Table of Contents

GPM.1.7.2.0022.15 Determination of the authenticity and purity of immunobiological medicinal products using the Western blot method

GENERAL PHARMACOPOEIAN ARTICLE

Introduced for the first time

This general pharmacopoeial monograph applies to the Western blot method, one of the variants of immunoblotting, which is used to assess the authenticity and purity of immunobiological medicinal products (IMPs) based on highly purified proteins, including those obtained using recombinant DNA technology.

At the first stage, electrophoresis is carried out in polyacrylamide gel with sodium dodecyl sulfate (so-called SDS-PAGE electrophoresis) to separate proteins. The proteins are then transferred to a nitrocellulose membrane or PVDF membrane. Detection is carried out using antibodies specific to the protein being detected, conjugated with alkaline phosphatase or horseradish peroxidase (direct version of ELISA), or sequentially using the first antibodies to the protein being detected, and the second antibodies (the so-called antiglobulin serum), the first antibodies specific to immunoglobulin, conjugated with alkaline phosphatase or horseradish peroxidase (indirect version of ELISA).

This OFS describes a detection method based on a color reaction, as one of the most widely used at present. For detection, chemiluminescent methods can also be used, including enhanced chemiluminescence, and methods of radioactive or fluorescent labels (RIA or RIF).

The test is carried out with pharmaceutical substances or finished products.

When assessing authenticity, at the electrophoresis stage, in addition to the tested samples, the following solutions should be added to the gel wells: a negative control sample (1-fold sample preparation buffer), a mixture of molecular weight markers (the use of pre-stained molecular weight markers is recommended), a standard sample of the protein being tested (if available), certified in the prescribed manner. When assessing purity (impurities), additional provision should be made for the addition of a sample with a protein concentration corresponding to the limit of detection or quantification of the impurity (depending on the purpose of the method) and established based on the results of method validation. When assessing purity, it is necessary to compare the blot results with the results of polyacrylamide gel electrophoresis; the technique should detect 1% impurity.

Methodology

To separate proteins by their molecular weights, electrophoresis is first carried out in accordance with the procedure outlined in the General Pharmacopoeia Monograph “Electrophoresis in polyacrylamide gels.”

To carry out protein transfer, a protein transfer device is used. All work is carried out using rubber gloves. The volume of all solutions used must be sufficient to completely immerse the membrane to which the proteins are transferred.

After electrophoresis to prepare the gel for immunoblotting, it is filled with a buffer solution for protein transfer (unless otherwise indicated in the pharmacopoeial monograph or regulatory documentation), placed on an orbital shaker and kept for 10-15 minutes. A sheet of nitrocellulose or PVDF membrane corresponding to the size of the gel is then prepared. When using a nitrocellulose membrane, the sheet is kept for 5 minutes in deionized water, and then 5 minutes in a buffer solution for protein transfer (unless otherwise indicated in the pharmacopoeial monograph or regulatory documentation). When using a PVDF membrane, it must be kept in a solution of methanol or alcohol for 10-15 minutes, and then the same steps must be performed as for a nitrocellulose membrane.

To transfer proteins to a nitrocellulose or PVDF membrane, a “sandwich” is assembled. To do this, a special sponge included with the protein transfer device is moistened with protein transfer buffer solution and placed on a special plastic frame, onto which one to three sheets of filter paper (for example, Whatman 3MM), pre-cut to fit the size of the membrane and the proteins soaked in the transfer solution. A gel is placed on top, on the surface of which a prepared sheet of nitrocellulose or PVDF membrane is carefully placed (without air bubbles). From one to three sheets of filter paper, pre-moistened in a protein transfer solution, are placed on the membrane, and covered on top with a second special sponge soaked in a protein transfer solution. The frame is secured with clamps and slowly immersed in an electrophoresis device filled with a protein transfer solution, the membrane sheet facing the anode. Turn on the device. Electrophoretic transfer of proteins is carried out at room temperature for 25 minutes, setting the current limit at the rate of A max = 2 mA/cm 2 (for example, for a gel measuring 10 × 10 cm 2 A max = 200 mA), unless otherwise indicated in the pharmacopoeia article or regulatory documentation.

