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Department of Pathology and Laboratory Medicine, University of Pennsylvania, 3400 Spruce St., Philadelphia, PA 19104. Fax 215-662-7529; e-mail larry_kricka{at}path1a.med.upenn.edu
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Abstract |
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 Top Abstract IntroductionEtiology of Anti-Animal...HAMAAntibodies to Other SpeciesStrategies to Eliminate Anti...ConclusionReferences |
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Issues: Anti-animal antibodies (IgG, IgA, IgM, IgE class, anti-isotype, and anti-idiotype specificity) arise as a result of iatrogenic and noniatrogenic causes and include human anti-mouse, -rabbit, -goat, -sheep, -cow, -pig, -rat, and -horse antibodies and antibodies with mixed specificity. Circulating antibodies can reach gram per liter concentrations and may persist for years. Prevalence estimates for anti-animal antibodies in the general population vary widely and range from <1% to 80%. Human anti-animal antibodies cause interferences in immunological assays. The most common human anti-animal antibody interferent is HAMA, which causes both positive and negative interferences in two-site mouse monoclonal antibody-based assays. Strategies to prevent the development of human anti-animal antibody responses include immunosuppressant therapy and the use of humanized, polyethylene glycolylated, or Fab fragments of antibody agents. Sample pretreatment or assay redesign can eliminate immunoassay interferences caused by anti-animal antibodies. Enzyme immunoassays, immunoradiometric assays, immunofluorescence, and HPLC assays have been designed to detect HAMA and other anti-animal antibodies, but intermethod comparability is complicated by differences in assay specificity and lack of standardization.
Conclusions: Human anti-animal antibodies often go unnoticed, to the detriment of patient care. A heightened awareness on the part of laboratory staff and clinicians of the problems caused by this type of interference in routine immunoassay tests is desirable. Efforts should be directed at improving methods for identifying and eliminating this type of analytical interference.© 1999 American Association for Clinical Chemistry
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Introduction |
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TopAbstractIntroductionEtiology of Anti-Animal...HAMAAntibodies to Other SpeciesStrategies to Eliminate Anti...ConclusionReferences |
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The Food and Drug Administration (FDA) has recognized the importance of anti-animal antibodies such as HAMA. In its "review criteria for assessment" documents, the FDA recommends that the labeling (e.g., package insert) of an in vitro diagnostic device list as a limitation the following: "As with any assay employing mouse antibodies, the possibility exists for interference by human anti-mouse antibodies (HAMA) in the sample" (19). In more recent documents, the FDA recommends the following: " If the assay kit employs mouse monoclonal antibodies, include a warning that specimens from patients who have received preparations of mouse monoclonal antibodies for diagnosis or therapy may contain human anti-mouse antibodies (HAMA) and may show either falsely elevated or depressed values when tested" (20).
This review surveys the scope and extent of human anti-animal antibody interferences, and examines methods to eliminate formation of these antibodies and reagents and sample pretreatment protocols designed to combat analytical problems attributable to their presence in biological fluids. It focuses on human antibodies reactive with animal immunoglobulins (e.g., mouse and goat), and the reader is referred to previous reviews for additional information on this general topic (2)(3)(8)(21)(22).
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Etiology of Anti-Animal Antibodies and Mechanism of Interference in Immunoassays |
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TopAbstractIntroductionEtiology of Anti-Animal...HAMAAntibodies to Other SpeciesStrategies to Eliminate Anti...ConclusionReferences |
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) (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34). In addition, some recombinant proteins
are affinity purified on immobilized monoclonal mouse antibody columns,
and the possibility exists for some of the mouse monoclonal antibody to
detach and copurify with the protein (35).
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Blood transfusion is also associated with an increased incidence of anti-animal antibodies. A study of 2829 participants in a population health survey revealed that 14.4% of the participants who had been transfused were anti-animal positive, compared with 10.4% of the participants who had never received a blood transfusion. This difference was presumably attributable to infusion of preexisting human anti-animal antibody or as a result of infusion of a foreign antigen present in the unit of blood (36).
