SIMULTANEOUS SCREENING OF VIRUSES WITH R-PHYCOERYTHRIN AND FLUORESCEIN ISOTHIOCYANATE USING A SINGLE FLUORESCENT FILTER SET. Carl Shaw, April Brandon, Brad Newsome, Mat Longiaru, PhD., and Jimmy Page, PhD. Diagnostic Hybrids, Inc., Athens, OH 45701, USA. Abstract Introduction: Differentiation of related viral infections or viral infections with similar symptoms can be time consuming and labor intensive. Current products that use a dual fluor approach allow rapid differentiation based on a second fluor, other than fluorescein, which identifies one particular virus from the other possible viruses that could be present. Brighter, more readily distinguished stains in a dual format are desirable that utilize a single fluorescent filter set. Objective: To develop user friendly, dual fluor MAbs for the rapid detection and identification of similar or related viruses. Methods: Possible secondary fluors tested were Tetramethyl Rhodamine Isothiocyanate (TRITC) and R-Phycoerythrin (R-PE). TRITC labeling of antibodies employs the same amine binding sites of Fluorescein Isothiocyanate (FITC). Experiments for both TRITC and R-PE included varying amounts, concentrations and conjugation times of the labeling process. R-PE labeling involves altering both the fluorescent marker and antibody for proper coupling. Strategies involved reducing MAbs to expose free sulfhydryls to bind to maleimide groups on activated R-PE and protein modification of the unactivated R-PE to bind to amine groups on the detection antibodies. Optimum results were determined by fluorescent intensity of the resulting stains in comparison to current available products. Results: The apple green fluorescence of FITC is easily distinguished from the golden yellow color emitted from excited RPE. Experiments using TRITC labeled MAbs yielded less than desirable results than those same MAbs labeled with R-PE. TRITC labeling also requires the addition of a filter set for use in detection. Our R-PE labeling protocols have established repeatable results of brighter detection antibodies that utilize the same filter required for the detection of FITC. Conclusion: We have developed multiple MAbs that employ R-PE and FITC as our dual fluors. R-PE and FITC labeled MAbs used in a system for simultaneous virus screening allows for prompt virus detection with the simplicity of a single filter set. Current MAbs developed for detection of respiratory viruses and the Herpes Virus family have been labeled with the R-PE technology. This approach has yielded excellent prospects for future kit development.
• The “color” observed using R-PE is a yellow gold; thus there is no confusion with the red “color” observed with the cell counter-stain using Evan’s Blue
•The absorption spectrum is outside the range of the fluorescein filter set (see Figure 1), i.e. the intensity of TRITC at 495nm is compromised substantially without using a second, TRITCspecific filter set; as can be seen from the comparison of excitation and emission spectra for TRITC in Figure 1, TRITC has a very low absorbance at 490 nm, which is the excitation wavelength for fluorescein. •If a second, TRITC-specific filter set was used, the “fluorescent signal” observed (i.e. red-like color) is difficult to distinguish from the red counter-stain in the monolayer produced by the excitation of Evan’s Blue. In contrast, R-Phycoerythrin (R-PE), the most intensely fluorescent of the phycobiliproteins, with quantum efficiencies probably in excess of 90% with a secondary peak at 495nm, could be an outstanding candidate chemistry to overcome the deficiencies of using TRITC.
R-PE Label Flu A
other 6 major respiratory viruses
Flu A/Flu B
other 6 major respiratory viruses
•A single filter set is required when using FITC and R-PE, as opposed to a dual filter set required to optimize when using a combination of FITC-TRITC
* The technology name for Diagnostic Hybrids MAbs that utilize both FITC- and R-PE labeled reagents is D3uet™
Co-infections of respiratory viruses stained with D3uet Flu A/Respiratory MAb Screen Reagent A
B Flu A R-PE
Flu B FITC
Flu A R-PE
The ability to distinguish types of viruses, or a particular group of viruses, within a single test sample is desirable in both direct specimen testing and rapid cell culture formats because it reduces the steps and reagents required to perform a diagnostic test. When used in a respiratory virus screening motif prior to the onset of influenza, for example, this testing strategy greatly reduces the labor associated with processing a large number of respiratory virus “negative” specimens. In cases where a mixed infection may occur, detection of both in a single specimen is desirable as well.
FITC is a commonly used fluorescent label in clinical diagnostic applications and can be readily and sensitively viewed using fluorescent microscopy and a fluorescein filter set. Unfortunately, TRITC as a second label is sub-optimal because:
Testing Format Flu A/Respiratory screen
•An increased signal intensity and brightness for the MAb(s) labeled with R-PE than MAb(s) labeled with TRITC
Dual detection of viruses with virus-specific MAbs requires the use of a dual, or multiple, fluorescent labeling scheme. The only current commercial version of a dual label and dual analyte detection strategy (Chemicon SimulFluor™) employs chemical fluors from the isothiocyanate class of compounds known as FITC and TRITC.
