• Efficient immunogen preparation with highly soluble, environmentally friendly mcKLH
• Easy coupling chemistry - complete kits include easy-to-use pre-activated carriers and chemistries
• Variety - six different carrier proteins including the new BLUE CARRIER Immunogenic Protein
• Measure specific antibody responses - kits provided with or without non-relevant carriers (BSA or OVA) •Increased immunogenicity - unique SuperCarrier Protein improves immunogenicity of large proteins
• No time wasted - kits combined with both antibody production and antibody purification tools

Pierce Imject Immunogen Preparation Kits and Carrier Proteins make it easy to prepare antigens for immunizing animals. Small molecules called haptens (<5,000 M.W.) cannot elicit an antibody response without being attached to a carrier protein. An immunogen is created by conjugating a hapten to a carrier protein. Pierce technology makes it possible to couple a hapten, such as a synthetic peptide, to the carrier via primary amines
(-NH₂) or sulfhydryl (-SH) or carboxyl (-COOH) groups.

If your peptide is synthesized with a terminal cysteine residue (i.e., a free sulfhydryl group, -SH), you can use any maleimide-activated carrier protein to create an immunogen. One-step chemical reactions are easy with Pierce Sulfo-SMCC Pre-Activated Carrier Proteins (Figure 1). If your hapten does not have a sulfhydryl group, you can use EDC chemistry (Figure 2) to couple amines (lysine) or carboxylic acids (glutamic, aspartic acid or 5'-phosphate) to the carrier protein.

Keyhole limpet hemocyanin (KLH), which is isolated from the giant sea mollusc Megathura crenulata,is one of the most popular carrier proteins used to create an immunogen for injection. KLH induces a strong antibody response because of its large mass (>10⁶ M.W.) and because it is a non-mammalian protein. Pierce recently introduced mariculture KLH (mcKLH), a product that is less damaging to marine aquatic environments because the molluscs are harvested and maintained in salt water tanks. The characteristic blue, opalescent color of mcKLH is highly soluble, making coupling reactions easier to complete.

Figure 1. Peptide Carrier Conjugation using Maleimide Activated Carriers

Figure 2. EDC-Mediated Peptide-Carrier Conjugation

 

Product #	Description	Pkg. Size	Features/Benefits
77653T	Imject mcKLH MES Buffer	2 mg	•Convenient - ready for EDC chemical coupling reactions to carboxyl (-COOH)- or amine (-NH2)-containing haptens •Provided in an acidic buffer (pH 4.5) •M.W. is 8 x 106
77600T	Imject mcKLH in PBS Buffer	5 x 20 mg	• Highly soluble - provided in neutral pH coupling buffer
77609T	VACMUNE cGMP KLH Subunits Packaged as a sterile, lyophilized powder with 10 ml of sterile water for reconstitution	10 mg	• Sterile - manufactured under cGMP guidelines for vaccine research • M.W. of the subunits range from 300,000-350,000 daltons
77130T	BLUE CARRIER Immunogenic Protein Supplied as a sterile liquid at 200 mg/ml	100 mg	• Economical - a hemocyanin isolated from the mollusc Concholepas concholepas • Large M.W. (6 x 106) protein • Appears as a dark ink-like liquid
77606T	Imject Maleimide Activated mcKLH	2 mg	• Efficient conjugation - carrier protein is pre-activated with Sulfo-SMCC • Specific - reacts with sulfhydryl (-SH)- containing haptens •Convenient - kit contains everything needed to produce and purify five immunogens for injection
77605T	Imject Maleimide Activated mcKLH	10 mg
77611T	Imject Maleimide Activated mcKLH Kit Sufficient material for five conjugation reactions Includes: Imject Maleimide Activated mcKLH, 5 x 2 mg Imject Maleimide Conjugation Buffer, 30 ml Dextran Gel Filtration Columns, 5 x 5 ml Imject Dry Blend Purification Buffer Salts, 5 x 5 gm	Kit
77614T	Imject Sulfhydryl Reactive Antibody Production and Purification Kit with mcKLH Sufficient material for five conjugation reactions Includes: Imject Maleimide Activated mcKLH, 5 x 2 mg Imject Maleimide Conjugation Buffer, 30 ml Dextran Gel Filtration Column, 1 x 5 ml Imject Dry Blend Purification Buffer Salts, 5 x 5 gm SulfoLink Column, 2 ml column SulfoLink Sample Preparation Buffer, 7.5 ml SulfoLink Wash Solution, 25 ml SulfoLink Coupling Buffer, 100 ml SulfoLink Reductant, 6 mg L-Cysteine • HCl, 100 mg Polyacrylamide Gel Filtration Column,.1 x 5 ml	Kit	• Efficient conjugation ­ carrier protein is pre-activated with Sulfo-SMCC • Specific - reacts with sulfhydryl (-SH)-containing haptens • Convenient - SulfoLink Gel included to create immobilized hapten column for anti-peptide antibody purification • Precise - purified antibodies are not contaminated with anti-carrier protein antibodies

