Design and Synthesis of Haptens for Chloramphenicol

Low molecular weight haptens (<1000 Da) are not immunogenic and they can only be recognized by the immune system by binding to larger carrier molecules. With more than 10 years of experience in small molecule antibody development, Creative Biolabs has specific expertise in designing small molecule/protein carrier adducts, which allows us to provide a variety of small-molecule hapten design and synthesis services to customers worldwide.

Introduction of Chloramphenicol

Chloramphenicol (CAP) is an antibiotic with a wide spectrum of activity against Gram-positive and Gram-negative cocci and bacilli (including anaerobes), Rickettsia, Mycoplasma, typhoid fever, and Chlamydia. It mainly plays a bacteriostatic effect because it binds to the 50S subunit of the ribosome and inhibits the synthesis of bacterial proteins. CAP is one of the most highly absorbed antibiotics and penetrates through cell membranes and purulent material.

The structure of chloramphenicol is shown in Fig.1. It has a paranitrobenzene ring attached to a propanediol group with a dichloracetamide side chain. Among four optical isomers in the chiral carbon atoms, the only isomer of D-(-)-threo possesses an antibacterial effect. The chemical structure of CAP is stable for 5 h in boiling water and maintains biological activity for 25 h in pH 2-9 at room temperature, and the crystals can even be stored over the years.

Structure of chloramphenicol. Fig.1 Structure of chloramphenicol. (Xu, 2015)

Haptens of Chloramphenicol

Chloramphenicol succinate (CAP-HS), a carboxyl derivative of CAP, can be directly used for synthesis of the immunogen. The immunogen was constructed by coupling CAP-HS containing the carboxyl group to carrier proteins (KLH and BSA) by the mixed anhydride (MA) method (Fig.2). The coupling ratio between the drug and the carrier protein is 2500: 1 and 100: 1. These immunogens (CAP-HS-BSA and CAP-HS-KLH) were injected subcutaneously into multiple sites and showed better sensitivity and lower IC50 values.

In recent years, a CAP mAb called 6F11 has been developed which shows the best sensitivity and high specificity with negligible cross-reactivities against CAP analogs (FF, FFA, and TAP) in milk. Moreover, the benzene ring of the CAP structure has a nitro group, and this nitro group can be reduced to an active aromatic amine to generate specific antibodies. Besides, immunogen (CAP-BSA) and coated antigen (CAP-OVA) can be obtained by the diazotization method.

Synthesis of chloramphenicol antigen by MA method. Fig.2 Synthesis of chloramphenicol antigen by MA method. (Xu, 2019)

Strategies of Hapten Design for Chloramphenicol

Haptens are distributed everywhere in our environment and are widely used as pharmaceuticals, hormones, and antibiotics. These small molecular weight haptens cannot be efficiently recognized by the mammalian immune system unless conjugated with larger immunogenic carrier molecules. Because CAP has a small molecular weight, it needs to be coupled to a carrier protein to be immunogenic. Creative Biolabs provides comprehensive services of hapten design and synthesis for CAP. We have extensive experience and expertise in:

  • Select the most suitable position on the haptens to connect the linker, which will maximize the specificity, sensitivity, and immunogenicity of the immunogen;
  • Perform full synthesis to make the hapten have a linker when the hapten molecule does not have a suitable functional group for conjugation synthesis;
  • Design and synthesis of the linker with a most suitable length;
  • Precisely control the most appropriate ratio of drug to a carrier protein to enhance the performance of immunoassay.

We have our own professional techniques for antigen/immunogen design and synthesis. Creative Biolabs is capable of meeting quality requirements for custom services of the design and synthesis of haptens for chloramphenicol and producing high-performance customized antigen/immunogens. If you want to know more, please feel free to contact us.


  1. Xu, N.; et al. Development and characterisation of an ultrasensitive monoclonal antibody for chloramphenicol. Food and agricultural immunology. 2015, 26(3): 440-450.
  2. Xu C.; et al. Food Immunoassay. Springer Singapore. 2019.
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