Modern methods for diagnosing HIV infection. Detection of antibodies to the virus Immunoblotting

IMMUNOBLOT(western blot) - a method of laboratory testing of blood serum for the presence of antibodies to HIV; it is a more accurate test than ELISA and is used to confirm ELISA results. ELISA - enzyme immunoassay(ELISA) - a laboratory test that allows you to determine the presence of HIV antibodies in the blood; HIV antibody test.

According to WHO recommendations, immunoblotting (Western blot) is used in the diagnosis of HIV infection as an additional expert method, which should confirm the results of ELISA. Typically, this method double-checks a positive ELISA result, since it is considered more sensitive and specific, although more complex and expensive.

Immunoblotting combines an enzyme-linked immunosorbent assay (ELISA) with preliminary electrophoretic separation of viral proteins in a gel and their transfer to a nitrocellulose membrane. The immunoblot procedure consists of several stages (). First, HIV, previously purified and destroyed into its constituent components, is subjected to electrophoresis, and all antigens included in the virus are separated by molecular weight. Then, using the blotting method, the antigens are transferred from the gel to a strip of nitrocellulose or nylon filter, which now contains a spectrum of proteins characteristic of HIV, invisible to the eye. Next, the test material (serum, blood plasma of the patient, etc.) is applied to the strip, and if there are specific antibodies in the sample, they bind to the antigen protein strips that strictly correspond to them. As a result of subsequent manipulations (similar to ELISA), the result of this interaction is visualized - made visible. The presence of bands in certain areas of the strip confirms the presence in the tested serum of antibodies to strictly defined HIV antigens.

Immunoblotting is most often used to confirm the diagnosis of HIV infection. WHO considers positive sera in which antibodies to any two HIV envelope proteins are detected by immunoblotting. According to these recommendations, if there is a reaction with only one of the envelope proteins (gp160, gp120, gp41) in combination or without a reaction with other proteins, the result is considered doubtful and re-testing is recommended, using a kit from a different series or from a different company. If even after this the result remains doubtful, studies are continued every 3 months.

To carry out immunoblotting, the first step is to separate the proteins contained in the blood serum in a gel according to their molecular weight and charge using an electric field (gel electrophoresis). Then a nitrocellulose or nylon membrane is placed on the gel and “blotted” (this is blotting). This is carried out in a special chamber, which allows for complete transfer of material from the gel to the membrane. As a result, the pattern of protein arrangement that was on the gel is reproduced on the membrane (blot), which can then be easily manipulated. Initially, the membrane is treated with antibodies to the desired antigen, and after washing off the unbound material, a radioactively labeled conjugate is added that specifically binds to the antibodies (as in ELISA). The location of the resulting antigen-antibody-labeled conjugate complex is determined by autoradiography using X-ray film. After its manifestation, everything becomes clear whether there are antigens in the blood or not.

Immunoblotting (immunoblot, Western Blot, western blot)- combines an enzyme-linked immunosorbent assay (ELISA) with preliminary electrophoretic transfer of virus antigens onto a nitrocellulose strip (strip).

In this beautiful scientific name, “blot” is most likely translated as “blot,” and “western” as “western” reflects the direction of spread of this “blot” across the paper from left to right, that is, to geographical map this corresponds to the direction from west to east.” The essence of the “immune blot” method is that the immunoenzyme reaction is carried out not with a mixture of antigens, but with HIV antigens, previously distributed by immunophoresis into fractions located according to molecular weight on the surface of the nitrocellulose membrane. As a result, the main HIV proteins, carriers of antigenic determinants, are distributed over the surface in the form of separate stripes, which appear during an enzyme-linked immunosorbent reaction.

Immunoblot includes several stages:

Strip preparation. The immunodeficiency virus (HIV), previously purified and destroyed into its constituent components, is subjected to electrophoresis, and the antigens that make up HIV are separated by molecular weight. Then, using the blotting method (analogous to squeezing out excess ink onto a blotting pad), the antigens are transferred to a strip of nitrocellulose, which now contains a spectrum of antigen bands characteristic of HIV, which is still invisible to the eye.

