Immunoenzyme method and kit for determining iga1-protease activity

FIELD: medicine.

SUBSTANCE: method involves absorbing myeloma IgA1 on micro panel, incubating solutions containing IgA1-protease and determining enzyme activity from IgA1 sorbate quantity reduction determined by conjugate with polyclonal IgA antibodies peroxidase. Determining IgA1-protease activity with inhibitors available in various concentrations enables one to calculate its inhibition constants. Kit has microplate containing absorbed myeloma IgA1 preparation, peroxidase conjugate with antibodies against human IgA and substrate buffer.

EFFECT: simplified method and kit for determining IgA1-protease activity and studying its inhibition process.

2 cl, 1 dwg, 1tbl

 

The invention relates to medical immunology, and in particular to methods for determining the functional activity and inhibition IgAl-proteases. IgAl protease is a bacterial enzymes with extremely narrow specificity. They are able to cleave only the immunoglobulin subclass IgAl humans and some primates. These proteases are one of the main factors of virulence of several pathogens, such as Hemophilus influenzae, Neisseria meningitides, Neisseria gonorrheoeae, Streptococcus pneumoniae and some other bacteria infecting the oropharynx and upper respiratory tract. IgAl protease used in laboratory practice for ittipiboon immunoglobulin A. the detection of these proteases can be used to characterize the virulence and pathogenicity of bacterial pathogens. The search for inhibitors IgAl-proteases necessary for the creation of medicines, protection from pathogenic microorganisms. For these purposes may be used in the invention.

The closest prototype is an immunoassay method for determining the activity of IgAl protease [1], in which IgA substrate adsorb on the polystyrene microplate coated with antibodies to light chains of the antibodies to bind IgA in his Fab-fragments.

The disadvantages of the considered method is the necessity of using two types of antibodies: against the easier the x circuits and against α -chains.

Objective of the claimed invention is to develop a method and kit for determining the functional activity of IgAl protease and study of its inhibition using only the available antibodies against IgA, which simplifies and significantly reduces the set.

This object is achieved by developing methods to determine and set. The method involves sorption on microarrays myeloma IgAl, incubation in cell microarrays solutions containing IgAl-protease, and determination of enzyme activity to reduce the amount of sorbed IgAl-defined conjugated with peroxidase polyclonal antibodies to IgA. The determination of the activity of IgAl protease in the presence of various concentrations of inhibitor can be used to calculate its inhibition constant.

Set to something called a microarray contains adsorbed drug myeloma IgAl, peroxidase conjugate with antibodies against IgA person and substrate buffer.

The technical result of the claimed invention to provide a simple method and kit for determining the activity of IgAl protease and study of its inhibition.

Example 1. Determination of enzyme activity IgAl protease. The method based on a determination of the residual amount of IgA enzyme-linked immunosorbent assay. To do this, hold the sorption of the drug myeloma IgAl on the surface is rnost holes microarrays in 0.05 M sodium carbonate buffer, pH of 9.5. In wells contribute solutions of the enzyme obtained from the culture fluid of Neisseria meningitides, in various concentrations. After incubation for selected depending on the activity of the enzyme time and temperature of the washing of the wells with phosphate-saline buffer, pH 6.5, with twin last contributed strength conjugate anti-IgA with horseradish peroxidase. After incubation for 1 h and similar washing the wells contribute substrate buffer with tetramethylbenzidine. After incubation for 20 min, the process is stopped by acidification with sulfuric acid and measuring the light absorption at 450 nm on a spectrophotometer with a vertical beam. The difference of optical density of solutions in the experiment and the control (without enzyme) calculate the quantity of hydrolyzed for 1 h IgA, which characterizes the activity of IgAl protease. The drawing shows the line number of the active enzyme to the difference of optical densities.

Example 2. The determination of the constants of inhibition of the enzymatic activity IgAl proteases. Carried out analogously to example 1, making holes IgAl-protease in the same concentration and inhibitors (benzamidine, bacitracin and specific IgG against Neisseria meningitides) in various concentrations. Determine the difference of optical densities at 450 nm in each well of experience and control without enzyme, mistaking them for relative activity IgAl protease in which each hole experience. Change of enzyme activity depending on the concentration of inhibitor calculate the inhibition constant. The results are shown in table 1.

Example 3. Kit for determination of enzyme activity IgAl protease. Set to something called a microarray contains adsorbed drug myeloma IgAl, peroxidase conjugate with antibodies against IgA person and substrate buffer. This set is used in accordance with examples 1 and 2.

As expected, the highest inhibition was observed for antibodies specific to Neisseria meningitides, and received constant typical dissociation constants of specific antibodies. Good binding showed bacitracin, a known inhibitor of proteolytic enzymes. Weakly inhibits benzamidine - proteinase inhibitor trypsinogen type.

Table 1
The determination of the constants of inhibition by bacitracin; benzamidine and IgG antibodies against Neisseria meningitides measurement activity lgAl-protease in the presence of different concentrations of inhibitors
Inhibitor
BacitracinIgG antibodies against Neisseria meningitidesBenzamycin
The concentration of the inhibitor, mg/ml The relative activity of the enzyme, cu
500.00450.00240.0054
100.00470.01090.0051
20.02430.01540.0063
Toig/mlAt 39.644.53609.8
ToiM2.79·10-053.02·10-080.0039

LITERATURE

1. Reinholdt j, Kilian M.A sensitive enzyme-linked immunosorbent assay for IgA protease activity // J. Immunol. Methods. 1983. V.63. P.367-376.

1. The immunoassay method for determining the activity and inhibition of IgAl protease, characterized in that the holes microarrays absorb the drug myeloma IgAl, then in wells make the solution under test IgAl protease, and also for studying the inhibition of the inhibitor at various concentrations, spend incubation for implementation of proteolysis and after drying the tablet and wash the wells contribute peroxidase conjugate with antibodies against IgA person and after incubation and washing of the substrate buffer, followed by the calculation of activity IgAl protease losses on defined IgA in experience compared to a control not containing IgAl protease, and in the study of inhibition calculated activity IgAl protease at various concentrations of inhibitor COI is lsout to calculate the inhibition constants.

2. The kit for immunoassay determination of activity and inhibition of IgAl protease, characterized in that it contains to something called a microarray adsorbed drug myeloma IgAl, peroxidase conjugate with antibodies against IgA person and substrate buffer.



 

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