Vaccination against infectious diseases is another route by which animal protein antigens may be inadvertently presented to the immune system and trigger antibody formation. In the US, chick embryo or egg cultures are frequently used in vaccine production, and residual chicken protein may be present in vaccines, whereas in Europe, some vaccines contain rabbit serum, e.g., rubella vaccine in France, and multimicrobial vaccine (Bruschettini vaccine) in Italy (32)(34).
The administration of unconventional therapies is also a route to immunization with animal protein. For example, a patient developed anti-rabbit antibodies following injections of "antireticulocytoxique", which is a lyophilized serum obtained from rabbits injected with homogenates of human bone marrow and spleen and is intended as a tonic to improve senescence and to reduce fatigue and debilitation (34).
Noniatrogenic causes of anti-animal antibodies include maternal transfer across the placenta to the unborn child (37)(38), animal husbandry or the keeping of animals as pets (39), and the transfer of dietary antigens across the gut wall in conditions such as celiac disease (40)(41). Anti-animal antibodies are also more common in multiparous females (36), and a high incidence of human anti-animal antibodies has also been observed in association with certain disease states, e.g., idiopathic cardiomyopathy (42).
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HAMA |
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TopAbstractIntroductionEtiology of Anti-Animal...HAMAAntibodies to Other SpeciesStrategies to Eliminate Anti...ConclusionReferences |
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A mouse monoclonal antibody is a foreign protein, and in vivo it can
trigger an immune response to produce HAMAs. Consequently, it is not
surprising to find that in the vast majority of clinical trials with
mouse monoclonal antibodies, many of patients were found to have
developed a HAMA response following administration of the antibody
(Table 2
) (50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65)(66)(67)(68)(69)(70)(71)(72)(73)(74). The specificity of a monoclonal antibody
permits targeting of a particular cell type or tissue. For example,
monoclonal anti-OKT3 is widely used in transplantation as an
immunosuppressant because it binds to the CD3 surface antigen on T
lymphocytes and interferes with the ability of the cell to recognize
foreign antigens. A further refinement is to attach drugs, toxins, or
imaging agents to a monoclonal antibody and to use the resulting
conjugates for the targeted delivery of these agents in high
concentration to specific sites in the body (e.g., tumor tissue). Other
proposed applications of mouse monoclonal antibodies include the use of
antibodies with enzyme activity ("abzymes") as antiviral,
anticancer, and thrombolytic therapeutic agents (75).
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antibody type and specificity
Human anti-animal antibody responses can be of the IgG, IgA, IgM,
or rarely, the IgE class (76)(77)(78)(79). In the case of
anti-animal antibodies elicited by animal immunoglobulins, the human
anti-animal antibody can have anti-idiotype or anti-isotype
specificity. Anti-idiotype antibodies are directed against the
hypervariable region of the immunoglobulin molecule, and anti-isotype
antibodies are directed against the constant regions (Fig. 1
). Anti-anti-idiotype antibodies can also be produced. These
recognize the binding region of the anti-idiotype antibody; thus, the
antigen-binding region of an anti-anti-idiotype antibody resembles the
antigen that elicited the original anti-idiotype HAMA
(78)(80). Additionally, the possibility exists
for the formation of antibodies with specificity for antigens or
neoantigens on the conjugated monoclonal antibody, e.g., anti-ricin
antibodies (54), or with specificity for a chimeric antibody
(81)(82).
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Generally, isotype antibodies may be more common than idiotype antibodies. For example, in one study involving 141 patients, 29% were positive for HAMA after treatment with 99 mTc-BW 431/26. In 80% of these positive patients, the HAMA response was predominantly anti-isotypic, and in 20% it was predominantly anti-idiotypic (57). In contrast, a study of a group of nine patients who developed HAMA 7–15 days after beginning treatment with B-E8, an IgG1 directed against interleukin-1 revealed that all nine of the patients developed IgG anti-idiotype antibodies against B-E8. Four of the patients also developed IgM anti-idiotype antibodies (50).
magnitude and duration of response
The magnitude and duration of an HAMA response shows great
variability, and serum concentrations in the microgram per liter to
gram per liter range have been detected
(83)(84). Anti-animal antibodies can persist in
blood for several months after exposure to mouse immunoglobulin. For
example, in one study (85), an IgG HAMA was still detectable
after 10 months, and in another study (86), it was
detectable up to ~30 months after immunoscintigraphy. In patients who
have developed an human anti-animal antibody response, B memory cells
that express the antibody would presumably remain for years and would
be activated upon reexposure to the antigenic stimulus.
prevalence of hama and anti-animal antibodies
The true number of people positive for anti-mouse antibodies is
not known, and estimates vary widely (<1–80%) (87)(88)(89)(90)(91)(92)(93).
