Development of Serological Techniques for Identifying Weak Antigens

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Table of Contents

Introduction
Old serological techniques
Precipitation
Immunodiffusion
Immunoelectrophoresis
Fluorescence antibodies technique
New serological techniques (column agglutination techniques)
Conclusion
References

Introduction

The developments of the serological test have taken some time to revolute from tiresome manual procedures to simpler techniques, which can allow automation during diagnosis and when reading results. This antigen antibodies detection is now a mandatory laboratory practical test usually performed to determine and identify various disease causative agents such as; viruses, bacteria’s, fungi’s and other parasites capable of penetrating the body and stimulating an immune response such as humoral or cell-mediated immunity which is very specific to the invaded antigen. (1) Samples required for the diagnosis include; body fluids such as blood, urine, and saliva which have the capability of harboring antigens. The detection and identification of the disease causative agents are very important when it comes to treatment administration. The physician may also do some preliminary examinations aided by visual symptoms on the patients before a confirmatory antigen detection diagnosis is done. (1, 2)

Old serological techniques

Conventional Tube method

This is an essential technique precisely used for blood cross-matching tests which should always be performed before any blood transfusion takes place. This technique is also referred to as the spin tube method, basically used for blood compatibility confirmatory test and which requires potentiation with the bovine albumin enzyme together with anti-human globulin (AHG). (1) The technique requires a drop of 5% suspension of antibody screening cell, added to 4 drops of the polyspecific AHG test serum and two drops of LISS-ADDS; all in their appropriate tubes. (1)

The tubes are then incubated at 37oC for 15 min then the cells are completely washed three times using normal saline and the supernatant drained out leaving only cellular residual at the bottom of the tube; “the anti-globulin test follows using the DiaClone polyspecific IgG (DiaMed) together with compliment AGH reagents”. (1) After centrifugation for 15 minutes at 3400r/ min, hemolysis or agglutination is observed macroscopically for positive tests, and the negative results are confirmed microscopically. (1)

Immunoprecipitation

This is another manual method of antibody detection; it is also scientifically referred to as IP (Immunoprecipitation). (2) During laboratory Immunoprecipitation tests, the patient blood is first drawn and exposed to a specific test antigen which is known (2) and if the patient body had produced antibodies against that test antigen, then the antibodies which are in the blood will bind to all the test antigens present forming antigen-antibody complexes. (2, 3) Notable to mention is that all the antibodies will remain unbound if the antibody specific to the test antigen is absent in the patient blood and this would give a negative result. (2) On the contrary, if the test is positive, the bounded antigen and antibody are thereafter isolated and removed from the sample for analysis using other more advanced lab methods used for specificity confirmation. (2) Presence of the antibody confirms that the patient is sick and should be subjected to immediate treatments; this technique helps diagnose diseases like hepatitis c virus infections. (2)

ELISA test

ELISA test is one of the immunological techniques applied in serology for manual antibody detection; ELISA (enzyme-linked immunosorbent assay) (2) is a technique frequently employed for the detection of antibodies in blood samples. The principle behind the test is that it relies basically on the fact that there will be an antigen-antibody reaction which will result in antigen-antibody complex formation. (2) During the ELISA test, a known strain of test antigen is bound to a membrane which is then treated with the blood sample collected from the patient. (2)

As a confirmatory macroscopic examination result, the presence of the antibody specific to the test antigen will automatically result in antigen-antibody binding, in that the antibody will bind onto the test membrane antigen. (2) Positive results will show that the patient’s body has already reacted by producing that specific antibody against the specific test antigen thus the patient will be referred to the doctor for immediate treatments (2)

Cell screening method

This is another manual method for antigen-antibody detection employed in immunology (serological technique); this method allows doctors or researchers to detect antigens antibodies present in cells under a light microscope. (3) The technique involves screening cells that contain specific antigens, where thereafter they will be incubated together with the patient’s serum sample. (2) If the patient serum contains the specific antibody for that antigen present, there would be antigen-antibody binding and this will further be analyzed using advanced tests to identify and confirm the presence of the antibody produced by the patient’s blood (serum). (2) This technique is commonly applied when screening pregnant mothers and patients who must undergo blood transfusion just to ensure that no antibody is presented to cause a late negative immunological response. (2, 3)

