Detection of colorectal cancer K-ras mutations using a simplified oligonucleotide ligation assay.

It has been suggested that some mutations in codons 12 and 13 of the K-ras gene are associated with the progression of colorectal adenomas to carcinomas. The aim of this study was to develop a rapid, colorimetric assay for K-ras point mutations commonly associated with colorectal cancer. K-ras exon 1 was amplified from colorectal tumor DNA and K-ras activating mutations detected using an oligonucleotide ligation assay (OLA) in combination with immunological and colorimetric detection. Using the OLA with oligonucleotides specific to individual K-ras mutations, 6 (of 17 total colorectal adenomas/carcinomas) were found to have K-ras mutations. The assay could detect as little as 10% mutant allele. A simplified OLA designed to test for either the presence (+) or absence (-) of any of the K-ras activating mutations was developed. The assay was further streamlined by use of a dipstick methodology for colorimetric development. If required, assay sensitivity can be increased by the use of the recently described EDNA-ELCA detection system. The simplified (+/-) mutation OLA in combination with a dipstick or EDNA-ELCA detection system provides a rapid, sensitive assay for K-ras point mutations suitable for use as part of the clinical assessment of colorectal cancer.

Enzyme linked fibrinolytic assay (ELFA). A new method for the measurement of t-PA in plasma using enzyme labelled fibrin.

We present an assay for components of the fibrinolytic system based on hydrolysis of solid-phase associated enzyme-labeled fibrin. This Enzyme-linked Fibrinolytic Assay, (ELFA) permits the measurement of less than 1 IU/ml of t-PA in 50 microliters of plasma diluted to 1:80 within six hours, in a microtiter plate format, with a colorimetric endpoint. High levels (greater than 10 IU/ml) of tissue plasminogen activator can be measured in less than 30 minutes. The assay was approximately 100 times more sensitive than a clot lysis assay performed in microtiter plates and used less reagents. By comparison with the parabolic rate, coupled assay using chromogenic substrates and soluble fibrin, ELFA performed assays with equal sensitivity and in less time. The ELFA assay uses solid phase fibrin as the substrate, activator and indicator for the assay. For this reason, it has the advantage of the clot lysis assays in that it is more analogous to the physiological hydrolysis of fibrin but with the sensitivity and convenience of the parabolic rate, coupled assay.

A comparison of four methods for the serotyping of group B streptococci.

Group B streptococci are implicated in a wide range of clinical conditions in human adults and neonates. The Group is subdivided into five serotypes Ia, Ib, Ic, II and III, which are differentiated on the basis of capsular polysaccharides. In the interests of epidemiology and efficiency a cheap, rapid method which is easily interpreted would be advantageous. In this study four methods of serotyping, namely, counter-immunoelectrophoresis (CIEP), microimmunodiffusion (MID), coagglutination (COA), and the Lancefield capillary precipitin (CP) test were compared in terms of ease of operation and interpretation, accuracy and rapidity. Todd Hewitt Broth (THB) cultures and acid extracts of the group B streptococcal strains were used as antigens for these methods. It was concluded that COA using THB cultures allows cheap and rapid screening for presumptive serotyping, having a 93-96% correlation with the CP test. MID gives an accurate (100% correlation with the CP test) and unambiguous confirmatory diagnosis of serotype.

The distribution of group B streptococcal serotypes among isolates obtained in three Australian cities.

One hundred and sixty three isolates of group B streptococci from human carrier and disease conditions in Brisbane, Melbourne and Adelaide were serotyped by the Lancefield capillary precipitin test, coagglutination and microimmunodiffusion. Serotypes I and III were most common; serotype III appeared to occur uniformly but serotype Ia varied in distribution. No significant difference was found in serotype distribution among isolates from pregnant and non-pregnant women, or rectal and vaginal swabs; however, type Ia was found to occur significantly more often as an infectious agent than as a commensal (P less than 0.01).

Solubilization of hyaluronic acid synthetic activity from streptococci and its activation with phospholipids.

