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A Technical Publication of SCANTIBODIES Laboratory, Inc.

Volume 1 Number 1

9336 Abraham Way Santee, CA 92071 USA (619) 258-9300 fax (619) 258-9366

In This Brief Scantibodies Heterophilic

Qualification Test for
Application of HBR
Blocking Reagent (HBR)
Heterophilic Antibody Introduction
InterferenceWhat is it? The existence of heterophilic antibodies and their potential for causing
The Unique Aspects of interference in immunoassays has been known for many years. The
HBR Over Conventional potentially devastating effects of false positive assay results on the
Blocking Methods patient and the medical community have more recently been delineat-
ed. The increasing use of the susceptible 2-site immunometric (sand-
Heterophilic Interference wich) assay format has led to growing concern over the problem.
What is Known? For this reason, Scantibodies Laboratory has developed a unique
Heterophilic Antibodies heterophilic blocking reagent (HBR) that minimizes the occurrence of
Sources heterophilic antibody interference.
Heterophilic Interference PresentingScantibodies HBR
How is it Detected? A Qualification Test for the Application of HBR
1. Do you develop sandwich samples that affects your assay?
What is the Difference
immunoassays? 6. Does your assay require a
between Heterophilic
2. Have you ever tested any blocking reagent? If so, should
Antibody and HAMA?
samples which have caused a the blocking reagent be a
What is a Blocking false positive result? HAMA blocking reagent or a
Reagent? 3. How is it possible to confirm heterophilic blocking reagent?
Characteristics of an Ideal that a sample is a false positive 7. Do you require a universal
Heterophilic Blocking sample and not a true positive blocking reagent which can be
Reagent sample? added to all assays?
4. Is your assay subject to HAMA 8. What criteria have you
The Scantibodies or heterophilic interference? established to evaluate a
Heterophilic Blocking 5. Have you identified the heterophilic blocking
Reagent (HBR) category of false positive reagent?
Performance Characteristics HeterophilicAntibody InterferenceWhat is It?
False Positive Identification Heterophilic antibodies are en- elevation of measured value that is
dogenous antibodies found in independent of the true analyte
Availability patients serum/plasma which can concentration, thus potentially
bind to immunoglobulins of other misclassifying samples. Although
species, including the species used they can affect various assay
to generate the antibodies used as formats, their main effect is on
reagents for immunoassays. These 2-site immunometric assays.
See Page 4. antibodies can interfere in im- These sandwich assays use at
munoassay, causing a spurious least two antibodies directed
SCAN-BRIEF/Heterophilic Blocking Reagent (HBR)

against different epitopes of an stances (mouse IgG, mouse analyte-specific monoclonal

