Acute autoimmune encephalomyelitis in mice. II. Susceptibility is controlled by the combination of H-2 and histamine sensitization genes.

The expression of acute experimental autoimmune encephalomyelitis (EAE) in mice is controlled by several dominant genes, H-2 and histamine sensitization genes. SJL/J and SWR/J, which are H-2s and H-2q, respectively, are susceptible to EAE and sensitive to Bordetella pertussis histamine-sensitizing factor (HSF), which produces a vasoactive amine hypersensitivity. Other H-2s or H-2q strains such as A.SW, B10.Q and several others do not develop acute EAE and are not sensitive to B. pertussis HSF. One strain tested, DDD (KsIsD?) is HSF sensitive but does not develop EAE (presumably because it lacks the appropriate responder H-2 haplotype). However, F1 hybrids between B10.S and DDD are sensitive to HSF and develop EAE. The induction and effector phases of acute EAE are apparently controlled by the combination of H-2 and HSF genes. A combination of the correct H-2 hapotype and histamine sensitivity is required for the development of acute EAE.

ABGEN: a knowledge-based automated approach for antibody structure modeling.

Immunoglobulin (Ig) amino acid sequences are highly conserved and often have sequence homology ranging from 70 to 95%. Antigen binding fragments (Fab), variable region fragments (Fv), and single chain Fv (scFv) of more than 50 myeloma proteins and monoclonal antibodies (mAb) have been crystallized and display a high degree of structural similarity. Based on this observation, several homology modeling approaches have been developed for the prediction of Fab and Fv structures prior to their experimental determination. We have extracted features from existing Ig sequences, 44 known Fab and Fv structures to create an automated AntiBody structure GENeration (ABGEN) algorithm for obtaining structural models of antibody fragments. ABGEN utilizes a homology based scaffolding technique, and includes the use of invariant and strictly conserved residues, structural motifs of known Fab, canonical features of hypervariable loops, torsional constraints for residue replacements and key inter-residue interactions. The validity of the ABGEN algorithm has been tested using a five-fold cross validation with the existing Fab structures. Molecular mechanics and dynamics methods have been implemented with ABGEN models to accurately predict two Fab structures of anti-sweetener antibodies prior to crystallographic determinations.

Local and transmitted conformational changes on complexation of an anti-sweetener Fab.

Crystal structures of an Fab (NC6.8) from a murine IgG2b(kappa) antibody and its complex with a sweet-tasting, N-,N'-,N"-trisubstituted guanidine compound (NC174) have been determined by X-ray analysis. Both crystal forms are produced by a microseeding technique in polyethylene glycol (PEG) 8000 but the habits and space groups are very different. The native protein crystallizes as plates in the monoclinic space group C2 and the complex crystallizes as prisms in the orthorhombic space group P2(1)2(1)2. The structures were solved by molecular replacement methods, with the Fab fragments from the 4-4-20, HyHel-5 and BV04-01 antibodies as starting models. On binding of the ligand, N-(p-cyanophenyl)-N'-(diphenylmethyl)-N"-(carboxymethyl)g uan idine, the protein exhibits significant local conformational changes in the active site, particularly in the third complementarity-determining region (CDR3) of the heavy chain. The ligand enters the small crevice by end-on insertion with the cyanophenyl group in the lead and the diphenyl rings partially protruding from the entrance. No strict pi-pi stacking interactions are observed. However, tyrosine L32 (CDR1), tyrosine L96 (CDR3) and tryptophan H33 (CDR1) help immobilize the cyanophenyl ring and guanido group, and tyrosine H96 moves about 4.5 A to lie between the rings of the diphenyl group. The positive charge on the guanido group is compensated by glutamic acid H50 (CDR2) while the negative charge on acetic acid is neutralized by arginine H56 (CDR2) and by hydrogen bonding with asparagine H58 (CDR2). Water molecules participate in the binding process by hydrogen bonding with the cyano and guanido groups. The mechanism of binding is a clear example of induced fit. Like hemoglobin, the NC6.8 Fab can be classified as an allosteric protein, since its overall structure is altered by the binding of a small ligand. In crystals of the native Fab the elbow bend angle is 184 degrees while in crystals of the complex the elbow angle is 153 degrees. There is also a reciprocal push-pull type of change where the heavy chain is flexed and the light chain is extended. The tail of the heavy chain, which would be connected to the Fc in an intact antibody, is displaced 19 A relative to its position in the unliganded Fab. Within the limited series of sweetener-Fab complexes we have thus far examined, only the NC174 hapten has produced such results.(ABSTRACT TRUNCATED AT 400 WORDS)

Ligand-induced domain movement in an antibody Fab: molecular dynamics studies confirm the unique domain movement observed experimentally for Fab NC6.8 upon complexation and reveal its segmental flexibility.

