Performance comparability between AZDye and Alexa Fluor^® dyes for fluorescent based applications.

Introduction

Sulfonated fluorescent dyes or fluorophores have revolutionized the field of fluorescence labeling, offering numerous advantages over traditional dyes like fluorescein and rhodamines. These benefits include superior water solubility, higher fluorescence intensity, reduced self-quenching, enhanced photostability, and lower pH sensitivity¹. The Alexa Fluor^® family of dyes, developed in the late 1990s and now a registered trademark of Thermo Fisher Scientific, has long been considered the gold standard in this category.

However, with the expiration of original patents covering Alexa Fluor^® dyes, Vector Laboratories has developed and commercially introduced AZDyes, which are structurally identical to Alexa Fluor^® dyes but offer significant cost savings. On average, AZDyes are 2.5 times less expensive per milligram compared to their Alexa Fluor^® counterparts. In depth experimentation was done to show the performance comparability of AZDyes relative to Alexa Fluors.

Materials and methods:

To assess the relative performance of AZDyes (AZDye 350-NHS ester FP-1002, AZDye 488-NHS ester FP-1013, AZDye 555-NHS ester FP-1166, AZDye 594-NHS ester FP-1646, AZDye 647-NHS ester FP-1121, all from Vector Laboratories), we conducted a comprehensive comparison with selected Alexa Fluor^® dyes (Alexa Fluor^® 350-NHS ester cat# A10168, Alexa Fluor^® 488-NHS ester cat# A20000, Alexa Fluor^® 555-NHS ester cat# A20009, Alexa Fluor^® 594-NHS ester cat# A20004, Alexa Fluor^® 647-NHS ester cat# A20006, all from Thermo Fisher Scientific) at specific nm wavelengths of 350, 488, 555, 594, and 647, respectively. Dye-NHS esters were conjugated with Goat anti-Mouse IgG using different NHS:IgG ratios to achieve degree-of-labeling (DOL) values within the optimal range for each Alexa fluorophore. These AZDyes and Alexa Fluor^® conjugates were then compared side-by-side in two different antibody-based detection systems: ELISA and immunofluorescence (IF) in tissue.

The following protocols were used to prepare reagents and test the performance comparability between the fluorophores.

Preparation of IgG-Dye Conjugates

Materials:

• Dye-NHS esters (AZDye or Alexa Fluor^®)
• Goat anti-Mouse IgG
• Phosphate-buffered saline (PBS), pH 7.8
• 1 M Tris-HCl buffer, pH 8.5
• PBS + 0.08% Sodium Azide

Also Used: 5 mL HiTrap Desalting Column on ÄKTA avant^™ chromatography system

Procedures:

Preparation of IgG solution:

1. Dissolve 10 mg of Goat anti-Mouse IgG in 2 mL PBS (pH 7.8) to achieve a concentration of 5 mg/
2. Confirm concentration of IgG by either Nanodrop or UV-Vis.

Preparation of dye-NHS ester conjugate:

1. Dissolve the dye-NHS ester in anhydrous DMSO to a concentration of 10 mg/mL immediately before use.
2. Conjugation reaction: Add the appropriate volume of dye-NHS ester solution to the IgG solution to achieve the desired NHS:IgG molar ratio.
3. Mix gently by pipetting.
4. Incubate the reaction mixture for 2 hours at room temperature (20-25°C), protected from light.
5. Quenching of excess NHS ester: Add 1 M Tris-HCl (pH 8.5) to achieve a final concentration of 10 Incubate for 30 minutes at room temperature, protected from light.
6. Purification of IgG-dye conjugates: Equilibrate the 5 mL HiTrap Desalting Column with PBS + 0.08% Sodium Azide using the ÄKTA avant^™ chromatography system.
7. Load the quenched reaction mixture onto the column. Elute the conjugate with PBS + 0.08% Sodium Azide at a flow rate of 1 mL/min. Collect 0.5 mL fractions and monitor absorbance at 280 nm and the maximum absorption wavelength of the dye. Pool the fractions containing the IgG-dye conjugate.
8. Alternatively, IgG-dye conjugate can be purified using desalting spin columns, ultrafiltration, or dialysis.
9. Store the purified IgG-dye conjugates at 4°C, protected from light.

