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.