One problem has been the choice of method to detect anti-animal
antibodies. There is no universal assay for this type of antibody
because the antigen causing the human anti-animal antibody response in
any given patient is usually unknown. For example, although HAMA assays
may be able to detect anti-isotype antibodies, anti-idiotypic
antibodies may escape detection. Table 3
(87)(88)(89)(90)(91)(92)(93) summarizes the results of a series of
studies designed to detect HAMA and other anti-animal antibodies.
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clinical significance and consequences of hama
It is not surprising that the administration of a foreign protein
may be accompanied by some adverse reactions. Although uncommon, the
spectrum of reported adverse reactions to intravenous, intraperitoneal,
or subcutaneous mouse-derived agents includes allergic reactions
(incidence, 0.3 in 1000) (78)(94), anaphylactic
shock (95), generalized pain, hyponatremia, fever, rigors,
chills, rash, paresthesias, weakness, chronic refractory postural
hypotension (70), and serum sickness
(96)(97). No relationship has been found between
the adverse reactions and the development of a HAMA (IgG or IgM)
(78).
Preexisting HAMAs can also interfere with mouse monoclonal antibody therapy or imaging by inactivation or by complexation with the administered antibody. Rapid clearance of the complexed agent neutralizes its therapeutic effectiveness (53)(79)(98). Sensitization to OKT3 has been studied and a relationship established between sensitization and mortality and/or allograft loss. The concentrations of OKT3 were followed, and failure to achieve steady-state or declining concentrations was equated with sensitization, subsequently demonstrated by the detection of HAMAs in six of the seven patients tested (53). A further consequence of anti-animal antibodies is unnecessary medical intervention or medical or surgical procedures because of false-positive test results, particularly from tests for cancer markers (see the section on immunosuppressant therapy) (8).
One potential benefit of a HAMA response is the induction of anti-anti-idiotype antibodies (86)(99)(100). These should be reactive with the original target antigen of the infused antibody and thus would be reactive with antigen-expressing cells (e.g., tumor cells) and provide a therapeutic effect.
assays for hama
The measurement of HAMA is important for the identification of
specimens that may give falsely increased results in two-site assays
and is also therapeutically important for assessing possible
complications of repeated administration of mouse monoclonal
antibodies. In it's "points to consider" document on monoclonal
antibody products for human use, the FDA makes specific recommendations
on monitoring of the development of HAMAs (101), i.e.,
"develop assays to detect human immunoglobulins against humanized or
primatized antibodies, immunonuclides, immunotoxins, their individual
components, and neoantigens formed by the linked
antibody/toxin/nuclide".
HAMA assay designs vary widely and include direct assays for immune complexes, immunofluorescence tests, immunofluorescence inhibition tests, IRMAs, ELISAs, reverse ELISA assays (53)(83)(102)(103), and dot blotting (104). Direct assays use HPLC to measure the immune complexes formed when serum is incubated with radiolabeled antigen (monoclonal antibody) infused into the patient. Other assays use either the same monoclonal antibody or a polyclonal antibody for capture and detection. Alternatively, a mouse antibody (monoclonal or polyclonal) is used for capture HAMAs, and an anti-species antibody is the detection antibody (105).
