So we can separate antigens into their components and allow them to react with their antibodies

Transcription

1 Ag-ab reactions As for single immunodiffusion, double immunodiffusion can be also combined with electrophoresis to speed up the reaction, and in this case the test is called immunoelectrophoresis. So electrophoresis combined with double diffusion is called immunoelectrophoresis The test is composed of 2 stages, the first is separation of antigens into their components by electrophoresis, so if we have serum, and we want to separate antibodies in serum, we run serum electrophoresis (by currency supplying electrical current), to separate these antigens based on their molecular size and charge, then after electrophoresis completely cut it, along longitudinal well, in the agarose the gel and we place antibodies to the antigens, in this case it will be anti-antibody (anti-human immunoglobulins), where we can prepare by injecting immunoglobulins to rabbits for example, and the product will be antibodies directed against all of these (anti-igg, anti-igm, anti-kappa..etc.), and then we place these antibodies in the trough which is cut in the well, and incubate and look for precipitation Lines of precipitate that form against serum proteins are shown in the figure in slides, showing that albumin is the fastest, then alpha globulin, then igm, and finally gamma globulin is the largest and slowest in migration. So we can separate antigens into their components and allow them to react with their antibodies The third figure shows Example where immunoglobulins reside, this test is a qualitative test, and it can be semi-quantitative, where we can detect missing antibodies, increased amounts of an antibody or abnormal proteins, but we cannot use it like the single immunodiffusion to quantitate immunoglobulins for example, here if an immunoglobulin is missing, we can detect that, and if one of the components of serum is increased in large amounts we can detect that (sharp or heavy bands form in this test), so its just a qualitative test that can be applied for serum, urine, or CSF.

2 Also used in Diagnosis of multiple myeloma, where we have one of the immunoglobulins, depending on the abnormality (but most commonly igg) this test has been largely replaced by a new test, which is immunofixation, and immunofixation is also a combination of electrophoresis and precipitation, proteins are separated by electrophoresis and the antibody is exposed directly instead of being exposed through a diffusion, so here the immunofixation test first, relies on separation of proteins whether in serum, urine, or CSF, by electrophoresis on gel, then paper strips embedded with specific antibodies are brought into the gel, and here the antigen transfers to paper to bind to the antibody, the gel contains the antigen, blotting will allow the migration of the antigen from the gel to the paper strip, if that antigen which migrates finds its antibody it will form a band that can be seen and this will demonstrate the presence of certain antigens, and so strips that are stained can reveal protein bands. Here, we can apply one antibody at a time per strip, we cannot apply all antibodies, and if the antibody finds its antigen it will form a precipitate, if not no line of precipitation will form. And by this we can detect abnormal proteins. So electrophoresis separates a mixture of proteins on gel, and then we have the antibody present on a paper strip, we apply the antibody on the paper strip, the antigen will migrate from the gel to the paper, and if it finds the antibody it will react, otherwise no reaction can take place, so in the figure in slides, the band indicates that there was an antigen in the gel separated which reacted with the antibody present on the strip, this antibody could be anti-igg, anti-lambda..etc., so each strip has one antibody, and if we have a positive reaction, this can be used to evaluate the situation, so if we are looking for example, for multiple myeloma, we should detect one immunoglobulin, usually sharp very condensed band, and tha could be igg for example, with one of the light chains, because it s a single molecule of an antibody, it will be either kappa or lambda, so we can make a diagnosis, as the antibody will react with an anti-fc to detect the class, and the antibody against the light chain, we use 2 antibodies

