Instruction for use

IFU (Article # 10-0001-02) IFU (Article # 10-0001-01)

Material Safety Data Sheet

DMSO (EU) (Article # 20-0002-01) DMSO (USA) (Article # 20-0002-01) PBS-T (EU) (Article # 20-0002-01) PBS-T (USA) (Article # 20-0002-01) Quenching Buffer (EU) (Article # 20-0005-01) Quenching Buffer (USA) (Article # 20-0005-01)

FAQs About this Product

1. We plan to conjugate an oligo-dT primer to an unmodified detector antibody for use in our sandwich ELISA. As a starting point, what molar excess of the ClickChem label and the oligo-dT primer should we use relative to the antibody?

   The optimal molar excess of the ClickChem label and oligo-dT primer relative to the detector antibody can vary depending on the specific detector antibody used, so it’s important to optimize conditions experimentally. However, based on our experience, a good starting point is to use a 5- to 20-fold molar excess of the ClickChem label relative to the detector antibody, followed by a 5- to 20-fold molar excess of the oligo-dT primer relative to the ClickChem-labeled antibody. Example on how to calculate molar ratios can be found in the IFU for Exazym® Conjugation Kit.

   We typically achieve good functionality of the detector antibody by using a 20-fold molar excess of the ClickChem Label relative to the antibody. This is followed by a 5-fold molar excess of the oligo-dT Primer relative to the ClickChem-labeled detector antibody.However, we have observed increased background levels when the oligo-dT Primer is used in 20-fold relative to the labeled antibody during conjugation. This background is partly due to unreacted, free oligo-dT Primers that bind non-specifically to the surface, resulting in unwanted signal.To reduce this background, we recommend a purification step after conjugation to remove free oligo-dT Primers. This can be achieved using Amicon® Ultra – 0.5 mL centrifugal filters with a 100K molecular weight cutoff (REF UFC510024). Note: For conjugates prepared with a lower molar excess of oligo-dT Primer (e.g. 5-fold) relative to the ClickChem-labeled detector antibody, purification using Amicon® spin filters does not result in a noticeable reduction in background levels.

2. Do sugar stabilizers (e.g., trehalose) interfere with conjugation?

   Although we haven’t conducted a dedicated study on the impact of trehalose, but we do not expect it to significantly interfere with the conjugation reaction. However, for best performance, we recommend removing trehalose prior to conjugation. This can be easily achieved using one of the spin columns included in the Clickchem Conjugation Kit.

3. What substances can interfere with the conjugation reaction?

   To ensure successful conjugation, certain components should be removed for best performance from your antibody sample beforehand. These include, glycerol, preservatives such as azide (N₃⁻) and thimerosal, and buffers with free amines such as for example Tris, Bis, and glycine.

   These should be removed via buffer exchange prior to the reaction. Removal is performed using one of the spin columns provided in the Clickchem Conjugation Kit.
   Additionally, carrier proteins like BSA or gelatin should not be present, as they will compete with the conjugation of the oligo-dT Primer to the detector antibody. For best results, we recommend using a carrier-free preparation of the unmodified detector. If carrier proteins are present, they should be removed prior to conjugation. Although we do not have direct experience with carrier protein removal, we suggest considering the use of a BSA Removal Kit, such as ab173231 from Abcam, for removal of BSA prior to conjugation.

4. What type of conjugation chemistry is used to attach the oligo-dT primer to the unmodified detector antibody?

   We use a two-step conjugation strategy. In the first step, the detector antibody is modified with an azide group using an amine-reactive NHS-PEG4-azide ester (Exazym® ClickChem Label), which targets primary amines such as those on lysine residues. In the second step, a DBCO-modified oligo-dT primer (Exazym® Primer) is added and incubated overnight at room temperature. This enables a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction between the azide on the antibody and the DBCO moiety on the primer, resulting in a stable conjugate.

5. We do not have an unmodified detector antibody, we only have access to a biotinylated detector antibody. Can we still use BOLD for signal amplification.

   No, to effectively amplify the signal with BOLD, you need an unmodified detector antibody to which you will conjugate the oligo-dT Primer. Using a biotinylated detector antibody will interfere with the detection of the DNA/RNA hybrid strand. This detection is performed using Exazym® Antibody Biotin (a biotinylated tertiary anti-DNA/RNA antibody) and a streptavidin-enzyme conjugate of your choice, such as streptavidin-HRP.