The type of HAMA detected will depend on the assay design. For example, if the capture, detection, and infused antibodies are identical, then the assay will detect isotypic and idiotypic HAMAs. If an irrelevant monoclonal antibody of the same isotype as the infused antibody is used as the capture antibody, then the assay will detect predominantly isotypic HAMAs. The variability in HAMA results between different types of assays has been assessed by the distribution of panels of specimens to laboratories in the US and Europe, and these surveys revealed significant intermethod and interlaboratory differences in HAMA results (106)(107)(108). The calibrators for HAMA assays also vary, and include baboon anti-mouse IgG, serum, or plasma from patients infused with monoclonal antibodies. Lack of standardization is a key factor in the poor intermethod and interlaboratory comparability of HAMA data.
Currently, there are six HAMA assays available in kit form: ImmuSTRIP HAMA (Immunomedics), ETI-HAMAK (Sorin Biomedica) (109)(110), HAMA-ELISA medac (Medac), HAMA RIA (Scantibodies Laboratory), Ideal HAMA ELISA (AIPCO), and Enzygnost HAMA (Behringwerke). All except one of the assays are ELISAs. The Immunomedics assay uses mouse IgG immobilized to a plastic surface as the capture antigen and a mouse IgG-horseradish peroxidase conjugate to detect captured HAMA. The Sorin Biomedica test uses an immobilized mouse monoclonal antibody (IgG1) and a goat anti-human IgG-horseradish peroxidase conjugate. In the Behring test, either IgG or IgM HAMA can be detected. The mouse monoclonal antibody (IgG1) supplied with the kit or the mouse monoclonal antibody administered to the patient is immobilized on the inside surface of a plastic microwell (capture antigen), and the HAMA is detected with a goat anti-human IgG- or IgM-horseradish peroxidase conjugate. A simple point-of-care type test would be useful for the rapid assessment of specimens suspected of containing HAMAs. At one time, Sangstat produced such a device, but it has since been withdrawn from the market (111). An alternative strategy now is to utilize a pregnancy test kit. A qualitative HAMA result can be obtained using the Tandem® ICON® ImmunoConcentration® human chorionic gonadotropin (hCG) assay. This has a negative control zone that is coated with mouse IgG specifically to detect anti-animal antibodies that might invalidate the hCG test; if this zone develops a color (positive response), this indicates that the sample is positive for HAMA. However, this assay may be relatively insensitive to HAMA because blocking agents (mouse, rat, or bovine) are included in the sample diluent. It should also be remembered that the device was not specifically designed for this purpose.
mechanism of interference in immunoassays
In two-site (sandwich) immunoassays, HAMAs present in a serum
sample can interfere in clinical assays by bridging between the mouse
immunoglobulin capture antibody and the mouse immunoglobulin conjugate
(Fig. 2
); this produces a false-positive result (Table 4
) (112)(113)(114)(115)(116)(117)(118)(119)(120)(121)(122)(123)(124)(125)(126)(127)(128)(129)(130)(131)(132)(133)(134)(135)(136)(137)(138)(139)(140)(141)(142)(143)(144)(145)(146)(147)(148)(149)(150). False-negative results attributable to
HAMA are also encountered in two-site assays, and this presumably is
the result of the HAMA reacting with one of the assay reagents
(immobilized antibody or the conjugate) and preventing reaction with
the analyte (Fig. 2
). A correlation (r = 0.885) has
been shown between HAMAs and false positivity in a CA 125 assay.
However, there were some outliers in which an increased HAMA
concentration was not associated with a increased concentrations of CA
125, indicating a more complex mechanism for the interference (e.g.,
HAMA may have greater reactivity with the monoclonal antibody in the
HAMA assay than with monoclonal antibody in the CA 125 assay).
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Interference has also been reported in solid-phase competitive binding assays as a result of blocking of the capture antibody binding site (93). The high affinity of the antigen and labeled antigen for the capture antibody, compared with the human anti-animal antibody, minimizes interference in competitive binding assays. However, interferences can occur if the anti-animal antibody is present in a high concentration or if a large amount of sample is used in the assay. An interference has also been reported in a competitive binding assay that used a double antibody technique for separation of the bound from the free labeled fractions (93).
