A portable reflectometric photometer for quantitative enzyme immunoassay

A portable optoelectronic reflectometric photocolorimeter was designed. The photocolorimeter implements two operating modes (transmission and reflection), and its performance was tested in two systems of enzyme immunoassay for testosterone: microplate ELISA and membrane dot-ELISA. The detection threshold for microplate ELISA and membrane dot-ELISA was 0.1 and 0.6 ng/ml, respectively. The coefficient of correlation between the photocolorimeter readings and conventional photometric methods is r=0.999. Relative standard deviation of the results of photocolorimetric measurements (n = 4) within the optical density range of 0.03 to 1.00 ranged from 3.4 to 0.7%. The simple and versatile design of the photocolorimeter makes it appropriate for testing various substances both in laboratory settings and in the field.     

Standard quantitative assays for apoptosis

The term apoptosis refers to a peculiar morphology of cell death. It is of special interest because it can be triggered physiologically (and pathologically), and it is regulated by the actions of specific gene products. Therefore, it can in principle be activated and suppressed by medical intervention. It thus is often important to determine whether cells are dying by apoptosis (or its less regulated counterpart, necrosis) and also to quantity the effect in a population of cells. Here the classic methods of apoptosis quantitation are described; they will be of particular use to those whose laboratories are set up for standard microscopical and biochemical techniques, who do apoptosis assays infrequently but wish them to be widely accepted and reproducible. A simple microscopic observation, using blue light illumination and a pair of fluorescent dyes, is recommended for most applications.     

A simple image analysis program for quantitative dot assays

Summary Dot assays are versatile, and are widely used for determining antigens and proteins. Because they require expensive equipment to be quantitative, often only qualitative dot results are reported. However, because the dot pattern is so regular, a simple image analysis program can determine mean dot grey levels, while handling irregular dot outlines, radial variations in colour within a dot, and varying or noisy sheet backgrounds. We describe a dot analysis program that runs on PCs under Windows, and permits quantitative dot assays to be run with inexpensive grayscale scanner input. The program is available from us as source and executable files. We present demonstration results for antigen and protein determinations.     

A dot-immunobinding assay for monoclonal and other antibodies

A procedure for the assay of antibodies in sera based on the application of the antigen as a spot to nitrocellulose filters is described. The method has the merit of being simpler in operation and more sensitive than comparable existing procedures. Applications for screening supernatants of hybridomas making monoclonal antibodies, and the use of such antibodies in the determination of the tissue distribution of the corresponding antigens, are described. An application for the screening of human pathological sera for multiple antibodies in one operation is also described.     

A convenient quantitative synthesis of methylglyoxal for glyoxalase I assays

A simple, convenient, and quantitative method for the preparation of methylglyoxal (pyruvaldehyde) solutions is described. The method involved acid hydrolysis (5% H₂SO₄) of pyruvaldehyde dimethyl acetal at 100°C for 25 min. The hydrolysis method is quite reproducible and does not require standardization of the methylglyoxal solutions. The preparations of methylglyoxal by the procedure described have been used in the study of glyoxalase I activity.     

A flow cell photometer for bacterial growth monitoring

A flow cell photometer is described with automatic cleaning of the photometric cell, denasimetric separation of air bubbles and precipitates, and a constant sensitivity from 0 to 10 mg/ml of bacterial dry weight.     

A novel multi-walled carbon nanotube-based antibody conjugate for quantitative and semi-quantitative lateral flow assays

In this study, the multi-walled carbon nanotubes (MWCNTs) were applied in lateral flow strips (LFS) for semi-quantitative and quantitative assays. Firstly, the solubility of MWCNTs was improved using various surfactants to enhance their biocompatibility for practical application. The dispersed MWCNTs were conjugated with the methamphetamine (MET) antibody in a non-covalent manner and then manufactured into the LFS for the quantitative detection of MET. The MWCNTs-based lateral flow assay (MWCNTs-LFA) exhibited an excellent linear relationship between the values of test line and MET when its concentration ranges from 62.5 to 1500 ng/mL. The sensitivity of the LFS was evaluated by conjugating MWCNTs with HCG antibody and the MWCNTs conjugated method is 10 times more sensitive than the one conjugated with classical colloidal gold nanoparticles. Taken together, our data demonstrate that MWCNTs-LFA is a more sensitive and reliable assay for semi-quantitative and quantitative detection which can be used in forensic analysis.     