antigen; one antibody is bound serum, nonspecific monoclonal antibody is used for blocking.
(or becomes bound) to a solid- antibodies, aggregated IgG, etc.)
phase, while the other is in to block the binding of the
Heterophilic Interference
solution and tagged with a signal human heterophilic antibody. All What is Known?
moiety such as 125-I, enzyme, of these approaches rely on the 1. The interfering factor is an
fluorophore, CLIA label, etc. affinity of the human heterophilic immunoglobulin, and both
Normally, antigen present in the antibody to affect the blocking. IgG and IgM heterophilic
sample bridges the two antibod- The affinity of the human het- antibodies have been reported.
ies so that the amount of labeled erophilic antibody is typically in 2. They occur at a high
antibody which becomes bound the K-value range of 105106. incidence. Depending upon
to the solid-phase is proportional HBR accomplishes its binding patient population, up to 40%
to the antigen concentration in by a totally different approach. incidence has been reported,
the sample. (See figure 1). The HBR is a specific binder that and the existence of at least
is directed against the human het- 10% incidence has been
A True Positive erophilic antibody. When HBR documented.
Label binds to the human heterophilic 3. The magnitude of the inter-
Antibody ference varies from sample to
antibody, the blocking is accom-
plished by steric hinderance. The sample, and may vary within a
HBR blocking is effected by the patient over time.
specific binder which has an 4. The heterophilic antibodies
affinity in the range of K = 109. are not species specific, but
Analyte The specific binding action of can bind to a variety of animal
the HBR, coupled with the thou- antibodies. Thus, the
sand times higher affinity in the interference is not limited to
Figure 1
reaction, results in the following monoclonal antibody-based
However, heterophilic anti- advantages of HBR over conven- assays.
bodies can also bridge the two tional blocking methods: 5. The interference is probably
antibodies independently of anti- 1. With HBR, less protein is mediated via the Fc region of
gen, resulting in an increase in required for blocking (no the antibodies used in the
bound labeled antibody concen- decrease in assay signal). assay.
tration. (see Figure 2). 2. HBR blocks more false 6. Heterophilic antibodies are
A False Positive positives than are blocked not a single, specific entity,
with conventional methods. but are a multi-component
3. HBR blocks all anti-species phenomenon. They comprise a
No (anti-rabbit, anti-goat, anti- mixed population of antibodies
Analyte sheep, as well as anti-mouse). which can cause interference,
4. HBR blocks all anti-subtypes some or all of which may be
of mouse monoclonals present in a particular sample.
(example: anti-mouse IgG1, 7. EIAs appear to be more
anti-mouse IgG2, etc.); susceptible than RIAs,
whereas, the use of one possibly because of the
Figure 2
monoclonal antibody such as increased modification of the
The Unique Aspects of IgG1, will only block human Fc region during conjugation.
antibodies to that subspecies of
HBR over Conventional Heterophilic Antibodies
monoclonal antibodies.
Blocking Methods 5. The use of HBR does not have Sources
Conventional, passive block- to be avoided in certain tests, There is a variety of possible
ing methods use nonspecific sub as in the case in which an causes for inducing heterophilic
SCAN-BRIEF/Heterophilic Blocking Reagent (HBR)
antibodies in patients, including: antibodies which can bind to Some are active blocking
Exposure to animals (e.g. animal antibodies and cause reagents in that they are directed
animal technicians, interference in immunoassays. specifically against the interfering
veterinarians, animal HAMA (human anti-mouse heterophilic antibodies, allowing
handlers) antibody) is one type of het- them to be used at lower concen-
Alternate animal contact erophilic antibody which can trations, (thus minimizing any
therapy (e.g. thymic cells, bind to mouse antibodies. adverse effects on the immunoas-
sheep cells, embryonic cells) say reaction), and with enhanced
Exposure to animal products
What is a Blocking effects on blocking kinetics.
(e.g. food preparation) Reagent?
Special diets (e.g. cheese) A blocking reagent is a prepara- Characteristics of an Ideal
Deliberate immunization (e.g. tion which, when added to Heterophilic Blocking
therapies, vaccinations, certain immunoassay reagents, prevents Reagent
imaging treatments). non-analyte mediated bridging 1. It should have the ability to
Rheumatoid factors can also of antibodies by heterophilic correct interference from all
act as heterophilic antibodies. interference. samples, irrespective of
Blood transfusions. There are two main types of whether it is caused by specific
Autoimmune diseases blocking reagent: anti-species activity,
Dialysis 1. HAMA type. rheumatoid factor, etc.
Patent medicines (OKT3) 2. It should be effective at low
These block only one specific
Maternal transfer concentrations so as not to
human anti-species antibody
Cardiac Myopathy interfere with the dose-
