Increasing Sensitivity of ELISA Sandwich Assay

[Elisadeveloper]

Hi,

I'm fairly close to being able to start validation of a regular sandwich ELISA for a large protein (mwt > 1000000) using a directly labelled peroxidase secondary antibody, and can target an LLOQ of ~ 2 ng/mL which is in itself highly respectable. But I need more. Selectivity in blank individual lots of matrix is consistent and spike in at close to the target LLOQ backcalculate well in multiple lots, so I am thinking that simple signal amplification might work. Coating and detection concentrations are optimized and I don't have flexibility to change the assay format itself.

TMB (KPL) and Quantablu give similar sensitivity, OPD is inferior. Any suggestions on how to boost the low end signal?

[NickWarner]

Have you considered a biotinylated secondary antibody and a strepavidin-HRP? you'll get multiple HRP's onto your secondary potentially boosting the signal.

[Elisadeveloper]

Thanks for the suggestion Nick, that may have given us a bit of a push at the low end. As it turned out, we got the green light to go into validation with the original format and are going to target a 1 ng/mL LLOQ with a back up at 1.6 ng/mL. I still think this is highly respectable for such a large molecule.

[Zagami]

NickWarner suggestion is good for to implement signal, while if you want to use a more chromogen powerful I recommend to you the leuco-malachite green as under suitable :
Leuco-malachite green HRP-substrate : dissolve 0.1 leuco-base of malachite green (tetramethyldiamidotri-phenyl-methane) in 5 ml glacial acetic acid. A minimal green color will develop in most cases even when a completely achromatic preparation is used; the green color is eliminated by adding an equal volume of chloroform. Distilled water is then added drop by drop while the mixture is carefully agitated until the chloroform precipitates entirely. The green chloroform is then separated from the supernatant reagent. The eventual clouding of the reagent, which can be caused by the precipitation of the leuco-base, is eliminated by adding glacial acetic acid. If traces of green color are still evident in the reagent, these are removed by shaking the reagent with a small quantity of chloroform. The reagent, prepared as indicated, should be entirely achromatic. For to prepare working solution, mix 10 ml of leuco-malachite achromatic solution with 0.1 ml 3% hydrogen peroxide and dispense 100 µl in wells and allowed for 10-20 min. at room temperature. The color solution turned in green and her intensity is proportional to concentration of HRP presente on surface wells. Should some of the leucobase precipitate during this process, the precipitate can be dissolved by adding a small amount of glacial acetic acid.

[Elisadeveloper]

Interesting Zagami....

Do you have any evidence that sensitivity can be increased using this approach over TMB while still maintaining acceptable intra and inter precision and accuracy at the reduced LLOQ concentration?

[Zagami]

Leucomalachite green (LMG) in peroxidase reaction system is an acceptor, while hydrogen peroxide a substrate. In this reaction the temperature coefficient of peroxidase acting on leucomalachite green is > 25 °C and hydrogen peroxide concentration is about 2.5 mg per litre. A stock solution of LMG reducing substrate was prepared by saturating N/20 acetic acid with the leuco-base in vacuo. The resulting solution contained approximately 10 mg of leucomalachite green per 100 ml, and remained colourless for several weeks if kept in vacuo. The reaction mixture was made up of 10 ml of this solution, 0.2 ml of 0.166 N sodium acetate (0.05 M) which adjusted the reaction to pH 4.1, containing 1.3x10-6 M of hydrogen peroxide. The reaction was carried out in a water-bath at 30-37 °C for a time varying between 2 and 8 minutes, at the conclusion of which the enzyme was destroyed by the addition of 1 ml of N sulphuric acid, which after 30 seconds was neutralised with an equivalent amount of sodium carbonate solution, and the mixture was shaken to remove carbon dioxide, and the malachite green produced (oxidized blue-green color) by the reaction is estimated in a colorimeter at 620 nm against a standard solution prepared by dissolving 10 mg of malachite green in a litre of N/20 acetic acid. The acetic acid inhibits the hydrolysis of the dye and thus prevents fading of the standard solution. LMG can be dissolved in acetone and then diluted with five volumes of methanol, and, in suitable proportion, mixed, just to the use, with sodium acetate buffer 0.05 M at pH 4.1 containing 1.3x10-6 M of hydrogen peroxide. In my foregoing research on human hemoglobin electrophoresis on agarose gel, the support has been incubated for 10 min in a 1:1 dH2O:LMG-peroxidase buffer (0.2 g leucomalachite green ad 0.02 g EDTA in 25 ml 40% [v/v] acetic acid with 0.06% [v/v] H2O2) with good black bands detection. Formulation and application of this reagent has been developed for different purposes and, although Leucomalachite green not true peroxidase test is, high sensitivity "nonspecific" oxidizing system showed. At any rate, is needed to be revalidated to ensure that the LLOQ and the QC samples in the actual concentration range to be used for sample analysis perform accurately and precisely.
To be on hand in future