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Purpose
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An increase in the sensitivity of immunoassays has always been considered one of the major trends in the development of IVD technologies. A plenty of analytes relevant to both clinical laboratory and research diagnostic applications should ideally be measured with highest possible sensitivity performance. In assaying many infectious antigens, such as for example HBsAg or microbial toxins, environmental pollutants, tumor markers, hormones, growth factors, etc., sensitivity is the most important parameter that ultimately determines an informative value of the test.
Cytokine research has become especially indicative of above trend. The fact that many cytokines are distributed in natural biological fluids in low picogram - femtogram concentrations challenges researchers and IVD manufacturers.
Over the last decade, several detection enhancement technologies have been developed. User labs set up additional requirements essential for their acceptance in practice. As example, recent advances in ELISA include enhanced chemiluminescence based substrate systems for both Alkaline Phosphatase (AP) and Horseradish Peroxidase (HRP), enzymatic labels that remain the most commonly used non-radioactive tracers. The are of cost control and limited budgets has made many end user labs hesitant towards purchasing newer insrtrumentation required with these latter techniques. Actually, well established colorimetric ELISA is still recognised as state of the art method in the marketplace of both routine Clinical Lab Diagnostics and Life Science Research.
PolyHRP is an enhanced enzymatic label comprising covalent HRP homopolymer. Proprietary synthesis process has been developed to produce HRP homopolymers of different size. Three standartized intermediates are currently made. These are PolyHRP20, PolyHRP40 and PolyHRP80 named according to the average polymerization range characteristic of each item. PolyHRP is stable in aqueous solutions and has a loose linear-branched structure which maintains 100 % activity of the composite enzyme. In the capacity of enhanced label PolyHRP can be covalently coupled to virtually any anti-analyte.
PolyHRP conjugates quantitatively deliver many signal-generating catalyst molecules to one bound analyte molecule. This results in multiple detection enhancement which is directly proportional to HRP polymerization range.
Three different Streptavidin-PolyHRP (SA-PolyHRP) conjugates have made a product line of universal reagents currently available for ultrasensitive detection in ELISA and related assay methods.
SA-PolyHRP conjugates are built up from on an average five identical HRP homopolymer blocks chemically coupled to multiple streptavidin molecules distributed mainly on their outer surface. The estimated average number of HRP monomer molecules in SA-PolyHRP20 conjugate is 100 (20 X 5), in SA-PolyHRP40 - 200 (40 X 5) and in SA-PolyHRP80 - 400 (80 X 5).
Thus, PolyHRP brings in reaction with substrate development system much larger number of enzyme label molecules (per one bound analyte molecule) than conventional conjugates do. In contrast to other methods, such as bridge, complex, multilayer and catalysed reporter deposition techniques, PolyHRP detection enables the achievement of much higher sensitivities in one or two (if biotin-streptavidin is used) steps, i.e. assay enhancement is set up by the design of the detecting conjugate itself.
Technically PolyHRP detection is very simple. There are no changes of principle that would affect an assay scheme (number of steps, incubation intervals). Composite kit reagents are essentially the same as in conventional ELISA using standard HRP conjugates. Similarly, PolyHRP detection may be used with any of currently applicable HRP substrates including OPD, TMB, luminol, enhanced luminol, DAB, AEC, newer fluorimetric substrates, etc. This also means perfect compatibility with existent routine and emergent modern (e.g. luminometric) ELISA instrumentation. Being also sufficiently stable (with, again, no difference of principle as compared to conventional HRP conjugates) PolyHRP meets the most practicable requirements. Thereby it gains a strong advantage over competing technologies, such as AP based enzymatic cascade amplification using cyclic cofactor regeneration (NADH/NAD+) system.
With the PolyHRP, there are actually no technical limitations, neither patent restraints. This makes the PolyHRP detection a very open developer friendly item. PolyHRP detection technology has been known since the early 90s. Enhanced SA-PolyHRP conjugates were commercially available for non-restricted research and bulk OEM applications since 1991 and are currently used by several IVD manufacturers in High Sensitivity (HS) cytokine ELISA test kit lines. Frequently there is no disclosure of polymeric nature of Streptavidin-HRP present in the kits, neither mentioning SDT as original conjugate producer.
Recently, a similar reagent, i.e. conjugate exploiting the same simple and effective approach to detection enhancement, but having a different, patented heteropolymeric design (HRP and Streptavidin conjugated to a dextran backbone), has been offered to assay developers.
To compare the performance of the newer SA-dextran-HRP conjugate with our current SA-PolyHRP product line, we have done a series of experiments in ELISA using antibodies and antigen standard from Pelikine Compact ELISA Kit of CLB Reagents (Amsterdam), originally designed for quantitation of human IL-13 in 0,5 - 50 pg/ml concentration range using detection with SA-PolyHRP20 diluted 1:10.000, 3.5-hour shaker protocol and TMB substrate system. Specially developed for (SA-)PolyHRP detection proprietary biotin-free Casein Buffer was used for diluting all reaction participants. Four conjugates of comparison were applied at different dilutions equalized on HRP content. Weaker chromogen, OPD, 15 min., was used instead of more sensitive 30-min. TMB development.
The results are summarized in three selected series which show PolyHRP vs. dextran conjugate performance at working dilutions 1:1.000, 1:5.000 and 1:8.000. In all three series, covering respective hIL-13 concentration ranges of 0.25-16 pg/ml, 0.5-32 pg/ml and prolonged 0.5-64 pg/ml, PolyHRP80, 40 & 20 show distinctive distribution in detecting activities. The given pattern is generally reproducible in different assay systems. HRP polymerization range accounts for the assay sensitivity. PolyHRP80 features the most sensitive detection, enabling reliable quantitation of analyte in low picogram- femtogram concentrations, with the best discrimination between lower calibrator points. PolyHRP40 secures the second place after PolyHRP80. PolyHRP20 shows naturally smaller activity in the given PolyHRP rank. Dextran-HRP occupies a position rather close to PolyHRP20. It offers no superior detection efficacy by similar background levels. No visible advantage over SA-PolyHRP20 could be observed with newer SA-dextran-HRP conjugate, as both reagents yield comparable overall sensitivities by approximately the same level of signal-to-noise ratios
Based on available experimental data, one has to conclude that SA-PolyHRP80 & 40 show superior detection performance over both SA-PolyHRP20 and SA-dextran-HRP. Neither of two latter conjugates outperform each other. Although all four reagents of comparison are certainly applicable in HS cytokine ELISAs, PolyHRP80 & 40 appear to be the better options. They are apparently more effective when precise measurement of analytes in femtogram concentrations is really concerned.
Other analytical methods, where PolyHRP detection can have an advantageous use in the capacity of a universal tool for sensitivity increase, include highly sensitive DNA/RNA-hybridization assays, ligand-receptor assays and IHC applications.
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