To date all hyaluronic acid synthetic systems have been of a particulate nature, and attempts at solubilization have been unsuccessful. This has hampered attempts to elucidate the mechanism by which hyaluronic acid is produced. In this paper we demonstrate that the hyaluronic acid synthetic activity from group C streptococcal membranes was solubilized using 2% digitonin and that the activity was optimized by reconstitution with cardiolipin at an optimum phospholipid/protein ratio (microgram/microgram) of 5:1. Furthermore, chromatography of the solubilized synthetase demonstrated that it eluted after the void volume of a Sepharose CL-6B column. CHAPSO, octyl glucopyranoside, sodium cholate, Triton X-100, and zwittergent 314 either inhibited or failed to solubilize the synthetic activity. Phospholipids other than cardiolipin also reconstituted the activity from the digitonin extract, particularly phosphatidylethanolamine and phosphatidylserine. In our system, the specific activity of hyaluronic acid synthetase was increased up to 63 times that of the system of the intact membrane. Furthermore, the total activity of the reconstituted system was 4.9 times greater than that of intact membranes. The soluble enzyme system showed similarities to the membrane-bound synthetase in the kinetics of production of trichloroacetic acid-soluble and -insoluble hyaluronic acid, and the hyaluronic acid produced was of comparable molecular weight.

Polysaccharase activity in Streptococcus agalactiae (group B streptococci).

Of 300 recently isolated strains of Streptococcus agalactiae from human sources, 97% degraded starch. Representative strains also degraded glycogen, pullulan, amylopectin and amylose. The polysaccharase activity is easily detected by clearing around growth on Columbia agar base medium. The activity is weaker than that of some S. pyogenes strains, and it does not appear to produce fermentable products but is inhibited by the presence of easily used sugars.

Extrinsic-pathway enzyme-linked coagulation assay (EP-ELCA). A clot-based alternative to prothrombin time for measurement of extrinsic pathway factors in plasma.

This solid-phase colorimetric microtiter-plate clotting assay is much more sensitive than standard clotting tests. In enzyme-linked coagulation assay (ELCA), enzyme-labeled fibrinogen and solid-phase fibrinogen are the substrate for thrombin generated in the clotting cascade. We used this assay to measure the factors of the extrinsic pathway by an extrinsic pathway-specific assay (EP-ELCA) and to determine the individual factors of the extrinsic pathway (VII, X, V, II) in plasmas of coumadin-treated and heparin-treated patients, with prothrombin time (PT) values used as a reference. In the ELCA method, samples and controls are incubated on the same plate, eliminating the requirement for pre-standardization of the substrate "plasma" before the factor assay is done. Concentrations of factors are determined by serially diluting sample and control plasmas to yield equivalent activity at given dilutions, a more direct approach for measuring specific factors than determining log concentrations vs log clotting time. Changes in the concentrations of clotting factors are seen before changes are apparent by PT. For coumadin-treated patients, all vitamin K-dependent factors were significantly (P less than or equal to 0.001) less than in normal controls, whereas factor V concentrations were normal, as expected. For patients treated with heparin, concentrations of factors X and VII were less than in normal controls (P less than 0.01) and results for EP-ELCA, II, and V assays were normal. This methodology can readily be automated.

Enzyme-linked immunosorbent assay-enzyme-linked coagulation assay for detection of antibodies to Clostridium botulinum neurotoxins A, B, and E and solution-phase complexes.

The solution-phase complex assay for toxins A, B, and E from Clostridium botulinum (Elcatech, Inc., Winston-Salem, N.C.) was modified to measure antibody. The addition of unlabeled polyclonal antibodies to a mixture consisting of toxin with chicken antibody and RVV-XA-labeled horse antibody reduces the sensitivity of detection of neurotoxin. This reduction in sensitivity can be used as a measure of the specific antibody titer.

A microtiter plate assay using cascade amplification for detection of nonisotopically labeled DNA.