Two molecular dynamics simulations were carried out for the antibody Fab NC6.8, both with and without the guanidinium sweetener ligand NC174, in order to assess the segmental flexibility as well as the conformational changes upon ligand binding. Trajectory analyses of the simulation of the uncomplexed Fab suggest low-amplitude motions of the Ig domains with respect to each other, most clearly reflected by a periodic alteration of the elbow angle within a range of 11 degrees. Upon insertion of the hapten into the binding site, the quaternary structure of the Fab exhibits considerable rearrangements: the elbow angle changes by almost 30 degrees, the light chain is elongated and the heavy chain becomes more flexed. Comparison with experiment reveals some interesting agreements with X-ray crystallographic results published previously.

The three-dimensional structure of a complex of a murine Fab (NC10. 14) with a potent sweetener (NC174): an illustration of structural diversity in antigen recognition by immunoglobulins.

The three-dimensional structure of a complex of an Fab from a murine IgG2b(lambda) antibody (NC10.14) with a high potency sweet tasting hap- ten, N-(p-cyanophenyl)-N'-(diphenylmethyl)-N"-(carboxymethyl)guan idine (NC174), has been determined to 2.6 A resolution by X-ray crystallography. This complex crystallized in the triclinic space group P1, with two molecules in the asymmetric unit. In contrast to a companion monoclonal antibody (NC6.8) with a kappa-type light chain and similar high affinity for the NC174 ligand, the NC10.14 antibody possessed a large and deep antigen combining site bounded primarily by the third complementarity-determining regions (CDR3s) of the light and heavy chains. CDR3 of the heavy chain dominated the site and its crown protruded into the external solvent as a type 1' beta-turn. NC174 was nested against HCDR3 and was held in place by two tryptophan side-chains (L91 and L96) from LCDR3. The diphenyl rings were accommodated on an upper tier of the binding pocket that is largely hydrophobic. At the floor of the site, a positively charged arginine side-chain (H95) stabilized the orientation of the electronegative cyano group of the hapten. The negative charge on the acetate group was partially neutralized by a hydrogen bond with the phenolic hydroxyl group of tyrosine H58. Comparisons of the modes of binding of NC174 to the NC6.8 and NC10.14 antibodies illustrate the enormous structural and mechanistic diversity manifest by immune responses.

Variable region sequence and characterization of monoclonal antibodies to a N,N',N"-trisubstituted guanidine high potency sweetener.

A library of monoclonal antibodies (mAb) was made against a trisubstituted guanidinium sweetener (N-(p-cyanophenyl)-N'-(diphenylmethyl)guanidine acetic acid) that is 200,000 times sweeter than sucrose on a molar basis. The mAb were characterized in terms of their ligand affinities, H- and L-chain isotypes and V-region amino acid sequences. Nine of these mAb were found to have dissociation constants in the nanomolar range. The H-chain V-regions were cloned, sequenced and found to be derived from five different families (Q52, X24, J558, 7183 and 36-60). L-chain V-regions were found to be derived from three kappa families (V kappa-4/5, V kappa-19/28 and V kappa-1) and one lambda family (V lambda-1). Amino acid homologies with these family sequences ranged from 51-91% for heavy chains and 69-97% for light chains. Sequence comparisons with Ig structures solved by X-ray diffraction were made in order to identify canonical structures. Identification and localization of combining region tryptophans (L:96W and H:33W) for two mAb (NC10.8 and NC6.8) supported previous ligand-induced tryptophan fluorescence quenching observations.

Conservation of cys-cys trp structural triads and their geometry in the protein domains of immunoglobulin superfamily members.