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Antibodies to Other Species |
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TopAbstractIntroductionEtiology of Anti-Animal...HAMAAntibodies to Other SpeciesStrategies to Eliminate Anti...ConclusionReferences |
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human anti-rabbit antibody
Rabbit anti-thymocyte globulin is an immunosuppressant, and
anti-rabbit antibodies develop in patients who receive this therapy. In
one study of a group of 32 renal transplant patients, all developed
human anti-rabbit antibodies (HARAs; 144.6 ± 33.7 mg/L IgA,
187.5 ± 100 mg/L IgG, and 44.9 ± 12.6 mg/L IgM), and
in the case of the IgG anti-rabbit antibody, these persisted for 2–12
months (152). The adverse consequences of an unrecognized
human anti-animal antibody interference is dramatically illustrated by
two case reports of a HARA interference (39). In the first
case, increased concentrations of serum hCG in a 41-year-old woman led
to an unnecessary laparoscopic examination. Her serum hCG had been
measured with an RIA based on antibodies raised in rabbits, and
reanalysis of the patient's specimens with a goat antibody-based assay
revealed normal concentrations of hCG in all samples. A more disturbing
case was that of a woman who presented because of infertility and
amenorrhea. High serum follicle-stimulating hormone (FSH) values were
noted, and this led to a series of unnecessary diagnostic procedures,
including laparoscopy, laparotomy, and an ovarian biopsy. Reanalysis of
her samples with a goat antibody-based assay gave normal values for
FSH. Both patients kept rabbits as pets, and it was surmised that this
was the source of antigen.
Other cases of HARA interferences (blockable with rabbit serum or IgG) include falsely increased thyrotropin (TSH) attributable to placental transfer from HARA-positive mothers (37)(153), increased luteinizing hormone and FSH attributable to vaccination (32), and unusual treatments [e.g., subcutaneous injections with antireticulocytoxique, a serum obtained by injecting rabbits with homogenates of human bone marrow and spleen (34)].
human anti-goat antibody
An interference attributable to serum human anti-goat
antibodies was uncovered in serum from an 84-year-old woman following
discordant creatine kinase (CK) isoenzyme results obtained with an
immunoassay (Stratus CK-MB assay result, 12–15 µg/L) and by
electrophoresis (>95% MM isoenzyme, no detectable MB). The addition
of mouse IgG was without effect on the assay results (indicating that
the sample probably did not contain HAMA), but the addition of normal
goat serum reduced the measured CK-MB to <1.7 µg/L, suggesting that
a human anti-goat antibody was the most likely cause of the
interference. The Stratus CK-MB assay includes goat IgG as a component
of the anti-CK-MB-alkaline phosphatase conjugate reagent. Presumably
the human anti-goat antibody reacted with the goat IgG, and the
resulting immune complexes trapped conjugate on the Stratus assay tab
to give a false-positive result. This was supported by the finding that
removal of the goat IgG from the conjugate eliminated the interference
(154). Anti-animal antibodies reactive with goat as well as
mouse IgG have also been described (118).
human anti-sheep antibody
DigibindTM is a sheep anti-digoxin-Fab
widely used to treat digoxin poisoning, but there are no reports of the
formation of anti-sheep antibodies following this type of treatment.
This may be because it is a Fab fragment and is rapidly removed from
the circulation. An interference in a RIA for 
-fetoprotein
because of human anti-sheep antibody has been described
(93). A 7% prevalence of human anti-sheep antibody in a
blood donor population was found, and it was suggested that this was
not attributable to occupational exposure (shepherds, slaughterhouse
workers, or butchers), but to immunization via the gut with bovine
immunoglobulin, which is cross-reactive with sheep immunoglobulin.
human anti-cow antibody
Human anti-cow antibody interferences have been reported in
the serum of three patients tested for thyroxine, free thyroxine, and
TSH with the enhanced chemiluminescent Amerlite assays. Addition of
bovine 
-globulin (final concentration, 10 g/L) eliminated the
interferences (155).
human anti-pig antibody
Human anti-porcine antibodies have been detected in hemophiliacs
receiving porcine factor VIII, but no assay interferences were noted
(156).
