Branch migration inhibition in PCR-amplified DNA: homogeneous mutation detection.

A novel method for detection of any mutation located within a PCR-amplified DNA sequence was demonstrated. The method is based on the inhibition of spontaneous DNA branch migration. Partial duplexes produced by PCR amplification of a test and a reference genomic DNA sample anneal to form four-stranded cruciform structures. Spontaneous DNA branch migration results in dissociation of these structures when the test and reference sequences are identical. Any base substitution, deletion or insertion inhibits branch migration and produces stable cruciform structures. When suitable ligands are attached to the PCR primers, the cruciform structures can be detected by standard immunochemical methods. This approach was tested using several commonly occurring mutations within the human cystic fibrosis gene. New methods for increasing the specificity of PCR amplifications are described that were used for successful mutation analysis.

Monitoring of phospholipid vesicle fusion by fluorescence energy transfer between membrane-bound dye labels.

A sensitive method which utilizes fluorescence energy transfer to assay Ca2+ -or Mg2+ -mediated fusion of phospholipid vesicles is reported. More than 85% quenching results when phosphatidylserine vesicles labelled with dansyl phosphatidylethanolamine (donor) are fused with vesicles labelled with rhodamine phosphatidylethanolamine (acceptor) in the presence of 5 mM CaCl2 or 10 mM MgCl2. Higher concentrations of divalent cations are required to obtain maximal quenching when phosphatidylserine is partially replaced with phosphatidylethanolamine or phosphatidylcholine. The rate of vesicle fusion is dependent upon the concentrations of both cation and vesicles. Maximum quenching occurs within 5 min using phosphatidylserine vesicles and 5 mM Ca2+, but quenching is incomplete even after 20 h with 0.8--2 mM Ca2+. This probably reflects the heterogeneous size distribution of these vesicles, since the extent of fusion was found to correlated with vesicle size. Binding of antibody to membrane-localized phenobarbital hapten effectively blocks Ca2+ -mediated vesicle fusion. This effect can be inhibited by preincubation of the antibody with phenobarbital. Leakage of tempocholine from intact vesicles induced by 5 mM Ca2+ occurs even when fusion is prevented by bound antibody. This demonstrates that fusion is not a necessary requirement for Ca2+ -induced leakage.

Inhibition of malate dehydrogenase by thyroxine and structurally related compounds.

The inhibition of pig heart mitochondrial malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37) by the thyroxine and structurally related compounds was studied to resolve a longstanding question about the exact nature of the inhibition. Thyroxine, in freshly prepared solution, was found to be a "pure" competitive inhibitor relative to the nucleotide cofactor. Upon standing in diffuse daylight, solutions of thyroxine showed increased ability to inhibit the enzyme, presumably as a result of oxidation of enzyme sulfhydryl groups by free iodine that is released photochemically. This behavior probably accounts for earlier reports of irreversible inactivation by thyroxine. Comment is made on the implications of these findings to the mechanism of thyroid hormmone action.

Action of beta-galactosidase on novel synthetic macromolecular substrates. A processive enzymic reaction controlled by coulombic interactions.

Macromolecular beta-galactosidase substrates were prepared by attaching o-nitrophenyl-beta-galactoside to carboxymethyldextran with positively charged linking groups. Almost all of the substituents were susceptible to enzymic hydrolysis by two distinct pathways. Under some conditions, there was random reaction to give a soluble product. In other conditions, in the initial stages of the reaction, most of the substituents of some, but not all, of the substrate polymers were hydrolyzed to give a product which precipitated as a second aqueous phase. Kinetics of hydrolysis were studied with respect to charge and molecular weight of both the enzyme and substrate. Factors that caused a decrease in Km favored formation of the second phase product. The reaction has similarities to the processive catalytic reactions found in naturally occurring enzyme systems with polymeric charged substrates.

Mechanism of inhibition of malate dehydrogenase by thyroxine derivatives and reactivation by antibodies: homogeneous enzyme immunoassay for thryroxine.

