Method of pre-treating samples in peroxidase-catalyzed enzyme assays

Medicilon provides various enzyme activity assays for kinases, phosphatases, proteinases, deacetylase, peptidase, esterase, and other enzymes. Our line of well-characterized immunoassays and biochemical kits ensures accurate and reproducible results.Email:marketing@medicilon.com.cn web:www.medicilon.com

A method for reducing the occurrence of falsely elevated results in a peroxidase-catalyzed, enzyme assay is described. Interference in an assay caused by blood or bood products in a clinical specimen is eliminated or reduced by reacting the specimen with an oxidizing agent such as sodium hypochlorite, hydrogen peroxide or sodium meta-periodate.

The use of enzyme immunoassays (EIAs) to determine whether a particular analyte is present in a patient sample has aided the expansion of diagnostic medicine. In a typical enzyme immunoassay, an assay reagent is labeled with an enzyme, the reagent becoming bound to a solid support in an amount that depends upon the amount of analyte in the sample. Enzyme substrate, generally added in a final step of the assay, reacts with the enzyme to generate a detectable signal which is related to the amount of analyte present in the sample.

However, present in some patient samples are compounds which may interfere with enzyme activity. One form of interference occurs when an interfering compound acts to catalyze a signal generating reaction in the absence of the specific enzyme. The presence of such compounds can result in incorrect assay interpretation owing to the generation of signal in an amount unrelated to the presence of the analyte in the patient sample.

Blood or blood products are common sources of assay interference in enzyme assays which employ peroxidase as the enzyme label. The blood or blood products help convert the enzyme substrate to its oxidized products, giving rise to positive assay interference. Specifically, the heme component of hemoglobin can bind to solid supports used in EIAs to immobilize reagents. The heme moiety (also called "microperoxidase") present during signal development in peroxidase-catalyzed EIAs generates non-specific signal and is a source of positive interference.

It has been discovered that the positive interference in peroxidase-catalyzed enzyme assays caused by blood or blood products can be eliminated by the addition of oxidative compounds, such as sodium hypochlorite, hydrogen peroxide and sodium meta-periodate, to the patient specimen. The treated patient specimen can be used in the enzyme assay with no interfering effects.

The methods of this invention can be used with patient specimens obtained by conventional methods, such as swabs, washes, aspirates and the like, taken from the eye, the nares at the back of the nose, cervix, vagina, urethra, rectum, throat, blood, serum, plasma and the like.

In one embodiment of the invention, the oxidizing agent may be added directly to the patient sample. For example, a solution of oxidizing agent may be added to a patient sample just prior to taking an aliquot of the sample for use in the assay.

In another embodiment, the oxidizing agent solution may be added to a reagent, such as a detergent solution, that is used to extract the patient sample from a swab or the like. This extraction solution is added to a tube containing a swab sample, mixed, and allowed to incubate for a predetermined length of time, followed by expulsion of liquid from the swab. The liquid containing the patient sample is then evaluated using an EIA or other enzyme assay such as a peroxidase catalyzed enzyme assay.

In yet another embodiment, a solution of oxidizing agent is added to the liguid extracted from a patient swab using a detergent or the like. This solution of oxidizing agent and patient sample is allowed to incubate for a predetermined length of time, following which an appropriate aliquot is taken for use in the enzyme assay.

The method of this invention may be used in any enzyme assay but is particularly useful in a passive or active, capture-dependent, peroxidase-catalyzed, sandwich-type EIA. The sandwich-type EIA is preferably one in which an antibody:antigen:antibody sandwich tethered to a solid support is formed. Typically, antigen or antibody is bound to a solid support such as filter paper, test tubes made from polyethylene, polystyrene, polypropylene or other suitable materials, latex particles, glass or plastic beads, magnetic particles or the like. Patient samples are contacted with the solid support and the specific analyte being assayed binds to the anchored ligand on the solid support. A second antibody with specificity to the analyte and directly or indirectly labeled with peroxidase is added and, after the reaction mixture has been allowed to incubate for a sufficient time to allow the reaction to occur, the solid support is washed. If the second antibody is directly labeled, then, after the solid support is washed, enzyme substrate is added to the solid support and the resulting signal is measured by colorimetric or spectrophotometric techniques. If the second antibody is unlabeled, then a labeled antiglobulin directed against the second antibody may be added and the solution allowed to incubate for a predetermined time before washing and determination of the quantity of label by conventional techniques.

Although the sandwich-type assay described above utilizes antibodies and antigens, the methods of this invention may be used generally with peroxidase-catalyzed, sandwich-type assays involving ligand-receptor pair members. As used herein, "ligand receptor pair" refers to a pair of compounds of which one, a "receptor" is capable of recognizing a particular spatial and polar organization of the other ("ligand") or portion thereof, and is capable of binding to that compound. For various ligands, illustrative receptors forming the other half of a ligand-receptor pair include antibodies, enzymes, lectins, Fab fragments, complementary nucleic acids and the like.

The invention is useful in detecting a broad range of analytes. U.S. Pat. No. 4,374,925 and U.S. Pat. No. 3,817,837, the teachings of which are incorporated herein by reference, set out excellent lists of analytes which are part of specific binding pairs. Other examples of binding pairs include nucleic acids and complementary nucleic acids. Analytes of particular interest include viruses, bacteria, fungi and protozoans, specific products and assemblages thereof and macromolecules and products of living organisms.

Antibodies useful with enzyme assays may be raised in humans or non-human species such as guinea pig, goat, horse, rabbit, sheep, etc. by immunization with appropriate antigens in accordance with known methods. Monoclonal antibody to appropriate antigens may also be used with the methods of this invention.

Assay interference can be detected by comparing assay results with results obtained using other evaluation methods, i.e., culture techniques, microscopic analysis, and the like. A specific example of the interference by blood or blood products of a direct antigen EIA is described as follows in connection with an assay for the detection of Chlamydia trachomatis. Samples for the detection of chlamydia were taken from prospective patients. Direct fluorescent labeling techniques were used to determine whether chlamydia was present in a sample, as follows: Antibody that binds specifically to chlamydia and that was labeled with a fluorescent agent was obtained. Labeled antibody of this type is commercially available. A volume of specimen extract was obtained and centrifuged to form a pellet comprised of chlamydial organisms and debris from the sample. The pellet was resuspended in a minimal volume of buffer, spotted on a microscope slide, fixed with methanol and stained with the labeled antibody reagent. The antibody bound to the chlamydia, if any, on the slide. The slide was examined using an appropriate microscope to determine whether chlamydia were present. In samples not treated with the oxidizing agent prior to performing the EIA, a significant number of specimens that tested negative with the direct fluorescent method yielded positive EIA results in the absence of the additives described in this invention.