6. We get a high signal amplification but also a large increase in background. What can we do to reduce the background?

   Try decreasing the concentration of the oligo-dT detector conjugate. Typically, you will need to reduce the concentration of the oligo-dT detector conjugate with some factors compared to the concentration used in the standard immunoassay. Additionally, you can improve the quality of the oligo-dT detector conjugate by removing unconjugated oligo-dT primers. This can be done using an ultra-centrifugal filter unit (e.g., Amicon®) with a suitable cutoff (100 kDa) and volume (0.5 mL). Furthermore, you can evaluate an oligo-dT primer detector conjugate by applying different ratios of the ClickChem label and the oligo-dT primer compared to the detector antibody.

7. Does the PBS-T buffer supplied with the Exazym® ClickChem Conjugation Kit, used to prepare the detector oligo-dT Primer conjugate, contain any added preservatives?

   No preservatives are added to the PBS-T buffer. We have intentionally left it preservative-free to allow users to choose and add their own preservatives if needed.

8. Why are spin columns included in the Exazym® ClickChem Conjugation Kit?

   In the small kit, we provide two spin columns. One of these columns can be used for buffer exchange of the unmodified detector antibody. This is particularly useful if there are substances that interfere with amine coupling, such as buffer ions with primary amines (ex. Tris and Glycin) or preservatives like azide or thimerosal. The other spin column is used to remove free uncoupled/deactivated ClickChem label prior to conjugation of the oligo-dT Primer to the ClickChem labeled detector antibody.

9. How is the Exazym® oligo-dT Primer conjugated to the detector antibody of the standard immunoassay?

   Conjugation of the Exazym® oligo-dT Primer to the detector antibody in the immunoassay is performed using click chemistry. Specifically, we employ a Cu-free click reaction based on strain-promoted alkyne−azide cycloaddition (SPAAC). The conjugation process involves two steps:

   1. Labeling the Detector Antibody with azide: Initially, the detector antibody is labeled with the ClickChem Label (NHS-PEG4-azide). The ClickChem Label reacts with primary amines on the detector antibody. These primary amines are present at the N-terminus (α-amino group) of each polypeptide chain and in the side-chain of lysine amino acid residues (ε-amino group). Because of their positive charge at physiological pH, they predominantly occur on the external surfaces of native protein tertiary structures, making them readily accessible for conjugation.

   2. Conjugation of the DBCO-Modified Exazym® Oligo-dT Primer: In this step, we add the DBCO-modified Exazym® oligo-dT Primer, which reacts with the azide-labeled detector antibody. As a result, the primer becomes conjugated to the detector antibody.

10. We have BSA (bovine serum albumin) in the detector antibody solution. Will it have an impact on the conjugation reaction?

    BSA is typically used at a much higher concentration than the antibody it protects. Because BSA contains lysines, it will compete with the conjugation of the oligo-dT Primer to the detector antibody. For the best results, we recommend to use an antibody solution without BSA or remove BSA using a BSA Removal Kit, such as ab173231 from Abcam.

11. We have Thiomersal or sodium azide as preservatives in the antibody solution. Will it have an impact on the conjugation reaction?

    Typically, concentrations of sodium azide (≤ 3 mM or 0.02%) or thimerosal (≤ 0.02 mM or 0.01%) do not significantly interfere with NHS-ester reactions. However, higher concentrations can cause interference. We recommend you remove it prior to conjugation. Removal is performed using one of the spin columns provided in the Exazym® ClickChem Conjugation Kit.

Instruction for use

IFU (Article # 10-3011-01)

FAQs About this Product

1. The Exazym® Antibody Pair Human IL-4 Kit contains an IL-4 capture antibody and an IL-4 detector antibody conjugated to an oligo-dT primer. What standard do you recommend for calibration?

   The kit does not include an IL-4 standard, and unfortunately, we cannot recommend a specific product to purchase. However, we advise using an IL-4 standard that has been calibrated against the international standard provided by the National Institute for Biological Standards and Control (NIBSC). This ensures consistency and reliability in your quantification.

2. What concentrations do you recommend for coating the IL-4 capture antibody and for the IL-4 detector antibody in a BOLD-amplified ELISA?

   When using a MaxiSorp™ 96-well plate, we recommend coating with the IL-4 capture antibody at a concentration of 0.25–0.5 µg/mL in phosphate-buffered saline (PBS), pH 7.4. After the coating step, the wells should be washed with PBS + 0.05% Tween-20, followed by a blocking step. We recommend using  PBS + 1% casein as the blocking buffer (e.g., Thermo Scientific™ Blocker® Casein, Art. No. 37528). The coating is performed overnight at 2–8°C, followed by a wash step and a 1-hour blocking incubation at room temperature and a final wash step before adding sample or calibrators.

