Novocastra™ Liquid
Mouse Monoclonal Antibody
Insulin
Product Code: NCL-L-INSULIN
Leica Biosystems Newcastle Ltd
Balliol Business Park West
Benton Lane
Newcastle Upon Tyne NE12 8EW
United Kingdom
(
+44 191 215 4242
Instructions for Use
Please read before using this product.
Check the integrity of the packaging before use.
IVD
Novocastra™ Liquid Mouse Monoclonal Antibody
Insulin
Product Code: NCL-L-INSULIN
Intended Use
For in vitro diagnostic use.
NCL-L-INSULIN is intended for the qualitative identification by light microscopy of human insulin in paraffin sections. The clinical
interpretation of any staining or its absence should be complemented by morphological studies using proper controls and should be
evaluated within the context of the patient’s clinical history and other diagnostic tests by a qualified pathologist.
NCL-L-INSULIN is recommended for the assessment of insulin protein expression in normal and neoplastic tissues.
Summary and Explanation
The first immunohistoperoxidase technique was reported by Nakane and Pierce.1 Since then many developments have occurred, leading
to increased sensitivity over earlier techniques. A recent development has been the use of polymeric labeling. This technology has
been applied to both primary antibodies2 and detection systems. The Novolink™ Polymer Detection Systems utilize a novel controlled
polymerization technology to prepare polymeric HRP-linker antibody conjugates. Therefore, the problem of non-specific staining that can
occur with Streptavidin/Biotin detection systems due to endogenous biotin does not occur.
Principle of Procedure
Immunohistochemical (IHC) staining techniques allow for the visualization of antigens via the sequential application of a specific antibody
to the antigen (primary antibody), a secondary antibody to the primary antibody and an enzyme complex with a chromogenic substrate
with interposed washing steps. The enzymatic activation of the chromogen results in a visible reaction product at the antigen site. The
specimen may then be counterstained and coverslipped. Results are interpreted using a light microscope and aid in the differential
diagnosis of pathophysiological processes, which may or may not be associated with a particular antigen.
Reagent
NCL-L-INSULIN is a liquid tissue culture supernatant containing sodium azide as a preservative.
Clone
2D11-H5
Immunogen
Insulin conjugated to bovine serum albumin carrier protein.
Specificity
Human insulin. Also reacts with swine and bovine insulin, but not mouse or rat insulin.
Ig Class
IgG1
Total Protein
Total Protein Concentration
Refer to vial label for lot specific total protein concentration.
Antibody Concentration
Greater than or equal to 8.5 mg/L as determined by ELISA. Refer to vial label for lot specific Ig concentration.
Warnings and Precautions
This reagent has been prepared from the supernatant of cell culture. As it is a biological product, reasonable care should be taken when
handling it.
This reagent contains sodium azide. A Material Safety Data Sheet is available upon request or available from
Consult federal, state or local regulations for disposal of any potentially toxic components.
Specimens, before and after fixation, and all materials exposed to them, should be handled as if capable of transmitting infection and
disposed of with proper precautions.3 Never pipette reagents by mouth and avoid contacting the skin and mucous membranes with
reagents and specimens. If reagents or specimens come in contact with sensitive areas, wash with copious amounts of water. Seek
medical advice.
Minimize microbial contamination of reagents or an increase in non-specific staining may occur.
Incubation times or temperatures, other than those specified, may give erroneous results. Any such changes must be validated by the
user.
Storage and Stability
Store at 2–8 °C. Do not freeze. Return to 2–8 °C immediately after use. Do not use after expiration date indicated on the vial label.
Storage conditions other than those specified above must be verified by the user.
The signs indicating contamination and/or instability of NCL-L-INSULIN are: turbidity of the solution, odor development, and presence of
precipitate.
Specimen Preparation
The recommended fixative is 10% neutral-buffered formalin for paraffin-embedded tissue sections.
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Recommendations On Use
Immunohistochemistry on paraffin sections.
Epitope Retrieval: Not recommended.
Suggested dilution: 1:150 for 30 minutes at 25 °C. This is provided as a guide and users should determine their own optimal working
dilutions.
Visualization: Please follow the instructions for use in the Novolink™ Polymer Detection Systems. For further product information or
support, contact your local distributor or regional office of Leica Biosystems, or alternatively, visit the Leica Biosystems Web site, www.
LeicaBiosystems.com
The performance of this antibody should be validated when utilized with other manual staining systems or automated platforms.
Materials Provided
See Reagent.
Materials Required But Not Provided
See Novolink™ Polymer Detection Systems Instructions for Use.
Quality Control
Differences in tissue processing and technical procedures in the user’s laboratory may produce significant variability in results,
necessitating regular performance of in-house controls in addition to the following procedures.
Controls should be fresh autopsy/biopsy/surgical specimens, formalin-fixed, processed and paraffin wax-embedded as soon as possible
in the same manner as the patient sample(s).
Positive Tissue Control
Used to indicate correctly prepared tissues and proper staining techniques.
One positive tissue control should be included for each set of test conditions in each staining run.
A tissue with weak positive staining is more suitable than a tissue with strong positive staining for optimal quality control and to detect
minor levels of reagent degradation.4
Recommended positive control tissue is pancreas.
If the positive tissue control fails to demonstrate positive staining, results with the test specimens should be considered invalid.
Negative Tissue Control
Should be examined after the positive tissue control to verify the specificity of the labeling of the target antigen by the primary antibody.
Recommended negative control tissue is tonsil.
Alternatively, the variety of different cell types present in most tissue sections frequently offers negative control sites, but this should be
verified by the user.