human anti-rat antibody
This type of human anti-animal antibody was not detected during
the treatment of 15 allogeneic bone marrow transplant patients with an
anti-CD25 rat monoclonal antibody (33B3.1) for prevention of graft vs
host disease (74), and no analytical interferences
attributable anti-rat antibodies have been reported.
human anti-horse antibody
The formation of anti-horse antibodies as a result of treatment
with an equine anti-thymocyte globulin immunosuppressant has been
recognized for a long time. In one study, anti-equine antibodies
developed in 4 of 27 cardiac transplant patients treated with equine
anti-thymocyte globulin (157).
human anti-chimeric antibody
Human anti-chimeric antibodies have been detected in patients
treated with chimeric antibodies (81), although in other
studies (e.g., studies of the treatment of multiple myeloma patients
with chimeric human anti-interleukin-6 antibodies), this type of human
anti-animal antibody was not detected (82).
antibodies with mixed specificity
There is considerable protein sequence homology between IgG
molecules from different animal species. Thus, it is not surprising
that anti-animal antibodies are cross-reactive with a range of animal
immunoglobulins. Cross-reactivity of anti-animal antibodies has been
illustrated by studies in two healthy males with spuriously increased
serum luteinizing hormone concentrations (151). This
anti-animal interference was blocked with equivalent efficacy by mouse,
sheep, or goat serum. It was also blocked with mouse
IgG1, mouse IgG2a, and rat
IgG. Another study investigated interferences in a two-site CK-MB assay
and showed the broad reactivity of interfering globulins to nonimmune
serum from a diverse range of animals (Table 5
) (89).
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Anti-animal antibodies that cross-react with mouse IgG, causing a
false-positive result in an -fetoprotein assay, have been attributed
to treatment with unconventional drug preparations, specifically
Wobenzy (MUCOS Pharma), a formulation of partly plant and animal
origins. Repeated administration of this preparation was thought to
have immunized the patient to produce anti-animal antibodies that
cross-reacted with mouse IgG (158).
An intriguing type of anti-animal interference in an enzyme immunoassay for TSH (peroxidase label) has been described in a series of 14 specimens (159). No interference was found in a RIA using an identical mouse monoclonal antibody capture antibody. The interference was inhibited by high concentrations of mouse IgG and blocked by anti-human IgM. The authors speculated that the anti-animal antibodies recognized epitopes on the peroxidase label or epitopes on the antibody exposed or modified by enzyme labeling.
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Strategies to Eliminate Anti-Animal Antibodies and Antibody Interferences |
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TopAbstractIntroductionEtiology of Anti-Animal...HAMAAntibodies to Other SpeciesStrategies to Eliminate Anti...ConclusionReferences |
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prevention
Immunosuppressant therapy. One method of minimizing the
development of HAMA has been to treat patients with immunosuppressive
drugs such as cyclosporin A, cyclophosphamide, azithioprine, or
deoxyspergualin before, during, and after the administration of mouse
antibody agents
(79)(84)(160)(161).
In a recent study, a series of 13 patients were given cyclosporin A
starting 2 days before treatment with a 99 mTc-labeled
F(ab')2 or Fab (161). Six to 9 days later,
186Re-F(ab')2 or intact antibody was
administered, and cyclosporin A treatment was continued for an
additional 14 days. In 5 of 13 patients (mean cyclosporin A, 726
µg/L), no HAMA was detected for up to 8 weeks, whereas the remaining
8 patients with lower cyclosporin A concentrations (mean, 364 µg/L)
became HAMA positive. In control experiments involving patients who
were not given cyclosporin A, 86–100% of patients developed a HAMA
response (161). Similarly, in a series of patients treated
with two courses of radiolabeled anti-carcinoembryonic antigen (CEA),
the mean serum HAMA was 3.5 mg/L after 2 weeks in patients treated with
cyclosporin A vs 1998 mg/L in patients not given this immunosuppressant
(84). Deoxyspergualin is also effective in suppressing the
HAMA response. In patients with advanced cancers treated with the
antibody L6, two-thirds developed HAMAs in the original trial, but when
L6 was administered in combination with deoxyspergualin, only 2 of 24
patients developed HAMAs (both had low serum HAMA concentrations, 160
and 181 µg/L) (160).
