Quantification by image analysis of immunocytochemical reactions: application for determination of lysozyme content in individual smeared cells.

The objective of the present study was to develop a cytophotometric technique to quantitate immunocytochemical reactions. Cell antigens were detected after immunophosphatase alkaline staining procedure. The amount of reaction product was quantitated by computerized scanning cytophotometry. The technical conditions (dilution of primary antibody; incubation time of the three antibodies; volume and pH of the enzyme substrate reaction; storage of the slides) required for optimal cytophotometric determination of the reaction product were determined. Under these optimally defined conditions, a linear relationship between cell protein content (lysozyme) and microdensitometric measure of the colored reaction product was found. This method could be used for other cells, antigens, and enzymatic indicators.

Use of Enzyme-Linked Antibodies to Measure Serum Anti-DNA Antibody in Systemic Lupus Erythematosus

We describe a solid-phase adsorbent enzyme-linked immunoassay for measuring the amount of antibody in serum. The assay consists of adsorbing an excess of DNA to a polystyrene test-tube support, reacting the anti-DNA serum with the coated tube, following with an additional reaction of an anti-human γ-globulin peroxidase enzyme conjugate with the coated tube, and finally developing a colored reaction product by the action of peroxidase on substrate. In principle, the amount of IgG bound to the tube is proportional to the amount of anti-DNA contained in the serum, and the conjugate also reacts proportionally to the amount of human IgG that has reacted. The assay gives data consistent with that expected for serum from patients with systemic lupus erythematosus, and it is possible to titrate the serum for micrograms of DNA antibody. At present the method still has considerable variability, but offers a new approach to measurement of antibody in autoimmune diseases.

A COLORIMETRIC METHOD FOR MEASURING INDICAN

A qualitative test for indican (indoxyl-O-sulphate, usually as the potassium salt) has been adapted for the quantitative measurement of indican. The method involves the condensation of indican and p-dimethylaminobenzaldehyde in the presence of acid to yield an orange-colored compound. When an excess of sodium acetate is added, the orange-colored reaction product is converted to a cherry-red derivative. The effects of time, acids, temperature, and concentration of reagents on the reaction have been studied and optimum conditions have been selected to obtain a standard curve.Unsatisfactory analytical results are obtained when this method is applied directly to urine samples and a method for the quantitative elution of indican from charcoal columns has, therefore, been devised. With this modification quantitative recoveries have been achieved with indican alone and with indican added to urine samples.In addition a review of the literature pertaining to indican measurement and indigoid pigments is presented.

A COLORIMETRIC METHOD FOR MEASURING INDICAN

A qualitative test for indican (indoxyl-O-sulphate, usually as the potassium salt) has been adapted for the quantitative measurement of indican. The method involves the condensation of indican and p-dimethylaminobenzaldehyde in the presence of acid to yield an orange-colored compound. When an excess of sodium acetate is added, the orange-colored reaction product is converted to a cherry-red derivative. The effects of time, acids, temperature, and concentration of reagents on the reaction have been studied and optimum conditions have been selected to obtain a standard curve.Unsatisfactory analytical results are obtained when this method is applied directly to urine samples and a method for the quantitative elution of indican from charcoal columns has, therefore, been devised. With this modification quantitative recoveries have been achieved with indican alone and with indican added to urine samples.In addition a review of the literature pertaining to indican measurement and indigoid pigments is presented.