Determination of DOL and concentration:

Dilute an aliquot of the purified conjugate with PBS to fit into the optimal range of your UV-Vis spectrophotometer (2x in most cases). Measure absorbance at 280 nm and at the dye’s maximum absorption wavelength using a UV-Vis spectrophotometer. Calculate the degree of labeling (DOL) and protein concentration using the formulas below:

ε_dye – extinction coefficient of the dye

ε_target – extinction coefficient of the biomolecule

A_dye – absorbance maximum of the dye

A_target – absorbance maximum of the protein (280 nm)

CF – correction factor for the protein at 280 nm

Functional Performance: ELISA

Materials:

• 96-well high-binding ELISA plates
• Mouse IgG
• Carbonate ELISA buffer (100 mM carbonate-bicarbonate, pH 9.6)
• Phosphate-buffered saline with 0.05% Tween-20 (PBST)
• Bovine Serum Albumin (BSA)
• Goat anti-Mouse IgG-dye conjugates (AZDyes and Alexa Fluor^®)
• Fluorescence microplate reader

Procedure:

1. Plate Coating: Prepare a 0.3 μg/mL solution of Mouse IgG in Carbonate ELISA buffer and add 100 μL to each well in the ELISA plate. Include wells with buffer only as negative controls.
2. Seal the plate and incubate overnight (12-16 hours) at 4°C.
3. Wash the plate 3 times with PBST. After the final wash, invert the plate and tap it gently on absorbent paper to remove any remaining liquid.
4. Blocking: Add 200 μL of 1% BSA in PBST to each well. Incubate for 1 hour at room temperature (20-25°C).
5. Secondary Antibody Incubation: Prepare 2 μg/mL solutions of each Goat anti-Mouse IgG-dye conjugate in 1% BSA/PBST.
6. Aspirate 1% BSA in PBST and add 100 μL of each dye-conjugate solution to appropriate wells. Each conjugate was evaluated in triplicate. Incubate for 1 hour at room temperature, protected from light.
7. Final Washing: Wash the plate 3 times with PBST as in step 2, ensuring thorough removal of unbound conjugates.
8. Fluorescence Measurement: Immediately after washing, read the plate on a fluorescence microplate reader. Use appropriate excitation and emission wavelengths for each dye:
   • AZDye/Alexa Fluor^® 350: Ex 346 nm, Em 445 nm
   • AZDye/Alexa Fluor^® 488: Ex 494 nm, Em 517 nm
   • AZDye/Alexa Fluor^® 555: Ex 555 nm, Em 572 nm
   • AZDye/Alexa Fluor^® 594: Ex 590 nm, Em 617 nm
   • AZDye/Alexa Fluor^® 647: Ex 649 nm, Em 671 nm
9. Data Analysis: Calculate the average fluorescence intensity for each dye conjugate. Subtract the average background signal (from buffer-only wells) from all measurements.

Data:
A.

B.

Figure 1: AZDye and Alexa Dye conjugates show equivalent intensity of fluorescence at similar degree of labeling (DOL) in direct ELISA.
A. Absolute fluorescence intensity for Goat x Mouse IgG-dye conjugates at lower end of the optimal DOL range.
B.  Absolute fluorescence intensity for Goat x Mouse IgG-dye conjugates at higher end of the optimal DOL range.

Functional Performance: Immunofluorescence Analysis

Materials:

• FFPE bowel carcinoma tissue sections (5µm)
• Mouse anti-Cytokeratin (AE1/AE3)
• Goat anti-Mouse IgG-dye conjugates (AZDyes and Alexa Fluor^®)
• Xylene
• Ethanol series (100%, 95%, 70%)
• Antigen Unmasking Solution (Citrate-based), H-3300-250
• PBS (Phosphate-buffered saline) pH 7.4
• Animal-Free Blocker^® and Diluent (RTU), Cat# SP-5035-100
• TrueVIEW^® Autofluorescence Quenching Kit, Cat# SP-8400-15
• VECTASHIELD Vibrance^® Antifade Mounting Medium with DAPI, Cat# H-1800
• VECTASHIELD Vibrance^® Antifade Mounting Medium, Cat# H-1700
• Nikon Eclipse fluorescence microscope.