Antibody fragments.
The immunogenicity of an immunoglobulin
molecule can be reduced by removing the Fc portion. The resulting Fab
or F(ab')2 fragments have been shown to be less immunogenic
than the intact IgG molecule
(109)(162)(163), although
the incidence of HAMA positivity increases with multiple therapies for
intact or fragments of mouse monoclonal IgG (109).
Humanized and chimeric antibodies.
One way of overcoming the
antigenicity of mouse monoclonal antibodies has been to "humanize"
the immunoglobulin molecule. This can be achieved using genetic
engineering techniques to combine mouse complementary determining
regions and human framework and constant regions or human constant
regions with mouse framework and complementary determining regions
(164)(165)(166)(167)(168)(169). A difficulty encountered with the humanization
strategy is that an IgG molecule is still potentially antigenic; hence,
an immune response will produce human anti-human antibodies. In
one study, 2 of 53 patients given 88BV59, an IgGk directed against the
tumor-associated antigen CTA16.88 (homologous to cytokeratins 8, 18,
and 19), developed a low titer of human anti-human antibodies 1–3
months after a single infusion of the antibody (116). An
analytical interference by human anti-human antibodies may be possible
as a result of cross-reactivity, but is as yet unreported.
Polyethyleneglycolylation.
Coating the surface of a
macromolecule with water-soluble polyethylene glycol (PEG) or
monomethoxy PEG (mPEG) molecules can lead to beneficial alterations in
their properties, e.g., reduced clearance and reduced immunogenicity,
enhanced tissue localization, specificity, potency, and stability
(170)(171)(172)(173). For example, immunogenicity reduction through
mPEGylation has been shown in animal studies with the murine antibody
W3/25 (173). This chemical "stealth" type
technology offers a potential route to reducing or eliminating
HAMAs in patients receiving mouse monoclonal antibodies.
blocking and removal
Considerations for methods designed to block or to remove a
anti-animal interference are ease of use, effectiveness, applicability,
cost, and convenience. Many of the available methods have deficiencies
in one of these areas.
The blocking agent can be included in the assay (e.g., in the assay diluent), or the sample can be pretreated before assay. Nonimmune serum (4)(126)(174)(175), polyclonal IgG (121)(124)(126), polymerized IgG (88), nonimmune (irrelevant) mouse monoclonals (103), and a mixture of monoclonal antibodies (124) or fragments of IgG [Fc, Fab, F(ab')2] (142) from the same species used to raise the reagent antibodies are commonly used as blocking agents (103)(121)(174). The effectiveness of added blocking agent depends on the concentration and class or subclass, specificity, and valence of the human anti-animal antibody and the species and subclass of the blocker (142). There are examples of HAMA interferences that were either not blocked or only partially blocked by mouse IgG. In one case, blocking could only be achieved by low temperature incubation with a high concentration of the monoclonal antibody administered to the patient (125).
Several blocking reagents are available commercially: Immunoglobulin Inhibiting Reagent (IIR; Bioreclamation) (176), Heterophilic Blocking Reagent (HBR; Scantibodies), Heteroblock (mixture of active and passive blocking reagents; Omega Biologicals), and MAB 33 (monoclonal IgG1) and Poly MAB 33 (polymeric monoclonal IgG1/Fab; Boehringer Mannheim). IIR is a proprietary formulation of immunoglobulins with a high affinity for anti-animal antibodies (109 L/mol), and HBR is monoclonal mouse anti-human IgM. In conventional blocking procedures, the blocking depends on the binding constant of the human anti-animal antibody with the added reagent (typically 106 L/mol). In contrast, reagents such as IIR and HBR are directed specifically against any IgM, not only those with anti-animal specificity, and have a higher binding affinity (109 L/mol) for an human anti-animal antibody than does an anti-animal for a nonspecific blocking agent. Consequently, these reagents can be used at lower concentrations and have superior blocking kinetics compared with nonspecific blocking reagents. In a comparative study of IIR vs a polymerized nonimmune (irrelevant) monoclonal (MAK-33) and nonspecific mouse IgG in a CA 125 assay, only IIR eliminated all interferences (103). In a study of HBR, it was shown to be effective in blocking anti-animal interference in a serum CK-MB assay. HBR also caused small changes (range, -6.8% to 11.5%) in the concentration of CK-MB in control specimens, and this was attributed to the intrinsic anti-immunoglobulin reactivity of the reagent (177). One alternative to the HBR product are the Heterophilic Blocking Tubes (Scantibodies), which contain proprietary predispensed and lyophilized specific binders to inactivate anti-animal antibodies (178).