In almost all members of the immunoglobulin superfamily (IgSF) for which an experimental structure has been determined, a triad (C-CW) consisting of two cysteine residues that form a disulfide bond and a neighboring tryptophan can be found in the core of the protein fold. We analyzed the geometry of these C-CW triads among a database of 60 Fab crystal structures and found it to be remarkably conserved. We identified C-CW triads of a similar configuration in other members of the IgSF such as T cell receptor (TCR), major histocompatibility complex antigens (MHC), cell surface antigens CD4 and CD8, and cell-adhesion molecules. We used this C-CW pattern to search a database of non-IgSF proteins, and identified several proteins that contain a disulfide bridge associated with a tryptophan in a similar configuration. Examination of the distances and orientations between triads found in adjacent domains in Fab fragments and TCR also reveal a high degree of conservation, which reflects the invariance of the inter-chain domain packing. This high degree of conservation of the geometry of the C-CW triad in IgSF structures suggests that the Trp may contribute significantly to the stability of the disulfide bond. Knowledge of these geometric parameters may prove useful in the construction and validation of theoretical models of Ig, TCR, and other IgSF members.

Analysis of diversity of nucleotide and amino acid distributions in the VD and DJ joining regions in Ig heavy chains.

Nucleotide fill-in between the germ line V, D and J genes in the H3 loop of immunoglobulins contributes to the diversity of the antibody repertoire. This fill-in process is mediated by terminal deoxynucleotidyl transferase (TdT), which has been widely believed to insert nucleotides in a random fashion. Using a database of 2443 immunoglobulin sequences, we identified the regions of nucleotide fill-in between the V-D and D-J gene regions. We translated the fill-in nucleotides and measured the diversity within the two regions both at the nucleotide and amino acid level. We found that the nucleotide and amino acid distributions that resulted from nucleotide fill-in were in fact not random. Examination of the synonymous substitution rates of nucleotides revealed evidence suggesting that TdT plays a less significant role in generating antibody diversity than previously thought. We observed preferences for polar residues, which are more likely to encourage interaction with ligand than non-polar residues and are often found in loop regions in general. We also observed a preference for the insertion of smaller residues versus larger residues of similar biochemical properties, aiding in loop flexibility. We interpret these findings to reflect the significant influence of biochemical (i.e. folding) constraints and/or binding affinity constraints (at the cellular/selectional level) on the sequence diversity in the H3 region. These constraints act as a filter on the randomness generated by nucleotide addition by TdT, as well as other diversity generating processes such as recombination of VDJ gene segments and somatic mutation. The results of this study suggest that the antibody repertoire might be reduced from what is traditionally believed.

Mechanism of allergenic cross-reactions--IV. Evidence for participation of aromatic residues in the ligand binding site of two multi-specific IgE monoclonal antibodies.

The binding sites of two IgE monoclonal antibodies (mAbs), LA2 and LB4, were examined by absorption, fluorescence spectroscopy and computer-aided molecular modeling (CAMM). Absorption spectra revealed the formation of 1:1 molecular complexes for both LA2 and LB4 with a variety of structurally different ligands. For mAb LA2, the binding constants for ligands consisting of different amino acid derivatives coupled to DNP could be divided into two groups, suggesting that certain amino acid side chains (e.g. hydrophobic) of the derivatives were a contributing feature in ligand recognition. The presence of a charge-transfer band (320-340 nm) was also observed for complexation with several different ligands, indicative of aromatic ligand interactions with mAb binding site tryptophans. CAMM studies of the Fv region for both mAb support of the empirical observations and inspection of the Fv models reveal numerous binding site aromatic residues that are likely candidates for ligand recognition and complexation. The multi-specificity of these mAbs for different ligands may be due to a multitude of interactions with aromatic residues in the binding sites.

Heteroligation of a mouse monoclonal IgE antibody (La2) with small molecules, analysed by computer-aided automated docking.