Procedure

1. Deparaffinization and Rehydration: Clear sections in xylene and rehydrate through graded ethanol series. Rinse in water for 5 minutes.
2. Perform Antigen unmasking in a pressure cooker with citrate-based antigen unmasking solution.
3. Rinse slides in PBS for 2 x 5 minutes.
4. Blocking: Apply RTU Animal-Free Blocker^® and Diluent to cover the sections. Incubate for 30 minutes at room temperature.
5. Primary Antibody Incubation: Dilute mouse anti-cytokeratin antibody (AE1/AE3) 1:50 in RTU Animal-Free Blocker^® and Diluent. Apply diluted primary antibody to tissue sections. Incubate for 30 minutes at room temperature.
6. Washing: Wash slides in PBS for 5 minutes.
7. Secondary Antibody Incubation: Prepare Goat anti-Mouse IgG-dye conjugates in RTU Animal-Free Blocker^® and Diluent at 1 µg/ml. Incubate for 30 minutes at room temperature, protected from light.
8. Washing: Wash slides in PBS for 5 minutes.
9. Block Autofluorescence: Apply TrueVIEW^® Autofluorescence Quenching Reagent for 5 minutes.
10. Washing: Wash slides in PBS for 5 minutes.
11. Coverslip: Mount slides using VECTASHIELD Vibrance^® Antifade Mounting Medium with DAPI (except for AZDye 350 and Alexa Fluor^® 350 samples which are mounted in VECTASHIELD Vibrance^® Antifade Mounting Medium).
12. Image the slides.

Procedure:

Image Acquisition:

Figure 2: AZDye and Alexa Fluor^® Dye conjugates show equivalent intensity of fluorescence and background staining at similar degree of labeling (DOL) in immunofluorescence analysis of bowel carcinoma tissue sections. The primary antibody used was Mouse anti-AE1/AE3 at 3.27 µg/ml and secondary antibody was Goat anti-Mouse-dye conjugates at 1 µg/ml.  20x magnification, 500ms exposure.

Figure 3: AZDye and Alexa Fluor^® Dye conjugates show equivalent intensity of fluorescence and background staining at similar degree of labeling (DOL) in immunofluorescence analysis of bowel carcinoma tissue sections. The primary antibody used was Mouse anti-AE1/AE3 at 3.27 µg/ml and secondary antibody was Goat anti-Mouse-dye conjugates at 1 µg/ml.  20x magnification, 50ms exposure.

Figure 4: AZDye and Alexa Fluor^® Dye conjugates show equivalent intensity of fluorescence and background staining at similar degree of labeling (DOL) in immunofluorescence analysis of bowel carcinoma tissue sections. The primary antibody used was Mouse anti-AE1/AE3 at 3.27 µg/ml and secondary antibody was Goat anti-Mouse-dye conjugates at 1 µg/ml.  20x magnification, 80ms exposure.

Figure 5: AZDye and Alexa Fluor^® Dye conjugates show equivalent intensity of fluorescence and background staining at similar degree of labeling (DOL) in immunofluorescence analysis of bowel carcinoma tissue sections. The primary antibody used was Mouse anti-AE1/AE3 at 3.27 µg/ml and secondary antibody was Goat anti-Mouse-dye conjugates at 1 µg/ml.  20x magnification, 50ms exposure.

Figure 6: AZDye and Alexa Fluor^® Dye conjugates show equivalent intensity of fluorescence and background staining at similar degree of labeling (DOL) in immunofluorescence analysis of bowel carcinoma tissue sections. The primary antibody used was Mouse anti-AE1/AE3 at 3.27 µg/ml and secondary antibody was Goat anti-Mouse-dye conjugates at 1 µg/ml.  20x magnification, 70ms exposure.

Results and Discussion

Our comprehensive evaluation revealed that AZDyes perform equivalently to Alexa Fluor^® dyes in their performance in bioconjugation reactions, as well as in the conjugate performance in intended use workflows.

