Design and Synthesis of Class-specific Haptens for β-Lactams

Immunological assays are commonly used to screen food products for the presence of anti-microbial drug residues. For the development of antibiotics-specific immunological assays, monoclonal antibodies (mAbs) or polyclonal antibodies (pAbs) against the common structure of the specific class antibiotics are required. In order to obtain antibodies specific for various kinds of β-Lactams antibiotics, Creative Biolabs has made different attempts to develop class-specific haptens for β-Lactams for immunization. With years of experience in antigen design and antibody development, Creative Biolabs provides customized class-specific haptens design, modification as well as synthesis services for β-Lactams antibiotics for our worldwide customers.

Class-specific Hapten Design

The first step for the generation of class-selective antibodies is to design an immunogen that is specific for the group of antibiotics. Studies on the antigenicity of β-Lactams antibiotics have indicated three important epitopes, the acyl-side chain, the common thiazolidine ring, and newly formed structures that arise by coupling antibiotics to the carrier protein. Since β-Lactam antibiotics share a 6-aminopenicillanic acid structure (a β-Lactam ring structure coupled to a thiazolidine ring) and differ in their acyl-side chain, the strategy for class-specific hapten design thus focus on the nucleus structure. For compound-specific hapten design, the key lies on the acyl-side chain instead.

Examples of Class-specific Haptens for β-Lactams

1. Penicillin could be derived as a stable mercuric or copper mercaptide of penicillenic acid (MMPA or CMPA). The derivative of MMPA or CMPA has a unique oxazolone ring instead of the β-Lactamic ring in penicillin. The new antigens synthesized with modified penicillin products have good stability and could be used as an effective bifunctional agent to stimulate animals to yield high titer antibodies in serum. For example, CMPA-BSA can stimulate the immune system to produce antibodies specific to penicillin with high titrations, up to 150000.

Fig.1 Synthesis procedures for CMPA and CMPA-BSA from penicillin. (Xu, 2011)

2. Polyclonal penicillin-specific antibodies were obtained after the immunization of three rabbits with physiological penicillin-protein conjugates. The broad-specificity of the antisera was improved by alternate immunizations using immunogens with different penicillins as hapten. Adopted immunogen design methods are shown in Figure 2.

Fig.2 Chemical structures of ampicillin-protein conjugates using different coupling methods. (Cliquet, 2001)

Bi-hapten Immunogen Design

Successful cases about bi-hapten immunogen design have been reported before. For instance, a novel bi-hapten immunogen was prepared by coupling amoxicillin and chlortetracycline to carrier protein. The immunogen was then used to immunize rabbits to produce the pAbs. The obtained antibody simultaneously cross-reacted with six penicillins (penicillin G, penicillin V, ampicillin, amoxicillin, carbenicillin and sulbenicillin) and four tetracyclines (tetracycline, oxytetracycline, doxycycline and chlortetracycline) with cross-reactivities in a range of 24–100%. Creative Biolabs possesses advanced bi-hapten immunogen design techniques and platforms and offers our clients bi-hapten design, modification, and synthesis services.

General Guidelines for Immunizing Hapten Synthesis

• Distal to important haptenic determinants
• Avoid attachment to functional groups
• Appropriate length of handle
• Compatibility of reaction with target molecule functional groups
• Solubility of hapten
• Stability of hapten under coupling conditions and subsequent use
• Ease of synthesis
• Appropriate coupling ratio determines the strength and specificity of the immune response

Antibodies with a wide recognition profile of β-Lactams antibiotics have been produced based on chemical criteria, theoretical studies, and molecular modeling assisted hapten design. The immunizing hapten preserves the most important and characteristic epitopes of this antibiotic family. For compound-specific haptens design, please see the section entitled Design and Synthesis of Compound-specific Haptens for β-Lactams. If you are interested in our services, please feel free to contact us for more information.

References

1. Xu, W.; et al. Synthesis and characteristics of a novel artificial hapten using the copper mercaptide of penicillenic acid from penicillin G for immunoassay of heavy metal ions. Science China Life Sciences. 2011, 54(9): 813-821.
2. Cliquet, P.; et al. Generation of class-selective monoclonal antibodies against the penicillin group. Journal of agricultural and food chemistry. 2001, 49(7): 3349-3355.