Sample examination. The test material (serum, blood plasma of the patient, etc.) is applied to the nitrocellulose strip, and if there are specific antibodies in the sample, they bind to strictly corresponding (complementary) antigenic bands. As a result of subsequent manipulations, the result of this interaction is visualized - made visible.

Interpretation of the result. The presence of bands in certain areas of the nitrocellulose plate confirms the presence in the tested serum of antibodies to strictly defined HIV antigens.

Strip A - Positive Control

Strip B - Weak Positive Control

Lane C - Negative Control

Lane D - Positive sample (antibodies to HIV-1 detected)

Currently, immunoblotting (immunoblot) is the main method for confirming the presence of virus-specific antibodies in the test serum. In some cases of HIV infection, before seroconversion occurs, specific antibodies are more effectively detected by immunoblotting than by ELISA. When studying using the immunoblotting method, it was found that most often antibodies to gp 41 were detected in the sera of patients with AIDS, and the detection of p24 in individuals examined with for preventive purposes, requires additional research for the presence of HIV infection. Test systems for immunoblotting based on recombinant proteins obtained by genetic engineering, turned out to be more specific than conventional systems based on purified viral lysate. When using a recombinant antigen, not a diffuse, but a clearly defined narrow antigen strip is formed, which is easily accessible for recording and evaluation.

Sera from individuals infected with HIV-1 reveal antibodies to the following major proteins and glycoproteins - structural envelope proteins (env) - gp160, gp120, gp41; core (gag) - p17, p24, p55, as well as viral enzymes (pol) - p31, p51, p66. Antibodies to env are typical for HIV-2 - gp140, gp105, gp36; gag - p16, p25, p56; pol - p68.

Among laboratory methods necessary to establish the specificity of the reaction, the most widely recognized is the detection of antibodies to the envelope proteins of HIV-1 - gp41, gp120, gp160, and HIV-2 - gp36, gp105, gp140.

WHO considers positive sera in which antibodies to any two HIV glycoproteins are detected by immunoblotting. According to these recommendations, if there is a reaction with only one of the envelope proteins (gp 160, gp 120, gp 41) in combination or without a reaction with other proteins, the result is considered doubtful and re-testing is recommended using a kit from a different series or from a different company. If even after this the result remains doubtful, observation for 6 months is recommended (research after 3 months).

Availability positive reaction with the p24 antigen may indicate a period of seroconversion, since antibodies to this protein sometimes appear first. In this case, it is recommended, depending on the clinical and epidemiological data, to repeat the study with a serum sample taken at least 2 weeks later, and this is precisely the case when testing of paired sera is necessary in HIV infection.

Positive reactions with gag and pol proteins without a reaction with env proteins may reflect early seroconversion and may also indicate HIV-2 infection or a nonspecific reaction. Persons with these results after testing for HIV-2 are retested after 3 months (within 6 months).

The set of reagents “MPBA-Blot-HIV-1, HIV-2” is intended to confirm the detection of antibodies to individual proteins (antigens) of HIV-1 and/or HIV-1 group O and/or HIV-2 in serum or plasma human blood by immunoblotting method.

Distinctive features:

• The set of reagents “MPBA - Blot - HIV-1, HIV-2” contains purified lysate viral proteins of HIV 1 and a peptide - the antigenic determinant gp36 of HIV-2;
• Provides detection of antibodies to HIV-1, HIV-1 group O, HIV 2 on one strip;
• Simple procedure for preparing and conducting tests;
• Internal reaction quality control*
• Maximum speed of analysis (3 hours);
• Small volume of the test sample - 20 µl;
• Does not require additional equipment for research;
• The quality of the set is guaranteed by the use of Russian and international standard samples**

* Internal quality control is ensured by the presence of:

• internal control strips, provides control over the introduction of a serum or plasma sample;
• control negative serum (K-);
• control positive serum (K+), which allows the identification of bands detected on the strip;
• control weakly positive serum (K+cl), which provides control over the sensitivity of the reagent kit.