Pig heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) is about 90% inhibited upon labelling an average of two amino groups per subunit with an active ester of thyroxine. Inhibition is probably associated primarily with thyroxine binding to one specific group which is normally unreactive but becomes activated upon noncovalent binding of thyroxine derivatives to the enzyme. Enzyme inhibition is due to a decrease in the rate of association of NAD. Antibodies to thyroxine induce a slow conformational change with partial reversal of inhibition of more heavily labelled conjugates. The antibody-induced activation is not cooperative and does not require bivalent association of the antibody. Activation can be blocked by the presence of free thyroxine and is the basis for a clinically useful assay for serum thyroxine.

Improved sensitivity in homogeneous enzyme immunoassays using a fluorogenic macromolecular substrate: an assay for serum ferritin.

A new highly sensitive nonseparation enzyme immunoassay for human serum ferritin is described. Reagents include a beta-galactosidase-ferritin conjugate, sheep anti-ferritin, anti-sheep IgG, and dextran-linked beta-galactosylumbelliferone as enzyme substrate. The method is based on inhibition of enzyme activity when anti-ferritin binds to the enzyme-ferritin conjugate. Ferritin in the sample and enzyme-labeled ferritin compete for a limited quantity of anti-ferritin. The enzyme activity of the reaction mixture is directly related to the ferritin content of the sample. Some patients' samples caused strong interference in the assay due to the presence of antibody to beta-galactosidase. Several ways of eliminating the interference are presented. When measures were adopted to suppress sample interference, the assay results correlated well with those of other immunoassay methods.

Monoclonal antibodies to glucose-6-phosphate dehydrogenase (G6PDH) form cyclic 1:1 complexes with G6PDH and act as regulatory subunits.

A number of IgG monoclonal antibodies against L. mesenteroides glucose-6-phosphate dehydrogenase (G6PDH) have been prepared. Four of the antibodies form 1:1 enzyme-antibody complexes which are stabilized in the presence of glucose-6-phosphate (G6P) and have greatly reduced enzyme activity. In the absence of G6P, the 1:1 complexes convert gradually to a more active multimeric form. Reduction of the IgG inter-heavy chain disulfides partially relieves inhibition and removes the G6P requirement for stability. F(ab')2 fragments of one of the antibodies behave similarly to the intact IgG. Reduction of the disulfides in the G6PDH-F(ab')2 complex leads to complete recovery of activity. The activity of complexes of G6PDH with reduced antibodies or Fab with digoxin bound to the antibody or Fab sulfhydryl groups can be modulated with antibodies to digoxin. The anti-G6PDH antibodies bridge two identical epitopes of this two subunit enzyme and simulate the function of regulatory subunits in which anti-digoxin acts as an activator. The system can be used to provide a sensitive homogeneous immunoassay for digoxin.

Use of liposome encapsulation in a combined single-liquid reagent for homogeneous enzyme immunoassay.

A technique has been developed to permit mutually reactive macromolecular reagents used in immunoassays to be combined without premature reaction. A conjugate of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and theophylline has been encapsulated in 0.2-micron-diameter bi-lamellar liposomes. Suspensions of these liposomes had excellent stability. Whereas the enzyme activity of the free conjugate is rapidly inhibited by anti-theophylline antibody, a suspension of the encapsulated conjugate in a solution of the antibody and NAD+ (6.0 mmol/L) retained greater than 92% of the initial enzyme activity after standing for one year at 4 degrees C. At higher NAD+ concentrations the liposomes aggregated, and enzyme activity was inhibited by leakage of the NAD+ hydrolysis product, adenosine diphosphoryl 5-ribose (ADP-ribose), into the liposomes. Inhibition by ADP-ribose could be blocked and partly reversed by adding semicarbazide. The liposomes were efficiently lysed by Triton X-100, deoxycholate, or octyl glucoside, the kinetics and extent of lysis being affected by liposome size and correlating with the acid strength of various cholate derivatives. Addition of a serum sample and a solution of buffer, substrate, and detergent to a single reagent containing the liposomes and anti-theophylline antibody provided assay results equivalent to those obtained by conventional two-reagent EMIT homogeneous enzyme immunoassay for theophylline.

Fluorescent excitation transfer immunoassay. A general method for determination of antigens.

