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Critical factors in an enzyme immunoassay (ELISA) for antibodies to mouse thymic virus (MTLV)
1Rockefeller University, Laboratory Animal Research Center, New York, NY 10021.
Abstract:
Mouse thymic virus (MTLV; murid herpesvirus 3) is a naturally occurring herpesvirus of mice. Critical variables in an enzyme immunoassay (ELISA) for antibodies to mouse thymic virus (MTLV) were assessed. High protein binding plates proved unsuitable. For storing coated plates, the most consistent results were obtained when coated plates were washed and stored with coating buffer (phosphate buffered saline, PBS) at -70 degrees C. Storage of antigen at -20 degrees C was unsatisfactory, although coated plates could be stored at -20 degrees C for at least 1-2 weeks. In performing the ELISA, vigorous washing and blocking were critical. However, substituting isotonic (0.9%) saline for PBS in washing solutions gave reliable results at considerable cost savings. For blocking, and as sample diluent, a modification of 'BLOTTO' (5% nonfat dry milk and 0.2% Tween 20) was most satisfactory.
Insights
Optimizing enzyme immunoassays (ELISA) for mouse thymic virus (MTLV) antibodies requires specific protocols. Key findings include optimal storage conditions for coated plates and cost-effective washing solutions for reliable antibody detection.
Area of Science:
- Virology
- Immunology
- Biotechnology
Background:
- Mouse thymic virus (MTLV), also known as murid herpesvirus 3, is a common herpesvirus in mouse populations.
- Accurate detection of MTLV antibodies is crucial for research and diagnostics involving mouse models.
Purpose of the Study:
- To optimize critical variables for a robust enzyme immunoassay (ELISA) for detecting antibodies against mouse thymic virus (MTLV).
- To identify cost-effective and reliable methods for performing MTLV antibody ELISAs.
Main Methods:
- Assessment of different microplate types for protein binding.
- Evaluation of storage conditions for coated plates and antigen.
- Optimization of washing and blocking steps using various buffer solutions, including saline and modified BLOTTO.
Main Results:
- High protein-binding plates were found unsuitable for MTLV antibody detection.
- Optimal storage of coated plates involved washing and storing with phosphate-buffered saline (PBS) at -70°C.
- Antigen storage at -20°C was unsatisfactory, but coated plates were stable for 1-2 weeks at -20°C.
- Vigorous washing and blocking were critical; substituting isotonic saline for PBS in washing solutions offered significant cost savings.
- A modified BLOTTO solution (5% nonfat dry milk, 0.2% Tween 20) proved most effective for blocking and as a sample diluent.
Conclusions:
- Standardized protocols are essential for reliable MTLV antibody ELISAs.
- Cost-effective modifications, such as using saline for washing, can be implemented without compromising assay reliability.
- Proper storage conditions and blocking reagents are critical for assay performance.