   For the IL-4 detector antibody, we recommend a working concentration between  0.025-0.1 µg/mL and a 1-hour incubation at room temperature. We recommend the detector antibody to be diluted in PBS + 0.05% Tween-20 (PBS-T) + 0.1% casein, which corresponds to a 10-fold dilution of Blocker® Casein in PBS-T. This buffer can also be used to dilute calibrators and samples.

   The concentrations and buffer compositions provided are suggested starting points. Depending on your specific assay conditions, alternative blocking agents, such as bovine serum albumin (BSA) or non-fat dried milk (NFDM), may offer improved performance and are worth evaluating.

   Note: When working with plasma or serum samples, it may be necessary to use an ELISA diluent that blocks heterophilic antibodies to prevent false-positive signals. We have tested Mabtech ELISA Diluent (Art. No. 3652-D2), but further evaluation is needed before we can make a specific recommendation.

Instruction for use

IFU (Article # 10-3021-01)

FAQs About this Product

1. The Exazym® Antibody Pair Human IL-6 Kit contains an IL-6 capture antibody and an IL-6 detector antibody conjugated to an oligo-dT primer. What standard do you recommend for calibration?

   The kit does not include an IL-6 standard, and unfortunately, we cannot recommend a specific product to purchase. However, we advise using an IL-6 standard that has been calibrated against the international standard provided by the National Institute for Biological Standards and Control (NIBSC). This ensures consistency and reliability in your quantification.

2. What concentrations do you recommend for coating the IL-6 capture antibody and for the IL-6 detector antibody in a BOLD-amplified ELISA?

   When using a MaxiSorp™ 96-well plate, we recommend coating with the IL-6 capture antibody at a concentration of 0.25–0.5 µg/mL in phosphate-buffered saline (PBS), pH 7.4. After the coating step, the wells should be washed with PBS + 0.05% Tween-20, followed by a blocking step. We recommend using PBS + 1% casein as the blocking buffer (e.g., Thermo Scientific™ Blocker® Casein, Art. No. 37528). The coating is performed overnight at 2–8°C, followed by a wash step and a 1-hour blocking incubation at room temperature and a final wash step before adding sample or calibrators.

   For the IL-6 detector antibody, we recommend a working concentration of  0.5 µg/mL and a 1-hour incubation at room temperature. We recommend the detector antibody to be diluted in PBS + 0.05% Tween-20 (PBS-T) + 0.1% casein, which corresponds to a 10-fold dilution of Blocker® Casein in PBS-T. This buffer can also be used to dilute calibrators and samples.

   The concentrations and buffer compositions provided are suggested starting points. Depending on your specific assay conditions, alternative blocking agents, such as bovine serum albumin (BSA) or non-fat dried milk (NFDM), may offer improved performance and are worth evaluating.

   Note: When working with plasma or serum samples, it may be necessary to use an ELISA diluent that blocks heterophilic antibodies to prevent false-positive signals. We have tested Mabtech ELISA Diluent (Art. No. 3652-D2), but further evaluation is needed before we can make a specific recommendation.

Instruction for use

IFU (Article # 10-3031-01)

FAQs About this Product

1. The Exazym® Antibody Pair Human TNF-α Kit contains an TNF-α capture antibody and an TNF-α detector antibody conjugated to an oligo-dT primer. What standard do you recommend for calibration?

   The kit does not include an TNF-α standard, and unfortunately, we cannot recommend a specific product to purchase. However, we advise using an TNF-α standard that has been calibrated against the international standard provided by the National Institute for Biological Standards and Control (NIBSC). This ensures consistency and reliability in your quantification.

2. What concentrations do you recommend for coating the TNF-α capture antibody and for the TNF-α detector antibody in a BOLD-amplified ELISA?

   When using a MaxiSorp™ 96-well plate, we recommend coating with the TNF-α capture antibody at a concentration of 1-2 µg/mL in phosphate-buffered saline (PBS), pH 7.4. After the coating step, the wells should be washed with PBS + 0.05% Tween-20, followed by a blocking step. We recommend using PBS + 1% casein as the blocking buffer (e.g., Thermo Scientific™ Blocker® Casein, Art. No. 37528). The coating is performed overnight at 2–8 °C, followed by a wash step and a 1-hour blocking incubation at room temperature and a final wash step at room temperature before adding sample or calibrators.

