Does Monoclonal Mean Monospecific? A Crisis in Antibody Specificity

Highly spe­cif­ic anti­bod­ies are integ­ral to a large num­ber of excit­ing pro­jects. From under­stand­ing physiolo­gic­al pro­cesses to life-chan­ging anti­body-based dia­gnostics and ther­apy, anti­body-powered research is at the fore­front of innov­a­tion in the life sci­ences. We love anti­bod­ies because they offer unpre­ced­en­ted spe­cificity and selectiv­ity. But what if they don’t?

In this art­icle, we’re look­ing at the import­ance of anti­body spe­cificity, the repro­du­cib­il­ity crisis, and how anti­body val­id­a­tion can pre­vent enorm­ous time and money waste.

Are Your Antibodies Genuinely Specific?

Depend­ing on your meth­od and tar­get, there are three main types of anti­bod­ies:

Hybridoma mono­clonal anti­bod­ies are accur­ate and gen­er­ally offer sol­id repro­du­cib­il­ity. How­ever, they are prone to genet­ic drift and usu­ally come at a high­er cost.

Recom­bin­ant cloned anti­bod­ies ensure bet­ter repro­du­cib­il­ity but are cur­rently not as widely avail­able as oth­er types.

Finally, poly­clonal anti­bod­ies, which bind mul­tiple epi­topes, offer clonal and bio­phys­ic­al diversity. This increases their sens­it­iv­ity in cer­tain assays and boosts their sta­bil­ity. While poly­clonal anti­bod­ies are the most con­tro­ver­sial of the three types, ignor­ing their advant­ages means miss­ing out on a “fit-for-pur­pose” tool.

One ques­tion remains:

What hap­pens when a reagent known for its spe­cificity and repro­du­cib­il­ity turns out not to be spe­cif­ic enough?

We now have a wider range of anti­bod­ies than ever before. Our val­id­a­tion tools have also improved. But with the increas­ing pop­ular­ity of anti­body char­ac­ter­iz­a­tion and eval­u­ation meth­ods, we are find­ing that some of the reagents are not nearly as spe­cif­ic as we thought.

In a 2018 study, 185 hybrido­mas were examined to assess poten­tial genet­ic diversity (the exist­ence of which had been anec­dot­ally known for dec­ades). 31.9% of the hybrido­mas examined con­tained addi­tion­al pro­duct­ive chains that affect anti­body prop­er­ties such as spe­cificity, bind­ing sig­nal, and sig­nal-to-noise ratio. (Brad­bury et al., 2018)

Here at engine, we per­formed a sim­il­ar exper­i­ment using three com­mer­cially avail­able anti­bod­ies. Using our hEXse­lect arrays, we detec­ted mul­tiple pos­it­ive hits, identi­fy­ing both spe­cif­ic and non-spe­cif­ic bind­ing. One would assume that a mono­clonal anti­body is mono­spe­cif­ic – but for one in three reagents, this is not the case. To do good sci­ence, we need high-qual­ity data that we can trust. If anti­bod­ies, a reagent we rely on for spe­cificity, are not spe­cif­ic enough, the cost can be dev­ast­at­ing:

The Cost Of Off-Target Specificity

Anti­bod­ies are the back­bone of break­through research. A tre­mend­ous amount of effort, time, and money invest­ment depends on these high-qual­ity reagents. Off-tar­get spe­cificity can jeop­ard­ize the entire exper­i­ment or dia­gnost­ic pipeline.

Inaccurate Results

False-pos­it­ive and false-neg­at­ive res­ults are part of the research and dia­gnost­ic pro­cess. When an anti­body cross-reacts with an unre­lated pro­tein, the accur­acy of the immun­oas­say decreases. This can slow down your research and neg­at­ively impact journ­als when it is time to pub­lish. Just as prop­er anti­body val­id­a­tion improves the chances of an art­icle being accep­ted, its absence has a neg­at­ive impact. (Gautron et al., 2019) This is not sur­pris­ing, of course, since inac­cur­acy in raw data affects the over­all qual­ity of research.

Time and Money Sink

The fin­an­cial impact of repro­du­cib­il­ity can­not be over­stated. Dec­ades of research effort and invest­ment can go down the drain due to insuf­fi­cient val­id­a­tion.

The dis­cov­ery of the estro­gen recept­or β is a par­tic­u­larly strik­ing example. The recept­or was repor­ted to be homo­log­ous with the phar­ma­co­lo­gic­al tar­get for breast can­cer, ERα, and rep­res­en­ted a new hope for endo­crine treat­ment. Twenty years of research were in vain, how­ever, when it was found that of the 13 anti-ERβ anti­bod­ies, only one had suf­fi­cient spe­cificity. In oth­er words, ERβ pro­tein expres­sion was not asso­ci­ated with breast can­cer, which misled research­ers. (Andersson et al., 2017)

And this is not the only case where the lack of adequate val­id­a­tion has had dev­ast­at­ing con­sequences. One study estim­ated losses due to off-tar­get spe­cificity and non-repro­du­cib­il­ity at $1.7 bil­lion in 2019. (Ber­glund et al., 2008)

Ulti­mately, inad­equate anti­body reagents are det­ri­ment­al to a suc­cess­ful sci­entif­ic endeav­our. Wheth­er you are explor­ing dis­ease mech­an­isms or devel­op­ing bio­mark­er tests, prop­er char­ac­ter­iz­a­tion and val­id­a­tion pre­vents these dis­astrous out­comes.

