Anti-metatype antibodies in immunoassays

In this review anti-metatype antibodies are described invoking new principles in immunoassay development. Anti-metatype antibodies are immunological reagents specific for the conformation of the liganded antibody active site which do not interact with bound ligand or unliganded antibody. Relationships between anti-metatype antibody reactivity and the ligand-induced conformational state of monoclonal antibodies are reviewed with emphasis on the fluorescein hapten as a small molecule model system. One characteristic result of the interaction of anti-metatype antibodies with liganded antibodies is a significant delay in the dissociation rate (k₂) of the ligand bound within the primary immune complex. The latter is an important consideration for assay development. Polyclonal and monoclonal anti-metatype antibody reagents are characterized in terms of their differential effects on the ligand dissociation rate. Anti-metatype antibody reactivity is further discussed in terms of protein-protein specificity patterns and relative interactions with idiotype-family members, structural derivatives, and site-specific mutants. Incorporation of principles inherent in the anti-metatype concept and their application to assay development are summarized.Abbreviations D₂O deuterium oxide - Fab 50 kd antibody fragment containing V_HC_H1 + V_LC_L domains - FITC(I) fluorescein isothiocyanate (isomer I) - Fv 26 kd fragment of the antibody molecule containing the variable domains of the H and L chains - Ig immunoglobulin - IgG immunoglobulin G with a mol. wt. of 150 kd. - IgM immunoglobulin M with a mol. wt. of 10⁶d - Id idiotype - Ka antibody affinity (k₁/k₂) in M^–1 - k₁ second order rate of ligand association in M^–1s^–1 - k₂ first order rate of ligand dissociation in s^–1 - K_D dissociation constant or the reciprocal of the affinity constant (1/Ka) - Mab monoclonal antibody - Met metatype - NMR nuclear magnetic resonance - SCA single chain Fv derivative containing a synthetic linker between the two variable domains - V_H variable domain of the antibody H chain - V_L variable domain of the antibody L chain     

syn-2,3-Disubstituted-2,3-dihydro-1,4-benzoxathiin rings: preparation from quinone ketals and 2-mercaptoethanols

A general method for the preparation of syn-2,3-disubstituted-2,3-dihydro-1,4-benzoxathiin rings from 2-mercaptoethanols and quinone ketals is presented. This ring system is produced by Michael addition of a 2-mercaptoethanol to a quinone ketal, followed by cyclization of the initial Michael adduct, and subsequent aromatization to afford a syn-2,3-disubstituted-1,4-benzoxathiin in fair to good chemical yield. Several chiral syn-2,3-disubstituted-2,3-dihydro-1,4-benzoxathiin rings were prepared with this method from enantioenriched 2-mercaptoethanols. No loss of enantiopurity was observed.     

Donor—acceptor interactions of substituted benzenes with molecular chlorine and carbon disulfide

CNDO molecular orbital calculations have been performed to analyze donor—acceptor interactions between molecular chlorine and benzene, toluene, mesitylene and hexamethylbenzene and the, as yet, unreported chlorine—hexafluorobenzene and carbon disulfide—benzene pairs. The stabilization energy and the dipole moment and its derivative (?p/?R_CICI) calculated for the benzene—chlorine complex are in good agreement with the estimated experimental values. The trends in the experimental stabilization energies and the Cl-Cl vibrational frequencies with increasing methyl substitution appear to be well reproduced by the calculations. The charge transferred from the benzene donor is polarized toward the outer chlorine atom or sulfur atom. For hexafluorobenzene-chlorine the direction of electronic charge polarization is reversed from that of the benzene and methylbenzene complexes. The calculated results are discussed within the framework of Muliiken's simplified resonance theory for complexes.     

Synthesis of 1,2-dihydroindolo [1,7-AB] [1,5] benzodiazepines and related structures. A new heterocyclic ring system

The synthesis of the novel 1,2-dihydroindolo [1,7-ab][1,5]benzodiazepine ring system 4 is described. Condensation of 2-fluoronitrobenzene with indoline provided the starting material for the synthesis, 1-(2-nitrophenyl)indoline (1a) in high yield. The nitro group was reduced catalytically and the resulting amino function was acylated to afford the heterocycle percursor amide 3. Refluxing this amide in phosphorus oxychloride brought about a Bischler-Napieralski type cyclodehydration to form the target 1,2-dihydroindolo[1,7-ab][1,5]benzodiazepine ring system. Dehydrogenation of the latter led to the fully aromatic indolo[1,7-ab][1,5]benzodiazepine structure 5, while reduction with sodium borohydride provided the 1,2,6,7-tetrahydroindolo[1,7-ab]-[1,5]benzodiazepine tetracycle 6.     

Ascorbate-induced oxidation of formate by peroxodisulfate: product yields,kinetics and mechanism

The slow reaction between peroxodisulfate and formate is significantly accelerated by ascorbate at room temperature. The products of this induced oxidation, CO₂ and oxalate (C₂O^2– ₄), were analyzed by several methods and the kinetics of this reaction were measured. The overall mechanism involves free radical species. Ascorbate reacts with peroxodisulfate to initiate production of the sulfate radical ion (SO^– ₄), which reacts with formate to produce carbon dioxide radical ion (CO^– ₂) and sulfate. The carbon dioxide radical reacts with peroxodisulfate to form CO₂ or self-combines to form oxalate. Competition occurring between these two processes determines the overall fate of the carbon dioxide radical species. As pH decreases, protonation of the carbon dioxide radical ion tends to favor production of CO₂.     