We describe a microtiter-plate-based, colorimetric assay for DNA, the enzyme-linked DNA-enzyme-linked coagulation assay (EDNA-ELCA). The EDNA-ELCA uses amplification of the common pathway of coagulation for the ultrasensitive detection of DNA which is tagged by incorporation of functional groups such as biotin and fluorescein. The EDNA-ELCA enables detection of attomole amounts of DNA (< 1 pg per microtiter well), with a sensitivity 200-1000 times higher than other colorimetric techniques. The assay has been applied as an adjunct to PCR for quantitative determination of methicillin-resistant Staphylococcus aureus DNA at levels corresponding to 1-10(5) organisms. The EDNA-ELCA can also be used to assay DNA by hybridization; < 50 amol of an unlabeled DNA template is detected by hybridization to biotin- and fluorescein-labeled probes.

Enzyme-linked immunosorbent assay and enzyme-linked coagulation assay for detection of Clostridium botulinum neurotoxins A, B, and E and solution-phase complexes with dual-label antibodies.

The measurement of toxins A, B, and E from Clostridium botulinum was accomplished by use of a modified sandwich enzyme-linked immunosorbent assay (ELISA) employing labeled horse antibody and either chicken antibody or biotinylated horse antibody. The complexes formed in solution phase were captured onto solid phases coated with rabbit anti-chicken immunoglobulin G (chicken antibody) or avidin (biotinylated antibody). The assay was brought to the sensitivity of the mouse bioassay (5 to 10 pg/ml, or 0.03 to 0.07 pM) by employing as labeling enzyme the factor X activator of Russell's viper venom (RVV-XA) and a sensitive coagulation-based assay amplification system known as enzyme-linked coagulation assay. Complex formation was found to be a slower reaction than binding to the capture plate, and so the assay used a preincubation step to produce the solution-phase complexes before they were bound to the solid phase. Keeping the concentrations of Russell's viper venom factor X activator antibody and capture antibody constant for diluted samples and diluting complexes into buffer without keeping labeled antibody concentrations constant were equivalent in allowing the detection of low neurotoxin concentrations. This ELISA-enzyme-linked coagulation assay procedure is a convenient alternative to the mouse bioassay, which shows complete resolution of the neurotoxins in addition to the requisite sensitivity.

Sensitive enzyme-linked immunosorbent assay for detection of Clostridium botulinum neurotoxins A, B, and E using signal amplification via enzyme-linked coagulation assay.

A new immunoassay amplification method has been applied to the measurement of toxins A, B, and E from Clostridium botulinum. The technique is a modified enzyme-linked immunosorbent assay (ELISA) which relies on the detection of sandwich complexes on microtiter plates by a solid-phase coagulation assay known as ELCA, or enzyme-linked coagulation assay. In the method, a coagulation activating enzyme (RVV-XA) isolated from the venom of Russell's viper is conjugated to affinity-purified horse antibodies specific for toxin type A, B, or E. Plates are coated with affinity-purified antibodies, and standard captag (capture-tag) protocols using labeled antibody are employed to bind the toxin from solution. Complexes are detected by adding a modified plasma substrate which contains all the coagulation factors mixed with alkaline phosphatase-labeled fibrinogen and solid-phase fibrinogen; deposition of solid-phase, enzyme-labeled fibrin on the solid phase is then a reflection of formation of toxin-RVV-XA-antibody complexes on the solid phase. Because of the ability to detect RVV-XA by this coagulation assay at concentrations < 0.1 pg/ml, it was possible to measure C. botulinum toxins A, B, and E at mouse bioassay levels (< 10 pg/ml, or < 0.07 pM) for both purified neurotoxin and crude culture filtrates obtained from strains known to produce appropriate single toxins. ELISA-ELCA should be applicable to measurement of toxins in most of the materials (contaminated food, blood, and excreta) for which the comparably sensitive mouse bioassay is currently employed. This method has the potential of broad application to the measurement of low concentrations of any antigen for which appropriate immunochemical reagents are available, in a color test format.