Non-specific staining, if present, usually has a diffuse appearance. Sporadic staining of connective tissue may also be observed in
sections from excessively formalin-fixed tissues. Use intact cells for interpretation of staining results. Necrotic or degenerated cells often
stain non-specifically.5 False-positive results may be seen due to non-immunological binding of proteins or substrate reaction products.
They may also be caused by endogenous enzymes such as pseudoperoxidase (erythrocytes), endogenous peroxidase
(cytochrome C), or endogenous biotin (eg. liver, breast, brain, kidney) depending on the type of immunostain used. To differentiate
endogenous enzyme activity or non-specific binding of enzymes from specific immunoreactivity, additional patient tissues may be stained
exclusively with substrate chromogen or enzyme complexes (avidin-biotin, streptavidin, labeled polymer) and substrate-chromogen,
respectively. If specific staining occurs in the negative tissue control, results with the patient specimens should be considered invalid.
Negative Reagent Control
Use a non-specific negative reagent control in place of the primary antibody with a section of each patient specimen to evaluate
non-specific staining and allow better interpretation of specific staining at the antigen site.
Patient Tissue
Examine patient specimens stained with NCL-L-INSULIN last. Positive staining intensity should be assessed within the context of any
non-specific background staining of the negative reagent control. As with any immunohistochemical test, a negative result means that
the antigen was not detected, not that the antigen was absent in the cells/tissue assayed. If necessary, use a panel of antibodies to
identify false-negative reactions.
Results Expected
Normal Tissues
Clone 2D11-H5 detected the insulin protein in the cytoplasm in islet cells of the pancreas. (Total number of normal cases evaluated = 51).
Abnormal Tissues
No staining was detected in brain tumors (0/2), tumors of the esophagus (0/2), a tumor of the larynx (0/1), a tumor of the thymus (0/1),
thyroid tumors (0/4), breast tumors (0/3), stomach tumors (0/2), soft tissue tumors (0/2), tumors of the tongue (0/2), lung tumors (0/4),
metastatic tumors of unknown origin (0/2), renal cell carcinomas (0/2), liver tumors (0/4), ovarian tumors (0/4), tumors of the cervix (0/2),
testicular tumors (0/2), colonic tumors (0/3), tumors of the rectum (0/2) and skin tumors (0/2). (Total number of tumor cases evaluated = 46).
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General Limitations
Immunohistochemistry is a multistep diagnostic process that consists of specialized training in the selection of the appropriate reagents;
tissue selection, fixation, and processing; preparation of the IHC slide; and interpretation of the staining results.
Tissue staining is dependent on the handling and processing of the tissue prior to staining. Improper fixation, freezing, thawing, washing,
drying, heating, sectioning or contamination with other tissues or fluids may produce artifacts, antibody trapping, or false negative
results. Inconsistent results may be due to variations in fixation and embedding methods, or to inherent irregularities within the tissue.6
Excessive or incomplete counterstaining may compromise proper interpretation of results.
The clinical interpretation of any staining or its absence should be complemented by morphological studies using proper controls and
should be evaluated within the context of the patient’s clinical history and other diagnostic tests by a qualified pathologist.
Antibodies from Leica Biosystems Newcastle Ltd are for use, as indicated, on either frozen or paraffin-embedded sections with specific
fixation requirements. Unexpected antigen expression may occur, especially in neoplasms. The clinical interpretation of any stained
tissue section must include morphological analysis and the evaluation of appropriate controls.
Performance Characteristics
The performance of NCL-L-INSULIN has been validated on a range of normal and abnormal tissues. See Results Expected.
Bibliography - General
1. Nakane PK and Pierce GB. Enzyme labeled antibodies : Preparations and applications for the localization of antigens. Journal of
Histochemistry and Cytochemistry. 1967; 14:929–931.
2. Tsutsumi Y, Serizawa A and Kawai K. Enhanced polymer one-step staining (EPOS) for proliferating cell nuclear antigen and Ki-67
antigen-applications to intraoperative frozen diagnosis. Pathology International. 1995; 45(2):108–115.
3. National Committee for Clinical Laboratory Standards (NCCLS). Protection of laboratory workers from infectious diseases transmitted
by blood and tissue; proposed guideline. Villanova, P.A. 1991; 7(9). Order code M29-P.
4. Battifora H. Diagnostic uses of antibodies to keratins: a review and immunohistochemical comparison of seven monoclonal and
three polyclonal antibodies. Progress in Surgical Pathology. 6:1–15. eds. Fenoglio-Preiser C, Wolff CM, Rilke F. Field & Wood, Inc.,
Philadelphia.
5. Nadji M, Morales AR. Immunoperoxidase, part I: the techniques and pitfalls. Laboratory Medicine. 1983; 14:767.
6. Omata M, Liew CT, Ashcavai M, Peters RL. Nonimmunologic binding of horseradish peroxidase to hepatitis B surface antigen: a
possible source of error in immunohistochemistry. American Journal of Clinical Pathology. 1980; 73:626.
7. Ackerman’s Surgical Pathology. Vol 1. Ed: Jaun Rosai. Seventh Edition. Publisher: CV Mosby Company. P778.
Amendments to Previous Issue
Not applicable.
Date of Issue
20 May 2013
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Leica Biosystems Newcastle Ltd
Balliol Business Park West
Benton Lane
Newcastle Upon Tyne NE12 8EW
United Kingdom
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© Leica Biosystems Newcastle Ltd. NCL-L-ENG-U Rev B INSULIN-L-U 20/05/2013
(
+44 191 215 4242
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