Table 1. - Analytes/paradigms under study using D3uet™* labeling technology
The goal of this study was to determine the labeling efficiency and performance characteristics of a battery of virus-specific MAbs developed by Diagnostic Hybrids using R-PE. The expected benefits to users of a new FITC/R-PE dual labeling scheme includes:
D Flu A R-PE
Para 2 FITC
RSV FITC Flu A R-PE
Figure 1 Absorbance and emission spectra for TRITC (upper panel) and RPE (lower panel). The upper panel shows the absorbance spectra for TRITC (left-hand peak) with a maximum at ~ 550 nm. The emission peak occurs at ~580 nm. For the R-PE curves, blue shows the absorbance spectra and red the emission spectra. The approximate wavelength for fluorescein excitation is illustrated by the colored boxes. Note that the absorbance spectra for R-PE has a strong secondary peak at 495 nm (marked by a hatched box) allowing excitation with a FITC filter set, while TRITC has a very low absorbance in this area. A wide pass emission filter allows visualization of the emission of both R-PE and TRITC as a yellow/gold color.
Methods A number of monoclonal antibodies (MAbs), developed inhouse at Diagnostic Hybrids, were labeled under various conditions using R-PE as the fluorophore and maleimide chemistry. Labeled MAbs were re-purified on Protein G columns and tested for binding activity and fluorescent intensity on fixed and infected cell monolayers appropriate for the particular viral target. For example, R-Mix cells were inoculated with Flu A virus and incubated at 37°C for 48 hrs before being fixed and stained with the labeled Flu A MAbs. Fixation of cell monolayers was done with 80% acetone for the Diagnostic Hybrids MAbs. All testing using SimulFluor® reagents was done according to the package insert.
Figure 2 – Mixed Infection Prototypes Monolayers infected with influenza A (all panels) and Flu B (panel A), Adeno (panel B), RSV (panel C), or Para 2 (panel D) were stained with Diagnostic Hybrids D3uet Flu A/Respiratory screen reagent. Photomicrographs were taken of representative fields of view at 100x magnification using a FITC filter set. The same exposure time was used for each virus pair.
Results The signal generated by the D3uet MAbs was readily distinguishable from the fluorescence of fluorescein using s single filter set for fluorescein.
Monolayers infected with influenza A (panels A and B), VZV (panels C and D), or HSV2 (panels E and F) were stained with either Diagnostic Hybrids D3uet MAbs against the respective viral targets (panels A, C, and E) or with SImulFluor® reagents for the same targets (panels B, D, and F). Staining was performed in parallel on monolayers inoculated at the same time and with the same titer of virus following the manufacturers’ instructions. Photomicrographs were taken of representative fields of view at 100x magnification using a FITC filter set. The same exposure time was used for each viral pair.
Future Work • D3uet MAbs labeled with R-PE will continue to be tested in the paradigms listed in Table 1 •Clinical trials will be done to assess the clinical performance of D3uet MAbs in both direct specimen and R-Mix culture confirmation formats • Real time testing of D3uet MAbs for stability in storage is underway • Additional analyte targets/paradigms may be explored following customer feedback
Summary • D3uet MAbs labeled with R-PE retained binding activity against their target viral proteins
R-Mix cell monolayers co-infected with respiratory viruses were stained with a D3uet Flu A/Respiratory screen in which the Flu A MAbs were R-PE labeled and the MAbs to the other six viruses (Flu B, Adeno, RSV, Para 1,2 &3) were fluorescein labeled. Figure 2 is a representative example of the types of staining patterns and relative intensities observed with co-infections of Flu A and Flu B (panel A), Flu A and Adeno (panel B), Flu A and RSV (panel C), and Flu A and Para 2 (panel D). In all cases the R-PE signal was easily distinguished from the fluorescein staining.
• D3uet MAbs were easily visible using a fluorescein filter set on both cell monolayers and direct specimen prototypes • The yellow/gold color of the R-PE fluorescence is easily distinguished from the fluoresceinproduced apple green fluorescence • Of equal functional importance, the yellow/gold color of the R-PE fluorescence is easily distinguishable from the counter-stain produced by using Evan’s Blue
The D3uet MAbs were also tested in parallel with SimulFluor® and viewed using a fluorescein filter set on a fluorescent microscope. Figure 3 shows a side by side comparison of cell monolayers stained using Diagnostic Hybrids D3uet MAbs (left panels) and the corresponding SimulFluor® antibody product (right panels). The photomicrographs shown are representative of the entire monolayer. The D3uet MAbs were more easily identified as positive.
• Labeling of MAbs with R-PE provides greater benefit to the laboratory than using TRITC labeled MAbs • When used in combination with FITC-labeled MAbs, it provides the user the opportunity to readily test for various combinations of analytes in both a direct specimen and culture confirmation format using a single filter set.