Immunoextraction using murine monoclonal antibody adsorbed onto vinylidene fluoride floccules (179) or protein G immobilized on Sepharose beads (103) has also been effectively used to remove HAMA interferences from samples in a CEA and a CA 125 assay, respectively. Alternatively, anti-animal interferents can be removed by precipitation with PEG 6000 (136)(180)(181). Chromatography is also effective in removing interferents. For example, protein A, protein G, cation-exchange, or gel filtration chromatography was used in a CA 125 assay for samples that could not be blocked with mouse serum or purified mouse antibody (117).
A combination of heat and acid treatment of samples is of limited utility because few analytes are sufficiently stable to survive these antibody-denaturing conditions. This procedure is used mainly as a sample pretreatment procedure for CEA assays (70 °C or 90 °C and acetate buffer, pH 5) (181). Optimization of these conditions is required for individual cancer markers, and for a CA 72-4 assay, the combination of 90 °C and Bis-Tris, pH 6.5, was most effective (122)(182).
assay redesign
One solution to the problem of human anti-animal antibody
interferences in two-site assays is to use Fab or the
F(ab')2 fragment instead of intact immunoglobulin
as the capture and detection antibodies. This eliminates interference
from anti-animal antibodies with specificity for the Fc portion of an
IgG antibody reagent (126)(142). Another
strategy is to use chimeric monoclonal antibodies as assay reagents.
These are now used in some Boehringer Mannheim immunoassays (e.g., ES
and Elecsys TSH assays and the Elecsys CEA assay), either as the
capture antibody or the labeled antibody (183). These
chimeric antibodies are human antibodies in which the variable regions
are replaced with the corresponding parts of a non-human antibody
(e.g., mouse or rat) of the desired specificity. In this way,
interferences by anti-mouse and other anti-animal antibodies are
eliminated.
Another alternative is to use antibodies raised in chickens for one or both of the antibody reagents (91). Mammalian and chicken IgG have no cross-reactivity; thus, chicken antibodies are unlikely to react with anti-animal antibodies. Chicken antibody-based assays have been tested using a rabbit anti-mouse antibody (HAMA surrogate) and with sera from HAMA-positive patients (treated with monoclonal antibody 17-1A). No false positives were observed when at least one of the antibodies (capture or detection) was a chicken antibody. However, chicken antibodies have low affinities, and there are currently no monoclonals, thus preventing two-site monoclonal assay strategies.
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Conclusion |
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). Clinicians should ensure that patients known to have such
antibodies or at risk for developing such antibodies because of
administration of animal-derived agents are clearly identified to the
laboratory. They should also be aware that if the results of two-site
immunoassay tests, particularly cancer marker and hormone tests, do not
fit with the clinical picture, then this may be an indication of a
human anti-animal antibody interference. Manufacturers of two-site
immunoassay kits should take steps to minimize assay interferences, and
some have already responded by reformulating assay reagents and
including warnings about HAMA interferences in package inserts.
Laboratories should develop plans for investigating and confirming
anti-animal interferences. Dilution experiments will often flag a
possible interference because samples containing anti-animal antibodies
do not give proportional results. Reanalysis of samples after
incubation with animal protein (e.g., mouse IgG) or animal serum can
also help to confirm an interference. Another strategy to guard against
false-positive and false-negative results attributable to anti-animal
antibodies is to test all samples for the presence these antibodies.
This would be an expensive undertaking, and currently this type of
analysis is reserved for identifying the presence of an human
anti-animal antibody.
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References |
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-lactalbumin in the serum of children and adults. Pediatrics 1965;35:571-588.
