Antibody Epitope and Affinity Determination of the Myocardial Infarction Marker Myoglobin by SPR-Biosensor Mass Spectrometry.

Myoglobin (MG) is a biomarker for heart muscle injury, making it a potential target protein for early detection of myocardial infarction. Elevated myoglobin levels alone have low specificity for acute myocardial infarction (AMI) but in combination with cardiac troponin T have been considered highly efficient diagnostic biomarkers. Myoglobin is a monomeric heme protein with a molecular weight of 17 kDa that is found in skeletal and cardiac tissue as an intracellular storage unit of oxygen. MG consists of eight α-helices connected by loops and a heme group responsible for oxygen-binding. Monoclonal antibodies are widely used analytical tools in biomedical research and have been employed for immunoanalytical detection of MG. However, the epitope(s) recognized by MG antibodies have been hitherto unknown. Precise molecular identification of the epitope(s) recognized by antibodies is of key importance for the development of MG as a diagnostic biomarker. The epitope of a monoclonal MG antibody was identified by proteolytic epitope extraction mass spectrometry in combination with surface plasmon resonance (SPR) biosensor analysis. The MG antibody was immobilized both on an affinity microcolumn and a gold SPR chip. The SPR kinetic analysis provided an affinity-binding constant KD of 270 nM for MG. Binding of a tryptic peptide mixture followed by elution of the epitope from the SPR-MS affinity interface by mild acidification provided a single-epitope peptide located at the C-terminus [146-153] [YKELGFQG] of MG. The specificity and affinity of the epitope were ascertained by synthesis and affinity-mass spectrometric characterization of the epitope peptide.

Mass Spectrometric Analysis of Antibody-Epitope Peptide Complex Dissociation: Theoretical Concept and Practical Procedure of Binding Strength Characterization.

Electrospray mass spectrometry is applied to determine apparent binding energies and quasi equilibrium dissociation constants of immune complex dissociation reactions in the gas phase. Myoglobin, a natural protein-ligand complex, has been used to develop the procedure which starts from determining mean charge states and normalized and averaged ion intensities. The apparent dissociation constant KD m0g#= 3.60 × 10-12 for the gas phase heme dissociation process was calculated from the mass spectrometry data and by subsequent extrapolation to room temperature to mimic collision conditions for neutral and resting myoglobin. Similarly, for RNAse S dissociation at room temperature a KD m0g#= 4.03 × 10-12 was determined. The protocol was tested with two immune complexes consisting of epitope peptides and monoclonal antibodies. For the epitope peptide dissociation reaction of the FLAG peptide from the antiFLAG antibody complex an apparent gas phase dissociation constant KD m0g#= 4.04 × 10-12 was calculated. Likewise, an apparent KD m0g#= 4.58 × 10-12 was calculated for the troponin I epitope peptide-antiTroponin I antibody immune complex dissociation. Electrospray mass spectrometry is a rapid method, which requires small sample amounts for either identification of protein-bound ligands or for determination of the apparent gas phase protein-ligand complex binding strengths.

Dual-modality impedimetric immunosensor for early detection of prostate-specific antigen and myoglobin markers based on antibody-molecularly imprinted polymer.

A new dual-modality immunosensor based on molecularly imprinted polymer (MIP) and a nanostructured biosensing layer has fabricated for the simultaneous detection of two important markers including prostate-specific antigen (PSA) and myoglobin (Myo) in human serum and urine samples. In the first step, 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide ester) (DSP) was self-assembled on a gold screen printed electrode (SPE). Then, the target proteins were attached covalently to the DSP-SPE. The imprinted cocktail polymer ((MIP(PSA, Myo)-SPE)) was synthesized at the SPE surface using acrylamide as monomer, N,N'-methylenebisacrylamide as a crosslinker, and PSA and Myo as the templates, respectively. The MIP-SPE was specific for the impedimetric sensing of PSA and Myo. After that, a nanocomposite (NCP) was synthesized based on the decorated magnetite nanoparticles with multi-walled carbon nanotube, graphene oxide and specific antibody for PSA (Ab). Then, NCP incubated with (MIP(PSA, Myo)-SPE. The modified electrodes and synthesized nanoparticles were characterized using electrochemical impedance spectroscopy, dynamic light scattering, surface plasmon resonance and scanning electron microscopy. The limits of detections were found to be 5.4 pg mL-1 and 0.83 ng mL-1 with the linear dynamic ranges of 0.01-100 and 1-20000 ng mL-1 for PSA and Myo, respectively. The ability of proposed biosensor to detect PSA and Myo simultaneously with high sensitivity and specificity offers a powerful opportunity for the new generation of biosensors. This dual-analyte specific receptors-based device is highly desired for the integration with lab-on-chip kits to measure a wide panel of biomarkers present at ultralow levels during early stages of diseases progress.