3 against anti-kappa and anti-lambda that will react with the light chain that is being abnormally produced by multiple myeloma cells So in immunoelectrophoresis, multiple antibodies react with multiple antigens, here a single antibody reacts with a single antigen. Even if we have here multiple antigens the reaction will be with the antibody that suits in the strip because there is only one antibody that s directed against an antigen to make a band and a diagnosis can be made, this is a very specific test that is commonly used in the diagnosis of abnormal immunoglobulin production. It is faster and easier to interpret than immune electrophoresis which is crowded with the precipitation lines produced against the mixture of immunoglobulins. Western blotting Is similar to immunofixation, but instead of assay for the antigen, here we look for the antibody.in the first case, the antibody acted as the antigen because we separate serum proteins and then we use anti-antibodies, but here in western blotting we look for antibodies, so we separate the antigens, this is most commonly used to confirm diagnosis of HIV infection. So if screening test for HIV is positive (screening test uses ELISA), this is a very sensitive test that requires confirmation before treating the patient, we confirm it by western blotting. In the lab, there are HIV proteins available, we can separate each protein by electrophoresis, and then we apply the paper strips embedded with serum from patient on the different strips, and allow for blotting, then we stain and look for bands that may form. So viral proteins separated on different strips, We incubate these strips each at a time with antibody to each one of these antigens, so we react with anti-a, then anti-b etc. so A or B or other proteins could be the antigen we want, anti-p55, anti-p41..etc. (proteins of HIV). If the antibody is present it will react with their antigen that has been separated by electrophoresis, and a positive reaction can be demonstrated by staining of these proteins.

4 Gp-60 is the envelope precursor which splits into gdp-120 and gp-41 And the p66 is the reverse transcriptase P55 is the gag protein So here we run electrophoresis of the viral proteins that are available in the lab, then we add patient s serum to paper strips, blotting is applied, then we look for positive reactions, and individuals who have antibodies to these viral proteins will make a reaction. the reactions are in the form of a positive balance that can be stained with special stains. In the figure, this is a positive control, meaning we have antibodies to all viral components so these bands are all positive, and the next is a negative control. For a confirmed HIV infection we need at least 2-3 antibodies, and that depends on the criteria used. the cdc criteria is that we should demonstrate 2 antibodies to the virus, an envelope antibody and a capsid or an enzyme antibody. The WHF criteria (couldn t get the name from the record) requires the demonstration of 3 bands, meaning that the patient should have antibodies to 3 viral antigens in order for confirmed diagnosis. So 2-3 bands,depending on the criteria used, should be demonstrated in the patients serum in order for a confirmed diagnosis of HIV. A single antibody does not confirm the diagnosis. So this is similar to immunofixation, but it is the opposite in terms of looking for the antibody (western blotting) rather than looking for the antigen( immunofixation). But the technique is the same. Flocculation Flocculation is a test similar to precipitation or agglutination of insoluble particles, and is characterized by very sharp pro- and postzones, so it has a very narrow zone of equivalence, where precipitation forms only in the zone of equivalence and no precipitates are formed in antigen excess or antibody excess.

5 As we see in the diagram, the zone of equivalence is narrow as compared to precipitation or agglutination where the curve is flattened. 1-VDRL: is named after the lab in which the test was developed. It is used to detect a mixture of antibodies against treponema pallidum, its called the reagent, which is a mixture of igm and igg that are produced against cardiolipin. In syphilis, non-specific as well as specific antibodies are produced. Non-specific antibodies are produced against the lipid component of the organism, and these lipid components cross react with beef cardiolipin (extracted from heart of beef). So serum of individuals with troponema pallidum who have syphilis, will produce a reaction with beef cardiolipin, and then those antibodies that react are a mixture of igg and igm ( being produced early in the course of the disease), then individuals will produce specific antibodies, anti-treponema pallidum antibodies, that can be detected by immunofluorescence or haemagglutination or other reactions. 2-RPR: uses charcoal as a carrier for cardiolipin. so either test (VDRL or RPR is used to diagnose troponema pallidum early in the coarse). Agglutination Is the 2 nd type of the 2ry antigen-antibody reactions, and it takes place when a soluble antibody reacts with an insoluble antigen ( ex.bacteria, RBC (in this case, we call it haemagglutination), or inert particles carrying soluble antigens like charcoal or latex). In agglutination, the medium should have certain characteristics, because proteins are usually negatively charged, this causes repulsion between particles. To overcome the forces, low ionic strength solution is recommended (or albumin, or polyethylene glycol) to make the reaction possible, because of the zeta potential that results from the similarity in charge between particles. Although its an antigen-antibody reaction that is specific, but the charges that are present on these particles tend to create a repelling force rather than attracting forces which can impede penetration. So agglutination is optimal when we use low ionic strength solution to decrease zeta potential.