Agglutination reactions

This is another popular laboratory manual serological technique used to detect anti-quantitate agglutins, it is also relevant in detecting and identifying cellular antigens such as bacterial cells present in patient’s blood. (3) The principle behind the test is that when the bacterial cells are subjected invitro with the appropriate antibody, they clump together and eventually results to formation of visible masses. (3) Moreover, when antibody and bacterial cell agglutinates, opsonization must definitely occur. (3) This agglutination occurs simply because the antibody is bivalent; meaning it has at least two antigen binding sites also called epitopes. (4) Secondly, this agglutination occurs because the antigen also has multiple binding sites which results into a very strong antigen antibody lattice that forms a large complex called agglutination. (3, 4) This aggregate may be seen microscopically under a microscope or macroscopically on the test tube; one common application of this technique is during the Widal test for detecting typhoid. (3)

Figure 1: Lattice structure composed of antigen antibody. Source: (3).

Precipitation

This is a technique that focuses mainly on the soluble antigens principle; when a soluble antigen comes into contact with a specific antibody, they precipitate. (3,4) Notable to mention is that since the antigen is soluble instead of being cellular, a large number of antigen molecules will always be required for the lattice to be formed. (3) Moreover, again for a more visible precipitate to be formed, a large lattice must first be formed. Precisely, when the antigen concentration is lower than antibody (superabundant), the formation of smaller complexes will take place in this zone of the excess antibody also called prozone. (3) However, when the concentration of antigens is more than the antibody, larger aggregates are formed and this zone of high antigen concentration is called the zone of antigen excess. (3)

Immunodiffusion

This is a laboratory technique that is used in serology based on precipitation phenomena. (5) When an antigen-antibody reaction takes place on a semi-solid media, for example, agar gel medium, visible bands of precipitates will form at the point of antigen-antibody complex formation. (5) This technique is also referred to as the double diffusion method since there will be diffusion of two components towards each other contrary to single diffusion where only one component diffuses to only one direction. As part of this ouchterlony procedure, the antibodies and antigens are prepared by placing them onto separate wells dug on the semi-solid agar medium. (4,5) The reactants will diffuse through the semi-solid agarose gel medium toward each other forming a visible band of visible precipitate at the point they meet. (5)

Figure 2: Double immunodiffusion (Ag=antigen; ab= antibody). Source: (4,5).

One major advantage of this process is that, antigenic relationship can always be detected from the precipitation patterns formed. For antigens identification; two identical antigens will always diffuse at the same rate and their bands will emerge forming a solid chevron. (3) Moreover, reactions of non identity will occur when two different antigens are used, whereby the bands will cross. Lastly a spur is formed when two antigens of partial identity form, indicating cross reactive but non identical antigens (see fig 3). (3)

Figure 3: Patterns of immunodiffusion. Source: (4, 5).

Immunoelectrophoresis

For this test, the Immunoelectrophoresis technique was developed to resolve accurately cases of complex mixtures of antigens. (4,5) The technique involve placing the test antigen in a well dug on the semisolid agar gel on a glass slide and then subjecting it to electrophoresis via the application of an electric current. (4, 5) Under these subjective specific conditions, the individual antigenic components will automatically migrate through the agar at different variable rates. When the antibody is then placed in a well-running lengthwise to the slide and parallel to the antigenic migration path, then the reactants will diffuse towards one another forming separate arches of precipitation for each specific antigenic component. (4, 5)