Pigment Nephropathy: Novel Insights into Inflammasome-Mediated Pathogenesis.

Pigment nephropathy is an acute decline in renal function following the deposition of endogenous haem-containing proteins in the kidneys. Haem pigments such as myoglobin and haemoglobin are filtered by glomeruli and absorbed by the proximal tubules. They cause renal vasoconstriction, tubular obstruction, increased oxidative stress and inflammation. Haem is associated with inflammation in sterile and infectious conditions, contributing to the pathogenesis of many disorders such as rhabdomyolysis and haemolytic diseases. In fact, haem appears to be a signalling molecule that is able to activate the inflammasome pathway. Recent studies highlight a pathogenic function for haem in triggering inflammatory responses through the activation of the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome. Among the inflammasome multiprotein complexes, the NLRP3 inflammasome has been the most widely characterized as a trigger of inflammatory caspases and the maturation of interleukin-18 and -1ß. In the present review, we discuss the latest evidence on the importance of inflammasome-mediated inflammation in pigment nephropathy. Finally, we highlight the potential role of inflammasome inhibitors in the prophylaxis and treatment of pigment nephropathy.

A novel electrochemiluminescence resonance energy transfer system for simultaneous determination of two acute myocardial infarction markers using versatile gold nanorods as energy acceptors.

A novel electrochemiluminescence resonance energy transfer (ECL-RET) system using versatile gold nanorods as energy acceptors was introduced into the ECL biochemical analysis. A spatial- and potential-resolved platform coupled with the ECL-RET strategy was developed for simultaneous determination of two acute myocardial infarction markers.

Simultaneous detection of three biomarkers related to acute myocardial infarction based on immunosensing biochip.

An immunosensing biochip for simultaneous detection of three biomarkers related to acute myocardial infarction (AMI) was developed based on anionic soybean peroxidase (SBP) functionalized nanoprobe and chemiluminescent imaging. The nanoprobes (Ab2-SiO2-SBP) were fabricated by co-immobilization of SBP and one of the detection polyclonal antibodies, anti-cardiac troponin I antigen (anti-cTnI), anti-creatine kinase-MB (anti-CK-MB) and anti-myoglobin (anti-Myo), on the silica nanoparticle surface. The detection sensitivity was enhanced since the large surface area of silica carriers increased the loading of SBP for per sandwiched immunoreaction. The immunosensing biochip designed as 3 × 8 wells array was constructed by simultaneously immobilizing three capture monoclonal antibodies on the same one microtiter well with 2 × 3 active spots. In the presence of target protein, the nanoprobes will be attached onto the spots with high specificity through the sandwiched immunoreactions, which triggered the chemiluminescence (CL) signals on each sensing site of the microtiter plates and allowed to CL imaging of three biomarkers in one well at the same time. Therefore, the proposed biochip was a promising convenient strategy for simultaneous detection of cTnI, CK-MB and Myo, which showed potential application for multianalyte determination in clinical diagnostics.

Suppressing allostery in epitope mapping experiments using millisecond hydrogen / deuterium exchange mass spectrometry.

Localization of the interface between the candidate antibody and its antigen target, commonly known as epitope mapping, is a critical component of the development of therapeutic monoclonal antibodies. With the recent availability of commercial automated systems, hydrogen / deuterium eXchange (HDX) is rapidly becoming the tool for mapping epitopes preferred by researchers in both industry and academia. However, this approach has a significant drawback in that it can be confounded by 'allosteric' structural and dynamic changes that result from the interaction, but occur far from the point(s) of contact. Here, we introduce a 'kinetic' millisecond HDX workflow that suppresses allosteric effects in epitope mapping experiments. The approach employs a previously introduced microfluidic apparatus that enables millisecond HDX labeling times with on-chip pepsin digestion and electrospray ionization. The 'kinetic' workflow also differs from conventional HDX-based epitope mapping in that the antibody is introduced to the antigen at the onset of HDX labeling. Using myoglobin / anti-myoglobin as a model system, we demonstrate that at short 'kinetic' workflow labeling times (i.e., 200 ms), the HDX signal is already fully developed at the 'true' epitope, but is still largely below the significance threshold at allosteric sites. Identification of the 'true' epitope is supported by computational docking predictions and allostery modeling using the rigidity transmission allostery algorithm.