Antibodies made against an immunogen (mcKLH-peptide conjugate) will produce antibodies that bind to the peptide (hapten or epitope) and to the carrier protein. To measure the specific anti-peptide response, couple the peptide to a non-relevant carrier protein using the same coupling chemistry (maleimide or EDC) of the injected immunogen. Absorb this conjugate onto a 96-microwell plate (pH 9.0, overnight incubation) to perform an ELISA assay. Bovine serum albumin (BSA; 67,000 M.W.) and ovalbumin (45,000 M.W.) are often used as non-relevant carrier proteins to assess anti-peptide antibody titers. Nonspecific binding sites on the microwell plate are reduced by using a blocking buffer (see Assay Development Section) after the conjugate has been absorbed to the plate.

Immunized serum (e.g., where the carrier was mcKLH) is serially diluted (1:64, 1:128, etc.) in the coated microwell plate to bind anti-peptide antibodies. Any anti-mcKLH antibodies are washed away before the next assay step. A secondary antibody-enzyme conjugate is used to bind the anti-peptide antibody. After washing, the bound antibodies are detected by adding substrate. The dilution factor at which no anti-peptide antibody binding can be observed is called the antibody titer. The antibody titer describes the strength of the antibody response. One immunization protocol, for example, may produce an antibody titer of 1:10,000 dilution vs. a second protocol that may produce an antibody titer of only 1:5,000. The higher the dilution factors, the stronger the polyclonal immune response.

Product #	Description	Pkg. Size	Features/Benefits
77171T	Imject Bovine Serum Albumin in MES Buffer	2 mg	• Convenient - non-relevant carrier protein provided in an acidic buffer (pH 4.5); ready for EDC chemical coupling reactions to carboxyl- or amine- containing haptens
77110T	Imject Bovine Serum Albumin in PBS Buffer	5 x 20 mg	• Convenient - non-relevant carrier protein provided in a neutral pH (7.2) buffer
77115T	Imject Maleimide Activated Bovine Serum Albumin in PBS Buffer	10 mg	• Efficient coupling - pre-activated with Sulfo-SMCC to couple with sulfhydryl (-SH)-containing peptides • Non-relevant carrier protein
77109T	Imject Ovalbumin in MES Buffer	2 mg	• Convenient - non-relevant carrier protein isolated from chicken egg whites lyophilized in an acidic buffer (pH 4.5); ready for EDC chemical coupling
77120T	Imject Ovalbumin in PBS Buffer	5 x 20 mg	• Convenient - non-relevant carrier protein lyophilized in a neutral pH (pH 7.2) buffer
77125T	Imject Maleimide Activated Ovalbumin in PBS Buffer	10 mg	• Efficient coupling - pre-activated with Sulfo-SMCC to couple with sulfhydryl (-SH)-containing peptides • Non-relevant carrier protein
77607T	Imject Maleimide Activated Immunogen Conjugation Kit with mcKLH and BSA Sufficient material for 10 conjugation reactions Includes: Imject Maleimide Activated Bovine Serum Albumin, 5 x 2 mg Imject Maleimide Activated mcKLH, 5 x 2 mg Imject Maleimide Conjugation Buffer, 30 ml Dextran Gel Filtration Columns, 10 x 5 ml Imject Dry Blend Purification Buffer Salts, 5 x 5 gm	Kit	•Efficient coupling - carrier (mcKLH) and non-relevant carrier (BSA) proteins are pre-activated with Sulfo-SMCC • Specific - maleimide-activated carrier proteins react with sulfhydryl (-SH)-containing hapten • Easy - non-relevant carrier protein (BSA) is included with complete instructions for measuring specific anti-hapten antibodies in an ELISA
77601T	Imject Immunogen EDC Conjugation Kit with mcKLH and BSA Sufficient material for 10 conjugation reactions Includes: Imject mcKLH in MES Buffer, 5 x 2 mg Imject Bovine Serum Albumin, 5 x 2 mg Imject EDC Conjugation Buffer, 30 ml Dextran Gel Filtration Columns, 10 x 5 ml Imject Dry Blend Purification Buffer Salts, 5 x 5 gm EDC Conjugation Reagent, 10 x 10 mg	Kit	• Easy chemistry - EDC cross-linker supplied for coupling an amine- or carboxyl-containing hapten to carrier proteins • Convenient - non-relevant carrier protein (BSA) is included for measuring specific anti-hapten antibodies in an ELISA
77608T	Imject Maleimide Activated Immunogen Conjugation Kit with mcKLH and OVA Sufficient material for 10 conjugation reactions Includes: Imject Maleimide Activated Ovalbumin, 5 x 2 mg Imject Maleimide Activated mcKLH, 5 x 2 mg Imject Maleimide Conjugation Buffer, 30 ml Dextran Gel Filtration Columns, 10 x 5 ml Imject Dry Blend Purification Buffer Salts, 5 x 5 gm	Kit	• Efficient coupling - carrier (mcKLH) and non-relevant carrier (OVA) proteins are pre-activated with Sulfo-SMCC • Specific - maleimide-activated carrier proteins react with sulfhydryl (-SH)-containing hapten(s) • Easy-to-use - non-relevant carrier protein (OVA) is included for measuring specific anti-hapten antibodies in an ELISA
77602T	Imject Immunogen EDC Conjugation Kit with mcKLH and OVA Sufficient material for 10 conjugation reactions Includes: Imject mcKLH in MES Buffer, 5 x 2 mg Imject Ovalbumin in MES Buffer, 5 x 2 mg Imject EDC Conjugation Buffer, 30 ml Dextran Gel Filtration Columns, 10 x 5 ml Imject Dry Blend Purification Buffer Salts, 5 x 5 gm EDC Conjugation Reagent, 10 x 10 mg	Kit	• Easy chemistry - EDC cross-linker supplied for coupling an amine- or carboxyl- containing hapten to carrier proteins •Convenient - non-relevant carrier protein (OVA) is included for measuring specific anti-hapten antibodies in an ELISA

