FAQ (& Facts about Streptavidin-PolyHRP and related SDT products)
1) What is PolyHRP? Which polymer is used in making enhanced PolyHRP label?
What makes PolyHRP different from competitor’s products?
PolyHRP comprises a HRP homopolymer. It is made by covalent coupling of HRP
monomer molecules to each other. Proprietary process of controllable HRP
polymerization ensures that HRP molecules in the polymer maintain 100%
catalytic activity of the intact enzyme.
No other polymers or non-HRP molecules as e.g. dextran, polylysine or BSA used
in the manufacturing process. Inert backbone polymers used in the
competitor's products have no other function than a carrier function while
occupying a certain volume* in the structure of the conjugate. Consequently a
specific signal-generating activity of the homopolymer-based PolyHRP
conjugate is physically larger compared to the activity of heteropolymeric
HRP conjugates utilizing "cold" backbone polymers.
* consider that by the same molecular weight hydrodynamic radius of such
a typical backbone polymer as dextran is larger than radius of the most of the
regular proteins.
2) What do 20, 40 and 80 in the names and codes of SA-PolyHRP conjugates stand
for?
20, 40 and 80 indicate the number of HRP monomer molecules in a polymer
of respective size reflecting actual polymerization grade of the enhanced
PolyHRP label used in the manufacture of #SP20, #SP40 and #SP80 conjugate
items.
3) How many HRP molecules does one SA-PolyHRP conjugate have?
One SA-PolyHRP20 - all catalog items having code #SP20 with C (concentrate,
1 mg/ml stock) or different D (diluted to 50 µg/ml, 10 µg/ml or 2 µg/ml in SA1
or SA3-RHT stabilizers) extensions - has on the average 100 HRP molecules,
SA-PolyHRP40 (#SP40) has 200, and
SA-PolyHRP80 (#SP80) - 400.
20, 40 and 80 are multiplied by 5 because one SA-PolyHRP conjugate contains on
the average five identical HRP homopolymer blocks of the respective size.
4) How many HRP molecules are in Aminoethyl-PolyHRP40?
#AEP40 comprises aminated PolyHRP40 label. It contains on the average
40 HRP monomer molecules.
5) How do compare detection power of the different SA-PolyHRP conjugates?
Basically, SA-PolyHRP40 is approx. two times stronger than SA-PolyHRP20, and
SA-PolyHRP80 is approx. two times stronger than SA-PolyHRP40 and approx. four
times stronger than SA-PolyHRP20. This however suggests that the assay system
of comparison is well optimized with the each specific SA-PolyHRP item. In the
practice, depending on specific assay conditions difference between 20-40 and
40-80 may be smaller (more often) or larger (rather seldom) than factor 2.
6) Which SA-PolyHRP should I take aiming at maximum sensitivity in ELISA?
SA-PolyHRP80
7) What is the expected sensitivity level with PolyHRP (in double antibody
sandwich for an antigen)?
There is no straightforward answer on this question. Sensitivity of the assay
will strongly depend on the quality of the given capture and detector
antibody. For instance, one customer reported 0,5 pg/ml analytical
sensitivity (peptide hormone) with just SA-PolyHRP20 in routine colorimetric
ELISA with TMB. A substrate system and other assay reagents will play
important role, too. PolyHRP practically enables achieving low picogram and
sub-picogram/femtogram sensitivity levels. Typically, calibration curves in
Ultra-Sensitive (US) and/or High Sensitivity (HS) ELISA test kits using
SA-PolyHRP often begin with 10-20 pg/ml (giving with TMB signals >2,000 OD
units) going down to 100-50 fg/ml as the end calibrator points.
8) Which ImmunoPlates should I use when developing HS ELISA with PolyHRP
detection?
High Binding Capacity polystyrene plates as e.g. Corning/Costar Cat. # 3590
(transparent for colorimetric ELISA) or equivalent Certified Surface
Chemistry plates of Greiner or Nunc (that is what we tested; it is of course
possible that good High Binding immunoplates are currently available form
other manufacturers).
PolyHRP detection does not work properly with Medium and Low Binding plates.
On the Low or Medium Binding Capacity plates SA-PolyHRP may even work not
better than conventional SA-HRP conjugate.
9) Which coating conditions are optimal for PolyHRP detection?