Neuroprotective effects of antibodies on retinal ganglion cells in an adolescent retina organ culture.

Glaucoma, a neurodegenerative disease, is characterized by a progressive loss of retinal ganglion cells (rgc). Up- and down-regulated autoantibody immunoreactivities in glaucoma patients have been demonstrated. Previous studies showed protective effects of down-regulated antibodies [gamma (γ)-synuclein and glial fibrillary acidic protein [GFAP]) on neuroretinal cells. The aim of this study was to test these protective antibody effects on rgc in an organ culture model and to get a better understanding of cell-cell interactions of the retina in the context of the protective effect. We used an adolescent retinal organ culture (pig) with an incubation time of up to 4 days. Retinal explants were incubated with different antibodies for 24 h (anti-GFAP, anti-γ-synuclein and anti-myoglobin antibody as a control). Brn3a and TUNEL staining were performed. We also conducted glutamine synthetase staining and quantification of the retinal explants. Mass spectrometry analyses were performed as well as protein analyses via microarray. We detected a continuous decrease of rgc/mm in the retinal explants throughout the 4 days of incubation with increased TUNEL rgc staining. Immunohistochemical analyses showed a protective effect of anti-γ-synuclein (increased rgc/mm of 41%) and anti-GFAP antibodies (increased rgc/mm of 37%). Mass spectrometric, microarray and immunohistochemical analyses demonstrated Müller cell involvement and decreased endoplasmic reticulum stress response in the antibody-treated retinae. We could detect that the tested antibodies have a protective effect on rgc which seems to be the result of reduced stress levels in the retina as well as a shift of glutamine synthetase localization in the endfeet of the Müller cells towards the inner retinal layer. Loss of retinal ganglion cells (rgc) in glaucoma leads to blindness. Several antibodies are down-regulated in glaucoma patients. Our aim was to test if these antibodies have a protective effect of rgc in a retinal organ culture. This could be shown with an increase of rgc numbers. This effect results through reduced stress levels and the shift of glutamine synthetase localization.

Early markers for myocardial ischemia and sudden cardiac death.

The post-mortem diagnosis of acute myocardial ischemia remains a challenge for both clinical and forensic pathologists. We performed an experimental study (ligation of left anterior descending coronary artery in rats) in order to identify early markers of myocardial ischemia, to further apply to forensic and clinical pathology in cases of sudden cardiac death. Using immunohistochemistry, Western blots, and gene expression analyses, we investigated a number of markers, selected among those which are currently used in emergency departments to diagnose myocardial infarction and those which are under investigation in basic research and autopsy pathology studies on cardiovascular diseases. The study was performed on 44 adult male Lewis rats, assigned to three experimental groups: control, sham-operated, and operated. The durations of ischemia ranged between 5 min and 24 h. The investigated markers were troponins I and T, myoglobin, fibronectin, C5b-9, connexin 43 (dephosphorylated), JunB, cytochrome c, and TUNEL staining. The earliest expressions (≤30 min) were observed for connexin 43, JunB, and cytochrome c, followed by fibronectin (≤1 h), myoglobin (≤1 h), troponins I and T (≤1 h), TUNEL (≤1 h), and C5b-9 (≤2 h). By this investigation, we identified a panel of true early markers of myocardial ischemia and delineated their temporal evolution in expression by employing new technologies for gene expression analysis, in addition to traditional and routine methods (such as histology and immunohistochemistry). Moreover, for the first time in the autopsy pathology field, we identified, by immunohistochemistry, two very early markers of myocardial ischemia: dephosphorylated connexin 43 and JunB.

Construction of an antimyoglobin single-chain variable fragment with rapid reaction kinetics.

