What is a conjugate diluent?
     It is a buffer used to stabilize newly conjugated proteins, to reconstitute lyophilized conjugates, and to dilute concentrated conjugates.  ICT's conjugate diluents stabilize the immunoglobulin and HRP components of the conjugate - protein complex.  All conjugate diluents concentrated at 5x.  When stored unopened and protected from light, a shelf-life of 18 months can be expected at 2°-8°C.
     ICT has formulated a group of unique stabilizing buffers designed to prolong the shelf-life of HRP-conjugated proteins and enhance their utility in ELISA applications. These conjugate stabilizer diluents can be used to reconstitute lyophilized conjugates, and to dilute concentrated conjugates into the useful range of the assay.
     HRP-conjugated antibodies are often unstable and can lose a significant level of activity within a short time after rehydration and dilution unless properly stored. This loss of activity is based on two factors: denaturization of the HRP-IgG protein complex; and destabilization of the catalytic region of the HRP (which may or may not remain associated with the IgG). ICT’s proprietary formulations preserve the integrity of the protein conjugate component (for example, the IgG-HRP complex) while also maintaining the enzymatic functionality of the HRP enzyme.
     ICT’s conjugate diluents contain several unique buffers that provide the optimal pH and ionic conditions during storage to maintain the native tertiary structure of the conjugate proteins. These buffers contain a proprietary protein additive to stabilize the reconstituted conjugated proteins, and magnesium and calcium salts to stabilize the catalytic site of the HRP porphyrin ring structure.
     These reagents were also designed to minimize nonspecific binding interactions between the conjugate and the plate well surface, and to confer additional stability to the overall conjugate structure. Selected buffers contain a proprietary detergent to further reduce nonspecific binding of the conjugate to the plate surface while maintaining HRP activity.
     When stored unopened and protected from light, a shelf-life of 18 months can be expected at 2°-8°C.  When used to stabilize a newly conjugated protein, the solution may remain active for several years at 2°-8°C (the shelflife must be determined for each conjugate).  When used to make a 1x diluted conjugate, the diluent may remain active up to 1 year at 2°-8°C. An anti-microbial agent is also included that will inhibit bacterial growth during storage, but will not inactivate the conjugate proteins.

How do I use a conjugate diluent?
     To stabilize newly conjugate proteins, simply add 1 part of the appropriate 5x conjugate diluent (that has not been diluted to 1x) to 4 parts of the new conjugate solution.  For example, if you just eluted 20 mL of an antibody-HRP conjugate off a column, simply add 5 mL of 5x CD6 Conjugate Stock Stabilizing Reagent to the conjugate solution.  If the conjugate was initially at 10 mg/mL, the stabilized conjugate will now be at 8 mg/mL in 25mL diluent (it's been diluted by 20%).
     To reconstitute lyophilized conjugates, simply pipette a known amount of a 1x conjugate diluent into the vial.  The liquid conjugate is now ready to be used directly, or further diluted to the required concentration.  For example, if you purchased a vial of goat anti-human IgG at 1mg, simply add 1mL of a 1x conjugate diluent to the vial.  The liquid conjugate is now at 1mg/mL goat anti-human IgG.
     To dilute concentrated conjugates for use in an assay, simply pipette a known amount of the conjugate stock into a 1x conjugate diluent and it’s ready to use.  For example, if you needed 100mL of conjugate at 10ug/mL, simply add 1 mL of the (above) concentrated goat anti-human HRP into 20mL of a 5x conjugate diluent and 79mL of diH₂O and it's ready to use.

Monoclonal / Polyclonal Conjugate Diluent & Stabilizer (CD1)
     CD1 is a unique formulation specifically designed for use in mouse monoclonal capture and polyclonal-HRP sandwich ELISA formats. This diluent contains mouse proteins which suppress nonspecific binding of the polyclonal-HRP conjugate to the mouse immunoglobulins adsorbed onto the plate. These protein additives also provide additional structural stability to the conjugate protein complex.  

Antigen-Down Conjugate Diluent & Stabilizer (CD2)
     CD2 is a unique formulation specifically designed for use with anti-IgG-HRP conjugates. This diluent contains proprietary non-mammalian protein additives which will not interfere with anti-IgG-HRP conjugates used in antigen-down and sandwich ELISA formats (it does not contain BSA). These additives also provide additional structural stability to the IgG-HRP conjugate protein complex. CD2 will also inhibit conjugate binding to nonspecific serum proteins bound to the plate, thus reducing conjugate bridging on the plate surface and lowering background noise. Antimicrobial agents have been added to retard bacterial growth. When stored unopened and protected from light, a shelf-life of 18 months can be expected at 2°-8°C.