Apart of the coating buffer and time (that are basically standard with the
most of the capture antibodies - 50-100 mM Na-CBB pH 9.5-9.6, overnight / ~18
hours incubation at +4°C) very important parameter is a concentration of the
capture antibody. Optimal concentration of the capture antibody in the
coating procedure is 1µg/ml that is effective for both whole IgG and F(ab')2
antibody fragments. Paradoxically, adsorption of the capture antibody from the
larger concentrations - as from often traditionally used 5 or 10 µg/ml - will
result in the essentially lower sensitivity.
10) What is the best buffer applicable with SA-PolyHRP?
Best products are Stabilizers SA1-HTS, SA1-HTS-E, SA3-RHT and Universal
Casein Diluent/Blocker UCDB. Said ready to use products are certified biotin
free products that were specially validated to ensure highest obtainable
sensitivities with SA-PolyHRP conjugates. Dedicated SA-PolyHRP Stabilizers
SA1-HTS, SA1-HTS-E and SA3-RHT will moreover maintain activity of the
prediluted and/or final working strength conjugates beyond levels compliant
to the regular IVD stability requirements. UCDB has no extra stabilizing
function and is recommended for making daily work dilutions.
11) Why other blocking buffers and stabilizers are not recommended for
working with SA-PolyHRP?
Other liquid products may contain (and as a rule do contain) endogenous
biotin while SA1-HTS, SA1-HTS-E, older SA1 and UCDB are TRUE biotin free
products - endogenous biotin is completely removed in proprietary
process of their manufacturing. SA3-RHT is 100% synthetic product
and does not contain biotin by definition.
Presence of endogenous biotin in conventional casein, gelatin, BSA and -
most pronouncedly - animal serum based liquid products is often underestimated,
especially with SA-PolyHRP. Reason is that due to the strongly shifted
towards enzymatic moiety HRP/SA ratio molar concentration of
Streptavidin (or biotin binding sites) in SA-PolyHRP conjugate is essentially
smaller than in the conventional SA-HRP. Consequently, SA-PolyHRP is essentially more
sensitive to the inhibiting through endogenous biotin compared to the
conventional SA-HRP. Diluents having smaller/trace biotin burden may work
well with SA-HRP but will strongly inhibit SA-PolyHRP.
Besides, very slow kinetics of reacting between streptavidin and biotin in
the range of the lower and/or trace endogenous biotin concentrations
adversely affects robustness of the assay test. Thus, results of the
test will vary depending on the time interval between diluting of the conjugate
(into final working strength form) and applying it to the test
device/immunoplate. Half an hour exposition before work in the assay may
not yet cause any visible inhibition, while conjugate diluted in 3 hours
prior to detection may show appreciable drop in activity through slow
inhibition by endogenous biotin present in diluent. Next day, after 24-hour
exposition drop in activity may appear dramatic. This does never occur with SA3-RHT,
SA1-HTS, SA1-HTS-E, SA1 and UCDB.
12) What is the optimum dilution of SA-PolyHRP conjugates in ELISA?
Dilution of the stock SP20/40/80C 1 mg/ml items giving maximum sensitivity is
about 1/4,000-1/5,000 (250-200 ng/ml). This corresponds to saturation in the
most of the sandwich assays. Saturating concentration may differ depending on
specific assay design and reagents. Some customers prefer to work beyond
saturation going down to 40-25 ng/ml which concentrations correspond to
1/25,000-1/40,000 dilutions. In the latter case the goal is obviously a cost
saving effect rather than higher sensitivity - smaller working strength
concentrations of SA-PolyHRP may yield still higher or equal sensitivity of
the assay compared to the larger concentrations of conventional SA-HRP.
13) Is SA-PolyHRP applicable in the assays on a microarray platform?
Yes, all SA-PolyHRP conjugates are very well applicable in the multiplex
microchip assay systems for both amplicon detection and direct DNA
hybridization, as well as for the determination of antibodies and antigens.
14) What is the difference between your SA1-HTS, SA1-HTS-E, SA1 and SA3-RHT stabilizers?
SA1-HTS, SA1-HTS-E (enhanced version of SA1-HTS) and older SA1 are
biotin free casein based stabilizers. They have excellent
NSB-blocking power towards true protein-protein NSB.