Figure 3: Immunogen Preparation and Immunization
Couple sulfhydryl-containing peptide (hapten) to Maleimide Activated mcKLH (Product # 77605).

Separate unreacted peptide from immunogen by gel filtration.

Mix immunogen with adjuvant.

Inject the animal on day one with immunogen in Freund's Complete Adjuvant (FCA) and again on day 14 using Freund's Incomplete Adjuvant (FIA).

Test bleed the animal on day 21 to measure antibody response by ELISA.

Typically, antibodies can be generated against any protein with a M.W. greater than 5,000. However, coupling a large protein to the SuperCarrier Immune Modulator can result in twice the antibody response (Figure 4). The SuperCarrier Protein is a cationized BSA (cBSA) molecule that stimulates a stronger immune reaction to any compound coupled to it. Both peptides and proteins elicit increased antibody titers when conjugated to the SuperCarrier Protein. The SuperCarrier Protein is supplied pre-activated with Sulfo-SMCC to react with sulfhydryl-containing proteins or is supplied with the EDC cross-linker to couple to carboxyl- or amine-containing proteins and peptides.

Figure 4. Enhanced Antibody Response using SuperCarrier Modulator

Table 4.

Product #	Description	Pkg. Size	Features/Benefits
77175T	Imject Activated SuperCarrier Immune Modulator in PBS	2 mg	•More antibodies - coupling proteins to the SuperCarrier Protein induces a stronger immune response than immunizing with the protein alone • Convenient chemistry - pre-activated to couple to proteins containing sulfhydryl groups (-SH) •Lyophilized into phosphate, EDTA, NaCl buffer, pH 7.2
77155T	Imject Activated SuperCarrier Immune Modulator in PBS	10 mg
77151T	Imject SuperCarrier EDC System for Proteins Sufficient material for five conjugation reactions Includes: SuperCarrier Immune Modulator, 5 x 2 mg Dry Blend Purification Buffer Salts, 5 x 5 gm EDC Cross-linker, 5 x 10 mg Conjugation Buffer, 30 ml Dextran Gel Filtration Columns, 5 x 5 ml Imject Alum, 50 ml	Kit	•More antibodies - stronger antibody response seen when protein is coupled to the SuperCarrier Modulator compared to immunizing with the protein alone • Easy chemistry - EDC cross-linker supplied to react with carboxyl- or amine-containing proteins • Convenient - everything needed to create and purify a SuperCarrier Immunogen
77656T	Imject Maleimide Activated SuperCarrier System with SuperCarrier Immune Modulator and mcKLH Sufficient material for 10 conjugation reactions Includes: Imject Maleimide Activated SuperCarrier Immune Modulator, 5 x 2 mg Imject Maleimide Activated mcKLH, 5 x 2 mg Maleimide Conjugation Buffer, 30 ml Dextran Gel Filtration Columns, 10 x 5 ml Dry Blend Purification Buffer Salts, 5 x 5 gm Imject Alum, 50 ml	Kit	•Convenient - everything needed to create and purify an immunogen and an antigen for ELISA analysis • Specific chemistry - carrier (SuperCarrier Modulator) and non-relevant carrier (mcKLH) proteins are pre-activated with Sulfo-SMCC to react with sulfhydryl (-SH)-containing proteins and peptides • More antibodies - stronger antibody response seen when protein is coupled to the SuperCarrier Modulator
