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I am working on developing lateral flow immunoassays for drugs of abuse and needed some advice on detecting multiple drug compounds in one immunoassay.

As an example, an existing LFIA on the market tests for amphetamines, and detects the following compounds as positive at varying levels:

  • d-Amphetamine
  • d,l-Amphetamine
  • l-Amphetamine
  • 3,4-Methylenedioxyamphetamine (MDA)
  • β-Phenylethylamine
  • Phenylpropanolamine
  • Tyramine
  • p-Hydroxynorephedrine
  • (±)-Phenylpropanolamine
  • p-Hydroxyamphetamine
  • d,l-Norephedrine

These compounds are all tested in one test strip — not multiplexed; the presence of any one of those compounds above the cut-off level will cause the single test strip to show a positive.

I don't quite understand how antibodies work and have a few theories:

  1. Polyclonal antibodies have been used on the test line, which means all these compounds are detected
  2. A mixture of monoclonal antibodies have been used, to detect all these compounds
  3. These compounds are all chemically similar enough to be reliably detected with a single monoclonal antibody

Which of the above would be correct? Or am I misunderstanding this entirely and the answer is something different?

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2 Answers 2

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Background: I worked for a number of years as a product scientist for a fairly well-known antibody company, designing and producing polyclonal and monoclonal antibodies from several species.

Of your three theories, any one of them could be correct. Polyclonal antibodies are the cheapest, easiest, and generally fastest way of producing antibodies to a particular target, and I would imagine that they're used pretty extensively in LFIAs and other clinical immunological tests. However, polys have a major limitation: reproducibility. Animals' immune responses can change over time, even with regular booster immunizations, so it can sometimes be difficult to maintain the exact same specificity and sensitivity over multiple lots or batches, especially if you have to change animals. One way around this is to make very large lots, but they may end up degrading over time, even if kept at very low temperatures.

One way to get the recognition of multiple epitopes that polyclonals have with the epitope stability that monoclonals have is to make a "multiclonal" mix of monoclonal antibodies. This could be anywhere from 2 to a dozen or more clones, depending on the number of targets you're trying to analyze.

A single multiclonal antibody is also possible in this circumstance. While a monoclonal does only recognize one epitope, that epitope could be rather general and exist on multiple molecular configurations. An example of this type of antibody is Cell Signaling's Phospho-Akt Substrate (RXXS*/T*) rabbit monoclonal antibody (clone 110B7E). Instead of recognizing phosphorylated Akt/Protein Kinase B, this clone recognizes its phosphorylated substrates that have an Arg-X-X-pSer/pThr motif in them. It has been shown in tandem mass spec studies to bind to hundreds of unique peptides with this motif.

In the context of this particular question, it is perfectly reasonable to believe that the mixed amphetamines LFIA could rely on a single clone that recognizes a generalized "amphetamine-like" epitope that is present in all of the molecules listed, but (hopefully) not in other chemicals that are not drugs of abuse.

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In general, monoclonal antibodies are used as primary antibodies and conjugated to the nanoparticles due to their ability to bind specifically to a single epitope of an antigen (1). Even a small change in the structure of the epitope can affect the binding to antigen, so this automatically makes your last statement impossible because a single monoclonal antibody can only recognize one epitope of the same antigen (2). In contrast, polyclonal antibodies are mostly used at the test band because they can recognize a host of antigenic epitopes, meaning that they are able to recognize highly similar compounds that vary in a single or small number of epitopes, making them ideal for generating signal for detection (1,2). But keep in mind that use of polyclonal antibody can also lead to increased risks of cross-reactivity (false positive result) (1,3).

Resources

(1) Qriouet Z, Cherrah Y, Sefrioui H, Qmichou Z. Monoclonal Antibodies Application in Lateral Flow Immunochromatographic Assays for Drugs of Abuse Detection. Molecules. 2021 Feb 18;26(4):1058. doi: 10.3390/molecules26041058. PMID: 33670468; PMCID: PMC7922373.

(2) Neil S. Lipman, Lynn R. Jackson, Laura J. Trudel, Frances Weis-Garcia, Monoclonal Versus Polyclonal Antibodies: Distinguishing Characteristics, Applications, and Information Resources, ILAR Journal, Volume 46, Issue 3, 2005, 258–268, https://doi.org/10.1093/ilar.46.3.258

(3) Reschly-Krasowski JM, Krasowski MD. A Difficult Challenge for the Clinical Laboratory: Accessing and Interpreting Manufacturer Cross-Reactivity Data for Immunoassays Used in Urine Drug Testing. Acad Pathol. 2018;5:2374289518811797. Published 2018 Nov 21. doi:10.1177/2374289518811797

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  • $\begingroup$ This is not correct, especially your bolded statement. It is perfectly possible for a single monoclonal antibody to recognize multiple targets, even if they are composed of different amino acid sequences (in the case of protein targets) or atoms, in the case of small molecule targets. Any one of the OP's proposed mechanisms could be correct. $\endgroup$
    – MattDMo
    Jun 9, 2021 at 18:51
  • $\begingroup$ @MattDMo if this is the case, I would accept that comment as an answer – that there isn't enough information to tell which is the case. Certainly would appreciate any other feedback to improve the question. $\endgroup$
    – Anonymous
    Jun 10, 2021 at 0:48

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