   For the TNF-α detector antibody, we recommend a working concentration of 0.15 µg/mL and a 1-hour incubation at room temperature. We recommend the detector antibody to be diluted in PBS + 0.05% Tween-20 (PBS-T) + 0.1% casein, which corresponds to a 10-fold dilution of Blocker® Casein in PBS-T. This buffer can also be used to dilute calibrators and samples.

   The concentrations and buffer compositions provided are suggested starting points. Depending on your specific assay conditions, alternative blocking agents, such as bovine serum albumin (BSA) or non-fat dried milk (NFDM), may offer improved performance and are worth evaluating.

   Note: When working with plasma or serum samples, it may be necessary to use an ELISA diluent that blocks heterophilic antibodies to prevent false-positive signals. We have tested Mabtech ELISA Diluent (Art. No. 3652-D2), but further evaluation is needed before we can make a specific recommendation.

Instruction for use

IFU (Article # 10-1001-02) IFU (Article # 10-1001-01)

Material Safety Data Sheet

Polymerase Buffer (EU) (Article # 20-1003-01) Polymerase Buffer (USA) (Article # 20-1003-01) Reaction-Solution (EU) (Article # 20-1002-xx) Reaction-Solution (USA) (Article # 20-1002-xx)

FAQs About this Product

1. We get a high signal amplification but also a large increase in background. What can we do to reduce the background?

   Try decreasing the concentration of the oligo-dT detector conjugate. Typically, you will need to reduce the concentration of the oligo-dT detector conjugate with some factors compared to the concentration used in the standard immunoassay. Additionally, you can improve the quality of the oligo-dT detector conjugate by removing unconjugated oligo-dT primers. This can be done using an ultra-centrifugal filter unit (e.g., Amicon®) with a suitable cutoff (100 kDa) and volume (0.5 mL). Furthermore, you can evaluate an oligo-dT primer detector conjugate by applying different ratios of the ClickChem label and the oligo-dT primer compared to the detector antibody.

2. Can the incubation steps be done at 37 degrees instead of room temperature?

   For the polymerization step it is not recommended to use 37 degrees since it will lead to a less efficient polymerization with the provided RT polymerase. A custom-made solution with another polymerase reaction kit optimized for 37 degrees is possible. Please contact us if you are interested in such a solution.

3. Is it possible to increase the amplification by increasing the polymerization time to longer than 1 hour?

   This depends on the amplified assay and can be tested. However, if low signal amplification is achieved, it could also be due to other factors within the assay, such as an unsuccessful conjugation of the Exazym® oligo-dT Primer to the detector antibody.

4. Is it possible to increase the amplification by increasing the concentration of polymerase?

   The Instructions for Use (IFU) of the Exazym® Polymerase Reaction Kit recommend specific concentrations and incubation times for the polymerase reaction to achieve signal amplification. However, for optimal performance of a BOLD amplified immunoassay, the concentration of polymerase and the polymerization time may need to be adjusted.

5. Can I re-use left-over buffers and reagents in another analytical run?

   We recommend not to re-use mixtures prepared from more than one component/reagent, such as Polymerase working solution, and Reaction Solution and Exazym® Template mix.

6. Can lids be used instead of adhesive plate covers?

   We recommend using adhesive plate covers as they prevent evaporation and contamination. However, you are welcome to test both options to see which works best for your needs.

7. Can smaller or larger volumes of the BOLD reagents than 100 µL/well be used?

   Yes, other volumes can be used as long as the concentrations of the reagents or reagent mixtures in the volume you will use are as outlined in the Instructions for Use (IFU). For example, if a smaller volume of a reagent mixture is required, the volume of each component should be scaled proportionally to maintain the same concentration, as described in the IFU.

8. We use PBS-Tween as a wash buffer in our standard immunoassay. Can we use PBS-T as a wash buffer for washing after the BOLD steps?

   Yes, you can use PBS-T, containing 0.05 % Tween™ 20, without any negative effect on BOLD or other steps after BOLD.

9. Is it possible to prepare the Reaction solution with Template in advance or does it have to be made shortly before use?

   We recommend that the mixture of Reaction solution and Template is prepared shortly before use, since we have observed for some assays that the signal decreases if the solution is prepared in advance.

10. Is it possible to prepare the Exazym® RT Polymerase working solution in advance or does it have to be made shortly before use?

    We recommend preparing the working solution shortly before use. However, we have tested storing it overnight in the refrigerator (2-8°C) and found it has very little effect on the assay signal.

