rev: June 15, 2005
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for Researchers and Manufacturers
Need more sensitivity in your protocol?
-DNA PROBE DETECTION
? What is POLY-HRP-STREPTAVIDIN: A proprietary polymer of HRP
(horseradish peroxidase) conjugated to Streptavidin in various polymerization
ranges. (20, 40 & 80). The POLY-HRP-SA product line can be used with
virtually any biotin labeled product including probes & primary and
secondary antibodies. It can replace the conventional streptavidin-HRP
conjugates currently in use. It will activate your current HRP substrates
including the sensitive TMB products (ELISA and blotting), DAB, luminol etc....
? What is POLY-HRP80-GAM:
A new specialty conjugate made with our POLY-HRP80 base. It is a goat
anti-mouse (IgG + IgM) affinity absorbed antibody that is specially conjugated
with our POLY-HRP 80 molecule. Its applications include sensitive histochemical
applications and for use in various immunoassays.
? How does it work: With conventional conjugates, you have only
one or two HRP molecules available to react with your substrate. With POLY-HRP
conjugates you increase the number of HRP molecules by 5X, 10X and even more
(depending upon the polymerization range used). The more HRP molecules
available, the greater the activation of your substrate=the more sensitivity
? How is it supplied: The POLY-HRP-SA is supplied in a
concentrated form of approx 1 milligram conjugate in 1ml of 50% glycerol/
buffer. It should be stored at -20 DEG C and fresh dilutions made daily. The
undiluted stock is good for at least one year. The working dilution required
will depend upon your assay characteristics. We recommend starting dilutions of
1:500 for blotting and 1:2000 for ELISA work. Working titers can then be
derived to optimize your assay.
-POLY-HRP80-GAM is supplied as approx 200ug conjugate in 1ml of
50% glycerol/buffer. It should also be stored at -20 DEG C with fresh dilutions
made daily. It must be titered for each application with initial 1:100-1:1000
dilutions for histochemistry.
authors: Dr. Dmitry Yu Plaksin & Elena T. Gromakovska-SDT Inc
We have currently developed detection technology using proprietary
covalent horseradish peroxidase (HRP) homopolymers as enhanced enzyme labels.
The enhanced signal generation by our POLY-HRP conjugates provides highly
increased sensitivity, short assay times and/or reduced detecting reagent
consumption in a variety of applications.
Universal covalent conjugates of Streptavidin & Protein A with HRP polymerized in different ranges including, -20, -40 & -80 offer a wide range of amplifying possibilities. They are available now as truly versatile tools for ultra-sensitive detection in immunoassays (of both microwell and membrane formats), DNA probe detection, ligand-receptor assays,immuno-histochemistry and cytochemical applications.
Performance of Streptavidin-POLY-HRP's from our current product line in comparison with a standard conjugate is shown in Figure 2 (click to view figures). Two fold dilutions of a biotinylated mouse monoclonal antibody (IgG isotype) were incubated for 1 hour in an immunoplate which had been coated with an affinity purified sheep anti-mouse IgG antibody. After washing, bound IgG was detected using either a standard Streptavidin-HRP conjugate or with a series of Streptavidin- POLY-HRP conjugates (30 minutes at ambient temperature on a shaker, standard color development with OPD for 15 minutes with acid stopping).
Conjugate concentrations were equalized to specific streptavidin concentration and applied in buffer with Casein Colloid, biotin free. POLY-HRP detection is optimized using a strong colloidal blocker to prevent non-specific interactions in detection systems that might suffer from high backgrounds due to protein-protein interactions Diluents made of raw, non-dialyzed animal sera are not compatible with Streptavidin-POLY-HRP's due to endogenous amounts of free or bound biotin that can bind the conjugate and make it unavailable to react with the desired biotinylated receptor (antibody, probe etc).