6 Direct agglutination is a reaction that results when we directly mix an antigen with its antibody so if we have for example streptococci and mix it with anti-streptococcal antibodies they will perform agglutination. If we speak of haemagglutination, if we have an individual with blood group A, and we add anti-a, this will result in direct haemagglutination ( the RBC is the antigen). Indirect or passive agglutination, here the insoluble particle acts as a carrier, it has nothing to do with the specificity of the reaction, so its latex particle that is used to carry soluble antigens (attached to each other by a cross linker), and you add antibodies to the antigen in this case agglutination will take place. If we use RBC as carriers, we can attach an antigen to RBC, the reaction will be passive haemagglutination. Agglutination inhibition, we can utilize 2 forms of the same antigen, soluble and insoluble forms, and we react the antigen to the soluble, so the soluble will not be able to react, or vise verca. Coombs test is a test that s used to make diagnosis of either blood groups or detection of antibodies to RBC s and this is commonly utilized in blood banks. So in the figure, there is a bacterial cell, for example, and there are antibodies (igm) that can bridge bacteria forming large complexes of bacterial cells in the form of direct agglutination. Another figure, is agglutination being carried out against bacterial suspension, there is a negative test and a positive test. O: somatic antigen, H:flagella antigen, K:capsular antigen, these are the antigenic determinants on the bacterial cell, which are used as targets and agglutination can be demonstrated in this case. On the 3 rd figure, reaction takes place where we have antigens exposed on the carrier in the case of latex agglutination, we can attach the antibody on the latex and assay for the antigen, and in this case it s a reversed passive agglutination.

7 Tube agglutination It can be performed as a titer can be measured in many bacterial infections like typhoid fever, we look for antibodies against salmonella typhi in gruberwidal test. In these tests, individuals develop antibodies in response to infection, and these antibodies can be demonstrated by this test in vitro. The antigen is available commercially, and we demonstrate a titer for these bacterial antigens. The absolute number is not important because antibodies to certain bacterial infections may be present in different populations, for example a titer of 80 against widal may be normal in our part of the world because of frequent exposure to salmonella, its not in swedan for example. The most important thing in infectious disease is to demonstrate a 4-fold rise in titer, so we notice the individual at acute phase of infection, so for example if the individual had a titer of 20 and titer raises to 80 after 2 weeks, that s diagnostic, so its not the absolute number that matters (in this example 20), it is the rise in titer that determines a positive test. In the figure in slides, the 1/100, and 1/200 are positive, and the 1/400 is negative. in this case, the titer is 200 and it is The total of highest dilution that gives a positive reaction. Coombs test utilizes anti-humanglobulin antibodies, and in figure in slides, if we want to make diagnosis of the Rh group of an individual (whether they are Rh ve or Rh +ve) we mix RBC with anti-rh antibodies (which are of the igg class), and igg is too short to make clumps, so the reaction may not be evident, so this individual might be misdiagnosed as Rh negative, that s why here we use anti-human-immunoglobulin (anti-igg) it will cause bridging of the different antibodies attached to RBC s creating a large clump (lattice) indicating a positive reaction. This is called direct coombs. So if we assay for an anti-rbc-antigen (ex. A, B, O, Rh..etc), if we have an antibody against that antigen, the antibody will not be able to make clumps (because its igg) and addition of anti-human-immunoglobulin will make it visible. So in this case, when we type for Rh, we should always use

8 coombs test If the first step of adition of antibody to antigen is negative (because it is not conclusive), and if after that the test is negative, then the individual is truly negative. This is direct coombs. Direct coombs detects in vivo, sensitization against RBC, in cases of Rh antibodies produced in the mother, these antibodies will cross placenta and bind to fetal erythrocytes, so fetal erythrocytes will be sensitized, so if we want to perform direct coomps we just take fetal erythrocytes and add antigen to antibody, this will give this reaction if antibodies are present, if its positive, that indicates sensitization of fetal erythrocytes by the mother s, but if it is negative, that indicates that no antibodies are attached to fetal erythrocytes. Indirect coombs detects sesitizatin in vitro, it is used to detect antibodies in mother s serum, so we take erythrocytes from fetus, and mix them with serum from mother, if antibodies are present in mothers serum, they will bind to antibodies of the fetus, and because igg are short, they will not make agglutination, so we add anti-humanglobulin (which is called coombs reagent) so it becomes positive. So if we use coombs reagent to detect : 1- antibodies on erythrocytes direct coombs 2-antibodies in serum of the mother indirect coombs So direct and indirect coombs are used to detect hemolytic disease of newborn. Agglutination inhibition During pregnancy, the human chorionic gonadotropin hormone(hcg) is produced, it is a small soluble hormone that is secreted in urine. If we want to diagnose pregnancy by using antibodies to the hormone, primary complexes will be formed and it will not be visible (because the hormone is small) so in order to make it visible, we use another form of the hormone, which is the insoluble form, which is latex attached to the hormone (hapten-carrier conjugate).