Antibodies with rapid reaction kinetics (high association and dissociation rates), named reversible antibodies, are used to perform continuous monitoring of sensitive disease biomarkers. In cases of acute myocardial infarction (AMI), continuous monitoring and early diagnosis are important. Human myoglobin (Myo) is a useful biomarker for AMI during the early stage after the onset of symptoms. In this study, a single-chain variable fragment (scFv) specific to Myo was derived from an IgG antibody that has rapid reaction kinetics. Enzyme-linked immunosorbent assay revealed that recombinant scFv exhibited 3.8-fold reduced affinity compared with the parent IgG antibody based on the antibody concentration necessary for 50% of the maximum signal. The scFv retained the rapid reaction kinetic mode with average kon and koff of 2.63 × 10(5) M(-1) Sec(-1) and 3.25 × 10(-3) Sec(-1) , respectively, which were reduced to 10- and 2.3-fold compared with those of the parent antibody. The equilibrium constant for the association of the scFv (KA = 8.09 × 10(7) M(-1) ) was 4.6-fold lower than that of its parent IgG antibody. This scFv may be a starting point for further mutagenesis/kinetic and structural analyses providing valuable insight into the mechanism of reversible antibodies.

IgE epitope proximity determines immune complex shape and effector cell activation capacity.

BACKGROUND: IgE-allergen complexes induce mast cell and basophil activation and thus immediate allergic inflammation. They are also important for IgE-facilitated allergen presentation to T cells by antigen-presenting cells. OBJECTIVE: To investigate whether the proximity of IgE binding sites on an allergen affects immune complex shape and subsequent effector cell activation in vitro and in vivo. METHODS: We constructed artificial allergens by grafting IgE epitopes in different numbers and proximity onto a scaffold protein. The shape of immune complexes formed between artificial allergens and the corresponding IgE was studied by negative-stain electron microscopy. Allergenic activity was determined using basophil activation assays. Mice were primed with IgE, followed by injection of artificial allergens to evaluate their in vivo allergenic activity. Severity of systemic anaphylaxis was measured by changes in body temperature. RESULTS: We could demonstrate simultaneous binding of 4 IgE antibodies in close vicinity to each other. The proximity of IgE binding sites on allergens influenced the shape of the resulting immune complexes and the magnitude of effector cell activation and in vivo inflammation. CONCLUSIONS: Our results demonstrate that the proximity of IgE epitopes on an allergen affects its allergenic activity. We thus identified a novel mechanism by which IgE-allergen complexes regulate allergic inflammation. This mechanism should be important for allergy and other immune complex-mediated diseases.

Electrosynthesis and binding properties of molecularly imprinted poly-o-phenylenediamine as artificial antibodies for electroanalysis of myoglobin.

Molecularly imprinted poly-o-phenylenediamine with template myoglobin molecules (i.e., polymeric antibodies to myoglobin, molecularly imprinted polymer, MIP) was synthesized via electropolymerization. Electropolymerization, washing, and the interaction of the polymeric antibodies with myoglobin was examined by square wave voltammetry and microgravimetry. The analysis of myoglobin was carried out through direct electrochemical detection of the reduction peak of Fe(3+) of the hemeprotein on screen-printed graphite electrodes modified by the MIP. According to the electrochemical analysis, MIP surfaces demonstrated remarkably higher ability to bind the protein compared to that of surfaces prepared by the same route under the same conditions but in the absence of myoglobin (surfaces of the non-imprinted polymer, NIP). The imprinting factor I max(MIP)/I max(NIP) was found to be 2-4. The equilibrium dissociation constant K d of the interaction of myoglobin with MIP electrodes was evaluated as (2.4 ± 0.5) × 10(-8) M. The lower detection limit of myoglobin by a MIP sensor was determined as 0.5 × 10(-9) M, the range of detectable concentrations being 10(-9)-10(-5) M.

[Preparation and application of monoclonal antibody against human myoglobin].

