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In accordance with FDA, can a biotinylated mAb be replaced by a DNP marked mAb?

 

As we explained on past year in our article FDA ALERT ON IMMUNO-ASSAYS « Don't let Biotin interfere with your lab tests», the Food & Drug Administration alerted clinical laboratory developers that exogenous biotin can significantly interfere with different immunoassays platforms and cause incorrect test results: biotin-induced interference is likely to affect many assays using Streptavidin-Biotin coupling and causes lower values (sandwich type assays) or higher values (competition type assays).

 

In fact, biotin or B vitamin is a common component of daily supplement diet and its ingestion is becoming an important growing phenomenon.

 

As recommended by the FDA, the easiest solution is to use an alternative assay that does not utilize biotin as a reagent. At SYnAbs, we figure that DNP seems to be the adequate molecule to do the job. 

 

 

DNP stands for Di-Nitro-Phenol, 2,4-Dinitrophenol (2,4-DNP or simply DNP), an organic compound with the formula HOC6H3(NO2)2. Dinitrophenyl (DNP) is a hapten, not found endogenously in tissues, so serves as a control for immuno-assay.

 

            But as passionated scientists we were eager to prove what we were saying. So we decide to verify that a biotinylated mAb revealed with streptavidin -HRP can be replaced by a DNP labelled mAb revealed with the mouse anti DNP MADP2–HRP.

 

ELISA description

  1. The antigen (here a mouse IgG2a) was coated on an ELISA plate that was saturated with gelatin. As a negative control, the antigen was omitted.
  2. Various concentrations of the rat anti mouse IgG2a LO-MG2a biotinylated or DNP labelled were added (the DNP labelling was performed with the N-Succinimidyl N-(2,4-dinitrophenyl-6-aminocaproate from Sigma).
  3. The biotinylated or DNP labelled LO-MG2a were revealed respectively with Streptavidin -HRP or MADNP2-HRP at 1 µg/ml.

 

These are the ELISA outcomes we obtained.

LOMG2a1

conc. ng/ml

LOMG2a1-DNP

L0MG2a1-biot

LOMG2a1 -DNP

L0MG2a1 -biot

1000

1,860

1,260

0,290

0,260

500

1,540

1,000

0,180

0,180

250

1,060

0,830

0,150

0,120

125

0,820

0,500

0,140

0,100

62,5

0,540

0,320

0,140

0,100

31,25

0,350

0,240

0,120

0,080

15,6

0,250

0,140

0,120

0,070

7,8

0,200

0,110

0,110

0,070

3,9

0,170

0,100

0,110

0,070

1,95

0,170

0,080

0,100

0,070

0,97

0,150

0,070

0,100

0,070

0

0,080

0,060

0,100

0,070

 

antigen

coated

nothing

coated

       

 

Conclusion

 

The detection of a biotinylated a mAb with streptavidin HRP can be advantageously replaced by the detection of a DNP labelled mAb with an anti DNP-HRP.

 

DNP is an excellent alternative to biotin for use in applications requiring biotin-free systems. Probes or antibodies labeled with DNP can be detected using the SYnAbs anti-DNP antibodies.

 

SYnAbs has developed anti-DNP in mice (MA-DNP) and in rats (LO-DNP), offering 16 different monoclonal antibodies references. Since small molecules (hormones, antibiotic, peptide, chemical therapeutic…) induce a better immune response in rat specie, LO-DNP are high-performance and well-known references in Pharma and IVD industries. LO-DNP-2 is part of SYnAbs Top10 sales chart.

 

About SYnAbs

 

SYnAbs' vision is to fill the gap in the marketplace for innovative mAbs against poor immunogenic compounds and complex antigens. Targeting steroids, peptides, small molecules, toxins, lipids and polysaccharides (on fungi and bacteria), SYnAbs has developed strong expertise in immunology to break immunotolerance in rat and guinea pig species to generate unique monoclonal antibodies of extreme specificity and higher affinity.

 

SYnAbs (https://www.synabs.be) was set up in September 2015 out of Professor Pierre Gianello’s experimental surgery laboratory (CHEX) in order to develop greater expertise in the custom production of monoclonal antibodies, of rats but also of guinea pigs.

 

The UCL spin-off was set up as a result of coordinated action by the laboratory, the Louvain Technology Transfer Office (LTTO), Wallonia Biotech Coaching and a French group, Biotech Investissement (which owns shares in RD Biotech, Diaclone and QVQ companies under the brand name mAbexperts https://www.mabexperts.com/en/ ).

SYnAbs is a CRO based on the innovative BioPark of Gosselies-Charleroi in Belgium, has currently 130 m2 of production laboratories and generates earnings beyond 1 million euro thanks to its diligent team. 

 

About the author

 

Julien Isoard,

SYnAbs - Business & Marketing Director

mAbexperts - Digital Marketing Manager

 

Julien is 35 and has a M.Sc. in biotechnology and a M.S. in management of bio-industries. He was Business Director of cGMP CMO Henogen, before becoming Market Manager of Novasep Group.

In 2012, he set-up the business department of clinical CRO ImmuneHealth, finally sold to Canada-based company Caprion Biosciences. 

 

Julien has then successfully implemented the business and investment strategies of CER Group, AmplyCell, Spectralys, MaSTherCell, Univercells, SATT, and many other service companies.

He’s co-founder and board member of Brasserie Saint LazareFreelance BiotechIsy Bio and SYnAbs.