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This paper discusses the detection of antibody catalysis using soluble test substrates. Antibodies raised against a transition state analog of a chemical reaction typically show dissociation constants for the antibody-hapten complexes in the range of 10-10 M. If hapten binding is transferred to catalysis as measured by the transition-state dissociation constant K TS = K M /( k cat / k uncat )= k uncat /( k cat / K M ), this corresponds to the concentration of antibody necessary to double the apparent rate of the reaction. This sets a lower limit for the detection of catalysis if no additional effects are present to induce catalysis. The concentration of antibodies in hybridoma cell culture supernatants (5-50 μg/ml) meets this requirement. The use of high-throughput screening (HTS) for catalysis together with ELISA to select hybridomas leads to the isolation of not only one, but also many catalytic antibodies. As examples, the application of HTS for catalysis using fluorogenic reactions to isolate retro-Diels-Alderase and pivalase catalytic antibodies useful for prodrug activation chemistry are discussed.

The T cell receptor (TCR) α and β chains are encoded by a series of stochastic rearrangements between variable (V), diversity (D) for TCR β chain only, and joining (J) gene segments, creating hypervariable complementarity-determining region 3 (CDR3) regions that contact the peptide/MHC complex and confer specificity. In the present paper, we applied the recently developed real-time quantitative RT-PCR technique to the detection of rearranged TCR β chain mRNA transcripts. We designed BV- and BJ-specific primers together with TaqMan™ probes specific for the CDR3 regions of the clones of interest. As an external reference, we used plasmids containing the entire TCR β chains, making it possible to normalize the number of specific rearranged BV-J mRNA copies among the total number of TCR β chains. Here, we present data validating this fluorogenic PCR-based method for the quantification of several TCR clonotypes characteristic of the CD4 T cell response to hen egg white lysozyme (HEL) in mice of the H-2 haplotype. This accurate and sensitive procedure permits the precise determination of T cell clone frequencies ranging from 10 to less than 10 in normal biological samples; it may provide an alternative approach when frequencies are too low to be assessed by flow cytometry.

Highly active anti-retroviral therapy (HAART) has reduced the plasma load of HIV-1 to undetectable levels. It has however failed to eliminate the virus from other body compartments. Current methods for monitoring persistent viral replication in HIV-1+ patients require a large amount of blood and/or repeated tissue biopsies. Furthermore, some of the viral reservoirs, such as brain and eye, are inaccessible for sampling. The detection of episomal HIV-1 DNA 2-LTR circles in CD4+ cells is indicative of recent, acute infection events. This paper describes a reliable and reproducible LightCycler-based assay for the quantitative measurement of HIV-1 DNA 2-LTR circles in human peripheral blood mononuclear (PBMN) cells. It details the modifications to the DNA extraction procedure and to the LightCycler PCR procedure that were required to achieve this. This new surrogate marker of persistent viral replication can now be reliably, reproducibly and robustly used to study the clinical progress of large numbers of patients whose plasma HIV-1 RNA has been reduced to undetectable levels by anti-retroviral drugs.

Complement receptor II (CD21) is the receptor for C3d fragments on immune complexes. It also serves as a receptor for Epstein-Barr virus (EBV) and on B-lymphocytes CD21 amplifies signalling through the B cell receptor. CD21 is shed from the surface of the cell and is found circulating in plasma. There, soluble CD21 (sCD21) binds to CD23 and complement fragments, thereby modulating the immune response. sCD21 activates monocytes through binding to membrane CD23. The clinical significance of sCD21 is shown by the increased levels found in the sera of patients with B lymphomas, EBV infections and other lymphoblastoid tumors. In this paper, we report the isolation of soluble CD21 from human plasma using affinity chromatography and density gradient centrifugation. sCD21 was found to be a single 126 kDa molecular species. By determining the sedimentation coefficient, we have calculated the partial specific volume, diffusion coefficient and frictional coefficient of the protein. These values show that the sCD21 isolated from human plasma is an elongated rod-shaped molecule.