How Our Protein Arrays Can Help!

Pro­tein arrays allow us to under­stand pro­tein inter­ac­tions without bias or high cost. Our anti­body spe­cificity ser­vice screens more than 10,000 poten­tial anti­gens that your anti­body reagent could bind to. These pro­teins are not selec­ted based on hypo­thes­is, so you get an over­all view of poten­tial off-tar­get spe­cificity. We can also include the anti­gen you want to detect in the array to fur­ther val­id­ate the anti­body.

And we already know that it works. High-dens­ity pro­tein array tech­no­logy has proven to be a fast and effi­cient val­id­a­tion meth­od in the past. (Kijanka et al. 2009) When we per­formed our own ana­lys­is of the bind­ing pat­terns of com­mer­cially avail­able anti­bod­ies. The res­ults using our hEXse­lect array con­firmed once again that pro­tein arrays are a valu­able tool for detect­ing off-tar­get activ­ity.

The assays are per­formed in a single run, requir­ing only µl of sample. We provide full access to data and sci­entif­ic dis­cus­sion and keep our ser­vice afford­able in the interest of a bet­ter, more accur­ate land­scape in immun­o­his­to­chem­istry.

Final Thoughts

The under­stand­ing, dia­gnos­is, and treat­ment of dis­ease are increas­ingly depend­ent on anti­bod­ies. Anti­body screen­ing is an excel­lent way to ensure con­sist­ent assays and reduce the fin­an­cial bur­den of off-tar­get react­iv­ity. With our engine pro­tein array ser­vice, we can test your reagent against >10,000 poten­tial anti­gens. And we only need some µl of your anti­body!

Val­id­at­ing anti­bod­ies using pro­tein arrays pro­tects you from devel­op­ing flawed assays, pro­du­cing unre­li­able data, com­prom­ising aca­dem­ic repu­ta­tion, and invest­ing time and money in your research. Let us help you achieve bet­ter, more accur­ate, and more repro­du­cible res­ults!

Inter­ested in more inform­a­tion? Down­load our res­ult present­a­tion detail­ing the spe­cificity of com­mer­cial anti­bod­ies, or con­tact our experts to sched­ule a free brain­storm­ing ses­sion.

  • Brad­bury, A., Trinklein, N. D., Thie, H., Wilkin­son, I. C., Tan­don, A. K., Ander­son, S., Bladen, C. L., Jones, B., Aldred, S. F., Bestagno, M., Bur­rone, O., Maynard, J., Fer­rara, F., Trim­mer, J. S., Görne­mann, J., Glan­ville, J., Wolf, P., Fren­zel, A., Wong, J., Koh, X. Y., … Dübel, S. (2018). When mono­clonal anti­bod­ies are not mono­spe­cif­ic: Hybrido­mas fre­quently express addi­tion­al func­tion­al vari­able regions. mAbs, 10(4), 539–546.
  • Gautron L. (2019). On the Neces­sity of Val­id­at­ing Anti­bod­ies in the Immun­o­his­to­chem­istry Lit­er­at­ure. Fron­ti­ers in neuroana­tomy, 13, 46.
  • Andersson, S., Sun­d­berg, M., Pristovsek, N., Ibrahim, A., Jonsson, P., Katona, B., Claus­son, C. M., Zieba, A., Ram­ström, M., Söder­berg, O., Wil­li­ams, C., & Asplund, A. (2017). Insuf­fi­cient anti­body val­id­a­tion chal­lenges oes­tro­gen recept­or beta research. Nature com­mu­nic­a­tions, 8, 15840.
  • Ber­glund, L., Björling, E., Oks­vold, P., Fager­berg, L., Asplund, A., Szig­yarto, C. A., Persson, A., Ottos­son, J., Wernérus, H., Nils­son, P., Lun­d­berg, E., Siverts­son, A., Navani, S., West­er, K., Kampf, C., Hober, S., Pontén, F., & Uhlén, M. (2008). A gene­centric Human Pro­tein Atlas for expres­sion pro­files based on anti­bod­ies. Molecu­lar & cel­lu­lar pro­teo­m­ics : MCP, 7(10), 2019–2027.
  • Kijanka, G., Ipcho, S., Baars, S., Chen, H., Had­ley, K., Beveridge, A., Gould, E., & Murphy, D. (2009). Rap­id char­ac­ter­iz­a­tion of bind­ing spe­cificity and cross-react­iv­ity of anti­bod­ies using recom­bin­ant human pro­tein arrays. Journ­al of immun­o­lo­gic­al meth­ods, 340(2), 132–137.

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