Dedicated SA-PolyHRP stabilizers of SA1 series ares good for the real time
storage of SA-PolyHRP in both pre-diluted and ready-to-use formats
at +4°C (providing stability far beyond one year) and room/ambient
temperature (many months - up to one year depending on specific item
and specific storage conditions - newer SA1-HTS/SA1-HTS-E better than older SA1).
SA3-RHT is a fully synthetic stabilizer that will maintain stability of
SA-PolyHRP at elevated (+37°C) temperatures during longer
time - up to several months. It will also work well with diluted to
the ready-to-use working strength conjugates giving sufficient
NSB blocking in e.g. double antigen sandwich ELISA provided that
both capture antibody and detector antibody-biotin are clean
and well balanced reagents. It may not solve all background problems
in more complex assay systems using composite immunosorbents (like
HIV-1/2 or HCV Ag/Ab 4G tests) or gen-chips (amplicon detection).
In the latter cases customers using SA-PolyHRP in their IVD products
(where high temperature stability is a kind of mandatory requirement) do prefer to incorporate
in the assay test kits pre-diluted (100X-20X) conjugates in SA3-RHT that will
eventually be diluted to the final working strength concentration in
SA1-HTS/SA1-HTS-E or SA1.
SA3-RHT--->+SA1-HTS combination works well when high temperature
stability is a requirement and assay background is an issue.
15) I obtain with SA-PolyHRP80 higher sensitivity in my ELISA for an antigen
but background also becomes larger. Is there any way to improve
signal-to-noise ratios? What should I do to optimize performance?
When managing backgrounds with SA-PolyHRP it is important to understand that
their major reason is a non-specific binding (NSB) of the first biotinylated
detector reagent that triggers NSB of SA-PolyHRP. As a rule SA-PolyHRP
alone - with no detector-biotin applied - does not cause backgrounds provided
it is used with an effective PolyHRP diluent/stabilizer/blocker as
UCDB, SA1, SA1-HTS, SA1-HTS-E or SA3-RHT and the capture reagent is clean.
Therefore blocking of the NSB of biotinylated detector antibody is the first
method of choice in eliminating backgrounds.
Specific practical recommendations troubleshooting background and
allowing for improvement of the overall performance in a double
antibody sandwich ELISA for an antigen are as follows -
- run differential controls in order to sort out (i) possible matrix effects
with your given specimen, (ii) direct NSB of SA-PolyHRP - without incubation
with detector Antibody-biotin (again, this should be very low, if capture
antibody is good enough) and (iii) aggregate background in system with both
Antibody-biotin and SA-PolyHRP incubations. With negligible (ii) any
significant value of (iii) will actually reveal an impact of the NSB of
biotinylated detector Antibody on cumulative backgrounds
- apply detector Antibody-biotin reagent in a diluent system containing
effective blocker of amorphous protein-protein NSB such as e.g. casein.
Consider our CBC2 based diluents, blockers and stabilizers for biotinylated
antibody (AADB, AA1, AA1-HTS and AA1-HTS-E)
- optimize working strength concentration of detector Antibody-biotin reagent
in checkerboard titration experiments. Aiming at true HS performance try to
hold working strength concentration of SA-PolyHRP at basically optimal level
= 200-250 ng/ml which is a saturating concentration that will yield best HS
performance in the most ELISA systems with 30-min incubation at room
temperature and shaker mode. Running away from the background by means of
lowering SA-PolyHRP concentration is not an expedient method as it will
make the detection weaker
- optimize biotinylation grade of your given detector antibody. Excessive
biotinylation of the IgG will adversely affect HLB of the labeled detector
antibody that may provoke higher backgrounds. Consider biotinylation
reagents with longer hydrophilic spacer arms as e.g. PEGn-biotin NHS esters
- presence of heavier than 150 kDa aggregates in the capture and detector
IgG antibodies may result in increased backgrounds. Complexes with trace
amounts of Protein A/G and/or polycations that may be present in IgG
preparations purified on Protein A/G- or DEAE-resins may cause especially
rigid backgrounds. Polishing of IgG antibody in SEC/GPC making it free
of > and < 150 kDa material may significantly improve HS performance.
