Immunoenzyme method and kit for determining iga1 and iga2 using polyclonal iga antibodies

FIELD: medicine.

SUBSTANCE: method involves determining total IgA quantity using immunoenzyme method based on polyclonal IgA antibodies as binding antibodies on the micropanel and the same antibodies in conjugate with peroxidase for detecting bound IgA in sample before and after treatment with specific IgA1-protease. Total IgA1 and IgA2 content is determined before being treated with enzyme. IgA2 content only is determined after being treated with enzyme. IgA1 is not determined after being disintegrated with IgA1-protease. Kit has microplate containing absorbed polyclonal IgA antibodies, conjugate of the same antibodies with peroxidase, reference sample having given IgA concentration, IgA1-protease and substrate buffer.

EFFECT: simplified method and kit for determining IgA1 and IgA2; improved results reproducibility; no specific monoclonal antibodies being used.

2 cl, 1 tbl

 

The invention relates to medical immunology, and in particular to methods of determining the content of immunoglobulins in drugs, serum and other biological fluids.

The invention can be used in medicine to determine the distribution of subclasses in drugs IgA, accurate diagnosis with multiple myeloma and changes in the ratio of IgA subclasses in various diseases. Determining the concentration of serum IgA2 can be used to detect pathology of mucous tissue. The change in the ratio of IgA1/IgA2 associated with specific diseases and conditions, such as anaphylactic transfusion reactions, chronic alcoholism, primary nephropathy.

To define subclasses IgA1 and IgA2 know the use of ELISA and other methods based on specific monoclonal antibodies to these proteins [1-3]. The disadvantages of these methods is the difficulty of obtaining specific antibodies to IgA1 and IgA2 due to the high homology of these proteins, and hence the cost of the respective antibodies and monoclonal antibodies to these proteins.

In order to distinguish between IgA1 and IgA2, apply specific IgA1-protease produced near pathogens and are able to split into two fragments exclusively IgA1, without affecting IgA2 molecules, but using specificeskich proteases to create methods for the quantitative determination IgA1 and IgA2 is not known.

Objective of the claimed invention is the development based on the use of specific IgA1-protease and immunoassay method and kit for determination of the content of IgA1 and IgA2 without the use of specific anti-IgA1 and anti-IgA2 antibodies.

This object is achieved by developing methods to determine and set. The method involves the determination of total IgA content using ELISA method based on polyclonal antibodies to IgA, used as binding antibodies on microarrays and the same antibodies conjugated with peroxidase for detection of bound peroxidase IgA in the sample before and after processing specific IgA1-protease. Before the processing enzyme is determined by the total content of IgA1 and IgA2, and after treatment with the enzyme is determined solely IgA2, and IgA1 after cleavage of IgA1-protease is not defined.

Set to something called a microarray contains adsorbed polyclonal antibodies to IgA, the conjugate of the same antibody with peroxidase, a standard sample with a known content of IgA, IgA1-protease and substrate buffer.

The technical result of the claimed invention is to develop a method and kit for determining IgA1 and IgA2, with good reproducibility and without using a specific monoclonal antibody.

the example 1. The definition of the content of IgA1 and IgA2 in serum samples. For each serum cook 2 sample: both contain 40 µl of serum diluted 10 times in phosphate buffer, pH 7.4, containing 0.15 M NaCl (PBS). To one sample, add 10 ál of the same buffer, to a second 10 μl of a solution IgA1 protease from Neisseria meningitides. After incubation of the samples for 18 h at 37°the reaction is stopped by introducing 1 ml of PBS and frozen at -20°C. a Solution of 5-20 µg/ml goat IgG antibodies against human IgA in 0.05 M sodium carbonate buffer, pH 9.5 to make 120 ál in each well microarrays. Close the lid and leave overnight at 4°Stri times washed to something called a microarray phosphate buffer, pH 7.4, containing 0.15 M NaCl (PBS) and 0.05% tween-20, then to something called a microarray dry by shaking out the remaining liquid. Then all wells contribute 100 μl of a solution of PBS and retitrement for IgA serum samples after incubation with specific IgA1-protease and without it. After incubation in an incubator at 37°C for 1 h on a rocking chair, a three-time washing PBS with 0.05% tween-20 and draining the tablet into each hole making 100 μl of peroxidase conjugate with antibodies against IgA in the same buffer at selected breeding. After incubation in an incubator for 1 h at 37°C, three times washing FBS with twin and drainage tablet in every hole making 100 ál sub is tretego buffer (250 ál of 0.02 M solution of tetramethylbenzidine in 10 ml citrate-phosphate buffer, pH 5.0, and 20 μl of 3% hydrogen peroxide). After 5-15 min of incubation in the dark, the reaction is stopped by introducing into each well of 75 μl of 7% sulfuric acid. The results of the reaction consider using a spectrophotometer with a vertical beam of measuring light absorption at 450 nm. The IgA concentration is calculated by comparing the results with the calibration curve for the control sample with a known content of IgA. In samples without enzyme determine the total content of IgA in the sera. In the samples after incubation with enzyme - content IgA2, and the content of IgA1 are calculated according to the difference of these values.

