Immunoglobulin VH determinants defined by monoclonal antibodies.

Hybridoma clones secreting antibodies against common VH determinants were readily produced by fusion of cells from mice immunized with isolated V mu fragments of human immunoglobulins (Ig), but not with intact Ig molecules or isolated heavy chains. Four monoclonal antibodies to the V mu fragments of different IgM paraproteins were selected for analysis: MH-44 (mu kappa), GB-24 (mu kappa), NF-11 (gamma 1 kappa), and SA-44 (gamma 1 kappa). Each antibody reacted with the homologous V mu fragment, homologous mu chain, and normal gamma chains, but not with the intact IgM molecules, intact IgG, or isolated light chains, as determined by radioimmunoassay. The VH reaction spectra with a panel of myeloma heavy chains showed overlapping but distinctive patterns for the four antibodies. Each of the four monoclonal anti-VH antibodies appeared to react with a different "hidden" VH determinant that is not exposed on undenatured, intact Ig molecules and differs from conventional VH subgroup determinants. In immunofluorescence studies, the monoclonal anti-VH antibodies did not bind to surface Ig on viable B lymphocytes, but visibly stained subpopulations of fixed B lymphocytes, pre-B cells, and normal plasma cells. The mean frequencies of VH+ plasma cells were 30% (MH-44), 17% (GB-24), 13% (NF-11), and 3% (SA-44), and similar frequencies were obtained for the VH+ B cell subpopulations. While subpopulations of B cells could be identified at all stages in differentiation by immunofluorescence with the anti-VH antibodies, neither resting nor activated T cells expressed these VH determinants in detectable amounts.

Genotype dependence of hepatitis C virus antibodies detectable by the first-generation enzyme-linked immunosorbent assay with C100-3 protein.

Hepatitis C virus (HCV) samples in 155 sera, from patients with chronic non-A, non-B liver disease and blood donors, were grouped into four genotypes (I, II, III, and IV) by amplification of core-gene sequences by polymerase chain reaction with type-specific primers. HCV genotypes were compared with various HCV-associated antibodies detectable by the first-generation ELISA (ELISA-1) with C100-3 protein and a second-generation immunoblot assay with four recombinant HCV proteins. Antibodies to C100-3 protein and those to its subsequence (5-1-1) were detected in 13 (93%) and 12 (86%), respectively, of 14 sera with genotype I HCV; 56 (79%) and 58 (82%) of 71 sera with genotype II; 13 (34%) and 6 (16%) of 38 sera with genotype III; and 11 (34%) and 4 (13%) of 32 sera with genotype IV. Amino acid sequences of C100-3 of genotype I HCV are conserved by approximately 90% in genotype II, but only by approximately 75% in genotypes III and IV. The sensitivity of ELISA-1, therefore, would be influenced by heterogeneity in C100-3 sequences of different genotypes.

IgM-mediated B cell apoptosis.