**Quality assurance:

The characteristics of the “MPBA-Blot-HIV-1, HIV-2” reagent kit were determined by testing samples of a random sample of donors, patients diagnosed HIV infection, commercial seroconversion panels, standard panels, and samples with “potentially interfering” components.

The set of reagents does not give false positive results when examining standard panel sera that do not contain antibodies to HIV 1,2 and HIV-1 antigen (“AT (-) HIV Standard”, No. FSR 2007/00953 dated 10.25.2007). Specificity - 100%.

Diagnostic specificity was determined by studying a random sample of 200 donors from various blood centers and clinics with previously confirmed absence of HIV-1 and HIV-2 infection. Specificity when studying a random sample of donors was 100%;

The specificity of the reagent kit was determined by testing 250 samples, including serum or plasma samples obtained from pregnant women, hospitalized patients, patients with hepatitis C and E, and samples with “potentially interfering” components. When using the MPBA-Blot-HIV-1, HIV-2 kit for these samples, no false-positive results were detected.

Diagnostic sensitivity was determined using:
- plasma samples from the Boston Biomedica, Inc HIV-1 panel (WWRB 301) from different regions containing different subtypes of HIV-1: group M (subtypes A, B, C, D, E, F), and group O; the sensitivity of the reagent kit was 100%;

The sensitivity of the reagent kit was determined in a study of international seroconversion panels of Boston Biomedica, Inc (SeraCare Life Sciences), cat. nrs. PRB 903, PRB 904, PRB 909, PRB 912, PRB 916, PRB 917, PRB 918, PRB 919, PRB 921, PRB 923, PRB 924, PRB 927, PRB 928, PRB 932, PRB 940.

The set of reagents detects antibodies to HIV-1 in sera of a standard panel containing antibodies to HIV-1 (“AT (+) HIV-1 Standard”, No. FSR 2007/00953 dated October 25, 2007), detects antibodies to HIV-2 in standard panel sera containing antibodies to HIV-2 (“AT (+) HIV-2 Standard”, No. FSR 2007/00953 dated October 25, 2007). Sensitivity - 100%.

Registration certificate No. FSR 2010/07958 dated July 13, 2011 (validity unlimited)

Compound:

• Immunosorbent. Nitrocellulose membrane strips white with individual HIV-1 proteins (gp160, gp120, p66, p55, p51, gp41, p31, p24, p17) sorbed onto them by electrotransfer and applied to the strip with a synthetic HIV-2 peptide, an analogue of the gp36 protein and anti-human IgG ( internal control) - 18 pcs;
• K- - control negative serum. Human blood serum that does not contain antibodies to HIV-1,2, HCV, HIV antigen, HBsAg, is inactivated by heating at 560C; transparent light yellow liquid - 1 test tube (0.08 ml). Contains preservatives: thimerosal and sodium azide;
• K+ - control positive serum. Human blood serum containing antibodies to HIV-1,2 (titer not less than 1:10000), not containing HBsAg, HIV antigen, antibodies to HCV, is inactivated by heating at 560C; transparent light yellow liquid - 1 test tube (0.08 ml). Contains preservatives: thimerosal and sodium azide;
• K+sl - control weakly positive serum. Human blood serum containing antibodies to HIV-1,2 (titer no more than 1:200), not containing HBsAg, HIV antigen, antibodies to HCV, is inactivated by heating at 560C; transparent light yellow liquid - 1 test tube (0.08 ml). Contains preservatives: thimerosal and sodium azide;
• RPOKk (x10) - solution for diluting samples and conjugate. Concentrate - Tris buffer containing pre-treated normal goat serum; opaque gray liquid - 1 bottle (10 ml). Contains preservative: thimerosal;
• PRk (x20) - washing solution. Concentrate - Tris buffer containing Tween-20; transparent colorless liquid - 1 bottle (70 ml). Contains preservative: thimerosal;
• Conjugate. Goat anti-human IgG antibodies conjugated to alkaline phosphatase; clear colorless liquid - 1 test tube (0.06 ml);
• Substrate (coloring solution). A solution of 5-bromo-4-fluoro-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT); transparent light yellow liquid - 1 bottle (50 ml);
• Powder for immunoblotting. Skim milk powder - amorphous white or light yellow powder - 5 pack x 1g;
• Tablet with a lid for setting up the reaction - 2 pieces;
• Plastic tweezers - 1 piece.