Polyclonal / Polyclonal Conjugate Diluent & Stabilizer (CD3)
     CD3 is a unique formulation specifically designed for use in polyclonal capture and polyclonal-HRP sandwich ELISA formats. Proprietary additives were specifically selected to provide optimal assay performance when using rabbit, goat, or rabbit and goat polyclonal antibodies in a sandwich ELISA format. CD3 contains a uniquely processed form of rabbit and goat IgG to suppress nonspecific binding of the polyclonal-HRP conjugates to the plate surface. To reduce nonspecific binding interactions, a detergent is also included. Antimicrobial agents have been added to retard bacterial growth. When stored unopened and protected from light, a shelf-life of 18 months can be expected at 2°-8°C.

Monoclonal / Goat-Polyclonal Conjugate Diluent & Stabilizer (CD4) 
     CD4 is a unique formulation specifically designed for use in monoclonal capture and goat polyclonal-HRP sandwich ELISA formats. Additives were carefully selected to specifically address the stability and nonspecific binding issues associated with this type of ELISA format. Goat proteins are included to reduce any nonspecific interactions between the monoclonal capture and goat polyclonal-HRP antibodies. CD4 also contains mouse IgG to specifically suppress nonspecific bridging of the goat polyclonal-HRP conjugate with the mouse capture IgG on the plate surface. To further reduce nonspecific binding of the conjugate to the plate surface, a detergent is also included. Antimicrobial agents have been added to retard bacterial growth. When stored unopened and protected from light, a shelf-life of 18 months can be expected at 2°-8°C.

Monoclonal / Rabbit-Polyclonal Conjugate Diluent & Stabilizer (CD5)
     CD5 is a unique formulation specifically designed for use in monoclonal capture and rabbit polyclonal-HRP sandwich ELISA formats. Rabbit proteins are included to reduce any nonspecific interactions between the monoclonal capture and rabbit polyclonal-HRP antibodies. CD5 also contains mouse IgG to specifically suppress nonspecific bridging of the rabbit polyclonal-HRP conjugate with the mouse capture IgG on the plate surface. To further reduce nonspecific binding of the conjugate to the plate surface, a detergent is also included. When stored unopened and protected from light, a shelf-life of 18 months can be expected at 2°-8°C.

Conjugate Stock Stabilizing Reagent (CD6)
     CD6 contains a BSA-based mammalian protein additive to stabilize conjugated proteins, and magnesium and calcium salts to stabilize the catalytic site of the HRP porphyrin ring structure.  When diluted to 1x, CD6 is an excellent HRP conjugate diluent and stabilizer base, to which additional  components can be added to for use in your specific assay.
     At 5x, CD6 is an excellent diluent for stabilizing newly conjugated proteins.  This allows the liquid conjugate stock to be stored, and then later diluted to make a working conjugate for use in the assay.  Simply add 1 part of 5x CD6 (that has not been diluted to 1x) to 4 parts of the new conjugate solution.   For example, if you just eluted 20 mL of an antibody-HRP conjugate off a column, simply add 5 mL of 5x CD6 to the conjugate solution.  If the conjugate was initially at 10 mg/mL, the stabilized conjugate will now be at 8 mg/mL in 25mL diluent (it's been diluted by 20%).
     Antimicrobial agents have also been added to CD6 retard bacterial growth.  When stored unopened and protected from light, a shelf-life of 18 months can be expected at 2°-8°C.

Help!  I can't get my assay to work!
     Just give use a call at 1-800-829-3194.  To help you develop your assay, ICT has provided some background information on ELISA technology (see What is an Immunoassay , What's in a test and what is an ELISA protocol? ), some calculation worksheets (see How Do I Coat Plates ), and offers consultation services in assay development (see Immunoassay Development ).

5x Conjugate Diluents 

Code

Product Name

Catalog Number

Size mL

Price

CD1-5x

5x Mono/Poly Conjugate Diluent

6168

25

698

100

699

500

6100

1,000

6101

10,000

CD2-5x

5x Antigen-Down Conjugate Diluent

6169

25

6102

100

6103

500

6104

1,000

6105

10,000

CD3-5x

5x Poly/Poly Conjugate Diluent

6170

25

6106

100

6107

500

6108

1,000

6109

10,000

CD4-5x

5x Mono/Goat-Poly Conjugate Diluent

6171

25

6110

100

6111

500

6112

1,000

6113

10,000

CD5-5x

5x Mono/Rabbit-Poly Conjugate Diluent

6172

25

6114

100

6115

500

6116

1,000

6117

10,000

CD6-5x

5x Conjugate Stock Stabilizing Reagent

6173

25

667

100

668

500

669

1,000

670

10,000