Cross-linking of surface immunoglobulin M (sIgM) on normal mature B cells induces different signaling consequences, including DNA synthesis (positive signaling) and cell cycle arrest and/or death by apoptosis (negative signaling). Presumably, the difference depends on the intensity of sIgM cross-linking: relatively weak cross-linking induces DNA synthesis, moderate cross-linking induces DNA synthesis with cell cycle arrest at the G2/M interphase, and intense cross-linking induces apoptosis. In vivo experiments with transgenic mice have shown that relatively weak cross-linking of sIgM by soluble antigens induces anergy in autoreactive B cells, whereas intense sIgM cross-linking by membrane-bound forms of antigens induces deletion of them. However, it is still unknown whether the different intensities of sIgM cross-linking generate qualitatively different signals responsible for DNA synthesis or cell death or whether they generate qualitatively the same but quantitatively different signals, and the quantitative difference is responsible for the induction of positive or negative signaling. The sIgM-mediated negative signaling presumably plays an important role in the induction and maintenance of B cell tolerance, and sIgD and sIgG also possess the machinery necessary for negative signaling. Negative signaling through sIgM is dependent on tyrosine kinase(s) and Ca2+ influx and is sensitive to cyclosporin A in certain types of B cells but not in all B cells. It has been suggested that there are different intracellular signaling pathways that transduce negative signaling via sIgM, and that activation-induced B cell death by sIgM cross-linking does not necessarily show DNA fragmentation and the morphology of apoptosis. On the other hand, sIgM-mediated B cell death may be inhibited in the presence of appropriate co-stimulators such as IL-4, alpha-, and beta-interferons and CD40-mediated signaling. The CD40-mediated signaling effectively inhibits sIgM-mediated B cell apoptosis in many but not all experimental systems. Although homotypic cell adhesion through the LFA-1/ICAM-1 dependent pathway was shown to be involved in certain types of CD40-mediated inhibition of sIgM-mediated negative signaling, it is still not known how the cytokines and CD40-mediated signaling inhibit sIgM-mediated B cell death. The molecular mechanisms responsible for sIgM-mediated negative signaling and for the inhibitory signaling against sIgM-mediated negative signaling need further elucidation.

Involvement of CD11b/CD18 in enhanced neutrophil adhesion by Fc gamma receptor stimulation.

Neutrophils showed a rapid and transient adhesion to immunoglobulin G (IgG)-coated plates compared with their adhesion to bovine serum albumin (BSA)-coated plates: the adhesion reached a peak after 15 min of incubation and then gradually returned to almost the basal state in 60 min. The addition of monomeric IgG or anti-Fc gamma RII monoclonal antibody (mAb) (IV.3) suppressed the increase in adhesion, whereas anti-Fc gamma RIII mAb (3G8) was hardly effective, indicating that the interaction of Fc gamma R, especially Fc gamma RII, with coated IgG is involved in the process. Adhesion was also blocked by cytochalasin B, suggesting that functional actin filament structures are crucial. Protein kinase inhibitors, erbstatin and genistein, inhibited the adhesion in a dose-dependent manner. The adhesion was inhibited by anti-CD11b (M1/70) and anti-CD18 (MHM23, TS1/18) mAbs. Moreover, neutrophils from a patient with complete leukocyte adhesion deficiency syndrome did not show increased adhesion to IgG-coated plates. The adhesion of neutrophils to fibrinogen- and BSA-coated plates was also increased when Fc gamma R was stimulated in the fluid phase with soluble aggregated IgG, which was also inhibited by anti-CD11b mAb. Stimulation of neutrophil Fc gamma R with soluble aggregated IgG enhanced the expression of CD11b in concert with the enhanced adhesion. These data collectively suggest that stimulation via Fc gamma R evokes a tyrosine kinase-dependent and actin filament-dependent intracellular signal that enhances the specific and nonspecific adhesive activity of neutrophils, presumably through the activation of CD11b/CD18.

Demonstration of two distinct antigenic determinants on human alpha-foetoprotein by monoclonal antibodies.

Mice were immunized with a purified preparation of human alpha-foetoprotein (AFP) and their spleen cells were hybridized with mouse myeloma cells. Eleven hybridoma cell lines secreting antibody to AFP were obtained. These antibodies were classified into two groups on the basis of different antigenic determinants they recognized. Seven cell lines produced antibodies directed to one determinant of AFP (determinant a), while the remaining 4 produced antibodies to another determinant (determinant b). A native AFP molecule bore one each of a and b determinants which were accessible by monoclonal antibodies; it was detected by a sandwich-type solid-phase radioimmunoassay only when antibody to a was used for coating wells of the microtitre plate and antibody to b as radiolabelled reagent, or vice versa. The presence of two different antigenic determinants on AFP was further confirmed by a chemical modification. When AFP was reduced in the presence of 2 mM dithiothreitol and then alkylated, determinant b was completely destroyed, but the determinant a remained unaffected. Furthermore, reduced and alkylated AFP was detected by the radioimmunoassay employing antibody to a both for coating wells and as radiolabelled reagent, thereby indicating that it bore two determinant as, one of which had been unaccessible in the native AFP.