OBJECTIVE: To prepare the monoclonal antibody (mAb) against human myoglobin (MYO) of high titer and specificity and develop double-antibody sandwich ELISA for detecting MYO in human serum samples. METHODS: The BALB/c mice were immunized with natural human MYO, and the hybridoma cell lines secreting anti-MYO mAb were established using cell fusion and hybridoma screening techniques. The characteristics of the mAb were identified after affinity purification from ascites. Then the best antibody pair was selected from mAb to establish a one-step sandwich ELISA method. Sixty human serum samples were detected by the homemade ELISA kit and the imported one, respectively. RESULTS: Nine strains of hybridoma cell lines stably secreted anti-MYO mAb. Four strains named 2M1, 3M4, 5M7 and 10M4 could secrete high-quality mAb and the titers of them were in the range of 1.0×10(6) to 2.6×10(6) (A450 value was about 1.0). Three antibody pairs (2M1/HRP-3M4, 5M7/HRP-3M4, 10M4/HRP-5M7) were selected by double-antibody sandwich ELISA. Among them, the 5M7/HRP-3M4 had higher sensitivity and larger linear range. The homemade ELISA kit had a larger linear range (25-1000 ng/mL) than the imported one (25-500 ng/mL) and showed high accuracies in detecting human serum samples, being 95% (19/20) in positive samples and 100% (40/40) in negative samples. CONCLUSION: With the anti-human MYO mAbs of high specificity and affinity, a one-step sandwich ELISA for detecting human MYO has been established successfully, which provides a basis for the development of domestic ELISA kit.

Purification and characterization of Fab fragments with rapid reaction kinetics against myoglobin.

Myoglobin is an early biomarker for acute myocardial infarction. Recently, we isolated the antibody IgG-Myo2-7ds, which exhibits unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid dissociation kinetics are thought to be premature IgG forms that are produced during the early stage of in vivo immunization. In the present study, we identified the epitope region of the IgG-Myo2-7ds antibody to be the C-terminal region of myoglobin, which corresponds to 144-154 aa. The Fab fragment was directly purified by papain cleavage and protein G affinity chromatography and demonstrated kinetics of an association constant of 4.02 × 10(4) M(-1) s(-1) and a dissociation constant of 2.28 × 10(-2) s(-1), which retained the unique reaction kinetics of intact IgG-Myo2-7ds antibodies. Because a rapid dissociation antibody can be utilized for antibody recycling, the results from this study would provide a platform for the development of antibody engineering in potential diagnostic areas such as a continuous monitoring system for heart disease.

Sensitive point-of-care monitoring of cardiac biomarker myoglobin using aptamer and ubiquitous personal glucose meter.

Myoglobin (Myo), which is one of the early markers to increase after acute myocardial infarction (AMI), plays a major role in urgent diagnosis of cardiovascular diseases. Hence, monitoring of Myo in point-of-care is fundamental. Here, a novel assay for sensitive and selective detection of Myo was introduced using a personal glucose meter (PGM) as readout. In the presence of Myo, the anti-Myo antibody immobilized on the surface of polystyrene microplate could capture the target Myo. Then the selected aptamer against Myo, which was obtained using our screening process, was conjugated with invertase, and such aptamer-invertase conjugates bound to the immobilized Myo due to the Myo/aptamer interaction. Subsequently, the resulting "antibody-Myo-aptamer sandwich" complex containing invertase conjugates hydrolyzed sucrose into glucose, thus establishing direct correlation between the Myo concentration and the amount of glucose measured by PGM. By employing the enzyme amplification, as low as 50 pM Myo could be detected. This assay also showed high selectivity for Myo and was successfully used for Myo detection in serum samples. This work may provide a simple but reliable tool for early diagnosis of AMI in the world, especially in developing countries.

Reflectometric interference spectroscopy-based immunosensing using immobilized antibody via His-tagged recombinant protein A.

The proposed approach demonstrated in this study provides an immunosensing system based on reflectometric interference spectroscopy (RIfS) in combination with an antibody immobilization method using histidine-tagged recombinant protein A. Carboxymethyldextran (CMD) was immobilized on a 3-aminopropyltriethoxysilane-treated a silicon nitride-coated silicon wafer, followed by chelating histidine-tagged recombinant protein A with copper (II) ions. The CMD-layer was found to be advantageous in terms of not only immobilization of histidine-tagged recombinant protein A-mediated an antibody against myoglobin (anti-Myo) but also prevention of non-specific binding of myoglobin. Myoglobin was repeatedly detected, and the apparent detection limit was 0.1 µg mL(-1). The proposed RIfS-based protein sensing system, in conjunction with the easy preparation of silicon-based inexpensive immunosensing chips, is expected to be applicable for label-free optical detection for other proteins in various fields.