Immunoblotting (immunoblotting) is a highly specific and highly sensitive reference method that confirms the diagnosis for patients with positive or indeterminate test results obtained, incl. using RIGA or ELISA. Immunoblotting is a type of heterogeneous immunoassay.

This method of detecting antibodies to individual antigens of a pathogen is based on performing ELISA on nitrocellulose membranes, on which specific proteins are applied in the form of separate bands, separated by gel electrophoresis. If there are antibodies against certain antigens-a dark line appears in the corresponding locus of the strip. The uniqueness of the immunoblot lies in its high information content and reliability of the results obtained.

The material for the study is human serum or plasma. For research on one strip, 1.5-2 ml of blood or 15-25 µl of serum is required.

OOO " Laboratory diagnostics"uses immunoblotting kits to detect antibodies to pathogens various diseases companies "EUROIMMUN" (Germany), "MIKROGEN" (Germany):

HSV 1 and HSV 2 IgM/IgG(herpes virus infection)

CMV IgM/IgG(cytomegalovirus infection)

Rubella IgG

TORCH IgM profile(Toxoplasmosis, Rubella, Cytomegalovirus, HSV 1 and HSV 2)

EBV IgMTIgG(Epstein-Barr virus infection)

HCV IgG(viral hepatitis C)

Two types of sets are used - Western blot and line blot.

Western blot: The kits contain test membrane strips with electrophoretically separated native antigens of the corresponding infectious agents, That. antigens are arranged in order of molecular weight. 1-2 additional lines with clinically significant antigens (Western line blot) can also be applied to the membranes. This is a reliable confirmatory method, eliminating false positive responses and cross-reactions.

Line blot: In this case, only clinically significant antigens (native, synthetic or recombinant) are applied to the test strip membranes in a certain order. This approach is used when differential diagnosis several infections on one strip.

Its essence lies in the transfer of molecules of the substance under study from one solid carrier used for fractionation of biopolymers to another, where they are specifically identified using an immunochemical reaction. The modern highly sensitive method consists of identifying proteins, including viral antigens. The method is based on a combination of gel electrophoresis and antigen-antibody reaction. High degree resolution is achieved through electrophoretic separation of proteins, glyco- and lipoproteins and the maximum specificity of detecting immune sera or monoclonal antibodies. Under optimal conditions, immunoblotting can detect antigen in quantities of less than 1 ng in the test volume. Technically, immunoblotting is performed in three steps:

1) the proteins to be analyzed are separated in a polyacrylamide gel in the presence of denaturing substances: sodium dodecyl sulfate or urea, this process is often referred to as SDS-PAGE; separated proteins can be visualized after staining and compared with reference samples;

2) separated proteins are transferred from the gel by overlay (blotting) onto
nitrocellulose filter and fixed on it; in many cases, but
Quantitative ratios of proteins are not always preserved during transfer;

3) poly- or monoclonal detectors are applied to the filters
antibodies containing a radioisotope or enzyme label; For
to detect bound antibodies, anti-species assay is also used
labeled serum, in other words, at the final stage of blotting
similar to solid-phase immunoassays.

It should be borne in mind that in this immunoblotting setup, proteins are in a denatured state, and therefore may not be recognized by antibodies specific to the native protein, but in the presence of sera to all constituent peptides, the entire antigenic spectrum of the test protein is simultaneously detected. Immunoblotting is quite widely used in studies of the structure of hepatitis viruses, in particular, to establish the antigenic relationship between individual strains. The high resolution of immunoblotting allows one to obtain good results in diagnostic practice, when it is necessary to identify the virus in the tissues or excreta of a patient.