A dot-immunobinding assay (dot-Iba) for rapid diagnosis of pulmonary tuberculosis

IgG antibody to Mycobacterium tuberculosis from the sera of patients with 'definite' pulmonary tuberculosis (PT) was isolated and coupled with Cyanogen bromide-Sepharose 4B. Using an immunoabsorbent affinity chromatography, 14 kDa antigen was recovered from the culture filtrates of M. tuberculosis. With this mycobacterial antigen, a dot immunobinding assay (Dot-Iba) was developed for the detection of specific antibody to M. tuberculosis in the sera of patients with PT and controls. The assay gave positive results in all the 12 sputum-smear positive [acid fast bacilli (AFB)] patients with PT and gave negative results in the 50 sera from control groups. The Dot-Iba as described in this study, is simple, rapid and specific for laboratory diagnosis of PT.     

An evaluation of protein assays for quantitative determination of drugs

We have evaluated the response of six protein assays [the biuret, Lowry, bicinchoninic acid (BCA), Coomassie Brilliant Blue (CBB), Pyrogallol Red-Molybdate (PRM), and benzethonium chloride (BEC)] to 21 pharmaceutical drugs. The drugs evaluated were analgesics (acetaminophen, aspirin, codeine, methadone, morphine and pethidine), antibiotics (amoxicillin, ampicillin, gentamicin, neomycin, penicillin G and vancomycin), antipsychotics (chlorpromazine, fluphenazine, prochlorperazine, promazine and thioridazine) and water-soluble vitamins (ascorbic acid, niacinamide, pantothenic acid and pyridoxine). The biuret, Lowry and BCA assays responded strongly to most of the drugs tested. The PRM assay gave a sensitive response to the aminoglycoside antibiotics (gentamicin and neomycin) and the antipsychotic drugs. In contrast, the CBB assay showed little response to the aminoglycosides and gave a relatively poor response with the antipsychotics. The BEC assay did not respond significantly to the drugs tested. The response of the protein assays to the drugs was further evaluated by investigating the linearity of the response and the combined response of drug plus protein. The results are discussed with reference to drug interference in protein assays and the development of new methods for the quantification of drugs in protein-free solution.     

A dot-immunobinding assay for the serodiagnosis of Pasteurella multocida infection in laboratory rabbits

A dot-immunobinding assay was developed to detect serum IgG specific for lipopolysaccharide of rabbit isolates of P. multocida. The assay detected serum IgG as early as 1 week after experimental subclinical nasal infection, whereas 8 weeks were required to detect antibody by a gel diffusion precipitin test. The assay was more reliable than nasal cultures, in that up to 46% of 16 weekly nasal washings of some infected rabbits failed to yield P. multocida. The bacterial antigen (proteinase k digested cell lysate) used in the assay reacted with IgG that did not cross-react with lipopolysaccharide antigens of B. bronchiseptica, P. pneumotropica or P. hemolytica. The assay is sensitive and specific, easily performed, cost effective, requires no special laboratory instruments and provides a permanent easily stored record.     

A microprocessor-controlled photometer for monitoring microbial growth in multi-welled plates

Multi-welled microtitre plates provide a convenient means of handling 'large block' multifactorial experiments with microbial cultures. An inexpensive instrument, termed a 'Biophotometer', has been designed to monitor microbial growth in each well, by transmitted light measurements. Optional microcomputer control is employed to facilitate scanning of plates and data handling. A unique method for agitating cultures is incorporated into the system. Typical results are presented to illustrate the versatility of this system.     

Centrifugation or filtration in quantitative radioreceptor assays

In quantitative radioreceptor assays the amount of a drug present in the medium to be assayed is inversely related to the amount of receptor-bound radiolabelled ligand. Usually, separation of the bound and free fractions of radiolabelled ligand is done by filtration, in which the bound fraction can easily be collected. However, the filtration disturbs the equilibrium between bound and free fractions, which may lead to erroneous results. Because the decrease in bound radiolabelled ligand is accompanied by an increase in free labelled ligand, we decided also to measure this free fraction after separation by centrifugation and to compare these data with the filtration data. In these experiments a radioreceptor assay for anticholinergics was employed. The results indicate that both methods are compatible in precision when appropriate conditions are used whereas each method has its specific features.     

A microprocessor-controlled photometer for monitoring microbial growth in multi-welled plates

Multi-welled microtitre plates provide a convenient means of handling 'large block' multifactorial experiments with microbial cultures. An inexpensive instrument, termed a 'Biophotometer', has been designed to monitor microbial growth in each well, by transmitted light measurements. Optional microcomputer control is employed to facilitate scanning of plates and data handling. A unique method for agitating cultures is incorporated into the system. Typical results are presented to illustrate the versatility of this system.