It is allowed to use equipment for semi-dry transfer of proteins from the gel to the membrane in accordance with the instructions for its use.

Upon completion of the transfer, the “sandwich” is disassembled. The membrane is washed with phosphate-buffered saline (PBS).

The completeness of protein transfer is assessed visually by the transfer of colored molecular weight markers, all of the main protein bands of which should be detected on the membrane.

Detection

To detect the results of the Western blot method, the test drug is treated (hybridized) with antibodies. To do this, the membrane with the proteins transferred to it is placed in a container with a blocking buffer solution and incubated on an orbital shaker for 30–60 minutes (unless otherwise provided in the pharmacopoeial monograph or regulatory documentation) to prevent nonspecific sorption of antibodies on the membrane.

After this, wash the membrane in a washing buffer solution three times for 5 minutes, unless otherwise specified in the pharmacopoeial monograph or regulatory documentation.

Then the membrane is treated with a solution of the first antibodies to the protein being analyzed, prepared using a buffer solution for antibodies (unless otherwise indicated in the pharmacopoeial monograph or regulatory documentation) immediately before use in accordance with the instructions for use. Antibody treatment is carried out at room temperature for 30–60 minutes (unless otherwise indicated in the pharmacopoeial monograph or regulatory documentation).

Next, the membrane is washed three times for 5 minutes with a buffer solution to remove unbound antibodies on an orbital shaker at room temperature, unless otherwise indicated in the pharmacopoeial monograph or regulatory documentation.

After washing the membrane, treatment (hybridization) is carried out with a solution of second antibodies. Polyclonal antibodies to immunoglobulins of the animal species that were used to obtain the first antibodies are used as second antibodies. These antibodies are conjugated to an enzyme (horseradish peroxidase or alkaline phosphatase). To perform this step, repeat the procedure described above, replacing the first antibody solution with a second antibody solution. Then the membrane is washed from unbound second antibodies in a manner similar to the first.

The development of the immunoblot pattern on the membrane is carried out with a solution of tetramethylbenzidine substrate (TMB) for antibodies conjugated to horseradish peroxidase, or a solution for the development of alkaline phosphatase - when using antibodies conjugated to alkaline phosphatase (unless otherwise indicated in the pharmacopoeial monograph or regulatory documentation).

The reaction is stopped by washing the blot with deionized water. Excess water is removed from the surface of the membrane with filter paper and dried in air in a dark place.

Evaluation of results

When assessing authenticity, the main colored bands on the developed membrane should correspond to the main colored bands on the electropherogram.

The amount of the analyzed substance and the content of impurities, as well as their ratio, are assessed densitometrically.

System suitability criteria

The results of the Western blot method can be taken into account if:

• molecular weight markers are distributed evenly along the entire length of the corresponding gel track;
• the blot shows all the main bands identified when the gel is stained with Coomassie bright blue R-250 or G-250;
• a sample with the minimum concentration established in the pharmacopoeial monograph or regulatory documentation is identified

Results Acceptance Criteria

• Matching of the blot of the test sample with the blot of the reference sample, for example, the corresponding standard sample based on purified protein (substance);
• compliance of the molecular weight of the main identified component with the specification requirements;
• compliance of the content of the detected impurity in the standard sample with the range established in the certificate for the standard sample.

Western blot testing of identity and purity should be used with proven validation characteristics for specificity and detection limit of the impurity.

Features of method validation

When using a methodology to assess authenticity, it is necessary to justify the criteria for the acceptability of the results; It is advisable to use a standard sample based on purified protein, certified in the prescribed manner.

When using a method to assess purity, it is necessary to justify the detection limit of the impurity. When making a quantitative determination, it is necessary to justify the limit of quantitative determination of an impurity, indicate the linear range when determining the main component and impurities, the precision and correctness of the method. For this purpose, it is advisable to use a standard sample based on purified protein. When changes are made to the method specified in the pharmacopoeial monograph or regulatory documentation, the validation characteristics of the previously approved method must be confirmed. The following changes are subject to validation:

• the use of different numbers of samples applied to the gel;
• changing the conditions for transferring proteins from the gel to the membrane, including changing the equipment used;
• changes in the composition of buffer solutions;
• changing the method of detection of the test substance.