The proliferative response of human white cells to stimuli such as foreign histocompatibility antigens or mitogens is traditionally assessed by measuring the amount of [3H]thymidine which the cells can incorporate in culture. In the present paper a new approach is described in which bromodeoxyuridine (BrdUrd) is used in place of thymidine and measurement of incorporation of this molecule is performed by an immunoenzymatic assay (using monoclonal anti-BrdUrd). The procedure avoids the expense, time and hazards associated with scintillation counting, and is simpler to perform. It also appears to be particularly sensitive to low levels of proliferation, and may thus detect such responses more effectively than thymidine incorporation.

Human α-fetoprotein (AFP) variants from cord sera were separated by isoelectric focusing in agarose gels under native conditions, transferred to nitrocellulose paper and detected with polyclonal and monoclonal antibodies (Moabs). Rabbit anti-AFP recognized up to 9 individual electrophoretic variants in the range of pH 4.5 to pH 5.2. The reactivity of 8 Moabs ranged from weak to strong and showed variability in the pattern of AFP bands recognized. Moabs were separated into 3 groups according to the number of bands detected: group 1 detected 6 to 7 bands; group 2 recognized only one band; and group 3 recognized 4 bands. The sensitivity of the system with polyclonal antibodies was 0.15 ng of AFP in complete cord serum and varied between 300 and 0.2 ng with Moabs.

A solid-phase radioimmunoassay (SPRA) has been developed to measure IgG antibodies to bee venom (BV) and phospholipase A2(PLA) in human sera. The principle of the test is similar to that of the radioallergosorbent test (RAST) measuring IgE antibody. Cyanogen-bromide-activated paper discs coupled with BV of PLA followed by supplementary coupling with human serum albumin were incubated with standard or test sera, washed, and incubated with 125I-labeled anti IgG. The serum levels of the IgG antibody have been temporarily expressed in arbitrary units. The reaction between the antigen and antibody was specific and the results were reproducible. Sera from 19 beekeepers, 42 beesting-sensitive patients and 20 blood donors (controls) were assayed by the SPRA. IgG antibodies to BV and PLA could not be detected (< 4 U/ml) in all control sera, in 25 of the 42 patients and in one beekeeper. The IgG antibodies in 17 patients ranged between 5 to 58 U/ml (mean 7.6 U/ml), and in the 18 beekeepers ranged between 8 to 160 U/ml (mean 59 U/ml).

This paper describes a dot immunobinding assay for determining total human IgE with a tandem of monoclonal anti-IgE antibodies. Minute quantities of monoclonal anti-IgE antibodies were adsorbed on nitrocellulose discs. IgE bound to this solid phase monoclonal anti-IgE antibody was detected by a second monoclonal antibody conjugated with horseradish peroxidase. Using 4-chloro-1-naphthol as a chromegen results in a stable colour reaction that can be semiquantitavely analysed by the naked eye. The colour intensities of the reaction were also analysed by densitometry, yielding a very reproducible quantitation of human serum IgE. Using a serum dilution of 1 : 50, IgE could be detected in the range of 12.5–2500 U/ml. Using non-diluted serum samples IgE levels between 0.05–50 U/ml were reproducibly measured. Total serum IgE as determined by this dot assay correlated very well with IgE determinations performed by the commercial PRIST assay.

This paper describes the proper statistical methods for the determination of the range of normal values of serum immunoglobulins in any population and for the conversion of those ranges, or any serum immunoglobulin value, from mg per ml to World Health Organization International Units. In determining the normal range, it is argued that the analysis should be based upon the logarithms of the serum immunoglobulin values and that the range of normal values about the mean should be determined by the application of tolerance limits, rather than the more familiar but incorrect confidence limits. For converting normal ranges from mg per ml to International Units, methods of parallel line bioassay (for IgG and IgA) and non-linear bioassay (for IgM and IgD) are used. For both bioassay methods, graphical techniques are described.