagents. Analysis of the positive and/or negative control tissues can help with determining the cause.
• Limitations: 1. Due to biological variability inherent to the expre ssion of certain antigens and immunohistochemical procedures, appropriate positive and negative controls should be used alongside the tissue specimen.; 2. This antibody, when used with the appropriate detection systems and reagents. detects antigen(s) that remain intact through the tissue fixation, processing and sectioning as described. Any deviation from these recommended procedures or improper handling may compromise the validity andlor analysis of the results. Do not use alcohol containing fixatives as those may result in a loss of staining activity.; 3. Provides prediluted antibodies in a ready-to-use, optimally diluted format for use as instructed. Due to the potential for variation in tissue processing and fixation, it may be necessary to adjust the incubation time of the primary antibody for different tissue specimens.; 4. Provides concentrated antibodies in a format that requires dilution with Antibody Diluent. Use of a diluent different than that specified in the package insert must be validated by the user to ensure proper compatibility with the antibody.; 5. False positive results may occur in tissue specimens due to the possibility of non-immmunological binding of substrate reaction products or proteins. False positive results may also occur subject to the type of immunostaining technique used, or due to the activity of pseudoperoxidase, endogenous peroxidase, or endogenous biotin.; 6. Due to the effect of autoantibodies or natural antibodies, normal sera from an animal source that is the same as the secondary antisera may result in false negative or false positive results when used in blocking steps.; 7. Non-specific staining with horseradish peroxidase may be observed when using tissues containing hepatitis B surface antigen due to the patient's infection with the hepatitis B virus.
• Preparation and Storage: Store at 2-8°C. To ensure stability, immediately replace vial back in the refrigerator after each use. When stored correctly, the antibody is stable until the expiry date indicated on the label. Positive and negative controls should be concurrently run with tissue specimens, to enable identification of any inadequacies with the antibody or reagents.

Materials and Methods

Application Data

(CTLA-4 on Tonsil)

Related Product Information for anti-CTLA-4 antibody

The CTLA-4 [IHC064] antibody is intended for qualified laboratories to qualitatively identify by light microscopy, the presence of associated antigens in formalin-fixed, paraffin-em~edded (FFPE) tissue sections using immunohistochemistry test methods. Use of this antibody is indicated as an aid in the identification of CTLA-4 strictly for research purposes.

Background: Cytotoxic T-Lymp ocyte-AssOCiated-Protein 4 (CTLA-4jlSa receptor on T helper cells that functions as an immune checkpoint and downregulator of immune responses. Mutations In CTLA-4 are associated with insulin-dependent diabetes mellitus, Hashimoto's thyroiditis, Graves' disease, systemic lupus erythematosis, celiac disease, primary biliary cirrhosis, thyroid-associated orbitopathy, multiple sclerosis, and other autoimmune diseases. The spliced varient of CTLA-4 in SLE is present in the patient's serum. Haploinsufficiency of CTLA-4 causes the immune system disorder known as CTLA-4 deficiency or CHAI disease (CTLA-4 haploinsufficiency with autoimmune infiltration).

References

1. Denizot F, et al. Nature. 1987; 328:267-70. 2. Dariavach P, et al. Eur J Immunol. 1988; 18:1901-5. 3. Krummel MF, et al. J Exp Med. 1995; 182:459-65. 4. Kuehn HS, et al. Science. 2014; 345:1623-7. 5. Walunas TL, et al. J Exp Med. 1996; 183:2541-50.