Spacer arms, haptens

Figure 8 Structure of immunogen haptens for pyrethroids with spacer arm attachment at the a-position of the alcohol moiety. Since the whole pyrethroid molecule is available for recognition by the antibody, assays resulting from these immunogens were selective for the parent pyrethroids...

The spacer arm length between the hapten and the carrier is in the range of 6 to 8 A, which should eliminate any steric interference with carrier side chains. According to the protocol described by Schultz the haptens were coupled to BSA and KLH via A-(3-dimethylaminopropyl)-A -ethylcarbodiimide or the N-hydroxysuccinimide ester at pH 5.5 in water. Other coupling strategies include substitution with diazonium salts and reductive amination. The ratio of hapten-carrier range between 8 and 15 haptens per carrier. 

The hemisuccinate of an alcohol derivative of allethrin s propene side chain (A) illustrates the use of a spacer arm between the carrier protein and the molecule of interest. Antibodies to this antigen demonstrated the greatest specificity for the chrysanthemate end of the molecule. The allethrin CMO derivative (B) was prepared at the V ketone. Haptens attached through a gem dimethyl group (C) or the isobutenyl group (D) would be expected to lead to antibodies with a greater specificity for the allethrelone end of the molecule. P indicates protein. 

Pioneering work on immunochemical assays for pesticides involved the synthesis of haptens for DDT and malathion. Haas and Guardia (24) used the acid chlorides of malathion half ester and DDA (2,2-bis-[j)-chlorophenyl]acetic acid) for conjugation while Centeno et al. (25) used the anhydrides of DDA and malathion diacid (0,0-dimethyl S-[l,2-bis-carboxyethyl]phosphorodi-thioate). In retrospect, more specific antibodies of a higher titer may have been obtained had a spacer arm been used. 

Labelling with enzymes drastically alters the properties of the labelled compounds, because of the large sizes of the enzjune labels, and the effect is more pronounced for small haptens than for larger compounds (e.g., peptides and proteins). A practice often used is to optimize the length of the spacer arm between the structure containing the antigenic determinants and the enzyme label, so that hindering effects are avoided [97,98]. 

Sometimes options for spacer arm location are limited, as is the case for EPTC (Figure 1). Many thiocarbamates contain the S-ethyl moiety thus only the dipropylamino group is unique to EPTC. Hence the spacer attachment for all our EPTC haptens (2a-2c) was at the sulfur, distal to the dipropyleunino group. These haptens elicited good titer antibodies, but the best EPTC assay was 100 fold less sensitive than the best assays for molinate or thiobencarb. It appears that this deficiency is due to the EPTC structure itself. 

IgE antibodies to trimethoprim were first demonstrated in the mid-1980s. Immunochemical investigations employing bimethoprim covalently coupled via a spacer arm to Sepharose in quantitative hapten inhibition experiments with carefully selected analogs provided insights into the precise structures of the drug recognized by... 

Dig-UTP (BMB) is a UTP analog containing a steroid hapten isolated from Foxglove plants. In the Dig-UTP molecule, digoxigenin is coupled to the C-5 of uracil via an 11-atom spacer arm. Because of the bulky size of the Dig molecule, Dig-UTP labeling of some transcripts is more efficiently performed at a Dig-UTP/ UTP ratio of 0.15/0.85 instead of the usual 0.35/0.65. Dig-labeled probes/products can be detected or quantitated in situ or on membranes by means of an anti-Dig antibody coupled with a reporter enzyme (e.g., alkaline phosphatase) and of the enzymatic reaction with chromogenic or (chemi)luminescent substrates. 

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