Hepatitis B surface antigen particles with all four subtypic determinants: point mutations of hepatitis B virus DNA inducing phenotypic changes or double infection with viruses of different subtypes.

Hepatitis B surface antigen (HBsAg) particles carry the common determinant, a, as well as d or y and w or r subtype determinants, and are classified into the four major subtypes, i.e., adw, adr, ayw and ayr. Rare sera contain HBsAg particles with all four subtype determinants (adywr). Target sequences (nucleotides 38-550) in the S gene of hepatitis B virus (HBV) DNA in two such sera were amplified by the polymerase chain reaction. Individual amplification products were cloned in an M13 phage vector. The HBV DNA clones obtained were subtyped by determining the second letters of codon 122 and 160 for lysine (AAA/AAG) or arginine (AGA/AGG), which specify the d or y and w or r determinants, respectively. From one serum (S-63), two adw, 10 adr and 58 ayr clones were obtained. When the two adw clones and two representatives each of the adr and ayr clones were compared against each other, for the sequence of 235 base pairs representing nucleotides 295-529 in the S gene, they differed only by 0.4-2.1% (average 1.2%). These results indicated multiple point mutations of a single HBV strain of subtype ayr and co-infection of hepatocytes with the original HBV strain and its mutant of subtype adw as the mechanism for the production of HBsAg/adywr particles. From the other serum (K-45), 1 adw, 73 adr and 4 ayw clones were obtained. The adw clone and two representative adr clones differed only by 0-1.7% in the S gene sequences, but they differed by 8.5% or greater from two representative ayw clones. HBsAg/adywr particles in this serum, therefore, could be explained by double infection of hepatocytes with two HBV strains of different subtypes (adr and ayw).

Antigenic sites on the arginine-rich carboxyl-terminal domain of the capsid protein of hepatitis B virus distinct from hepatitis B core or e antigen.

The capsid protein of hepatitis B virus (P19) is made of 183 amino acids and carries the antigenic sites of hepatitis B core antigen (HBcAg) and hepatitis B e antigen (HBeAg) on the amino-terminal domain. The carboxyl-terminal domain of P19 (amino acids 150-183) is arginine-rich (47%) and faces the interior of the nucleocapsid for the binding with DNA. Monoclonal antibody was raised against an antigenic site on this protamine-like region of P19, which was distinct from HBcAg or HBeAg sites, and the novel antigenic site(s) was provisionally designated as hepatitis B inner core antigen (HBicAg). When P19 in a low concn (150 ng/ml) was immobilized on the solid surface, HBicAg sites were preserved, while HBcAg or HBcAg sites were no longer available on it. This allowed the detection of antibodies against HBicAg (anti-HBic), by sandwiching them between immobilized P19 and anti-IgG labeled with horseradish peroxidase. Anti-HBic was detected in sera from HBsAg carriers, typically those seropositive for antibody to HBeAg. A synthetic arginine-rich decapeptide, with a sequence of Arg-Arg-Arg-Gly-Arg-Ser-Pro-Arg-Arg-Arg, representing amino acids 150-159 of P19 and conserved in the majority of reported hepatitis B virus, absorbed the activity to bind with P19 in seven (44%) out of 16 sera containing anti-HBic. These results indicate that the decapeptide carries an HBicAg epitope and the remaining amino acid sequence of the arginine-rich carboxyl terminal domain (160-183) may be responsible for the other HBicAg epitopes.

The loss of subtypic determinants in alleles, d/y or w/r, on hepatitis B surface antigen.