Notes

1. Buffer solution for protein transfer pH 8.3. If there are no other instructions in the pharmacopoeial monograph or regulatory documentation, use one of the methods for preparing solutions described below (see Option 1 and Option 2). The transfer solution can be used 2-3 times. The solution is stored for 6 months at a temperature of 4 to 8 0 C.

Option 1. 7.86 g of tris(hydroxymethyl)aminomethane, 33.75 g of glycine are placed in a graduated beaker with a capacity of 3000 ml, up to 2400.0 ml of deionized water is added and stirred on a magnetic stirrer until complete dissolution. The pH is measured, which should be equal to 8.3 - 8.4 (it is not allowed to adjust the pH of the solution with acid or alkali). Add 600.0 ml of alcohol and stir.

Option 2. 5.8 g of tris(hydroxymethyl)aminomethane, 2.9 g of glycine, 11.55 ml of 10% sodium dodecyl sulfate solution are placed in a graduated beaker with a capacity of 1000 ml, 800.0 ml of deionized water are added and stirred on a magnetic stirrer until complete dissolution, pH is measured (8.3-8.4). Add 200.0 ml of 100% methanol and mix. If a PVDF membrane is used, then sodium dodecyl sulfate is excluded from the buffer solution, and the amount of alcohol is reduced to 100.0 ml, thereby increasing the volume of added water to 900.0 ml.

1. Phosphate-buffered saline (PBS), pH 7.2(unless otherwise indicated in the pharmacopoeial monograph or regulatory documentation). 4500.0 ml of deionized water is poured into a graduated container and successively added: 40.0 g of sodium chloride, 1.0 g of potassium chloride, 5.75 g of sodium phosphate disubstituted 2-aqueous, 1.0 g of potassium phosphate disubstituted (each subsequent salt is added after complete dissolution of the previous one). Set the pH to 7.2 with a solution of 70% phosphoric acid or a solution of 45% sodium hydroxide. Adjust the volume to 5000.0 ml with deionized water. The solution is filtered and stored for 3 months at a temperature of 4 - 8 0 C.
2. Buffer solution for washing. Add 5.0 ml of a 10% Tween-20 solution to a 1000 ml volumetric flask, adjust the volume of the solution to the mark using PBS and mix. The solution is stored for 1 month at a temperature of 4 °C to 8 °C.
3. Blocking buffer solution. To 100.0 ml of buffer solution for washing, add 5.0 mg of bovine serum albumin (or other protein), unless otherwise indicated in the pharmacopoeial monograph or regulatory documentation, and mix until completely dissolved. The solution is prepared before use.
4. Antibody buffer solution. To 100.0 ml of buffer solution for washing, add 2.0 mg of bovine serum albumin (or other proteins), unless otherwise indicated in the pharmacopoeial monograph or regulatory documentation, and mix until completely dissolved. The solution is prepared before use.

1. TMB solution— solution for the development of horseradish peroxidase. Use TMB substrate, ready for use.
2. Alkaline phosphatase development solution. Dissolve 1 tablet of BCIP/NBT substrate in 10.0 ml of deionized water. The solution is prepared immediately before use.

Are common.

All Western staging methods consist of the following steps:

1) transfer of protein from the gel to the membrane;
2) blocking nonspecific sorption;
3) adsorption of I-antibodies;
4) washing;
5) adsorption of II-antibodies;
6) washing;
7) filter development;

Which type of membrane to use: NC or PVDV (they say the latter is more sensitive) depends only on your taste. The NC membrane is more fragile, but with careful operation this is not noticeable.

The points.

Whether to trim the gel, leaving only the area that you want to see in the picture before transfer, after transfer, or not to trim at all, depends on your desire to save membrane and antibodies.

Can be done in a gel bath.

But it’s better to apply everything at once without bubbles.