Analytes as hIgE, hTSH, hIL-6 (and other super sensitive cytokine assays) & Tetanus Toxoid have already been measured in femtogram levels using Poly-HRP conjugates. This enhanced sensitivity only requires 2.5 -3 hour assay protocols (two site ELISAs with monoclonal and polyclonal pairs) of routine colorimetric format. Based on currently available information and results, detection levels of 50-100 femtogram/ml are achievable with analytes as cytokines etc
The limit of detection in real DNA- hybridization with POLY-HRP coupled with precipitating chromogens achieves 10 fg target DNA/dot. As little as 10 picomoles\L of monobiotinylated Substance P can be detected on the ELISA-like model of a ligand-receptor assay. Both Streptavidin- & Protein A-POLY-HRP conjugates are suitable for immunohistochemical/cytochemical protocols, and are especially effective in the detection of minor surface membrane antigens. In comparison with other currently available technologies for high sensitive detection, POLY-HRP has an advantage of genuine simplicity, requiring only standard lab equipment and minor protocol variations. It is also compatible with other HRP activated chromogens (luminol, TMB, DAB, 4ClN etc)
PolyHRP Detection Users Guide: Ultra-sensitive two-site (sandwich) ELISA systems utilizing Streptavidin-PolyHRP conjugates. by D. Plaksin, SDT
POLY-HRP-STREPTAVIDIN DETECTION TECHNOLOGY
(BULK AVAILABLE FOR OEM USE).
cat#RDI-PHRP20-SA #RDI-PHRP40-SA2 #RDI-PHRP80-SA2
$410.00/1 mg $500.00/1mg $594.00/1mg
#RDI-PHRP20-SA2 #RDI-PHRP40-SA1 #RDI-PHRP80-SA1
$95.00/200ug $94.00/100ug $94.00/100ug
consists of 1 vial of PHRP20-SA2 (200ug), 1 vial of PHRP40-SA1 (100ug) & 1 vial of PHRP80-SA1 (100ug)
and 100ml specially prepared biotin-free casein diluent (cat#RDI-PHRP-DILX)
(limited one per customer)
strating dilutions of 1:5000 for ELISA recommended (most assays seem to work best at 1:5000-1:10,000, which would be the 1 milligram stock diluted to 1 Liter of conjugate!) See our HRP-conjugate stabilizer for long term diluted storage and our casein buffers for lower backgrounds.
-ideal for poly-HRP conjugates and conventional HRP conjugates diluted to working strength!
RDI-SA3-RHT is a novel product having 100% synthetic formula. It is absolutely protein-free reagent containing no organic substances of the animal, plant or microbial origin. It does also not contain phenol compounds or other volatile organic substances having distinctive "chemical odor". This product contains no listed hazardous or toxic products.
Code Number: #RDI-SA3-RHT $500.00/1Liter $438.00/liter 25+ Bulk on request
-limited number of 100ml evaluation sizes available for OEM use
Storage: +2�C/+8�C; neutral/borosilicate type I glass, PC/PET/PETG/PP/HD-PE
Expiration date: 04/2006+ (longer after re-check)
Appearance: Transparent liquid, 0.45/0.2/0.1�m-filterable
Odor: Weak, characteristic
Preservative/anti-microbial: 5-Nitro-5-Bromo-1,3-Dioxan (BND), 1500 ppm
QC release (ELISA-based NSB-eliminating and detection strength boosting activity use test): Passed
NOTE: This product is for in vitro research or further IVD manufacturing use only.
SA3-RHT is a novel product having 100% synthetic formula. It is absolutely protein-free reagent containing no organic substances of the animal, plant or microbial origin. It does also not contain phenol compounds or other volatile organic substances having distinctive "chemical odor". This product contains no listed hazardous or toxic products.
SA3-RHT will provide real-time stability to ready-to-use SA-PolyHRP conjugates at +2�C/+8�C (beyond one year) and room temperature (at least one year). SA3-RHT will support SA-PolyHRP conjugates also in the very low working strength concentrations down to 200-100-50 ng/ml. It will maintain activity of diluted SA-PolyHRP conjugates in working strength ready-to-use format at a level of 90-to-100% of the initial activity during at least 4 weeks stressing at 37�C. That way SA3-RHT makes SA-PolyHRP suitable for routine clinical IVD applications through finally making it compliant with mandatory IVD requirements on stressed/accelerated reagent stability at elevated temperatures.
RDI Division of researchd Industries Intl
San Jose, 95123 CA Snell ave 658
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