Hepatitis B surface antigen possesses the group-specific determinant called a and one or another member from each of two pairs of allelic determinants, d and y as well as w and r, thereby creating the four major subtypes, adw, adr, ayw and ayr. In the sequence of major surface antigen polypeptides made of 226 amino acid residues, lysine or arginine at amino acid position 122 specifies d or y determinant, and lysine or arginine at position 160 specifies w or r determinant, respectively. By means of site-directed mutagenesis and expression of mutant genes in cultured cells, the mechanism for the loss of subtypic determinants on surface antigens was investigated at the molecular level. A rare sample of surface antigen of subtype ad, devoid of w or r determinant, had asparagine at position 160. When it was converted to lysine, the surface antigen of subtype adw was obtained. Two samples of surface antigen were subtyped as ar. They lacked d determinant, despite having lysine at position 122 which usually specified it. They differed from all reported sequences of surface antigen in amino acid 144 or 145. They displayed d determinant when amino acid 144 was converted from glutamic acid to aspartic acid, or when amino acid 145 was changed from alanine to glycine. These results indicate that the key amino acid residue at position 122 or 160 is indispensable for the expression of subtypic determinants and that some distant residues are also crucially involved in conforming them.

Interferon and (2'-5')oligoadenylate enhance the expression of low affinity receptors for IgE (Fc epsilon R2/CD23) on the human monoblast cell line U937.

The regulation of low affinity IgE receptor (Fc epsilon R2/CD23) expression on the human monoblast cell line U937 was examined by an anti-Fc epsilon R2/CD23 monoclonal antibody (H107) and the cDNA probe for Fc epsilon R2/CD23. Alpha interferon (IFN-alpha) and its intracellular mediator, (2'-5')oligoadenylate (2, 5-A), induced Fc epsilon R2/CD23 expression on U937 with no significant increase of the Fc epsilon R2/CD23 mRNA. PMA and IFN-gamma increased both surface Fc epsilon R2/CD23 expression and the Fc epsilon R2/CD23 mRNA levels. IFN-alpha effectively induced 2, 5-A synthetase activity in U937 cells, whereas IFN-gamma induced little. The results suggest that the mechanisms of enhancement of Fc epsilon R2/CD23 expression on U937 cells by IFN-alpha and IFN-gamma are different and that 2, 5-A may play an important role in the Fc epsilon R2/CD23 expression on U937 cells induced by IFN-alpha.

A glycopeptide containing 15 amino acid residues derived from hepatitis B surface antigen particles: demonstration of immunogenicity to raise anti-HBs in mice.

The major polypeptides composing hepatitis B surface antigen (HBsAg) particles are P-I and P-II. P-II shares the same amino acid sequence as P-I and contains an additional carbohydrate moiety of mol. wt approximately 5000. When a purified preparation of P-II was digested with Nagarse and then with Pronase P, it gave rise to a glycopeptide containing 15 amino acid residues and the carbohydrate moiety of P-II. The N-terminal amino acid sequence of the glycopeptide was determined to be Lys-Pro-Thr-Asp-Gly-Asn-. The polysaccharide moiety contained 5 moles of N-acetylglucosamine and was connected with Asn at the sixth position from the N-terminus. When mice were immunized against this HBsAg glycopeptide, they raised humoral antibodies which bound to each of three preparations of P-I derived from HGsAg particles of subtypes adw, adr and ayw, thereby indicating that the sequence of 15 amino acids in the glycopeptide would constitute a common antigenic structure of HBsAg.

IL-4 and prostaglandin E2 inhibit hypomethylation of the 5' regulatory region of IFN-gamma gene during differentiation of naive CD4+ T cells.