Cyclopentadienyl-ruthenium and -osmium chemistry: XXVIII. Reactions and isomerisation of 1,2-bis(methoxycarbonyl)ethenyl complexes: X-ray structures of Ru{Z)-C(CO2Me)CH(CO2Me)} -(CO)(PPh3)(η-C5H5) · 0.5EtOH,Ru{(E)-C(CO2Me)CH(CO2Me)}(dppe)(η-C5H5) and Ru{C(CO2Me)C(CO2Me)C(CO2Me)CH(CO2Me)} - (PPh3)(η-C5H5)

A reinvestigation of the reaction between C₂(CO₂Me)₂ and RuH(PPh₃)₂(η-C₅H₅) and some related complexes is reported. Initial cis addition is followed by conversion into the trans isomer. In the case of the bis-(PPh₃) complex, isomerisation is followed by chelation of the ester CO group with concomitant displacement of one PPh₃ligand. The resulting chelate complex reacts with CO or CNBu^t to give the (Z)-RuC(CO₂Me)CH(CO₂Me) complexes; the (E)-isomer of the carbonyl complex is obtained by addition of C₂(CO₂Me)₂to RuH(CO)(PPh₃)(η-C₅H₅). The ^1Hand ¹³C NMR spectra are not a reliable guide to assignment of the stereochemistry of the vinyl group. Other products isolated from the initial reaction are the bis-insertion product $\text{Ru{C(CO}{}_2\text{Me)C(CO}{}_2\text{Me)C(CO}{}_2\text{Me)}$CH(CO₂Me)} -(PPh₃)(η-C₅H₅) and the 1/2 PPh₃/C₂(CO₂Me)₂ adduct. The molecular structures of Ru{(Z)-C(CO₂Me)CH(CO₂Me)}(CO)(PPh₃(η-C₅H₅) · 0.5EtOH, Ru{(E)-C(C₂Me)CH(CO₂Me)}(dppe)(η-C₅H₅) and $\text{Ru{C(CO}{}_2\text{Me)C(CO}{}_2\text{Me)C(CO}{}_2\text{-Me)}$CH(CO₂Me)}(PPh₃)(η-C₅H₅) have been determined. The cis isomer is monoclinic, space group P2₁,with a 9.328(8), b 17.385(10), c 10.356(7) Å, β 101.78(3)° and Z = 2; 2107 data with I ≥ 2.5σ(I) were refined to R = 0.076 R_w = 0.085. The trans isomer is triclinic, space group P$\text{1}$, with a 10.404(7) b 11.221(6), c 13.230(9) Å, α 92.67(5), β 110.56(5), γ 106.21(5)° and Z = 2; 2520 data with I ≥ 2.5σ(I) were refined to R = 0.055 R_w = 0.068. The butadienyl complex is monoclinic, space group P2₁/a, with a 19.655(8), b 8.674(4), c 21.060(5) Å, β 116.22(3)° and Z = 4; 2724 data with I ≥ 2.5σ(I) were refined to R = 0.042, R_w = 0.047.     

Quinazolines. VII. Synthesis of 1,3-Diamino-5,6-dihydrobenzo[f]quinazolines

Eighteen new 1,3-diamino-5,6-dihydrobenzo[f] quinazolines ( 6 , R. = alkyl, Cl, MeO) were synthesized via the condensation of appropriate 2-tetralones with cyanoguanidine under fusion conditions. Methods were developed for the preparation of a number of heretofore undescribed 2-tetralones as precursors. The final products can be viewed as conformationally rigid analogs of pyrimethamine ( 2 ), and are of interest as inhibitors of dihydrofolate reductase and as potential antimalarial and antitumor agents.     

Detection of DNA fragments separated by capillary electrophoresis based on their native fluorescence inside a sheath flow

A scheme for the separation and detection of native DNA fragments in capillary electrophoresis is presented. A UV laser at 275 nm excites the intrinsic fluorescence of the fragments, which is greatly enhanced at pH 2.8. To provide a compatible system, methylcellulose-based size separation is performed at the identical pH. A sheath-flow arrangement isolates the detection region from the linear polymer for a reduced background level. The performance is an order-of-magnitude enhancement in detectability over absorption detection. We also uncovered a selective degradation/ligation process at these pH conditions that may be useful as additional selectivity for DNA characterization.     

Resonance enhanced three-photon absorption of molecular iodine

D ← X resonance enhanced three-photon excitation spectrum of iodine was observed by a cw intracavity absorption technique. Vibrational quantum numbers of D ← X transitions are given for every major spectral feature. The corresponding one-photon enhancement from the B state is evident from the one-photon vibrational assignment. The spectroscopic constants for the D state are v₀₀ = 40 998 cm^?1 ω′₀ = 113 cm^?1 and ω′₀χ′₀ = 0.045 cm^?.