E-LLNA:  Enhanced Local Lymph Node Assay 

Enhanced Alternative Sensitization Assay Using Flow Cytometry

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The Animal Welfare Act (AWA) and government agencies require, when available, the use of alternative methods which address the Reduction, Refinement and Replacement (3R’s) of animal studies.

The Local Lymph Node Assay (LLNA) is an alternative to the guinea pig sensitization test which is employed by the pharmaceutical, chemical, consumer product and cosmetic industries to identify and characterize substances with immunotoxic properties.

A major limitation of the LLNA in its current form is that it cannot differentiate between subclasses of sensitizing agents (i.e., contact vs. respiratory allergens). Furthermore, the assay uses radioactivity and large numbers of animals.

Our team of toxicologists have developed and validated an Enhanced LLNA  that utilizes flow cytometry-based endpoints which enhance the sensitivity and discriminating power of the LLNA, while:

(1) Complying with the AWA by directly addressing the 3R’s
(2) Increasing the quality and quantity of data generated when compared to existing methods (guinea pig test)
(3) Substantially reducing the cost of analysis by avoiding the use of radioactivity
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The Mouse Local Lymph Node Assay is now accepted by the EPA, OECD, and FDA as the preferred "stand-alone alternative" to the Guinea Pig Sensitization Test. MB Research offers a "Standard LLNA" (OECD TG429 and EPA OPPTS 870.2600). In addition, for problem test substances and irritants, MB Research also offers an "Enhanced LLNA" using Flow Cytometry to allow the evaluation of optional immunophenotypic markers which discriminate "true sensitizers" from "false-positive irritants".

During the "induction phase" of sensitization, following exposure to a sensitizing test substance, lymphocyte proliferation occurs in the local lymph node. The LLNA measures increased proliferation of lymphocytes in the auricular lymph nodes (which drain the site of exposure; ears). Proliferation is assessed by determining the incorporation of the thymidine analog, bromodeoxyuridine (BrdU), into the DNA of lymph node cells using flow cytometric method. This "Enhanced LLNA" reduces background noise and false positives, by quantitatively assessing irritation, (via ear swelling) and by incorporating optional immunophenotype analysis to resolve and discriminate false-positive irritants(%B220+, CD3+, CD69+, I-Ak+ cells).

 

Note: Although EPA/OECD Test Guidelines specify a minimum or 3 test concentrations, in the case of substances with dermal irritation properties, or in the absences of previous mouse ear irritation data, the LLNA test may require testing of 4-5 test concentrations to conduct a proper evaluation.

The standard LLNA protocol can be modified to suit your budget and specific research needs. Please call for other custom LLNA study prices and schedules.  

To discuss any aspect of this assay, or if you would like information on other in vivo and in vitro assays that are conducted at MB Research Laboratories, please contact our Client Services or our in vitro staff at 215-536-4110 or .

 

MB Research Laboratories
Spinnerstown, PA  18968
voice:  (215) 536-4110
fax:  (215) 536-1816

Prices subject to change without notice.

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Other In Vitro and Alternative Toxicology Tests available at MB Research:  CORROSITEX.COM and 3T3NRU.COM