Or ~3mA per individual strip.

Sometimes it is useful to mark the position of gel boundaries, wells, etc.

You can control how much protein remains in the gel after transfer by coloring the gel.

Hybridization.

The filter should always be wet.

The dried PVDF membrane must be wetted with methanol.

Side P (to which the transfer took place) should be in contact with the solution.

Hybridization with a/t is carried out in a hybridizer at 26 o C in small cylinders or 50 ml CF tubes. The volume of the hybridization solution is ~3ml (as minimal as possible, but so that the membrane does not dry out).

Washing and filling: with NT, shaking in a bath (lid from types) V = 30-50ml.

Either throw away the solution or carry out hybridization in it, just do not drip concentrated a/t onto the filter.

For filling non-specific materials, it is best to use milk powder (BSA is also possible, but we like it less). Different batches differ slightly in background intensity.

Pour the hybridization solution into a 50ml CF tube, add the required amount of a/t, mix. Lay the filter along the wall of the test tube and place it in the hybridizer.

If you do not know at what dilution the a/ts work, then you will have to determine this experimentally.

The interaction time with antibodies can be increased or decreased depending on your antibodies.

Hybridization is carried out in 0.1-0.15M NaCl; a decrease in ionic strength causes strong nonspecific hybridization.

Very important!!! The solution with antibodies can be used several times (5-7). It is enough to freeze it after use at -20 o C. This procedure works at least for the hybridization buffer: 1x PBS (without Mg++/Ca++), 0.3-1.0% Dry milk, Antibodies.

When immunostaining after immunoprecipitation, the background can be removed by preconjugating the secondary antibodies to the primary ones.

The given procedure is not the only one. Options:

1. Do everything with NT on a swinging platform, hybridization and packing in plastic bags (V solution ~1.5ml, depending on the size of the filter), washing in a bath:
1. filling: 3% milk powder, 1x PBS, 30-40";
2. "I"a/t: 1h 0.3% milk powder, 1x PBS, antiserum;
3. wash 3 times x 5": 1x PBS, 0.05% Tween-20;
4. "II"a/t: 30" in 1x PBS;
5. washing as in step 3.

Despite the significantly lower content of dry milk in the hybridization buffer for “I” a/t and its complete absence in the buffer for “II” a/t, this method gave clear pictures. Apparently, success was determined by the quality of the vehicles used.

2. Washing and filling are carried out in a bath. Hybridization: put a piece of parafilm (larger than a membrane) on the table, and 0.5-1 ml of hybridization solution with a/t on it. Place the membrane with side (P) facing the solution, avoiding bubbles, and cover the top with a piece of parafilm the size of the membrane. Incubate 1h.

This method reduces the volume of the hybridization mixture, but may deteriorate the quality of hybridization.

The maximum glow occurs 4-5" after applying the solution (reasonable glow intensity is maintained for 15-20").

Western blot analysis to test the amount of calpastatin involved the separation of tissue proteins (30 μg per lane) by electrophoresis in 12% PAGE in the presence of SDS (Laemmli, 1970) followed by semi-dry transfer of polypeptides to a nitrocellulose membrane (buffer: 48 mM Tris-HCl, 39 mM glycine, 0.0375% SDS, 20% methanol, pH 9.2). After incubation (2 h, 20°C) of the membrane in TBS buffer (50 mM Tris-HCl buffer, 150 mM NaCl, pH 7.5), nonspecific binding sites were blocked with a 5% solution of skim milk in TBST buffer (TBS with the addition of 0.1% Tween 20, pH 7.5) for 1 hour. Next, the membrane was sequentially exposed to polyclonal antibodies to calpastatin (dilution 1: 2500 in TBST buffer; 1 hour) and with antibodies to rabbit IgG conjugated with peroxidase (dilution 1: 2000 in TBST buffer; 1 hour). 1 h); Each of these steps was completed by repeated washing with TBST buffer. The membrane was subjected to standard treatment with the Immune-Star system (Bio-Rad, USA).

