Specific cyclooxygenase antibodies: examples of applications

In the fifties Yalow & Berson discovered the principle of radioimmunology by using insulin radioactively labelled with iodine 131, a discovery which gained the 1977 Nobel prize for medecine. The immunoassay became a recognised method and antibodies used outside the living organism would be revealed as a tool of considerable importance.

Cyclooxygenase (Cox, also known as prostaglandin endoperoxide H synthase, EC 1.14.99.1) is a key enzyme in the biosynthetic process leading to the formation of prostaglandins and thromboxanes, deriving from arachidonic acid. This glycoprotein (approx 70 kDa) has two types of catalytic property. First, it allows the formation of PGG2 from arachidonic acid via cyclooxygenase activity. Then PGH2 is formed from PGG2 via its peroxydase activity. Thus Cox controls the synthesis of prostaglandins, which are an important group of molecules acting as mediators in many cell functions. Most clinical symptoms of inflammation are accompanied by an increase in the prostaglandin rate. Hence Cox is targeted by many non-steroidal anti-inflammatory drugs such as ibuprofen or indomethacin; the best known of these is still aspirin, which inactivates Cox via irreversible acetylation of the Ser530 residue.

Over the years only one form of Cox (known as constitutive Cox or Cox-1) had been identified. A second form (inducible Cox or Cox-2) has recently been detected in cell cultures in response to various stimuli such as interleukin-1alpha or phorbol esters. The complementary DNA of Cox-1 and Cox-2 has been sequenced for different animal species and demonstrates i) that the sequence is largely conservative for Cox-1 (>90%), ii) a large degree of interspecies homology for Cox-2 (about 80%), iii) approx. 60% homology between Cox-1 and Cox-2 within a given species. The two genes are carried by separate chromosomes and code for fibrous proteins of like molecular mass (about 70 kDa).

The sequences of homogeneity preserved between the two isoforms relate in particular to the residues associated with enzymatic activity (the site of acetylation by aspirin, the different histidines linking the heme and the sites of glycosylation). The main difference is the deletion of a sequence of 17 amino acids in the terminal amino section of Cox-2, as compared with Cox-1. This is compensated by the insertion of a sequence of 18 amino acids near the terminal carboxyl section of Cox-2.

In order to study the production and differential regulation of these two enzymes, the C.E.A.'s (French Atomic Agency) Pharmacology and Immunology Department raised polyclonal and monoclonal antibodies against these two isoforms.

For the constitutive form (Cox-1), monoclonal anti-bodies were raised against Cox-1 purified with ram seminal vesicles. In the case of the inducible form (Cox-2), to avoid all risk of cross-reaction with Cox-1, monoclonal antibodies were raised against a synthetic peptide reproducing part (580-598) of human Cox-2, the sequence which is absent from Cox-1.

These different monoclonal antibodies were used to study cyclooxygenase in human umbilical vein endothelial cell cultures in which Cox-2 synthesis had been demonstrated in response to different stimuli. The strict specificity of these monoclonal antibodies with regard to the isoform against which they were raised was verified. Their joint use makes it possible to compare the rates of expression and regulation of Cox-1 and Cox-2 activity in stimulated endothelial cells. This also permits to verify that modification of cyclooxygenase enzyme activity (measurement of 6-keto-PGF1alpha metabolite derived from arachidonic acid) is directly correlated with a proportional increase in Cox-2, while Cox-1 remains constant (figure 1). Finally, as shown in figures 2a and 2b, these antibodies can be used to distinguish the presence of the isoforms in various animal species. These antibodies should, therefore, be very useful tools for studying the regulation of the two Cox isoforms in many biological or pathological situations.

If you are interested in the studies described above, we should be happy to discuss protocols which could be put in place for your molecules

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