The results of determination are shown in table 1.

Example 2. Kit for determination of the content of IgA1 and IgA2. Set to something called a microarray contains adsorbed polyclonal antibodies to IgA, the conjugate of the same antibody with peroxidase, a standard sample with a known content of IgA, IgA1-protease and substrate buffer. This set is used in accordance with example 1.

According to literature data [4] is the concentration of total IgA in men is 1,49 of 2.68 mg/ml in women - 1,24-2,17 mg/ml; the ratio of IgA1/IgA2 for men of 4.44±1,81, for women to 4.81±1,85. The obtained data are shown in table 1, correspond to the literature. For normal sera ratio corresponds to the norm, and for myeloma violated in the matter myeloma a subclass IgA viewers what is.

Table 1
The definition of the content of IgA1 and IgA2 in serum samples (marked with an asterisk myeloma patients).
No. wheyIgA, Mr/MnIgA1, mg/mlIgA2, mg/mlIgA1, %IgA2, %IgA1/IgA2
1*1,040,200,8519810,23
2*0,890,390,5044560,79
31,080,890,198218to 4.68
4*was 2.762,700,0698246,22
5*0,360,310,058713for 6.81
60,960,750,2278223,48
7*3,360,472,8914860,16
8*1,000,590,415941the 1.44
9*0,160,070,0942580,74
10*5,880,735,1512880,14
11*1,110,650,465248the 1.44
12*0,170,000,170,001000,00

LITERATURE

1. Van den Wall Bake A.W., Daha M.R., Van der Ark, A., P.S. Hiemstra, Radi L. Van Es L.A. // Clin. Exp. Immunol. 1988. V.74. P.115-120.

2. Depelchin S., Dehennin J.P., A. Bottaro, A. Carbonara, Vaerman JP, Sibille Y. // Int. J.Clin. Lab. Res. 1994. V.24. P.154-161.

3. Mestecky J., Hamilton, R.G., Magnusson C.G., R. Jefferis, Vaerman JP, Goodall M. et al. // J.Immunol. Methods. 1996. V.193. P.103-148.

4. Berth M, Delanghe J., Langlois M., De Buyzere M. //Clinical Chemistry. 1999. V.45. P.309-310.

1. The immunoassay method of determining the content of IgA1 and IgA2 based on polyclonal antibodies to IgA, characterized by the fact that in the sample containing IgA, determine the total IgA content using ELISA method, using as binding antibodies on microarrays polyclonal antibodies to IgA and the same antibody conjugated with peroxidase for detection of bound peroxidase IgA, before and after processing of the sample-specific IgA1-protease; however, before treatment with the enzyme is determined by the total content of IgA1 and IgA2, and the Le processing enzyme is determined solely IgA2, and IgA1 after cleavage of IgA1-protease is not defined.

2. The kit for immunoassay determination of the content of IgA1 and IgA2 based on polyclonal antibodies to IgA, characterized in that it contains to something called a microarray with adsorbed polyclonal antibodies to IgA, the conjugate of the same antibody with peroxidase, a standard sample with a known content of IgA, IgA1-protease and substrate buffer.



 

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