2.3.6 Other methods

Protein concentration fractions were determined by the Bradford method (Bradford, 1976) using bovine serum albumin (BSA) as a standard.

Densitometry stripes on zymograms and X-ray film were carried out using the standard “Image J” program.

Statistical data processing were carried out using generally accepted methods of variation statistics using MS Excel and StatGraphics software packages. The significance of the differences was assessed using the nonparametric U test (Wilcoxon-Mann-Whitney test), as well as using one-way analysis of variance (Korosov, Gorbach, 2007).

Chapter 3.Research results and discussion

3.1. Calpain/calpastatin system in rats subjected to beta-amyloid-induced neurodegeneration during estrogen therapy

Neurodegeneration was induced in Wistar rats of older age groups - 12 and 24 months. The experimental effect consisted of intracerebral administration of a 42-membered fragment of the amyloid precursor protein - Abeta(1-42) (an experimental model of Alzheimer's disease), as well as combined intracerebral administration of beta-amyloid peptide and intranasal administration of a neuroprotector (estradiol). Among the animals, the following were identified: control group - sham-operated (2 μl of saline solution in the area of ​​the right hippocampus); first experimental group - 2 μl of Abeta(1-42) peptide solution (corresponding to 5 μg of peptide) into the area of ​​the right hippocampus; the second experimental group - after a similar injection of Abeta(1-42) peptide, daily intranasal administration of 0.1 mg of 17-beta-estradiol.

It was found that in the presence of amyloidogenic peptide in nervous tissue, activation of the calpain system occurs, and the degree of activation positively correlates with the intensity of cell death of nervous tissue (neuron density). Regulation of calpains can be carried out both at the level of synthesis of the enzyme protein (or its individual forms - products of different genes), and at the post-translational level due to the processes of autolysis, binding to the endogenous inhibitor calpastatin or to the allosteric regulator - calcium.

The increase in the pool of autolyzed calpains (118 kDa) detected by casein zymography in group of animals No. 2 reflects the activation of calpains in vivo. The observed activation of m-calpain (120 kDa), apparently, as in many other situations, is associated with excess calcium in the cytoplasm. Another confirmation of this pathway of regulation of calpain activity is the stable level of their inhibitor, calpastatin, detected in our study.

As it turned out, estradiol therapy reverses the effect of calpain hyperactivation, with both the overall activity of calpains in nervous tissue and the activity of individual fractions decreasing. In the presence of estradiol, a smaller proportion of the calpain precursor undergoes autolysis and, consequently, activation. Further studies of the mechanism of regulation of calpain activity in experimental animals are needed, in particular, experiments aimed at establishing the source of excess calcium and finding means to prevent these pathological currents are needed.

The level of proteasome synthesis in brain tissue is low compared to other organs and tends to decrease with age (when comparing indicators in 18, 24 and 30-month-old animals), as judged by the amount of alpha-1,2,3,5,6 ,7 proteasome subunits that form alpha rings of the core 20S particle, universal for 20S and 26S proteasomes.

Changes in the level of expression and activity of cathepsins in different areas of the brain in experimental animals were confirmed. In our experiment, intracerebral administration of beta-amyloid peptide led to a significant increase (p<0,05) экспрессии гена катепсина D в коре (правом полушарии) головного мозга крыс по сравнению с животными контрольной группы. В неокортексе крыс, которые после введения бета-амилоидного пептида получали эстрадиол, относительный уровень экспрессии данного гена не отличался от контрольных значений, то есть восстанавливался до нормального уровня. В области правого гиппокампа введение бета-амилоидного пептида привело к увеличению экспрессии гена катепсина D приблизительно в 7 раз (p<0,001). Последующее введение эстрадиола привело к еще большему возрастанию (в 30 раз) относительной экспрессии указанного гена (p<0,001).

Our data demonstrated significant effects of beta-amyloid and estradiol on cognitive performance in rats assessed using the Morris water maze. In previously trained female and male rats, after injection of beta-amyloid peptide into the hippocampus, there was a significant (p<0,05) увеличилось время поиска скрытой подводной платформы. Последующее введение эстрадиола сокращало время поиска подводной платформы у животных обоих полов по сравнению с таковыми, получившими только бета-амилоид. При этом у самок время поиска уменьшилось более значимо (p<0,01), чем у самцов (p<0,05).