77652T	Imject SuperCarrier EDC System for Peptides with mcKLH Sufficient material for 10 conjugation reactions Includes: SuperCarrier Immune Modulator, 5 x 2 mg Imject mcKLH, 5 x 2 mg Dry Blend Purification Buffer Salts, 5 x 5 gm EDC Cross-linker, 10 x 10 mg Conjugation Buffer, 30 ml Dextran Gel Filtration Columns, 10 x 5 ml Imject Alum, 50 ml	Kit	•More antibodies - stronger antibody response seen when peptide coupled to the SuperCarrier Modulator than when coupled to mcKLH • Convenient - kit contains everything needed to create and purify an immunogen and an antigen for ELISA analysis • Easy chemistry - EDC cross-linker supplied to react with carboxyl- or amine-containing peptides

Adjuvants enhance the immune response against the injected antigen. When mixed with the immunogen, they help deposit or sequester the injected material. The initial injection exposes the cells of the immune system to the immunogen. The second injection, called the "booster," is needed to initiate B memory cells to secrete antibodies. Adjuvants, however, cannot render haptens immunogenic. Haptens must be coupled to a larger protein to be recognized and processed by the immune system. The following chart provides information for the most popular adjuvants used for immunization.

Product #	Description	Pkg. Size	Features/Benefits
77140T	Imject Freund's Complete Adjuvant (FCA)* Warning: can cause tissue necrosis at injetion site	5 x 10 ml	• Strong immune stimulator - water-in-oil emulsion containing killed Mycobacterium • Most popular adjuvant that is used for initial injection
77145T	Imject Freund's Incomplete Adjuvant (FIA)	5 x 10 ml	•Compatible - used for subsequent "booster" injections after immunizing with FCA •Nontoxic - oil with no Mycobacterium present
77138T	AdjuPrime Immune Modulator	20 mg	•Nontoxic - carbohydrate-based adjuvant that does not cause tissue necrosis at injection site • Easy-to-use - water-soluble so it mixes easily with immunogen compared to FCA
77161T	Imject Alum	50 ml	•Nontoxic - a suspension of aluminum hydroxide and magnesium hydroxide • Easy-to-use - water-soluble so it mixes easily with immunogen compared to FCA

Small compounds such as drug molecules may not contain a classic functional group (e.g., -NH₂, -SH or -COOH) for chemical coupling. The PharmaLink Chemistry can be used to couple "active hydrogens" to a carrier protein via the Mannich reaction (Figure 5), a condensation reaction with formaldehyde. Antibodies to serotonin and estradiol have been produced successfully with this specialized chemistry. The small ligand must be able to withstand heating (56°C) for 24-48 hours. Please visit the Pierce web site at www.piercenet.com to view examples of active hydrogen species.

Figure 5. Mannich Reaction with "Active" Hydrogen

Product #	Description	Pkg. Size	Features/Benefits
77158T	Imject PharmaLink Immunogen Kit Sufficient reagents for 10 conjugations Includes: SuperCarrier Immune Modulator (cationized BSA), 10 x 2 ml PharmaLink Conjugation Buffer, 30 ml PharmaLink Coupling Reagent, 4 ml PharmaLink Purification Buffer Salts, 5 x 2.5 gm Imject Alum, 50 ml Desalting Columns, 10 x 5 ml	Kit	•Unique chemistry - reacts with active hydrogens on drug compounds to couple to a carrier protein • More antibodies - supplied with a SuperCarrier Immune Modulator as a carrier protein for a strong immune response • Forms a stable secondary amine linkage between the ligand and carrier protein

See Antibody Purification section.