A mouse monoclonal anti-TNP IgE antibody (IgE-La2) was screened by a competitive-binding ELISA with a random pool of over 2000 small molecules, mostly drugs, drug derivatives and metabolites. Thirteen of these (naproxene, beta-carboxy-chi-naphthol, oxolinic acid, hymecromone, 8-aminoquinoline, beta-naphthylamine, chi-nitrilo-cinnamic acid, 1,5-diaminonaphthaline, prolonium iodide, diaspirin, 3,4,5-trimethoxy-cinnamic acid, cycrimine, hemimellitic acid) were found to bind as strongly, or stronger, to the antibody as the immunizing hapten. We have used a Monte Carlo search technique for simulated docking of the DNP and non-DNP ligands to a model of the Fv region of IgE(La2). The validity of structural predictions made by the AutoDock program were tested on IgG(ANO2), the three-dimensional structure of which had been obtained previously by X-ray crystallography and 2D-NMR. The rms differences between the experimentally determined and auto-docked complexes in the energetically most favored binding modes were 0.31-0.44 A. Evaluation of structures of IgE(La2)-ligand complexes [including 2,4-dinitrophenol (DNP), 16 DNP amino acids, and the 13 non-DNP ligands listed above] obtained by computer-aided automated docking, suggested the existence of two subsites within an approximately 12 x 18 A2 groove extending between the H and L CDRs. Some of the ligands (DNP-Glu, 8-aminoquinoline, prolonium-I, beta-naphthylamine) were found to bind exclusively to subsite 1, others (DNP-Ala, chi-nitrilo-cinnamic acid, hemimellitic acid, beta-carboxy-chi-naphthol) to subsite 2. The majority of DNP amino acids and other ligands (oxolinic acid, 3,4,5-trimethoxy-cinnamic acid, diaspirin, [R]-cycrimine) were found to occupy an overlapping area including subsites 1 and 2, while some of the compounds (DNP-Asn, DNP-Pro, hymecromone, 1,5-naphthylenediamine) were predicted to interact with either of these subsites with comparable probabilities. When all of the docked La2-ligand complexes were taken into account, five tyrosine residues (H33, L32, L91, L92, L96) were found to provide the majority (53.4%) of all observed contact points. Thus, a multitude of interactions with aromatic residues, and a combinatorial type of interaction within the binding region, seem to be the major factors to explain the mechanism of heteroligation by IgE(La2).

Experimental autoimmune inflammatory myopathy.

We report an experimental model of autoimmune inflammatory myopathy. Splenic cells from two inbred murine strains (BALB/c and SJL/J) are activated (immunized) in vitro by co-culture with their respective syngeneic skeletal muscle myotubes. Subsequent injection of the activated splenocytes with or without B. pertussis into the respective syngeneic hosts results in inflammatory myopathy in the SJL/J mice but never in the BALB/c mice. The muscle inflammation is very similar in appearance to human autoimmune inflammatory myopathies. The myositis is not effector cell-skeletal muscle specific because splenocytes activated by co-culture with smooth muscle will also elicit skeletal muscle lesions. Both strains of skeletal muscle appear to express class II (Ia) antigens and the splenocytes from both strains appear to be equally activated. Thus we postulate that the difference in the expression of myositis between the two strains is in the effector phase of the disease. Since SJL/J mice have vasoactive amine sensitive vascular systems and BALB/c do not, it is likely that activated splenocytes emigrate from muscle microvessels in the SJL/J strain whereas they cannot do so in the BALB/c strain. The most significant contribution of this model may be in its potential for addressing a sine qua non of cellular autoimmune disease, i.e. lymphocyte migration from the vascular compartment into the target tissue. Finally, the data support a cellular more than a humoral pathogenesis in this model.

Absorption spectroscopy of the complexation between superpotent guanidinium sweeteners and specific monoclonal antibodies.

Spectroscopic studies of antibody-antigen interactions can provide useful information about the interactive motifs and energetics involved in the intermolecular association process. In this study we used absorption spectroscopy to examine the interactions between five different monoclonal antibodies (mAb) and four superpotent ligand sweeteners. Quantitative changes in the absorption spectra in the wavelength range of 230-800 nm were utilized for the determination of intrinsic association constants and thermodynamic parameters of the mAb-ligand complexes. The intrinsic association constants for the mAb-ligand complexes were found to be in the range of 10(7)-10(5) lM-1 and were in agreement with previous radioimmunoassay determinations. For two mAb, qualitative changes in the spectra in the 340 nm range could be identified and were related to the presence of charge-transfer interaction between the guanidinium ligand and aromatic residues in the binding site of the mAb. A charge transfer spectra was observed in mAb NC10.8 with two different sweetener ligands. The thermodynamic parameters of the ligand-mAb interactions were analyzed by van't Hoff plots and in almost all cases the reactions were found to be enthalpically driven. The determinations of intrinsic affinity and thermodynamic parameters may be useful in computer-aided molecular modelling studies of the antibody binding pocket and predicted ligand docking orientations. Antibody NC6.8 was found to react with this set of sweetener ligands in a rank order that is related to their sweetness potencies and the spectroscopic findings for NC6.8 are in agreement with the X-ray diffraction data of the Fab-ligand crystal structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Production and characterization of monoclonal idiotypes and anti-idiotypes for small ligands.