Depending on the substance being studied, DNA, RNA and protein are distinguished - blotting.

Immunochemical detection of antigens can be carried out using antibodies conjugated to a label. As a mark in lately either radioactive isotopes or enzymes (peroxidase, alkaline phosphatase, lactamase, etc.) are widely used.

Blotting time by diffusion is 36-48 hours. But the fastest and effective way transfer of proteins from gels - electroblotting, the time of which is generally 1-3 hours, for some high-molecular proteins - more than 12 hours.

The specific choice of sorbents for various modifications of blots (nitrocellulose or paper processed accordingly), the choice of blocking conditions and immunochemical detection of antigens completely depends on the antigen, its quantity, the immunoassay method and the purposes of the study.

The ability to detect antibodies to specific antigens of a pathogen allows one to assess the significance of these antibodies (specificity for a given etiological agent) and exclude a reaction to cross antigens. This distinguishes immunoblotting from ELISA, where various combinations of antigenic determinants can be used as an antigen - both specific and not, giving cross-reactions with other pathogens. Otherwise, upon receipt positive result in ELISA one can only assume that it is a consequence of cross-reaction, but in the case of immunoblotting this is conclusive

For a number of reasons, the information security method has become most widespread as a method suitable for use as a confirmation test.

The undoubted advantage of the method is the possibility of testing antibodies to weakly or completely insoluble antigens and eliminating the stage of introducing a radioactive label into the antigens.

Sensitivity in the case of IB is judged by the maximum amount of antigen applied to the gel, which, during protein fractionation, can be detected immunochemically after transfer from the gel to the solid phase (nitrocellulose). The overall sensitivity of the assay depends on a number of factors: conditions of fractionation and immobilization of the antigen on a solid carrier, background level, specificity and affinity of antibodies. The type of tag used and the method of identifying it are important.

Thus, the immunoblotting method makes it possible to identify antigen zones on the solid phase without binding the entire protein to specific serum antibodies. Immunoblotting and its modifications are mainly used for typing bacterial and viral antigens and antibodies, especially in the case of insufficient resolution of conventional systems, as well as in the analysis of immunoglobulins, nucleic acids or as a confirmation test in combination with other methods.

Great difficulties in interpreting cross-reaction results and in cases initial stages seroconversion. In the first situation, during a repeated study after a certain period of time, no antibodies are detected, but in the second immunoblot new bands appear, indicating the appearance of antibodies to HIV proteins or glycoproteins, characterizing the response dynamics of the immune reaction to virus antigens.

It is actually the final verification method in the chain of serological studies, allowing one to make a final conclusion about the patient’s HIV positivity or reject it. To perform IB, nitrocellulose strips are used, onto which HIV proteins are previously transferred by horizontal and then vertical immunophoresis in order of increasing molecular weights. Antibodies in the tested serums interact with proteins in certain areas of the strip. The further course of the reaction does not differ from that for ELISA, that is, it involves treating the strip with a conjugate and chromogen-substrate, washing off unbound components and stopping the reaction with distilled water. Preliminary electrophoretic separation of proteins and their fixation on nitrocellulose makes it possible to identify antibodies to specific proteins in accordance with the presence (or absence) of staining (grayish-blue) of the corresponding zones of the strip. Immunoblotting cannot be used for mass screening studies due to its high cost and is a method of individual arbitration for final stage serological research.

There is a fairly clear correlation between the results of serum testing in IB and ELISA. Sera that are twice positive in ELISA (in different test systems) are then interpreted in IB as HIV-positive in 97-98% of cases. Sera that are positive in ELISA in only one of the two test systems used turn out to be HIV-positive in IB no more often than in 4% of cases. When conducting confirmatory studies, about 5% of IB can give so-called “indeterminate” results, which, as a rule, correspond to positive ELISA, but not RIP. In approximately 20% of cases, “undetermined” IBs are caused by antibodies to HIV-1 gag proteins (p55, p25, p18). Upon receipt questionable results immunoblotting, the study must be repeated after 3 months and, if the result remains uncertain, after 6 months.