9 So during pregnancy, if we take urine, and add to it anti-hcg, if HCG is present, antibodies will bind to it and all antibody sites will be saturated, if we add after that the insoluble form to the mixture, so insoluble hormone will not react with the antibody because the antibody is already occupied so this is a negative reaction, but it is a positive test. But if the hormone is not present and we add the insoluble form, the antibody will be free to bind to latex and make agglutination, the reaction will be positive but it is a negative test. So that s why it is called agglutination inhibition. Hemagglutination inhibition test Certain viruses have hemagglutinin on their surface like influenza and rubella, during infection with a virus, anti-hemagglutinin antibodies are produced, so if we take serum of an individual who was infected with rubella or influenza and mix it with hemagglutinin, the antibody in the serum will saturate binding sites on the hemagglutinin, and when we add erythrocytes the hemagglutinin will not agglutinate erythrocytes added, because it was neutralized by the antibodies, so this is a positive test, but a negative reaction because hemaglutination was inhibited. But if patient wasn t infected, and we add erythrocytes to hemagglutinin, no antibodies are present, an hemagglutinin is free to react with erythrocytes, this is a positive reaction, but a negative test. In this case we detected antibodies. We can also use it to detect antigens, and we use 2 forms of the antigen, anti-hepatitisb surface antigen and the other is anti-hepatitis B surface antigen on erythrocytes. So when we add serum that has the antigen, and react it with the antibody, the antibody will bind to antigen, so when we add coated erythrocytes (coated with the antigen) no reaction will take place, because the antibody is already bound to antigen from serum. However, if serum does not contain HBsAg and we add anti-hbsag, then we add erythrocytes coated with antigen, agglutination will take place, here agglutination wasn t inhibited. Use of labels in Ag-Ab reactions when we have agglutination or precipitation the reaction is easy to become in a form that is visible, but in cases of hormones, drugs, and other small

10 molecular substances, the reaction cannot be visible, and the only way to make it visible is by use of labels. Use of radioactivity can be utilized to detect antigens or antibodies and to quantitate them. These tests are very sensitive, they can measure nanograms of a substance. And usually, we should use a solid plate to which the antigen is attached, and then we add antibody, antibody will bind to antigen, then we can use a second antibody that is labeled with a radioactive substance (radioisotope), and that antibody will bind to the second antibody via the Fc fragment, ( because the first antibody is bound to the antigen with its Fab, so we produce antibodies that can bind to its Fc, for example when we talk about an anti-igg we mean that its an anti-fc, an anti isotype not an anti-idiotype) and this antibody can be quantitative, we can construct a standard curve, and from that curve we can determine the amount of antibody. To detect bound antibody, we have to use an anti-antibody (which is labeled) that can be counted (and we can use beta or gamma counters). Instead of using a second antibody that is radiolabeled, we can use a second antibody that is labeled with an enzyme, then we add a substrate, that will be acted upon by the enzyme producing a color change. So radioimmunoassay, for example in measurement of a hormone or any other substance, we use 2 forms of the antigen, labeled, and unlabeled. And in competitive assay, both compete for binding to an antibody that is fixed to the plastic plate. So if we re looking for HBsAg, we attach antibody for this antigen onto plastic, and we use 2 forms of the antigen, one labeled, and other unlabeled, and we run different concentrations of the labeled substance, and fixed amounts of the unlabeled, then we run the reaction and construct a standard curve, that can be utilized later to measure the concentration of HBsAg in unknown patient serum. ELISA on the other hand will result in a color change, the test can be quantitative by measuring the intensity of the color, and also from standard curves, we can determine the concentration of the substance that is being assayed.