The results of confocal microscopy of brain sections showed that the administration of beta-amyloid peptide led to a significant increase in the level of its immunoreactivity in the tissues of both the right and left (to a lesser extent) hemisphere of the brain. These results, along with behavioral data, demonstrate the effectiveness of the administered amyloid peptide drug and provide evidence for the representativeness of the selected model of Alzheimer's disease. Subsequent administration of estradiol reduced the amount of beta-amyloid in the rats' brains to almost control levels. The reduction in amyloid deposits in rats treated with estradiol indicates the launch of certain adaptive processes in the nervous tissue aimed at their utilization.

The mechanism of the neuroprotective role of estradiol is not yet fully understood, although this phenomenon has been noted for a long time. The use of estradiol as a neuroprotector is dictated by several reasons. First, the neuroprotective effect of estradiol is quite well known and described in the literature (McEwen et al., 2001; Asimiadou et al., 2005; Lebesgue et al., 2009). Secondly, it has been shown that estradiol is a neurosteroid in the full sense of the word, since the brain contains all the enzymes necessary for its synthesis (Stoffel-Wagner, 2001; Reddy, 2010). Third, it is known that the neuroprotective effect of estradiol is associated with its antioxidant (Behl et al., 1997) and anti-apoptotic (Asimiadou et al., 2005) effects, that is, with effects opposite to those of Abeta peptide.

The results obtained may indicate an adaptive response of the nervous tissue to the introduction of a toxic peptide. Some publications indicate that the lysosomal autophagy system may be involved in the degradation of beta-amyloid peptide (Nixon, 2007). It is likely that estradiol stimulates autophagy of protein material; This is evidenced both by data on the content of Abeta peptide in brain tissue and by assessment of the activity and level of expression of lysosome proteinases. Fluorescent immunohistochemistry revealed a decrease in the amount of beta peptide in rats receiving neuroprotective therapy with estradiol.

Based on the data obtained in the experiment, the following conclusions can be drawn. 1. The level of expression of the genes of lysosomal proteinases CtsD, CtsB, CtsL and alpha subunits of proteasomes and the activity of the enzymes they encode in the cerebral cortex of rats decreases with aging. On the contrary, the activity of calpains in animals of older age groups increases. 2. The level of expression and activity of cathepsin D, as well as the intensity of calcium-dependent proteolysis, significantly increases in the hippocampus and cerebral cortex of rats after intracerebral administration of beta-amyloid peptide, and the cognitive function of the animals suffers. 3. Administration of estradiol against the background of beta-amyloid intoxication leads to a decrease in the content of this peptide in the hippocampus of rats and an improvement in biochemical and behavioral indicators in experimental animals. 4. Pharmacological activation of lysosomal function by estrogens may promote Abeta removal in Alzheimer's disease; normalization of calcium homeostasis in this situation prevents pathological activation of the calpain system, and, at the same time, the loss of neurons along calpain-dependent cell death pathways.

In the practice of laboratory diagnostics of infectious diseases, there is sometimes a need to determine antibodies not to a pathogen in general, but to certain of its proteins (antigens), that is, a spectrum of specific antibodies. If the method of enzyme-linked immunosorbent assay is used for this purpose, then in this case it is necessary to first isolate and purify the necessary antigens from the pathogen culture. The resulting proteins are applied separately to the solid phase. In the case of using a 96-well plate, one type of antigen is placed in each well. Then specific antibodies are determined by an indirect method.

By the presence of a positive reaction in a well with a particular antigen, one can judge the presence of corresponding specific antibodies. These types of immunoenzyme test systems are offered by manufacturing companies, but the immunoblotting method (Western blot) has become widespread due to its greater information content and ease of execution of the test itself.

Immune blotting allows one to determine antibodies in blood serum simultaneously and at the same time differentiatedly to all diagnostically significant proteins of the pathogen. Translation from English Western blot means Western transfer (literally - blotting). The history of this unusual term is as follows.

