Immunoassay Cross-Reactivity continued

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The page below is a sample from the LabCE course Drug Testing Methods in the Clinical Toxicology Laboratory. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Immunoassay Cross-Reactivity continued

While false positives are probably the first thing to come to mind when considering cross-reactivity, false negatives can also be a problem. With immunoassay drug screens we actually want an assay that can detect multiple drugs within the same class (for example, we want an opiate screen that can detect many different opiates, and we'd want many different benzodiazepines to be picked up by our benzodiazepine screen). If cross-reactivity within a drug class is too low, it can be a problem. In the screening of certain classes of drugs, each drug in that class will display varying degrees of cross-reactivity. For example, an amphetamine screen with a cutoff of 300 ng/mL may show high binding for amphetamine but much less for methamphetamine. And the cross-reactivity for MDMA, MDA, and MDEA will likely be very low compared to amphetamine. Let’s suppose amphetamine is present in a urine specimen at 325 ng/mL and MDMA is present at 975 ng/ml. Let's also presume that the cross-reactivity of MDMA in this particular immunoassay is 10 times less than that of amphetamine (which is not atypical). In this example then, the result would show that amphetamine is above the normal positive cutoff but MDMA would be below the cutoff (negative). So this assay would be a good one for detecting amphetamine but would not reliably pick up MDMA, even when present at a concentration of 975 ng/mL.

Immunoassays for opiates are another example where varying levels of cross-reactivity represent a challenge.

Morphine and codeine elicit a strong response for most opiate immunoassays, but there is lower sensitivity for semisynthetic opioids such as hydrocodone, hydromorphone, oxycodone, oxymorphone, heroin, and buprenorphine. Because of this cross-reactivity pattern, a negative result does not necessarily mean an opiate was not used. And synthetic opioids such as fentanyl and methadone may not be detected at all, even when present in very high concentrations. For this reason, specific immunoassays for some of these compounds have been developed. In the screening of opiate class drugs, there are separate assays for methadone, fentanyl, oxycodone/oxymorphone, tramadol, buprenorphine, and others. Below is a list of other drugs (some from entirely different drugs classes) that can interfere with some immunoassays.
Examples of cross-reacting compounds for certain immunoassay classes of drugs:
  • Opiates - Quinolone antibiotics (eg, levofloxacin, ofloxacin), levorphanol, naloxone
  • Fentanyl - Trazodone
  • Phencyclidine - Venlafaxine, dextromethorphan
  • Methadone - Quetiapine
  • THC- Antiretroviral efavirenz, proton pump inhibitors (eg, pantoprazole)
  • Amphetamine - Diet pills, promethazine, l-methamphetamine, phenmetrazine, selegilene
Different assays will often have different cross-reactivities based on the detection antibodies being used.