activity (e.g. human anti-mouse),
G.I. Disease (E. Coli) response curve.
and are typically normal serum,
3. It should not interfere with
Heterophilic normal IgG, monoclonal anti-
spike and recovery of the
InterferenceHow is it body not directed against the
analyte in human serum.
Detected? target analyte, etc.
4. It should not interfere with
They are passive blocking
A variety of methods have been linearity of dilution of a true
agents in that they are added in
proposed: positive patient sample.
excess concentration so that any
1. Discordant values from the 5. It should exhibit reproducible
specific anti-species antibodies
clinical picture or another performance with no lot-to-lot
present in the sample bind to
reference assay. variation.
these in preference to the specific
2. Poor dilution performance of 6. It should have long term
immunoreactants present in low
certain samples in an assay stability.
with normally satisfactory 7. Its cost should be such that
Such reagents are of limited use
performance. incorporation is economically
as they only remove one compo-
3. Removal of specific analyte viable.
nent of the heterophilic interfer-
from sample by affinity 8. It should be suitable for
ence, which is a multi-component
chromatography to see if signal incorporation into one of the
phenomenon. It is frequently
is abolished (if not, then it is a immunoreagents to obviate
observed that addition of mouse
false positive). the requirement for an
IgG (for example) to a double-
4. Addition of heterophilic additional reagent or a
monoclonal sandwich assay will
blocking reagent to see if separate sample pretreatment
only correct a portion of the
observed value is decreased. step.
heterophilic interference.
What is the Difference 2. True Heterophilic Blocking The Scantibodies
Between Heterophilic Reagents Heterophilic Blocking
Antibody and HAMA? These are formulations which Reagent (HBR)
Heterophilic antibody is a generic have the ability to remove all Scantibodies HBR is a novel
term used to describe all human types of heterophilic interference. reagent which has been specifi-
SCAN-BRIEF/Heterophilic Blocking Reagent (HBR)
cally designed to combat the e. rheumatoid factor False Positive Identification
problems of heterophilic antibody The broad specificity means Scantibodies has identified
interference in immunoassays. It that Scantibodies HBR is suit- donors which elicited false posi-
is a unique formulation of able as a Universal Reagent for tive results. These donors were
immunoglobulins targeted spe- 2-site immunometric assays using the most difficult to block with
cifically against heterophilic any of the commonly employed conventional methods (mouse
antibodies to neutralize their antibodies as solid-phase or IgG, etc.) These samples may be
interference. HBR is a defined labeled reagents. used to help identify false
reagent with a purity >95%. 2. Scantibodies HBR, being a positives and evaluate the vulner-
Unlike most of the non-specif- specific, highly purified ability of an assay format against
ic passive blockers which are reagent, can be used at very heterophilic interaction.
available from other suppliers low concentrations (sometimes Please note that false positive
(which need to be added in vast 100x or 1,000xs less than samples are very often assay spe-
excess to ensure that heterophilic normal mouse IgG) so that the cific. Therefore, these samples
antibodies will bind to them in assay signal is not adversely may not show false positives in
preference to assay-specific com- affected. certain assays.
ponents), Scantibodies HBR 3. Scantibodies HBR does not
is an active blocker. This for- interfere with spike and Availability
mulation of immunoglobulins is recovery of analyte in human Scantibodies HBR is supplied
targeted specifically against serum. with a data sheet detailing the
heterophilic antibodies and is 4. Scantibodies HBR does not following:
therefore able to neutralize their interfere with linearity of i. Description: IgG
interference. dilution of a true positive ii. Purification Method
patient sample. iii. Purity by SDS-PAGE
Performance 5. Scantibodies HBR is highly iv. Buffer composition
Characteristics reproducible from lot to lot. v. Storage conditions
1. HBR blocks false positive 6. Scantibodies HBR is stable for vi. Stability
reactions from a panel of: up to 5 years. vii. Appearance
a. human anti-mouse 7. The price of incorporation of viii. No preservatives
antibodies (HAMA) Scantibodies HBR in an assay HBR is supplied in liquid form.
b. human anti-goat antibodies is less than the cost of using For pricing details or to arrange
c. human anti-sheep mouse IgG or mouse serum. for a FREE sample of HBR,
antibodies 8. Scantibodies HBR can be please contact Scantibodies
d. human anti-rabbit added directly to assay buffers, Laboratory at the numbers
antibodies conjugates, etc. given below.


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9336 Abraham Way Santee, California 92071, U.S.A.
Phone: (619) 258-9300 Fax: (619) 258-9366 email:
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4 HBR Scan-Brief 4PAM08