In 1975, a scientist named E. Southern first proposed a method for transferring electrophoretically separated DNA fragments from a gel to a membrane. According to the author, the method was called Southern blot, which translated means “southern transfer”. The method of transferring RNA molecules, in turn, was nicknamed by experts Northern blot - “northern transfer”. At first as a joke, and then this name became established in the official scientific literature.

In 1979, G. Towbin published the results of the first experiments on protein blotting. In continuation of the traditions of “geographical” names for methods of transfer of biological macromolecules, this method began to be called “Western” transfer - Western blot.

The first step of this method involves electrophoretic separation of a mixture of pathogen proteins in a polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS). SDS, being a surfactant, evenly envelops protein molecules and gives them all a negative charge of approximately equal magnitude. Therefore, molecules move in an electric field in one direction, and the speed of movement depends only on the size of the molecule (molecular weight) of the protein.

As a result of the electrophoretic procedure, a gel plate is obtained, in the thickness of which proteins are located in the form of separate thin linear zones. According to the direction of movement, they are divided in the following order: proteins with a large molecular weight, about 120-150 kDa, are closer to the start, and proteins with a mass of 5-10 kDa have moved furthest to the finish line. In the second stage, the gel plate is placed on a sheet of nitrocellulose and this structure is placed between the electrodes of a direct current source. Under the influence of an electric field, proteins flow from the porous gel to a denser membrane, where they are firmly fixed.

The resulting blot is treated with a blocking solution containing antigenically indifferent proteins and/or nonionic detergents (Tween 20), which block antigen-free sites on the membrane. The membrane sheet is then cut into narrow strips so that each strip contains all antigenic fractions. The described steps are carried out by the manufacturer.

Commercial test systems for detecting antibodies using immunoblotting contain blots (strips, or strips) that are ready for testing. The user determines the entire spectrum of specific antibodies to pathogen proteins using the indirect method. A soluble colorless substance is used as a chromogen to carry out a color (enzymatic) reaction, the product of which acquires color, becomes insoluble and settles (precipitates) on nitrocellulose.

As a result of sequential immune and enzymatic reactions, in the presence of antibodies to pathogen proteins in the test sample, dark transverse stripes appear on the blot, the location of which is in the zone of certain pathogen proteins. Each such band indicates the presence of specific antibodies to the corresponding antigen. The result of a study carried out by immunoblotting is given in the form of a list of antibodies to specific proteins of the pathogen. For example: “antibodies to proteins p17 and p24 have been detected.”

Nitrocellulose blots can be stored dried for a long time after development. However, the intensity of the color weakens significantly. Wet blots can be photographed or their graphic images can be entered into the memory of personal computers using scanners. Special computer programs allow you to process the results and quickly monitor the dynamics of the antibody spectrum during dynamic observation

Western blotting is a method that searches for specific antibodies against the bacteria that cause Lyme disease. What is a Western blot test? How to interpret the results of the study?

Western blotting is a test that looks for antibodies that the body makes against the bacteria that causes Lyme disease. There are antigens on the surface of bacteria, against which the body has specific antibodies in the IgM and IgG classes. IgM.

Immunoglobulin M (IgM) - produced when our body first encounters a given pathogen. An increase in the amount of IgM against a given pathogen indicates the onset of the disease process.

Immunoglobulin G (IgG) is produced by the body after IgM, the highest level is reached about six months after infection, and unlike IgM, antibodies can remain in the blood for a very long time, even several years.

Western Blot test - indications for performing

Western blot is used in the second stage of diagnosing borreliosis - when the ELISA test (first test) gives a positive or equivocal result. However, it is not used when the ELISA test is clearly negative.

Western blotting - what is the test?

The Western Blot test for borreliosis (Lyme disease) accurately evaluates antibodies to various fragments of bacteria. Various antibodies
against individual bacterial fragments are graphically reflected as black stripes on nitrocellulose paper.

1. To perform the test, two main elements are required: the patient’s blood serum and killed and fragmented cultured borreliosis bacteria.

