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

Alert on biotin interference for immunoassays

 

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.

 

Replacing biotin-streptavidin by DNP

 

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

       

Labelled antibodies and probes with DNP

 

 

The detection of a biotinylated monoclonal antibody 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 drug…) induce a better immune response in rat specie, LO-DNP are high-performance and well-known references in Pharma and IVD industriesLO-DNP-2 is part of SYnAbs Top10 sales chart.