A general immunochemical method for the assay of haptens and proteins has been devised and applied to morphine, a morphine-albumin conjugate, and human immunoglobulin G. A fluorescein-labeled antigen and a quencher-labeled antibody are employed. By use of fluorescein and rhodamine as the fluorescer and quencher, respectively, dipole-dipole-coupled excitation energy transfer can occur within the antigen-antibody complex. The resulting quenching of fluorescence can be inhibited by competitive binding with unlabeled antigen, Alternatively, separate antibody samples can be labeled with fluorescein and rhodamine, respectively. Unlabeled antigen causes aggregation of the separately labeled components with resultant quenching. Using the latter method, experiments suggest that up to about 20 anti-morphine antibody binding sites will associate with morphine-albumin conjugates. When an excess of the conjugate is present the antibodies appear to assemble in clumps on the protein surface. Mathematical analysis of the quenching of fluorescein-labeled morphine by rhodamine-labeled anti-morphine gives an approximate fit to the quenching data, but the calculations are very dependent on the assumptions used.

Fluorescence protection immunoassay: a new homogeneous assay technique.

We describe a "fluorescence protection immunoassay," in which formation of an immune complex of a fluorescer-labeled antigen sterically protects the fluorescer from binding by antibodies to it. Competitive binding of unlabeled antigen by its antibody prevents formation of the fluorescer-labeled antigen immune complex, and allows anti-fluorescein to quench the fluorescence by binding to the fluorescer. This phenomenon is the basis of a new homogeneous assay technique that requires no separation step. The steric exclusion of anti-fluorescein from fluorescein-labeled human IgG immune complexes was altered by changing the molecular dimensions ob both antifluorescein and the immune complex. The assay did not require highly purified fluorescein-labeled human IgG. An assay is demonstrated in which was used a fluorescein-labeled human IgG conjugate containing IgG that was only 10% pure. Measurement of IgG in human serum samples correlated well with results by radial immunodiffusion. The method is applicable to the assay of both proteins and analytes of low molecular mass.

Specific antibodies to theophylline for use in a homogeneous enzyme immunoassay.

Bovine serum albumin conjugate of 1-methyl-3-(3'-carboxypropyl)xanthine elicits highly specific anti-theophylline antibodies when injected into sheep. When used in a homogeneous enzyme immunoassay for theophylline these antibodies show insignificant cross-reactivity (less than 1%) to 1-methyl- and 1,3-dimethyluric acid, 3-methylxanthine, caffeine, and theobromine. In contrast, immunogens prepared from the C-8 functionalized drug afford antibodies which show more serious cross-reactivity to these compounds. Plausible rationale for attachment of the drug to carrier proteins through its N-3 position which furnished specific antibodies are given.

Rapid, simple, and reliable doctor's office test for antibodies to human immunodeficiency virus 1 in serum.

This "Unit Test Method" assay for detecting anti-human immunodeficiency virus 1 antibody is suitable for nonlaboratory testing and has a sensitivity comparable with that of present enzyme immunoassay methods. The method does not require instrumentation, gives a result in less than 15 min, and incorporates a procedural control. Little technical expertise and hands-on time are required of the user.

Formation of chimeric DNA primer extension products by template switching onto an annealed downstream oligonucleotide.

Oligodeoxynucleotide sequences are described that anneal to a template downstream of a priming site. During polymerase-catalyzed extension of the primer, the extending primer shifts from the original template to a segment of the annealed oligonucleotide that acts as an alternative template. The resulting chimeric extended primer has one segment that is complementary to the template and a second segment that is complementary to the oligonucleotide. The influence of the sequence elements of the oligonucleotide and the reaction conditions on template switching have been explored. The sequence requirements for template switching are compared to those for transposon excision.

DELISA: sensitive nonisotopic assay for GAD65 autoantibodies, a key risk-assessment marker for insulin-dependent diabetes mellitus.