We have previously shown that prostaglandin E2 (PGE2) and IL-4 inhibit the priming of IFN-gamma-production during the differentiation of naive CD4+ T cells from human cord blood by different signal-transducing mechanisms. To compare and analyse the molecular mechanisms by which PGE2 and IL-4 inhibit the priming of IFN-gamma production, we investigated the effects of PGE2 and IL-4 on the methylation of the IFN-gamma gene during the in vitro differentiation of naive CD4+ T cells. In human naive CD4+ T cells, which produce primarily IL-2 and a little amount of IFN-gamma, the IFN-gamma gene was methylated. After stimulation via TCR, CD4+ T cells produced IFN-gamma and the CpG dinucleotide contained within the TATA proximal regulatory element of the IFN-gamma gene was partially hypomethylated. Both IL-4 and PGE2 inhibited the hypomethylation of this site and the acquisition of IFN-gamma-producing ability. In contrast to the SnaBI site in the TATA proximal regulatory element, the HpalI site in the first intron of the IFN-gamma gene of the CD4+ T cells from cord blood was completely methylated even after stimulation via TCR. 5-azacytidine restored the IFN-gamma-producing ability of these cells treated with IL-4 and PGE2. These findings suggest that, although the signal transduction that inhibits the priming of IFN-gamma-production is different for each reagent, the protection from hypomethylation of the regulatory region of the IFN-gamma gene is involved in the molecular mechanisms by which these reagents inhibit the priming of IFN-gamma-production during the differentiation of human naive CD4+ T cells.

Induction of phosphatidylinositol turnover and EGR-1 mRNA expression by crosslinking of surface IgM and IgD in the human B cell line B104.

We have previously shown that a human B lymphoma cell line, B104, expressed surface IgM (sIgM) and surface IgD (sIgD), and that crosslinking of sIgM and sIgD by anti-IgM antibody (Ab) and anti-IgD Ab, respectively, induced Ca2+ influx to almost the same degree, whereas only sIgM-crosslinking caused B104 cell death. Here, we investigated the accumulation of cyclic AMP (cAMP), the hydrolysis of inositol phosphates, protein kinase C (PKC) activity and the induction of Egr-1 and c-fos mRNA expression by sIgM- and sIgD-crosslinking to examine differences in the signals mediated through sIgM and sIgD in B104 cells. Both sIgM- and sIgD-crosslinking with antibodies induced elevation of cAMP levels, phosphatidylinositol turnover, PKC activation and expression of Egr-1 and c-fos mRNA, although sIgM-crosslinking was more effective than sIgD-crosslinking, presumably due to the higher expression of sIgM than of sIgD. Egr-1 mRNA expression induced by sIgM- and sIgD-crosslinking was inhibited by H7, erbstatin and genistein, but not by HA1004. Erbstatin and genistein inhibited the sIg-crosslinking-induced Egr-1 mRNA expression in a dose-dependent manner parallel to that observed in the inhibition of sIg-crosslinking-induced protein tyrosine phosphorylation. Phorbol myristate acetate induced Egr-1 mRNA expression but forskolin and dibutyryl cyclic AMP did not. These findings suggest that the Egr-1 mRNA activating signals through sIgM and sIgD are protein tyrosine kinase- and PKC-dependent, but protein kinase A-independent. Cyclosporin A (CsA) and FK506 rescued B104 cells from death induced by anti-IgM Ab, but did not affect the expression of Egr-1 and c-fos mRNA, showing that CsA and FK506 affect signal transducers differently from or downstream to these molecules. The difference in signals transduced through sIgM and sIgD in B104 cells is discussed.

Low burst-promoting activity (BPA) production by cord mononuclear cells is due to functional immaturity of monocytes.

Cord blood mononuclear cells produced lower burst-promoting activity (BPA) than adult peripheral blood mononuclear cells when stimulated with phytohemagglutinin (PHA). In order to examine the cellular basis of the low production of BPA by PHA-stimulated cord blood mononuclear cells in the context of the functional immaturity of T cells or monocytes, we studied BPA production by T cells or monocytes from cord blood and adult peripheral blood. Cord T cells produced as much BPA as adult T cells. Monocytes themselves did not produce significant BPA at the concentration used in this experiment (1 x 10(5)/ml). BPA production by adult T cells was significantly enhanced by the presence of autologous monocytes. BPA production by cord T cells was also enhanced by the presence of adult monocytes but not by that of cord monocytes. Cord monocytes did not enhance BPA production by adult T cells either. These results indicate that cord monocytes are primarily responsible for the low BPA production by PHA-stimulated cord mononuclear cells.