FUNDAMENTAL DIFFERENCES BETWEEN NATURAL ANTIBODIES AND POLYREACTIVE IMMUNOGLOBULINS.

A problem of similarity and differences between so-called polyreactive immunoglobulins (PRIGs) and natural antibodies (NAbs), capable of cross-reacting with some structurally dissimilar antigens, has been considered. The analysis of mechanisms of an unspecific interaction between PRIGs or NAbs and antigens evidences for the fact that essential differences exist between these substances. These differences permit classifying the abovementioned substances as different types of immunoglobulin molecules. The major difference between PRIGs and NAbs may include both the mechanisms of the above mentioned immunoglobulin molecules binding to antigens and their interaction affinity, as well as an absolutely different influence of some low-molecular substances on the efficiency of the interaction with antigens. Relying on the obtained data it can be assumed that, since PRIGs and NAbs have fundamental differences, they may perform not only similar but also different functions of the immune system.

Refolded scFv antibody fragment against myoglobin shows rapid reaction kinetics.

Myoglobin is one of the early biomarkers for acute myocardial infarction. Recently, we have screened an antibody with unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid reaction kinetics are thought to be an early IgG form produced during early stage of in vivo immunization. We produced a recombinant scFv fragment for the premature antibody from Escherichia coli using refolding technology. The scFv gene was constructed by connection of the V(H)-V(L) sequence with a (Gly4Ser)3 linker. The scFv fragment without the pelB leader sequence was expressed at a high level, but the solubility was extremely low. A high concentration of 8 M urea was used for denaturation. The dilution refolding process in the presence of arginine and the redox reagents GSH and GSSH successfully produced a soluble scFv protein. The resultant refolded scFv protein showed association and dissociation values of 9.32 × 10⁻4 M⁻¹·s⁻¹ and 6.29 × 10⁻³ s⁻¹, respectively, with an affinity value exceeding 107 M⁻¹ (k(on)/k(off)), maintaining the original rapid reaction kinetics of the premature antibody. The refolded scFv could provide a platform for protein engineering for the clinical application for diagnosis of heart disease and the development of a continuous biosensor.

Continuous immunosensing of myoglobin in human serum as potential companion diagnostics technique.

To attain early diagnosis of acute myocardial infarction (AMI) with enhanced accuracy, continuous immunosensing has been investigated to measure myoglobin concentration in real-time. To this end, a capture antibody showing rapid reaction kinetics was immobilized on the surface of a surface plasmon resonance sensor. Three problems associated with the continuous sensing of myoglobin in human serum needed to be overcome: non-specific binding of the analyte, aggregation of serum components, and drift of the sensor baseline. Non-specific binding was controlled by pretreating the sample with a detergent mixture consisting of sodium dodecyl sulfate and P20, and adjusting the micelle size and net charge. Aggregation was managed by inactivating certain serum constituents through chelation of heavy metals. Baseline drift perceived in the sensorgram was able to be corrected by compensating for the slope calculated by a linear regression. Under the optimal conditions, the continuous sensor reproducibly traced the varying doses of myoglobin over about 8h with periodic one-point calibration every 3h. The dose-response curve of the sensor was linear with acceptable variations (CVs<4.91% in average) between the detection limit (31.0 ng/mL) and about 2000 ng/mL in the arithmetic scale (R(2)>0.98), covering the clinical concentration range. The immunosensor performance correlated with the Pathfast reference system (R(2)>0.98) and analytical consistency could be maintained for longer than a month if appropriately calibrated. Such immunosensing could be used as a companion diagnostic means along with real-time electrocardiographic measurement, significantly enhancing the sensitivity of AMI diagnosis and thereby enabling treatment at an early stage.

[Avidity of polyreactive immunoglobulins].

An analysis of the mechanism of interaction between polyreactive immunoglobulins (PRIG) and antigen was conducted and it was shown that most of the traditional methods of antibody affinity evaluation are not applicable for PRIG affinity. The comparative assessment of the mouse and human PRIG avidity against ovalbumin and horse myoglobin and the avidity of specific monoclonal antibodies against ovalbumin have shown that the avidity of PRIG not only is much less than the avidity of monoclonal antibodies but even exceeds it.