We have produced novel bispecific antibodies by fusing the DNA encoding a single chain antibody (ScFv) after the C terminus (CH3-ScFv) or after the hinge (Hinge-ScFv) with an antibody of a different specificity. The fusion protein is expressed by gene transfection in the context of a murine variable region. Transfectomas secrete a homogeneous population of the recombinant antibody with two different specificities, one at the N terminus (anti-dextran) and one at the C terminus (anti-dansyl). The CH3-ScFv antibody, which maintains the constant region of human IgG3, has some of the associated effector functions such as long half-life and Fc receptor binding. The Hinge-ScFv antibody which lacks the CH2 and CH3 domains has no known effector functions.

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A genetically engineered chimeric HIRMAb has been produced, and the chimeric antibody has identical reactivity to the human and primate BBB HIR as the original murine antibody. This chimeric HIRMAb may be used in humans for drug targeting through the BBB of neurodiagnostic or neurotherapeutic drugs that normally do not cross the BBB.

Both IgM and IgA exist as polymeric immunoglobulins. IgM is assembled into pentamers with J chain and hexamers lacking J chain. In contrast, polymeric IgA exists mostly as dimers with J chain. Both IgM and IgA possess an 18-amino acid extension of the C terminus (the tail-piece (tp)) that participates in polymerization through a penultimate cysteine residue. The IgM (tp) and IgA (␣tp) tail-pieces differ at seven amino acid positions. However, the tail-pieces by themselves do not determine the extent of polymerization. We now show that the restriction of polymerization to dimers requires both C ␣ 3 and ␣tp and that more efficient dimer assembly occurs when C ␣ 2 is also present; the dimers contain J chain. Formation of pentamers containing J chain requires C 3, C 4, and the tp. IgM-␣tp is present mainly as hexamers lacking J chain, and ␥-tp forms tetramers and hexamers lacking J chain, whereas IgA-tp is present as high order polymers containing J chain. In addition, there is heterogeneous processing of the Nlinked carbohydrate on IgA-tp, with some remaining in the high mannose state. These data suggest that in addition to the tail-piece, structural motifs in the constant region domains are critical for polymer assembly and J chain incorporation.

In this article we show how the polymerase chain reaction (PCR) and primers designed for conserved sequences of leader (L), framework one (FR1) and constant (CONST) regions of immunoglobulin light and heavy chain genes can be used for the cloning and sequencing of rearranged antibody variable regions from mouse hybridoma cells. RNA was extracted from the mouse hybridoma cells secreting MAbs: IOR-T3a (anti-CD3), C6 (anti-P1 of N. meningitidis B385), IOR-T1 (anti-CD6), CB-CEA.1 (anti-carcinoembryonic antigen), and CB-Fib.1 (anti-human fibrin). First strand cDNA was synthesized and amplified using PCR. The newly designed primers are superior to others reported recently in the literature. Isolated PCR DNA fragments of C6 and IOR-T3a were sequenced after asymmetric amplification, or M13 cloning. The FR1/CONST primer combinations selectively amplified mouse lights chain of groups kappa II, V, and VI, and heavy chains of groups IIa and IIc. The L/CONST primers for light chains amplified light chains from all four hybridomas. These methods greatly facilitate structural and functional studies of antibodies by reducing the efforts required to clone and sequence their variable regions.

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The 18-amino acid carboxyl-terminal tailpiece from IgM (mutp) has now been added to the carboxyl-termini of IgG1, IgG2, IgG3, and IgG4 constant regions to produce recombinant IgM-like IgGs. Polymeric IgGs obtained by this approach possess up to six Fcs and 12 antigen-combining sites, greatly increasing the avidity of their interactions with other molecules. Not surprisingly, the C activity of normally active IgG1 and IgG3 and somewhat less active IgG2 Abs is shown to be dramatically enhanced upon polymerization. The multiple Fcs present in a single molecule apparently allow for more efficient interactions with the multiple C1q heads present in C1, the first component of the classical C cascade. An unexpected result however, is that IgG4, normally devoid of C activity, when polymerized in the same fashion directs C-mediated lysis of target cells almost as effectively as the other polymers. Interestingly though, IgG4mutp does not deplete C activity in a standard consumption assay using soluble Ag. The other gamma mutp isotypes are capable of depleting 100% of the serum lytic ability even in the absence of Ag, whereas IgG4mutp shows no evidence of activity in this assay under any of the conditions tested. Additionally, we show that, in contrast to monomeric IgG, polymeric IgGs bind with very high affinity to Fc gamma receptor II (Fc gamma RII), a low affinity receptor for wild-type antibodies; however, binding to Fc gamma Rl, the high affinity receptor, appears to be unaltered. Finally, the in vivo t1/2 of the gamma mutp proteins is decreased relative to wild-type IgG, apparently because of rapid clearance of the polymeric fraction.

We demonstrate that a mixture of four recombinant dengue virus E polypeptides corresponding to the N-terminal region of the envelope protein from all serotypes substitutes for standard antigens in two immunoglobulin M enzyme-linked immunosorbent assay formats with 100% concordance, making these polypeptides a useful and accessible reagent for serological diagnosis of dengue in endemic countries.Dengue is a major global public health problem with increasing numbers of cases and expanded geographic distribution (5). The four serotypes of the dengue flavivirus (DENV1 through DENV4) have a positive-sense RNA genome that is translated as a single polyprotein and posttranslationally cleaved into three structural proteins and seven nonstructural proteins (8). The envelope protein (E) is considered to be the immunodominant protein (10).Large-scale dengue diagnosis relies on serological testing. While analysis of the change in immunoglobulin M (IgM) or IgG antibody titer in paired acute-and convalescent-phase sera is considered the "gold standard," in practice, the most commonly used diagnostic test is the IgM capture enzymelinked immunosorbent assay (MAC-ELISA) (13) with a single specimen. Viral antigen, commonly prepared in cell culture or in suckling mouse brain, is often the limiting reagent in developing countries in which in-house ELISAs are used instead of expensive commercial kits. The simplified production of recombinant DENV antigen in bacteria avoids problems associated with the quality and standardization of conventional DENV antigen preparations (2,4,7,9,11,14). Previously, we identified and produced a candidate diagnostic polypeptide corresponding to the N-terminal portion (166 amino acids) of the immunogenic E protein of DENV2 (3). Here, we produced homologous recombinant E antigens from DENV1, DENV3, and DENV4, which, in combination with the DENV2 polypeptide, have great potential as a low-cost and accessible tetravalent dengue diagnostic reagent.A ϳ500-bp fragment from the N terminus (nucleotides 1093 through 1585) of the DENV E gene was amplified by reverse transcriptase PCR (3) (3), and sequenced. The identities of the products were confirmed by comparison to known sequences (GenBank). The cDNAs were then cloned in-frame into the pQE-30 expression vector (3) immediately downstream of the hexahistidine tag.Expression of the recombinant polypetide clones in transformed Escherichia coli M15 was assessed by induction with 1 mM isopropyl-D-thiogalactoside (IPTG) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of aliquots at various time points. Large-scale expression of the expected ϳ20-kDa recombinant proteins (200 ml SuperBroth) yielded enough soluble protein for nickel affinity purification. Bacterial lysis and binding to affinity resin were conducted under denaturing conditions, whereas elutions were performed under native conditions, according to the protocol previously established for DENV2 (Fig. 1A) (3). DENV2 clone pD2-3 (3) was used to prepare the DENV2 E polypepti...