Polyclonal Antibody or Monoclonal Antibody Selection for Lateral Flow Rapid Tests

By February 21, 2019 June 6th, 2019 Articles
polyclonal antibody, polyclonal antibodies

Written by: George Parsons, Ph.D.

Should you select polyclonal or monoclonal antibodies for your lateral flow rapid test? Some readers are probably wondering why polyclonal antibodies are not a thing of the past, like manual transmissions on cars and telephone landlines. However, the older models still have many advantages. This blog explores these advantages in the context of lateral flow assays.

Both monoclonal and polyclonal antibodies start out in much the same way: immunization of a live animal. Rabbits, goats and sheep are the most common subjects for making polyclonal antibodies and mice are the starting point for most monoclonals. Repeated immunizations are key for making good antibodies in any species. Detectable antibodies can be seen in a few months of boosting, but a really good polyclonal response can take 6 to 9 months to fully mature. Once the desired response has been achieved, production bleeds can begin. It is possible to take about 40 mLs of blood from a healthy rabbit once a month for several years with no ill effects on the animal’s health. This blood yields about 20 mLs of useable serum after processing. These sera are accumulated and tested for titer and other relevant parameters before being pooled if the individual sera are similar enough. A pool of 100 mL developed over a year is not unusual. With a coating titer of 1:10000 and a coating volume of 100 uLs, that size pool should be sufficient to produce 100,000 96-well microtiter plates or >1,000,000 lateral flow strips.

A company in Massachusetts recently quoted a cost of $1,000 to develop a rabbit polyclonal antibody to a small molecule in about a month. Additional booster injections and keeping the animals longer were offered at additional yet modest costs.

Production of monoclonals is more technically complex. Once the mouse has shown an adequate polyclonal response, the animal is euthanized and the spleen cells are fused with a mouse myeloma line to yield immortal cell lines. A single fusion can lead to several hundred individual cell lines, all of which must be screened and isolated so that a true monoclonal antibody with only one specificity can be assured.  Good manufacturing practices demand that cell banks be created and stored in different locations to ensure a continuing supply of a given monoclonal antibody.

A central European company offers a monoclonal antibody project for under $6,000 and cites a time frame of about 3 months from immunization to initial samples of antibody.

Monoclonal antibodies have several advantages over polyclonals for routine production of diagnostic tests. Their specificity is the most obvious one. They only react with one epitope on the target molecule. Polyclonals contain hundreds or even thousands of specificities that can lead to cross reactions that can compromise an otherwise excellent test. An example of this would be a test for hCG, the hormone that shows rapidly increasing levels in blood and urine during the early stages of pregnancy. hCG shares common subunit building blocks with TSH, LH and FSH.  Early polyclonal pregnancy tests could show false positives because the assay detected an LH surge during ovulation due to cross reactions between an antibody raised to hCG with LH. Ovulation and pregnancy are related but clearly different conditions.

Monoclonals have another important advantage over polyclonals in that once the monoclonal line is established, the supply of that particular antibody is essentially infinite. It is true that more antibody must be produced either in cell culture or in ascites, but the difficult and risky task of isolating the desired cell line never has to be repeated. That is not the case with polyclonals. Pools of polyclonals eventually run out and there is no guarantee that immunizing other animals will yield a useable antibody.

Estradiol is a good case study to illustrate some of these issues. Attempts to make mouse monoclonals to estradiol have been uniformly unsuccessful. Researchers have tried different immunogens with differing attachment chemistries, but the seemingly insurmountable problem is that mice appear to metabolize the estradiol immunogens to their sulfate and glucuronide derivatives and then they make antibodies to the derivatives. Since these metabolites are present in blood, these antibodies may cross react with estradiol but will give test results that are too high because they recognize both authentic estradiol and its sulfate and glucuronide derivatives. Ratios of analyte to metabolite also vary from patient to patient, further complicating that analytical problem.

Rabbits immunized with estradiol immunogens also produce antibodies to the metabolites in most cases, but brute force and pure luck can sometimes yield a good antibody. Campaigns in which over 500 rabbits have been immunized to get a useable estradiol antibody have failed to produce a single, useful antibody, but a European academic was lucky: he got a rabbit that produced an antibody that met the sensitivity and cross reactivity requirements. He built a pool of more than 100 mLs and then, figuring he had several lifetimes of antibody, he then exsanguinated the rabbit. That pool lasted about 5 years when it was used to manufacture tests for a high throughput automated analyzer. Finding a replacement antibody was a difficult and expensive process.

Monoclonals are also easier to label than polyclonals. The highest percentage of a specific polyclonal antibody I have ever seen was with a hyperimmunized rabbit antibody to Fluorescein Isothyocyanate (FITC). Roughly 0.4% of the total IgG was directed against FITC. The rest were the antibodies that rabbit had raised to defend itself from its environment. Affinity purification can be used to prepare a polyclonal antibody preparation that is suitable for labeling, but that step adds complexity and cost.

Sometimes monoclonals can be too specific. A good example of this is the silent LH problem that surfaced when a major manufacturer introduced a sandwich assay for LH that used a monoclonal antibody for the capture antibody and another monoclonal as the detector antibody[1]. It was a very good assay that seemed to work well until they tried it in Scandinavia. Using the test, it seemed to indicate that 25% of the women there did not produce any detectable LH. Tests that used at least one polyclonal antibody to LH had no problem detecting LH. Investigation soon showed that the women with LH undetectable with monoclonals to LH had a deletion mutation in their LH that had no effect on the function of the hormone. As luck would have it, the region that was deleted is immunodominant in the mouse. That means that immunizing mice with wild type LH without the deletion mutation is going to preferentially produce antibodies to that epitope that is missing in the Scandinavian women with the mutation. Either by accident or design an antibody company made a monoclonal to LH with the deletion mutation and the problem was solved.

LH is not the only analyte where monoclonals can be too specific. Modern troponin I assays use cocktails of monoclonals to overcome this problem. Sometimes two monoclonals are used as capture antibodies and two other monoclonals are used as detector antibodies[2]. Various subspecies of phosphorylated or oxidized troponin are thought to be in play.

Established practice can also keep a polyclonal antibody in the market when a more specific monoclonal antibody is available. I once developed a drug assay for an automated platform that used a monoclonal. The competition was an established assay that used a polyclonal antibody that cross reacted with a metabolite. Laboratories and their clinicians had grown accustomed to the results provided by the cross reacting polyclonal and would not accept the lower results provided by the analytically superior monoclonal.

Polyclonals were the first antibodies used in immunodiagnostics and their simplicity and relatively lower costs are going to keep them in play for some time to come. Monoclonals solve some of the supply and purity issues inherent in polyclonals but bring their own set of problems of narrow specificity, added costs and complexity. Landlines allow calls to specific locations and cannot be easily turned off. Manual transmissions dominate in areas where fuel costs are extremely high. As with most technological options, flexibility in choosing the right solution in a given environment is prudent and cost effective.

Our team of engineers and scientists can help you determine the specification for monoclonal or polyclonal antibody selection for your test. Feel free contact us with any questions and antibody selection.

[1] Heterogeneity of plasma gonadotropins. Consequences on immunological properties of LH. Roger MLalhou N., Nucl Med Biol. 21(3):349-57 (1994).

[2] Analytical Characteristics of High-Sensitivity Cardiac Troponin Assays, Apple FS and Collinson PO, Clin Chem 58:1 54–61 (2012)