SEC/GPC polishing is well advisable with both capture and detector antibody
- the same with replacing IgG - detector or capture or both - with the
smaller F(ab') fragments. This is also generally helpful in case of emerging
matrix effects associated with heterophilic and HAMA antibodies
- use High Binding Capacity immunoplates
- use ~1 µg/ml IgG and/or F(ab')2 in coating procedure with capture antibody
- do not incubate plate with SA-PolyHRP at temperatures higher than room
temperature. Incubation at 37°C will not improve but rather worsen
performance. Higher temperature and longer time do not make any sense with
SA-PolyHRP and will only increase backgrounds.
- shaker mode - at ~700(+/-) rpm on the orbital ELISA shaker - is strongly
recommended as it may give up to 2-fold sensitivity increase compared to
static incubation and helps to keep background at acceptable level
- hydromechanically effective stringent wash of the immunoplate after
incubation with SA-PolyHRP is important. We recommend washing 5 times.
Using wash buffer with smaller than isotonic or larger ionic strength
may help. Thus, for instance, we successfully use in our lab 1/5 (~30 mM)
PBS-Tween comprising 1 part 0,15M PBS pH 7,3 + 4 parts deionized water +
0,05% Tween-20 + 0,05% BND which also makes the wash work more cost effective
in terms of the salts consumption
16) Despite the higher positive signals with SA-PolyHRP I also see an
increased background in my assay system. I saw no or less background when
I worked with a conventional, non-polymeric SA-HRP conjugate. What is the
reason for emerging background?
The reason is simple - essentially stronger SA-PolyHRP makes visible
non-specific binding of the biotinylated detector antibody (or antigen) that
was not seen with the weaker standard conjugate.
17) What is the lot to lot variability of the PolyHRP conjugates?
None or negligible. Our core SA-PolyHRP product line is well recognized
in the IVD and Life Science Research industries for its very high lot to lot
consistency.
Each new master batch is subject to standardization at the end of the
production process. We very carefully adjust performance in our QC release
ELISA use tests allowing no lot to lot variations larger than regular CVs
appropriate to a well done colorimetric ELISA test. In other words we achieve
such a level of the lot to lot consistency that there are no differences in
performance that could be visible in ELISA. Since our QC release
ELISA use tests correlate with specific ELISA test systems very well our
customers are unlikely able to see any significant difference in performance
between different lots of SP20, SP40 and SP80 in their specific assay test
kits.
18) For the SA-POLYHRP40, do you mean it can be treated as a big molecule which consists of one
Streptavidin linked to a polyHRP40(40*5 HRP)? I am wondering what is the ratio of the SA to PolyHRP40? How is this ratio measured?
A conjugate with 200:1 mol HRP:SA ratio will unlikely work.
In SA-PolyHRP multiple SA molecules are coupled onto the surface of the core
HRP homopolymers.
Thus, the average molar ratio HRP:SA in e.g. SA-PolyHRP40 is
~5*40 HRP : ~50 SA
It has an aggregate nature, i.e. translating it into ~4:1 would
actually not be correct.
19) I have a question about the storage of #SP20C/#SP40C/#SP80C. Your CoA recommends
Storage: "-20°C (-15°C/-25°C). Do not store below -25°C!"
What is the implication for this storage requirement? Is product degraded below -20°C/ -25°C?
No, product will not degrade at temperatures below -25°C but we think there might be a risk that
freezing/thawing will adversely affect it.
All our cryopreserved items remain liquid at recommended storage regimen as indicated above. This
enables readily and accurately aspirating needed volume right from the original storage vial. It is an evident
advantage over the need of making aliquots with a material that turns frozen below 0°C.
Another reason for supplying conjugates in the liquid cryopreserved formulation is that it
provides excellent stability. Our QC has been monitoring backup samples of all manufactured
masterbatches beginning from the end of the '90s. We do not observe any drop in activity for over 15-16 years.
At the temperatures lower than -25°C product will become frozen.
We did not investigate influence of freezing/thawing on the activity and performance of our
PolyHRP conjugates. Giving their large size we assume that it would perhaps not be good for them. Therefore we just
recommend avoiding situations where material can get frozen, i.e. storage at temperatures below -25°C.
20) Would you be able to provide the molecular weights for the
Streptavidin-PolyHRP product line?
Average MW of SA-PolyHRP20 is about 5000 kDa,
Average MW of SA-PolyHRP40 is about 10000 kDa and
Average MW of SA-PolyHRP80 is about 20000 kDa.
21) What is the average molecular weight of casein in the UCDB solution?
I experienced steric hinderance of my protein when I used BSA as a blocker so I wonder if casein is smaller than BSA or not.