Nonisotopic assays for the measurement of autoantibodies to 65-kDa glutamic acid decarboxylase (GAD65) have not previously achieved performance equivalent to radiobinding assays (RBA). We have developed a modified ELISA protocol, DELISA, for measuring autoantibodies to GAD65 in serum. The method overcomes the problems of poor sensitivity and specificity associated with conventional ELISAs. Serum containing GAD65 autoantibodies is incubated with biotinylated GAD65 (bGAD65). Sufficient soluble Protein A-dextran conjugate is added to bind the immunoglobulins in the sample, including GAD65 autoantibodies to which GAD65 is bound. After incubation, the mixture is transferred to a streptavidin-p4ated microtiter well, which binds free bGAD65 but not bGAD65 bound to autoantibodies. Streptavidin-bound bGAD65 is detected by means of a peroxidase-GAS65MAb conjugate. The method appears to have comparable sensitivity and specificity to those of RBAs. Reaction of the antibodies with soluble antigen to increase the binding rate and the use of high serum concentrations and very low antigen concentrations to increase sensitivity are critical elements of the method.

Mechanism by which antibodies inhibit hapten-malate dehydrogenase conjugates. An enzyme immunoassay for morphine.

Antimorphine antibodies inhibit the activity of morphine conjugates of mitochondrial malate dehydrogenase. Conjugation of malate dehydrogenase through tyrosine and amino groups resulted in only moderate losses of enzyme activity. On conjugation through disulfide bonds the enzyme activity first increased but dropped sharply with increasing substitution. Only the former conjugates were inhibited by excess antibodies. The degree of inhibition (up to 86%) was directly related to the number of morphine residues bonded directly to amino groups. The maximum number of antibody binding sites that bind to enzyme was nearly equal to the number of haptens provided there were 16 or less haptens/enzyme. However up to 26 haptens/enzyme became completely bound by antibody on long incubation. Inhibition of enzyme activity was detectably reduced by 2 times 10 minus 9 M morphine or 2 times 10 minus 10 M codeine, thus providing a sensitive assay for these drugs. The data suggest that enzyme inhibition occurs by conformational freezing of the enzyme when antibody binds to a morphine residue attached to one specific amino group.

Enzyme-enhancement immunoassay: a homogeneous assay for polyvalent ligands and antibodies.

A homogeneous enzyme immunoassay for proteins has been developed that avoids the need for a labeled antigen. The technique involves antibody labeled with beta-galactosidase (EC 3.2.1.23), succinylated antibody, and a macromolecular o-nitrophenyl-beta-galactoside substrate. The enzyme-labeled antibody and the succinylated antibody form an immune complex in the presence of sample antigen. An enzyme within this negatively charged microenvironment produces a product that forms a second light-scattering phase, whereas the product produced by free enzyme remains soluble. Thus the antigen modulates the rate of increase in light scattering. The technique has been applied to assays for human immunoglobulin G and C-reactive protein as well as for specific antibodies.

Anti-immune complex antibodies enhance affinity and specificity of primary antibodies.

Antibodies have previously been described that enhance the binding of a second antibody to its antigen. The origin of this effect has been variously ascribed to binding to a neodeterminant on the Fc region, to a combined determinant representing portions of the second antibody and the immunogen, and to a ligand-induced conformation of the Fab fragment. This paper describes an antibody that recognizes an immune complex of an antibody to tetrahydrocannabinol (THC). The antibody binds the anti-THC antibody at an epitope recognized by an anti-idiotype antibody that is capable of blocking THC binding. The ability of various THC derivatives to enhance or inhibit binding taken together with equilibria and kinetic data support a model in which the anti-immune complex antibody interacts through adventitious binding to pendant groups on the THC derivatives. This type of interaction offers the opportunity to increase the sensitivity and specificity of immunoassays beyond the limits imposed by normal antibody binding. The implications of these findings with regard to earlier observations of anti-immune complex antibodies are discussed.

A non-flow cytometric system for detecting antibodies and cellular antigens in blood.

The system described here can distinguish between single and agglutinated erythrocytes by use of a non-flow fiber optic fluorometer. The method is capable of detecting cell-surface antigens and antibodies to cell-surface antigens present in blood. Significant features include high-efficiency fluorescent dyes that intercalate into cell membranes, a stretching membrane for transport and mixing of samples, charged colloidal magnetite for magnetic separation of erythrocytes, and an immersible fiber optic probe for measuring fluorescence associated with cells in a 1-nL volume of a bulk solution. We describe the application of the system to automation of ABO/Rh grouping and antibody screening.