11. Are there anything in the BOLD components that can disturb the antibody-antigen interactions?

    It is not expected that the BOLD components will disturb antibody-antigen interactions, as antigen binding has already occurred by the time signal amplification with BOLD begins. However, the conjugation of the Exazym® Oligo-dT Primer to the detector antibody of the immunoassay may affect its binding to the antigen. This potential impact should be assessed.

Instruction for use

IFU (Article # 10-2001-02) IFU (Article # 10-2001-01)

Material Safety Data Sheet

Antibody Buffer (EU) (Article # 20-2005-00) Antibody Buffer (USA) (Article # 20-2005-00) PBS-T (EU) (Article # 20-2005-00) PBS-T (USA) (Article # 20-2005-00)

FAQs About this Product

1. We get a high signal amplification but also a large increase in background. What can we do to reduce the background?

   Try decreasing the concentration of the oligo-dT detector conjugate. Typically, you will need to reduce the concentration of the oligo-dT detector conjugate with some factors compared to the concentration used in the standard immunoassay. Additionally, you can improve the quality of the oligo-dT detector conjugate by removing unconjugated oligo-dT primers. This can be done using an ultra-centrifugal filter unit (e.g., Amicon®) with a suitable cutoff (100 kDa) and volume (0.5 mL). Furthermore, you can evaluate an oligo-dT primer detector conjugate by applying different ratios of the ClickChem label and the oligo-dT primer compared to the detector antibody.

2. It looks like something has been sedimented in the Antibody Buffer. Is it ok to use and should I mix it before use?

   Some components of this solution may sediment. Try a few short/quick vortex steps to get a homogenous solution before use.

3. Can I re-use left-over buffers and reagents in another analytical run?

   We recommend not to re-use mixtures prepared from more than one component/reagent, such as Polymerase working solution, and Reaction Solution and Exazym® Template mix.

4. Can lids be used instead of adhesive plate covers?

   We recommend using adhesive plate covers as they prevent evaporation and contamination. However, you are welcome to test both options to see which works best for your needs.

5. Can smaller or larger volumes of the BOLD reagents than 100 µL/well be used?

   Yes, other volumes can be used as long as the concentrations of the reagents or reagent mixtures in the volume you will use are as outlined in the Instructions for Use (IFU). For example, if a smaller volume of a reagent mixture is required, the volume of each component should be scaled proportionally to maintain the same concentration, as described in the IFU.

6. We use PBS-Tween as a wash buffer in our standard immunoassay. Can we use PBS-T as a wash buffer for washing after the BOLD steps?

   Yes, you can use PBS-T, containing 0.05 % Tween™ 20, without any negative effect on BOLD or other steps after BOLD.

7. According to the IFU Exazym® Antibody Biotin should be incubated 1 hour in the Antibody buffer before use. Does it have to be exactly 1 hour?

   No, it does not have to be exactly 1 hour. However, in our tests, we have observed that pre-incubating the Antibody Biotin in the Antibody Buffer for 30 to 75 minutes reduces the background signal compared to using it immediately after mixing. There is no further improvement beyond 75 minutes. For consistency and reproducibility, we recommend using the same pre-incubation time for all assays.

8. Are there anything in the BOLD components that can disturb the antibody-antigen interactions?

   It is not expected that the BOLD components will disturb antibody-antigen interactions, as antigen binding has already occurred by the time signal amplification with BOLD begins. However, the conjugation of the Exazym® Oligo-dT Primer to the detector antibody of the immunoassay may affect its binding to the antigen. This potential impact should be assessed.

Instruction for use

IFU (Article # 10-2002-01)

Material Safety Data Sheet

Antibody Buffer (EU) (Article # 20-2005-00) Antibody Buffer (USA) (Article # 20-2005-00) PBS-T (EU) (Article # 20-2005-00) PBS-T (USA) (Article # 20-2005-00)

Instruction for use

IFU (Article # 10-2002-03)

Material Safety Data Sheet

Antibody Buffer (EU) (Article # 20-2005-00) Antibody Buffer (USA) (Article # 20-2005-00) PBS-T (EU) (Article # 20-2005-00) PBS-T (USA) (Article # 20-2005-00)

Instruction for use

IFU (Article # 15-2005-01)

Material Safety Data Sheet

Antibody Buffer (EU) (Article # 20-2005-00) Antibody Buffer (USA) (Article # 20-2005-00) PBS-T (EU) (Article # 20-2005-00) PBS-T (USA) (Article # 20-2005-00)