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Routine Quality Control on Rabies Diagnostic Conjugates Identified Recent Lots with Low Affinity and Inconsistent Rabies Virus Variant Recognition

Posted: August 11, 2009

The direct fluorescent antibody (DFA) test is a highly sensitive and specific test for primary rabies persons in lab looking through microscopediagnosis. The results of the test have clinical and public health impact regarding appropriate and timely rabies post-exposure prophylaxis when it is needed. Due to the critical nature of this testing, each new lot of commercial conjugate should be optimized prior to use. The working dilution of conjugate should demonstrate sparkling apple-green fluorescence (4+ intensity) and detect 100% of antigen compared to a reference conjugate when rabies virus variants endemic to the region are tested. The standard DFA test protocol [PDF - 2.15MB] in the United States requires at least 2 anti-rabies virus conjugates to maximize recognition of different antigenic sites.

The following lots may require more concentrated working dilution:

Millipore Corp. Light Diagnostics DFA Reagent (Monoclonal antibody FITC labeled Cat# 5100)

  • AW1472018
  • JH1529441
  • TR1500205
  • JH1561036
  • JH1561037
  • JH1586865*
  • JH1611393*

*May demonstrate more sensitivity

Millipore Corp. Light Diagnostics Polyclonal DFA Reagent (Goat lgG FITC conjugate Cat# 5199)

  • TR1480600
  • TR1443700

Variation in affinity and concentration (titer) of these conjugates was noted with different rabies virus variants. More concentrated dilutions of the above conjugates were necessary to obtain 4+ intensity and in some cases, this level of intensity was not demonstrated.

The monoclonal antibody conjugate (#5100; lots#: AW1472018, JH1529441, and TR1500205) at best detected only 25-50% of antigen from a bat, Eptesicus fuscus (big brown bat), variant, but did detect 100% of antigen in predominant terrestrial variants at a more concentrated dilutions. The polyclonal reagent (#5199; TR1480600 and TR1443700) at optimal dilution detected only 75% of antigen with some terrestrial and bat rabies virus variants compared to a reference lot of anti-rabies conjugate.

The inconsistencies among recent lots of anti-rabies diagnostic conjugates emphasize the critical need for thorough evaluation of new lots of rabies conjugates with multiple rabies virus variants prior to use. The manufacturer, Millipore, is aware of the problems with the rabies reagents and is working diligently to resolve the problems as quickly as possible.

Recommendations

  1. Strict adherence to the national standard protocol [PDF - 2.15MB] (optimize all reagents prior to use and use 2 anti-rabies conjugates in every test).
  2. Repeat (Confirmatory) DFA with 2 diagnostic conjugates and specificity control reagent (Negative Control Conjugate) when inconsistent results occur, on all weak positive and inconclusive test results.
  3. Prompt submission of samples to a reference laboratory if rabies can not be ruled-out or confirmed by repeat (Confirmatory) DFA testing. Provide information regarding exposures.
  4. Use of alternate confirmatory testing such as DRIT, RT-PCR and virus isolation as adjunct test procedures.
  5. The CDC Rabies program should be notified (404-639-1050) when inconsistent results with two or more rabies virus conjugates are recognized.
 
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