Do not do this test soon after a tick bite. Wait at least 4 weeks. The cost of a Western Blotting study in both classes of antibodies is about 2500-5000 rubles.

2. Under the influence of electric current, distribution occurs into factors, primarily bacteria obtained from a cell culture, including bacterial proteins (antigens). These proteins are then transferred to a nitrocellulose membrane. The membrane is cut into strips.

3. The antigen strip, in combination with the patient's blood serum, is stained using a special technique that detects antibodies specifically associated with Sprechete Borrelia antigens.

4. In areas where the patient's antibodies are bound to proteins (antigens) of the borreliosis bacteria, we notice characteristic stripes (indicating infection with Lyme bacteria). The test result is positive.

Each band corresponds to a bacterial protein (antigen). If the Borreliosis cell proteins and antibodies do not combine, the band will not appear. Then the result is negative.

Western Blot test - when should it be done?

Early diagnosis of Lyme disease is problematic due to the so-called serological window. This is the period from the beginning of infection to the start of the body producing detectable antibodies. For Lyme disease, the serologic window lasts an average of 4 weeks.

Performing tests less than 4 weeks after a tick bite poses the risk of obtaining a false negative result.

Western Blot test - positive result

Having antibodies against Borrelia means you have Lyme disease. However, it is difficult to answer the question of whether the infection is active or not.

IgG antibodies resulting from infection can be detected in the blood even 10 and sometimes 20 years after Lyme disease diagnosis.

It also happens that the detected IgM antibodies (usually considered an active marker of infection) may be persistent and also do not indicate an active infection.

Western Blot test - negative result

The test can give a negative result in the initial period of the disease, i.e. In the first few weeks after the bite.

Western blot testing can be performed not only if Lyme is suspected, but also if there is infection with H. pylori (which causes peptic ulcers) or HIV.

The Western blot test can also give a false negative result in another situation - when in an old chronic borreliosis the production of antibodies has been stopped or when the antibodies have been completely used up in the fight against this disease.

If the suspicion of Lyme disease is strong, the Western Blot test should be repeated several times, for example, every few weeks, to get to the point where antibodies are present in the blood.

The presence of antibodies in active Lyme disease varies, and a person who tests negative has a chance of testing positive when tested again a few weeks later. Sometimes the diagnosis is confirmed only after the fourth or fifth time.

In this case, some doctors try to get confirmation of the infection in a different way: they treat the patient with antibiotics for several weeks and after 5-6 weeks they send him for Western blotting.

Treatment with antibiotics for this amount of time will not cure the chronic disease, but it will alter the immune system enough that there will be enough antibodies in the blood to be detected. The result of a Western blot test must be interpreted by a physician who specializes in the treatment of Lyme disease

The WB test can also be performed during antibacterial therapy, but with antibiotics the likelihood of a positive result is somewhat less likely. The easiest way to diagnose the disease with this test is 6 weeks after stopping antibiotic therapy.

Important

The interpretation of the study is the interpretation of the bands. As a general rule, it should be assumed that the more bands, the more reliable the diagnosis. Three stripes are really a big confidence, and 5-6 stripes mean Lyme disease with almost 100% probability.

IgM bands are of greater diagnostic value because they suggest an active phase of tick-borne borreliosis, although detected antibodies in the IgM class (IgM bands) may be persistent and not indicate active infection. It turns out that IgM is high at the beginning of the infection and, contrary to logic, in chronic Lyme disease.

Elevated levels of IgG antibodies may be considered a residual infection, or it may indicate chronic active disease.

Even a negative test result does not mean that Lyme disease is not present. A negative test result only means that there are no antibodies in the blood against borreliosis bacteria - this can occur, for example, when the bacteria have entered the body and antibody production has not yet begun (doctors call this period the serological window).

Because of the variety of methods used to conduct this study, it is difficult to make universal recommendations regarding interpretation. Each laboratory uses its own criteria.

The disease can only be detected by a medical specialist based on symptoms and laboratory test results. The Wester Blot test cannot be interpreted without taking into account symptoms.