AZDyes were conjugated to Goat anti-Mouse IgG using the same protocol as Alexa Fluor^® dyes. The degree of labeling (DOL) achieved was equivalent between AZDyes and Alexa Fluor^® dyes, indicating similar conjugation efficiency. In addition, conjugate yields were comparable. AZDye conjugates showed comparable performance to Alexa Fluor^® conjugates in ELISA assays. Signal intensity and background levels were similar for both dye families across the tested wavelengths (350, 488, 555, 594, and 647 nm). In tissue immunofluorescence studies, AZDye conjugates again demonstrated performance equivalency to Alexa Fluor^® conjugates. Images captured using a Nikon Eclipse fluorescence microscope showed similar fluorescence intensity, signal-to-noise ratio, and overall image quality for both dye families. Specific staining patterns were observed for various cellular structures (e.g., cytokeratin) with both AZDye and Alexa Fluor^® conjugates. Exposure times used for imaging were the same for corresponding AZDye and Alexa Fluor^® conjugates, indicating similar brightness.

Conclusions

AZDyes are a valuable tool in the field of fluorescent labeling, offering performance equivalence to Alexa Fluor^® dyes at a fraction of the cost. Their superior optical and physical properties, combined with their broad spectral coverage, make AZDyes an excellent choice for researchers seeking high-quality, cost-effective fluorescent labels. The comparable performance of AZDyes in ELISA and tissue immunofluorescence analysis demonstrates their potential to replace Alexa Fluor^® dyes in various applications without compromising performance.

AZDyes are available with a wide range of reactive groups, allowing researchers to select the most appropriate chemistry for their specific bioconjugation needs. This flexibility in reactive group options ensures that AZDyes can be easily integrated into diverse experimental workflows, accommodating different biomolecules and conjugation strategies.

As the field of fluorescence microscopy continues to evolve, AZDyes are poised to play a crucial role in advancing scientific discovery across multiple disciplines, enhancing research capabilities in the life science, diagnostic, and biopharma communities. The combination of cost-effectiveness, performance, and versatility in bioconjugation chemistry – positions AZDyes as a valuable tool for researchers pushing the boundaries of fluorescence-based techniques.

Advantages of: AZDyes

1. Brightness and photostability: AZDyes exhibit excellent brightness and photostability, comparable to Alexa Fluor^® dyes, allowing for extended imaging times and improved signal detection².
2. pH insensitivity: AZDyes maintain their fluorescence intensity over a broad pH range (4-10), ensuring consistent performance across various experimental conditions².
3. Reduced background staining: The high water solubility of AZDyes minimizes aggregation and non-specific binding, resulting in lower background fluorescence.
4. Spectral compatibility: AZDyes are spectrally similar to Alexa Fluor^® dyes, allowing researchers to use existing filter sets and experimental protocols without modification³.
5. Versatility: AZDyes are suitable for a wide range of applications, including flow cytometry, confocal microscopy, and high-content screening.
6. Minimal spectral overlap: The distinct spectral properties of AZDyes facilitate multicolor imaging with minimal crosstalk between channels.
7. Cost-effectiveness: AZDyes offer significant cost savings, making them an attractive alternative for researchers and laboratories working with limited budgets.
8. Simplified licensing: Unlike Alexa Fluor^® dyes, AZDyes do not require complicated and/or time-consuming licensing agreements, streamlining the procurement process for academic and industrial researchers.

References

1. Mahmoudian, Jafar et al. “Comparison of the Photobleaching and Photostability Traits of Alexa Fluor^®568- and Fluorescein Isothiocyanate- conjugated Antibody.” Cell journal vol. 13,3 (2011): 169-72.
2. Panchuk-Voloshina, N et al. “Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates.” The journal of histochemistry and cytochemistry: official journal of the Histochemistry Society 47,9 (1999): 1179-88. doi:10.1177/002215549904700910
3. Berlier, Judith E et al. “Quantitative comparison of long-wavelength Alexa Fluor^® dyes to Cy dyes: fluorescence of the dyes and their bioconjugates.” The journal of histochemistry and cytochemistry: official journal of the Histochemistry Society 51,12 (2003): 1699-712. doi:10.1177/002215540305101214