UCDB is made of the highly purified bovine milk casein. It is IgG and biotin free and
is composed predominantly of the filterable alfa-casein (23 kDa) and beta-casein (24 kDa), although
kappa-casein (19 kDa) and gamma-casein are also present. Despite given MWs look smaller than MW of BSA (67 kDa)
casein molecules display both strong amphipathic and amphoteric (while as phosphoprotein clearly
more acid with pI ~4,1-4,5) features and in aqueous buffers are prone to
pseudo-micellar behavior actually building relatively stable multi-molecular complexes
(non-covalent homopolymers) that are larger than BSA in size.
Nonetheless, we do not know a single case where UCDB being an excellent blocker of the
true (protein-protein) NSB would inhibit specific antigen-antibody reactions. A risk of occurring "steric hindrance"
speculatively attributed to the molecular size of the blocker in essentially mechanistic/simplistic way is in the most
cases overestimated and/or exaggerated. Again, UCDB supports specific antibody-antigen binding very well, whereas
it effectively blocks non-specific amorphous protein-protein interactions.
22) I was hoping to get some information on your AEP40 product. I wondered what is the molecular mass? Or how can I work out the molar ratio of AEP40 to IgG antibody when performing conjugation?
I would typically use a 12x molar ratio of HRP to IgG antibody when carrying out standard conjugation, giving antibodies with ~1.5-3 HRP/molecule of Ab. How would I go about this with AEP40?
Molecular mass of AEP40 is on the average ~1,6 kDa. Size dispersion may vary from lot to lot in pretty broad limits (is not subject to QC Release).
With the whole IgG - applying 2-IT or SATA/SATP/SAT(PEG)n thiolation - mass ratio in coupling is generally 1 (mg, µg, whatever) IgG + 1 (mg, µg, whatever) Mal-HRP40;
molar ratio is respectively ~1:4.
This is an ave and /or starting point compromise compromise: <1+1 would give stronger conjugate at smaller yield loosing larger portion of antibody
remaining unbound whereas e.g. 1+1,5 or 1+2 would yield larger amounts of generally weaker conjugates.
Similarly, with 2-MEA mass ratio in coupling is generally 1 (mg, µg, whatever) rIgG-SH + 1 (mg,µg, whatever) Mal-HRP40.
It translates into molar rIgG-SH/HRP ratio ~1:2 assuming ~80kDa for 1/2IgG and ~40 kDa for
HRP monomer whereas molar ratio of starting IgG to HRP (in AEP40) is the same ~1:4.
23) Are you able to provide the host species (if applicable) of Streptavidin-PolyHRP40?
Streptavidin-PolyHRP40 is a synthetic product made in proprietary process of covalently coupling SA to HRP40 polymers.
HRP is a native glycoprotein of the plant origin derived from horseradish (Armoracia rusticana, syn. Cochlearia armoracia) roots.
Streptavidin is native (from Streptomyces avidinii) and/or recombinant (produced in E.coli) - we may use both.
24) Do you have any data or recommendations using these products for IHC, if using a biotinylated secondary antibody?
Are these products suitable for IHC procedures (if using a biotinylated secondary antibody)?
Are there issues penetrating the cell due to the large size of the compound?
Do you have any data if one would select the smaller poly HRP products over the more active HRP-80?
Are there any cell penetration concerns with the larger version that have been observed?
Could you supply titer/concentration info for these products?
For the Goat anti mouse IgG (H+L) Poly-HRP40 product, is there data on how many HRP molecules are on each antibody and is there reason to believe that this would perform better or worse in an IHC protocol than the products in question above, question 1?
If so do you have a recommendation for a secondary AB?
Are you able to provide the host species (if applicable) of Streptavidin-PolyHRP40?
SA-PolyHRP conjugates are practically not applicable in IHC staining and CISH. They may find limited use
in HS detection of low abundance membrane antigens on the whole cells and/or cell monolayers/arrays but
they are just bad at penetration in FFPE tissues due to their very large size. Also endogenous biotin would
be a nasty problem with extremely sensitive SA-PolyHRP compared to conventional LSAB. All in all, we do
basically not recommend using SA-PolyHRP20/40/80 in IHC applications.
For IHC staining and CISH we offer a specialty/dedicated product line featured by AmpliStain™ and StrongZyme™ items -