Dibutyryl cyclic AMP induces formyl peptide receptor expression and chemotactic responses in a human eosinophilic cell line, EoL-1.

We investigated actin polymerization and the increase of cytosolic free calcium concentration ([Ca2+]i) in a human eosinophilic leukemia cell line, EoL-1, in response to stimulation with chemotactic factors; we also investigated the effect of dibutyryl cyclic AMP (dbcAMP) on the responses. EoL-1 cells under normal culture conditions did not show either actin polymerization or an increase in [Ca2+]i when stimulated with formyl-methionyl-leucyl-phenylalanine (fMLP). Expression of formyl peptide receptors was not detectable on untreated EoL-1 cells, either. Dibutyryl cAMP induced the expression of formyl peptide receptors and the responsiveness to fMLP. The responsiveness of EoL-1 cells to the complement fragment C5a and platelet-activating factor (PAF) was also induced or enhanced by dbcAMP. The growth of EoL-1 cells was decreased and the proportion of cells in the G0/G1 phase of the cell cycle was increased by the treatment of EoL-1 cells with dbcAMP. The proportion of eosinophilic granule-containing cells and the content of eosinophil cationic protein (ECP) in EoL-1 cells was also increased when they were stimulated with dbcAMP for a longer period. The responsiveness of EoL-1 cells to fMLP, C5a, and PAF was shown to be regulated independently. EoL-1 cells and dbcAMP seem to be useful for examining chemotactic receptor expression and its signal transduction mechanisms.

Characterization of an eosinophilic leukemia cell differentiation factor (ELDF) produced by a human T cell leukemia cell line, HIL-3.

An adult T cell leukemia cell line, HIL-3, constitutively secretes a factor which induces the phenotypical and functional eosinophilic differentiation of a human eosinophilic leukemia cell line, EoL-1. Biochemical characteristics of the factor, termed eosinophilic leukemia cell differentiation factor (ELDF), were examined. ELDF was precipitated by 35 to 65% saturated ammonium sulfate from the culture supernatants of HIL-3 cells (HIL-3 sup). ELDF was eluted in a peak corresponding to a molecular weight of 30 to 40 kd by gel filtration. Isoelectric focusing in the Rotofor showed that ELDF had isoelectric points of 5 to 6. ELDF was trypsin-sensitive and stable to heat treatment at 65 degrees C for 30 minutes but labile at 80 degrees C or pH lower than 3. Half of the activity adhered to lentil-lectin but not to Con-A, indicating that a part of ELDF is glycoprotein with an N-linked carbohydrate moiety, which did not seem to be essential for ELDF activity. The biochemical characteristics of ELDF and blocking experiments using cytokine-specific neutralizing antibodies suggest that ELDF is different from gamma-interferon (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-5 (IL-5) and interleukin-2 (IL-2), which may exist in HIL-3 sup, and that ELDF may be a previously unrecognized leukemia differentiation factor.

Application of monoclonal antibodies to the isolation and characterization of a killer toxin secreted by Hansenula mrakii.

A strain of yeast, Hansenula mrakii, secretes a toxin that kills sensitive yeasts, such as Saccharomyces cerevisiae Monoclonal antibodies raised against the toxin had both binding and neutralizing activities. The toxin in culture media was isolated by an affinity column of monoclonal antibody. The toxin is a basic polypeptide with an isoelectric point at pH 9.1, and devoid of mannosides. It is composed of 88 amino acid residues with a molecular size of 10 721 Da. The monoclonal antibodies could be applicable to the analysis of biologically active sites on the toxin, in an attempt to synthesize chemically a small peptide with killer activity and little immunogenicity.