Radioimmunoassay (RIM) (Radioimmunoassay, RIA) Radioimmunoassay - method of quantitative determination biologically active substances, (hormones, enzymes, medicines etc.) in biological fluids, based on the competitive binding of the desired stable and similar radionuclide-labeled substances with specific binding systems. The latter are most often specific antibodies. Due to the fact that the labeled antigen is added in a certain amount, it is possible to determine the part of the substance that is bound to the antibodies and the part that remains unbound as a result of competition with the detected unlabeled antigen. The study is performed in vitro. For R. a. produce standard sets of reagents, each of which is designed to determine the concentration of a single substance. The study is carried out in several stages: mix biological material with reagents, incubate the mixture for several hours, separate the free and bound radioactive substance, carry out radiometry of the samples, and calculate the results. The method is highly sensitive, it can be used in the diagnosis of diseases of the cardiovascular, endocrine and other systems, to determine the causes of infertility, fetal development disorders, in oncology to determine tumor markers and monitor the effectiveness of treatment, to determine the concentration of immunoglobulins, enzymes and medicinal substances. In some cases, studies are performed against the background of load functional tests(for example, determination of insulin levels in blood serum against the background of a glucose tolerance test) or over time (for example, determination of sex hormones in the blood during the menstrual cycle).

Using a commercial kit from ABBOTT - Austria II-I 125, it is possible to detect HBsAg in concentrations up to 0.1 ng/ml. The advantages of the method include the possibility of standardizing and automating the method with obtaining answers in digital terms. The disadvantage of the method is the limitations determined by the mode of operation with radioactive material and the relatively short shelf life of the diagnostic kit, which is associated with the decay of the radioactive label.

Diagnostic kits for identifying various antigens of hepatitis A, B and D viruses and antibodies to them are produced by Izotop (Tashkent) and some foreign companies (for example, ABBOTT). Polystyrene balls (“EBBOTT”) or test tubes (“Isotope”) are used as the solid phase. To label antibodies or antigens, the isotope I 125 is most often used, which has a half-life of 60 days and high specific radioactivity. Measurement of a radioactive tracer, i.e. radiation, is carried out on special counters - radio spectrometers. Counting of radioactive pulses in both control and test samples is carried out at a single fixed time, usually within 1 minute. When analyzing the reaction results, it is necessary to take into account the presence of background radioactivity, which can affect the final result of the reaction. The reasons for the increased background may be: contamination of the sample container or nest; incorrect device settings; presence of a source of strong radiation near the device.

To confirm a positive result obtained during the initial screening of samples, repeat testing with RIA or an alternative test is recommended. If HBsAg is detected, a confirmation test must be performed.

Table 1. Classification of vaccines

References:

Mandatory:

1. Khaitov R.M., Ignatieva G.A., Sidorovich I.G. Immunology: Textbook.-M.: Medicine, 2000.- 432 p.: ill. (Textbook for students of medical universities).

2. Kovalchuk L.V. et al. Immunology: workshop: textbook. manual – M.: GEOTAR-Media, 2012. – 176 p.

3. Pozdeev O.K. Medical microbiology / ed. acad. RAMS V.I. Pokrovsky - M.: GEOTAR-Media, 2001. – 768 p.

4. Borisov L.B. Guide to practical classes in microbiology. M. 1997

Additional:

1. Genkel P.A., Microbiology with the basics of virology. M., 1974

2. Korotyaev A.I., Babichev S.A. Medical microbiology, immunology and virology: a textbook for honey. universities. - 3rd edition, revised. and additional – St. Petersburg, SpetsLit. 2002. – 591 p.

3. Borisov L.B., Smirnova A.M., Medical microbiology, virology, immunology, M., Medicine. 1994

4. Timakov V.D., Levashov V.S., Borisov L.B. Microbiology. M. 1983