PolyHRP Detection: a real Technology for UltraSensitive ImmunoAssays

Dmitry Plaksin and Elena Gromakovski

Stereospecific Detection Technologies (SDT) GbR

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 (Fig. 1)
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 (Fig. 1). 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). Fig. 2 shows how total number of active enzyme molecules in
different standard HRP (heterooligomeric to heavy heteropolymeric) and PolyHRP conjugates
may be compared.

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+) system1.

With the PolyHRP, there are actually no technical limitations, neither patent restraints.
This makes the PolyHRP detection a very open developer friendly item. Created by SDT,
PolyHRP detection technology has been known since the early 90s2,3,4,5,. Enhanced SA-
PolyHRP conjugates were commercially available for non-restricted research and bulk OEM
applications6,7,8,9 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

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

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 (Fig. 3), 1:5.000 (Fig. 4) and 1:8.000 (Fig.
5). 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. This can also be seen in
simplified use test described in our previous Application Note5 (Figs. 6, 7 & 8). 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 assays4, ligand-receptor assays and IHC applications.

1. R&D Systems 1996 Cytokine Catalog, p. 123.

2. European diagnostics companies taking note of Russian biotech startup, Diagnostics Intelligence, 3:5-6, 1991.

3. Vasilov RG, and Tsitsikov EN, An ultrasensitive immunoassay for human IgE measurement in cell-culture supernatants, Immunology
Letters, 26:283-284, 1990.

4. Kiseleva VI, Kolesnik TB, Turchinsky MF, et al., Trans-Diamminedichlorplatinum (II)- Modified Probes for Detection of Picogram
Quantities of DNA, Analytical Biochemistry, 206:43-49, 1992.

5. Plaksin DYu, and Gromakovska EG, Poly-HRP Conjugates: Novel Reagents for Ultrasensitive Detection in Immunoassays, Nucleic
Acid Hybridization and Ligand-Receptor Assay Systems, The Journal of NIH Research, 6:98, 1994.

6. Janssen Biochimica 1991-1992 Catalog, Cell Biology 3, item No. 28.005.69.

8. RDI Inc, PolyHRP detection in 93-97 ImmunoSite issues and in Internet: http:/www.researchd.com

9. RDI Inc, advertising in IVD Technology, May-June: 48, 1996.

10. Stanley C, and Lihme A, A High Performance Upgrade for ELISAs, European Clinical Laboratory, Feb.:8, 1995

Figure 1. Simplified (2D) schematic representation showing molecular design of SA-PolyHRP40 conjugate

Figure 2. Number of HRP monomer molecules in different standard and PolyHRP conjugates