Conjugation with haptens containing

Fig. 10. Different techniques for competitive immuno-polymerase chain reaction of small-molecule compounds. (A) Using biotinylated haptens and a DNA-streptavidin nanocircle conjugate [105], hapten-DNA conjugates were synthesized and used for a competitive assay in a sample containing free hapten and capture antibody-coated surfaces [92]. (B) Hapten-coated microplates were simultaneously incubated with a sample containing free hapten and a hapten-specific antibody. Following competitive coupling, the immobilized antibody was subsequently detected by a species-specific antibody-DNA conjugate [93, 94].

Bis-diazotized benzidine can be used as a bridging reagent between proteins and haptens containing aromatic groups that react with diazon-ium compounds. A conjugate of thyrotropin-releasing hormone (which contains a reactive histidine residue) was obtained in this way. ... 

The study shewed hapten protein molar ratios of 8.5, 25, and 71 for the BSA, OVA and THY proteins, respectively. These ratios ware obtained by infrared (IR) analysis as well as elemental chlorine analysis. Chlorine analysis was used as only the dieldrin molecule contains covalently bound chlorine. The conjugates with the highest hapten loadings, OVA and THY, were used for immunization in the dieldrin case. 

Fig. 2.4. Competitive, homogeneous EIA. The symbol E represents enzyme, A antigen or hapten, S and CO substrate and enzyme cofactor, and Av and B avidin and biotin. Enzymes indicated with have modulated (increased or decreased) activities. In (a) hapten is conjugated to the enzyme and reaction with the antibody (shaded structure) modulates the enzyme activity. This interaction is, however, prevented if antigen is present in the sample. Similar principles apply if substrate (b) or cofactor (c) are conjugated with the antigen. In (d) antibody is directly linked to the enzyme, and the reaction of this antibody, particularly with high-molecular weight antigens, will block the active site of the enzyme. In (e) antibody, directed to haptens labeled to avidin, prevents avidin from inhibiting the biotin-containing enzyme, unless antibody is neutralized by the same hapten present in the sample.

Maleimides (Section 11.2.3.2.1.2) are particularly useful for the conjugation of thiol-containing haptens. Other approaches are based on the activation of the protein with bromoacetamide groups (Singh et al., 1978) or on the formation of disulfide bonds between the carrier and hapten in acetate buffer, pH 4.0, in the presence of hydrogen peroxide, as used for the conjugation of penicillenic acid (De Week and Eisen, 1960). 

Nitrocellulose Hi-flow plus membrane was cut into sections (3.0 x 0.5 cm2), for the detection of small molecular (such as pesticide). The procedures of preparation of membrane strip were shown Step 1 The membrane was coated with hapten-OVA conjugates in a volume of 1 a.L containing 1 pg hapten conjugate as test line with Camag Linomat 5 automatic TLC sampler. The control line was coated with 0.5 pL of anti-rabbit IgG from goat diluted 1/100 in PBS buffer (pH 7.2). The distance between the two lines was 0.5 cm. 

---