A pitfall in two-site sandwich 'one-step' immunoassay with monoclonal antibodies for the determination of human alpha-fetoprotein.

Utilizing monoclonal antibodies directed to 2 distinct antigenic determinants of the human alpha-fetoprotein (AFP), Uotila, Ruoslahti and Engvall developed the 2-site sandwich immunoassay. They found that due to the different specificities of monoclonal antibodies, 2 antigen-antibody reactions, AFP with immobilized antibody and AFP with labeled antibody, could be accomplished in a single step. We have found, however, that above a certain concentration, AFP detectable by their method decreased because the labeled antibody tended to bind with AFP that failed to react with the immobilized antibody. Consequently, 2 different AFP concentrations produced the same result, and an extremely high, still clinically expectable concentration gave a false negative result. Such an inhibition in high AFP concentrations was not observed in the conventional '2-step' immunoassay within the range of concentrations tested (0.1 ng-3 mg/ml). On the basis of these observations, the 2-site '1-step' immunoassay for AFP would have to be applied on multiple dilutions of the serum to avoid an erroneous interpretation of the results.

Determination of the antibody to hepatitis B e antigen by the inhibition on electrophoresis of free e antigen.

A new method is described for the rapid and sensitive detection of the antibody to hepatitis B e antigen (anti-HBe) in serum. Twenty-five microliters of the test serum were incubated with 10 microliter of the purified small 'free' e antigen, and then the consumption of the added antigen was detected by the failure to precipitate with standard anti-HBe in counter-electrophoresis. When a total of 444 serums from asymptomatic carriers of hepatitis B surface antigen was tested for anti-HBe by this method, 348 (78.4%) were found to be positive with a detectability much higher than that of the passive hemagglutination method which detected the antibody in only 264 (59.5%) of the same serums.

A two-site sandwich radioimmunoassay of beta 2-microglobulin with monoclonal antibodies.

Among 21 batches of monoclonal antibodies raised against beta 2-microglobulin (anti-beta 2m), 6 reacted with soluble beta 2m, as well as with beta 2m present in association with HLA heavy chains on the surface of leukocytes. The remaining 15 anti-beta 2m antibodies bound with soluble beta 2m, but failed to react with beta 2m on the cell surface. No monoclonal anti-beta 2m antibodies revealed precipitin lines when they were tested against beta 2m in immunodiffusion. When 2 anti-beta 2m antibodies with different specificities were mixed together, however, they developed a precipitin line against beta 2m. Based on these observations, there was only 1 epitope on beta 2m that was available for the binding with a monoclonal anti-beta 2m antibody with either specificity. This allowed the development of a solid-phase radioimmunoassay in which beta 2m in test specimens was sandwiched between immobilized anti-beta 2m of 1 specificity and radiolabeled anti-beta 2m of a heterologous specificity in a single step. Monoclonal antibodies with at least 2 different specificities would be required for developing a sandwich-type immunoassay of polypeptides, such as beta 2m, that do not display 2 or more epitopes of the same specificity.

A radioimmunoassay that sandwiches human interleukin-2 between radiolabeled monoclonal antibody and the receptor on a hematopoietic cell line.

Two monoclonal antibodies were raised against human interleukin-2 (IL-2) produced by E. coli harboring recombinant complemental DNA. Both antibodies did not neutralize its activity, nor did they inhibit the binding of IL-2 to the receptor on target cells. Taking advantage of the ability of monoclonal antibodies to detect IL-2 that had bound to the receptor, a radioimmunoassay was developed that sandwiched IL-2 between the radiolabeled monoclonal antibody and the receptor on a hematopoietic cell line infected with human T cell leukemia virus Type I. The assay had the advantage of detecting only IL-2 with the ability to bind to the receptor, and displayed a linear dose-response relationship over concentrations ranging from 5 to 100 ng/ml.