Synthesis of olaquindox metabolite, methyl-3-quinoxaline-2-carboxylic acid for development of an immunoassay

Figures & data

Figure 1.  Synthesis of methyl-3-quinoxaline-2-carboxylic acid.

Figure 2.  Principles of connecting of spacer arm to methyl-3-quinoxaline-2-carboxylic acid.

Figure 3.  Standard curves of ic-ELISA for MQCA. Conditions: A: coating antigen (Hapten A–OVA, 25 ng/well), antibody (raised using Hapten C–BSA, 1:40,000); B: coating antigen (Hapten E–OVA, 50 ng/well), antibody (raised using Hapten E–BSA, 1:24,000); IgG-HRP (1:3000); each point represents the mean of 16 determinations; vertical bars indicate ±standard deviation about the mean.

Table 1. Coupling ratio of hapten–protein conjugates.

Conjugate	Hapten/protein mole ratio
Hapten A–BSA	24.6
Hapten A–OVA	12.6
Hapten B–BSA	28.4
Hapten B–OVA	10.4
Hapten C–BSA	29.6
Hapten C–OVA	12.3
Hapten D–BSA	29.1
Hapten D–OVA	9.3
Hapten E–BSA	25.6
Hapten E–OVA	14.8

Table 2. IC₅₀ values of ic-ELISA for determination of MQCA with different conditions.

	IC50 of MQCA (ng/mL)
Coating antigen (200 ng/well)	Antibody (1:25,000)
	Hapten A–BSA	Hapten B—BSA	Hapten C–BSA	Hapten D–BSA	Hapten E–BSA
Hapten A–OVA	66	31	15	27	47
Hapten E–OVA	556	180	138	151	327
Absorbencies were all measured after 15 min incubation with TMB at room temperature. IC50 values are means of duplicates. The relative deviations were all below 30%.