The procedures described in this chapter have enabled us to identify and characterize monoclonal antibodies and their respective anti-idiotypes. We have developed several different types of immunoassays which afford greater flexibility to the investigator, depending on the type of antibodies desired and the availability of labeled antigens. Use of the intrasplenic injection technique for the final booster immunization prior to the fusion protocol has enabled us to achieve more consistent results than the usual intravenous or intraperitoneal injection routes. Isoelectric focusing of tissue culture supernatant from monoclonal antibody-secreting clones can easily identify possible duplicate clones, and thereby reduces the amount of labor required for extensive characterization of a large number of clones. We have found that these techniques have enabled us to identify "sister clones" or redundancies in our collection of antiligand and anti-idiotype antibodies rapidly and accurately. These various techniques have allowed us to save much time, labor, and money in the search for specific antibodies with desired characteristics.

A simple 'free 3H-ligand' assay for rapid screening of hybridoma monoclonal antibodies against neurotransmitter analogs and anti-idiotype antibodies.

Effective, rapid screening of hybridoma supernatants for monoclonal antibodies against the dopaminergic antagonists pimozide and haloperidol, and the serotonergic antagonist ketanserin was performed using a 'free 3H-ligand' assay. Anti-mouse Ig-coated microtiter plates were incubated with hybridoma supernatants prior to incubation with excess 3H-ligand. After removal of free 3H-ligand, bound 3H-ligand was eluted with acid for liquid scintillation counting. With minor modification, the assay can be used to screen hybridomas for anti-anti-ligand (anti-idiotypic) antibodies.

Expression of mRNA for 55-kDa and 75-kDa tumor necrosis factor (TNF) receptors in mouse cerebrovascular endothelium: effects of interleukin-1 beta, interferon-gamma and TNF-alpha on cultured cells.

The interactions between tumor necrosis factor-alpha (TNF-alpha) and its receptors have been implicated to play an important role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). The effects of cytokines on the steady state levels of TNF receptor (TNFr) mRNA in cerebrovascular endothelial cells (CVE) cultured from EAE-susceptible (SJL/J) and EAE-resistant (BALB/c) mice were examined. Interferon-gamma and interleukin-1 beta up-regulated the levels of both the 55-kDa and 75-kDa TNFr mRNA, while TNF-alpha had no effect. No differences were observed in cytokine-induced TNFr mRNA levels between BALB/c and SJL/J CVE that might explain differences in EAE susceptibility.

Enhancement of histamine-induced vascular leakage by pertussis toxin in SJL/J mice but not BALB/c mice.

Pertussis toxin (PTX) from Bordetella pertussis is known to enhance inflammatory responses which involve histamine and serotonin, including cell-mediated delayed-type hypersensitivity reactions. In this study we examined the effects of PTX on histamine-modulated microvascular responses. The actions of histamine on arteriole diameter and post-capillary leaky site formation in the cremaster muscle were measured intra-vitally in two inbred strains of mice (viz. BALB/c and SLJ). In SJL mice the rate and extent of histamine-induced leaky site formation were greatly enhanced (from 8.3 to 21.0 leaky sites per 0.1 cm2) by pre-exposure to PTX. In sharp contrast, PTX did not alter histamine-induced leaky site formation in BALB/c mice. Histamine-mediated dilation in arterioles in both strains of mice were not enhanced by PTX. PTX may enhance the development of inflammatory responses by enhancing histamine-induced leaky site formation of the microvasculature.

Nicotine induces leukocyte rolling and adhesion in the cerebral microcirculation of the mouse.

