Microbial receptor assays

The CHARM II test for tissues is relatively fast, easy to perform, and requires limited laboratory equipment. However, for antibacterials with established tolerance levels, it can serve only as a screening test because the results are not quantitative and therefore should be supported by additional quantitative chemical methods. The microbial receptor assay, with its broad-spectrum capability, can enhance any existing monitoring system as a first-line monitoring test or as a confirmation for any program using microbial inhibition tests. 

Application of some kind of sample treatment may have the potential to improve substantially the detection of certain antibacterials in milk by microbial routine methods (59). Treatment, for example, of milk samples with ammonium oxalate solution prior to analysis can lead to lower limits of detection of tetracyclines by both microbial inhibition and microbial receptor assays. This is due to the fact that tetracycline residues tend to form chelates with divalent cations and bind to proteins, which reduce their antibacterial efficacy. However, the oxalate treatment causes splitting of complex and/or protein bonds without increasing the detection limits of other antibacterials commonly used in dairy cows. 

E Zomer, J Quintana, S Saul, SE Charm. LC-Receptorgram—a method for identification and quantitation of beta-lactams in milk by liquid chromatography with microbial receptor assay. J AOAC Int 78 1165-1172, 1995. 

From these data the potential of the receptor assay is evident. Comparison with the microbial diffusion assay system. Table I, indicates that the levels of detection and measurement are reasonably similar. The receptor assay has the added virtue of allowing for the completion by the analysis within an hour, generally, rather than several hours or the next day. 

Assays for detection of antimicrobial residues in foods are based on the microbial growth inhibition, microbial receptor, and enzymatic colorimetric formats. 

More versatile than the growth-inhibition assays and potentially applicable to determining the presence of different antibiotic residues in different matrices are the microbial receptor CHARM I and II test assays (19, 20). The Charm I test, developed exclusively for -lactams in milk, constitutes the first rapid test recognized by The Association of Official Analytical Chemists (AOAC) with a test time of 15 min (19). The speed and sensitivity of this test permitted testing of milk tankers before they unloaded at the processing plant (21). In 1984-1985, the CHARM I test was further developed to test for antibiotics beyond -lactams to include tetracyclines, sulfonamides, aminoglycosides, chloramphenicol, novobiocin, and macrolides. The extended version has been referred to as CHARM II test. 

The starting point in the technical development of a microbial inhibition assay is the sensitivity of the test microorganism to different antimicrobial compounds under different media conditions. Factors such as the pH and nutrient profile of the media can be varied to obtain the optimum performance. The sensitivity of MIAs may also be modulated by the addition of specific compounds to the test composition. For certain antimicrobial compounds an enhancement in sensitivity may be advantageous, whereas for others, a sensitivity decrease is required to avoid the number of false non-compliant results. For example, cysteine can reduce the overall sensitivity to the P-lactam class. Antifolates, such as ormethoprim or trimethoprim, are known to improve the sensitivity of the test organism to sulfonamide compounds. Sensitivity to sulfonamides can also be modulated by using the enzyme dihydropteroate synthetase that selectively inhibits the response of sulfonamides." Antibiotic receptors such as antibodies can also be employed to decrease the sensitivity of the test to specific compounds, as required. 

Okerman et al. compared the performance of a Tetrasensor with three microbial inhibition assays for the analysis of tetracycline antibiotics in tissue. The group concluded that when large numbers of samples have to be analyzed without the requirement for immediate results, classical agar diffusion tests with thin plates and performed as prescribed for the EPT still seem the most economical choice. However, the receptor-based test Tetrasensor was recommended for use in official surveys and also in cases when immediate results are required. Unlike the inhibition tests, this receptor test does not require a well-equipped laboratory for use and is more suited for the meat industry. ... 

In addition to these effects, neurotoxic effects of certain PAs have been demonstrated in in-vitro receptor assays [130]. However, the relevance of these rather sporadic effects in vivo needs clarification. In a broad screening in which the interaction of many compounds from different classes of alkaloids with various molecular targets were tested, in almost all cases PAs were found to be inactive [131]. There are a few reports that indicate sporadic and weak (i.e., EC5o>0.5 mg ml antibacterial and antifungal activity [132-134] of PAs. These effects do not indicate an efficient antimicrobial potential of PAs. In this context it should be recalled that in plants PAs are constitutively formed they seem not to be induced by wounding, herbivorous or microbial attack [135-137]. 

The classical microbial assay approaches to measuring antibiotic residues, diffusion, turbidimetric and acid production were described and the advantages and limitations reviewed. Other systems so discussed and reviewed were the affinity or receptor methods and the immunological approach using ELISA or EMIT assay techniques. The classical systems, in general, could measure antibiotic residues at the fractional ppm to the ppb levels. The potentials of the receptor and immunological assay system were discussed. 

Matsuda, Y. (1993). Screening of bioactive substances of microbial origin based on enzyme and receptor binding assays. Actinomycetology, 7, 110-118. 

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