Nicotine and several related metabolites were examined for their ability to induce leukocyte rolling and adhesion in the cerebral microcirculation of the mouse. A cranial window was surgically prepared for the visualization of the pial microcirculation using an intra-vital microscopy imaging system. Using this technique rhodamine-labeled leukocytes could be visualized and video-recorded as they traveled within the microvessels, and the quantitation of their rolling and adhesion along the pial venule walls was assessed during an off-line video playback analysis. Nicotine was found to produce significant dose-related increases in leukocyte rolling and adhesion. Cotinine, a major nicotine metabolite, did not induce the same degree of leukocyte rolling and adhesion. Mecamylamine, a nicotine antagonist, was found to inhibit the nicotine-induced leukocyte rolling and adhesion. Anti-P-selectin antibody blocked nicotine-induced leukocyte rolling, while anti-CD18 antibody effectively inhibited leukocyte adhesion, but not rolling in similar experiments. Nicotine-induced leukocyte rolling and adhesion were also inhibited by superoxide dismutase and catalase. These data suggest that nicotine, the principle pharmacological agent in cigarette smoke and related tobacco products, acts via a ganglionic-type nicotinic receptor to enhance leukocyte rolling via P-selectin and reactive oxygen radical-dependent mechanisms in cerebral microcirculation of the mouse.

Host genetic regulation of acute MHV-4 viral encephalomyelitis and acute experimental autoimmune encephalomyelitis in (BALB/cKe x SJL/J) recombinant-inbred mice.

In the present report we provide the strain distribution patterns of susceptibility to acute mouse hepatitis virus type-4 (MHV-4) encephalomyelitis, acute experimental allergic encephalomyelitis (EAE) and vasoactive amine sensitivity (VAAS) for 9 (CXJ) recombinant-inbred strains between BALB/cKe (C) and SJL/J(J) mice. We confirm that susceptibility to MHV-4 is not linked to the H-2 complex, and that all strains susceptible to acute EAE have both a responder H-2 haplotype (H-2s or H-2d) and induced (B. pertussis) VAAS. In addition, we provide evidence that susceptibility to acute EAE induction is controlled by an additional presently unmapped locus and that an EAE-like histopathological disease does not usually follow MHV-4 infection intracerebrally in animals susceptible to MHV-4, acute EAE and induced VAAS.

Preparation and characterization of antisera and monoclonal antibodies to serotonergic and dopaminergic ligands.

In an attempt to produce polyclonal antisera and monoclonal antibodies to serotonin, SKF 38393 (D-1 agonist), dopamine, and haloperidol (D-2 antagonist) several procedures for the preparation of immunogenic ligand-protein carrier conjugates were investigated. The Mannich reaction utilizing formaldehyde as the chemical linker was used to prepare serotonin-protein conjugates; antibodies raised to this conjugate reacted specifically to the conjugated serotonin moiety but did not react to native serotonin. Chemical conjugations involving dimethylpimelylimidate or N-carboxymethyl derivatives for the coupling of serotonin, dopamine and SKF 38393 to carrier proteins produced antibodies primarily directed against the 'chemical coupling arm' and very little antibody activity against the ligand itself could be detected. Synthesis of a haloperidol derivative suitable for chemical coupling to a protein carrier via oxobutyric acid produced an immunogen which was capable of eliciting both polyclonal and monoclonal antibodies specific for the hapten. The pitfalls of the various chemical conjugation procedures and the difficulties of producing antibodies to free ligands are discussed.

Detection of antibodies to myelin basic protein by solid-phase radioimmunoassay with [125I]protein A.

A solid phase radioimmunoassay (RIA) for the detection of antibodies to myelin basic protein (MBP) in sera has been developed employing MBP-coated flexible polyvinylchloride microtiter trays and [125I]protein-A as the radiolabel. [125I]Protein-A directly binds to the Fc region of serum IgG from several animal species and serves as an excellent reagent for detecting antibody. It can also be used to bind to a second antibody ligand, thereby making it useful even when it does not bind directly to the primary antibody Fc region. The use of one preparation of [125I]protein-A label allows sera from several species to be tested for antibodies to MBP simultaneously, thereby making this RIA technically simple, rapid and economical. This assay has been particularly useful in examining serum samples from animals with experimental autoimmune encephalomyelitis and hypomyelinogenisis congenita. In patients with multiple sclerosis low levels of antibody to MBP can be detected in cerebrospinal fluid (CSF) and acid-eluted extracts from brain plaque material; detection of low levels of antibody in human serum has not been possible due to non-specific binding of human serum Ig in this RIA.