Fluorescence antibodies technique

Fluorescent antibodies are used in laboratory diagnostic tests in which fluorescent dye is added to tissues that contain antigens; (7) and as a result, the targeted area will glow with ultraviolet light when viewed on a fluorescent microscope. (7) This technique has two ways of procedural approaches; the direct and indirect methods. Also, these fluorescent antibodies are known to be either monoclonal (made from the same parental cell) or polyclonal (derived from different cells). (7) direct fluorescent antibody tests are specifically performed to identify the presence of a specific microbe in an organism. It precisely involves “directing antibodies against antigens on the surface of a specific microorganism” (7) which have already been previously labeled using fluorescent dye. (7)

Incubation of the sample plus the fluorescent antibodies done at 37oC follows creating favoring conditions for antigen-specific binding to take place. (7) The excess and non-specifically bound antibodies are thereafter washed away from the sample then the final sample is viewed using a fluorescent viewer like a fluorescent microscope, plate reader, or even fluorescent-activated flow cytometer. (7)

For the indirect fluorescent antibody test, the antibody or microorganism itself is incubated together with the patient’s blood serum to demonstrate the presence of antibodies in the serum. (7) The excess serum is further washed away leaving only antibodies specific to the antigen which could either be an antigenic portion or fragment from the microorganism present in the serum. These samples are then incubated after labeling the antibodies with fluorescent dye specifically used on human antibodies called fluorescent antihuman antibodies. (7) This antibody is obtained by injecting human immunoglobulins into another species, which will trigger the production of anti human immunoglobulin antibodies. (7) The samples are further viewed under a fluorescent viewer either by using a fluorescent microscope, plate reader, or fluorescent-activated flow cytometer. (7)

New serological techniques (column agglutination techniques)

Ortho Biovue system technique

The Ortho Biovue test focuses on the sieving effects of the glass beads microparticles on density gradient diluents that are contained inside the microcolumns. (1) “Each specific cassette contains six mounted microcolumns and each microcolumn contains micro glass beads, diluents and also a specific reagent”; the micro glass beads will automatically trap any agglutinated blood cells after being subjected to centrifugation whereby all unagglutinated blood cells will be forced through the glass beads forming pellets at the bottom of the microcolumn. (1) Moreover, columns containing the active ortho Bioclone antihuman globulin (AHG) serum which acts as a macromolecule barrier will hold back serum during centrifugation allowing only RBC to pass through into contact with the anti-human serum in the column. (1)

Gel method for antibody detection

This was developed late1980 in Switzerland and is one of the newest standardized methods of obtaining visible agglutinations as a way of detecting red cell antigen antibodies reactions, specifically during blood transfusions. (8) Besides, it also offers a simple way of performing anti-globulin tests without having to wash them, consuming very small amounts of serum and cells, and also allows automated readings. (8) Unlike in the old convention tube methods, the gel agglutination method takes place only on a solid phase (gel), not a liquid phase contained in a special microtubule. (1, 8) The patient’s plasma (obtained from uncoagulated blood) is mixed to 1% suspension of red cells in a low ionic medium (LIM), dispensed into the microtubules, and incubated at an average temperature of 37oC for 15 minutes. (8) The card containing the microtubules is then subjected to centrifugation for about 10 minutes and then cells are separated from the serum and mixed with the ant-globulin serum contained inside the microtubule. (8) The cells will finally be trapped by the gel or pellets present that is if they have agglutinated and been deposited at the bottom of the tube. (8)

Red affinity column technology

Contrary to the gel agglutination technique which is based on the size exclusion of the agglutinated red blood cells in the inert matrix, the red affinity technology (ReACT) is primarily focused on the affinity adherence of the red blood cells in an immunologically active matrix. (8,9) The antibody sensitized red blood cells will automatically bind to the ligands attached to the agar matrix which is mainly composed of the protein G, known to have a higher affinity for all four immunoglobin subclasses, and the Protein A which binds to the IgG1, 2, and 4. (8)

Solid-phase adherence assay

The solid phase adherence assay (SPAA), is a technique employed in detecting and identifying unexpected antibodies based on the procedure set after research done earlier In the 1980s. (9) This method is rapid, more sensitive, and very specific; which just like the gel method, consumes very little amounts of serum and reagents, with also an added advantage of enabled automated reading. (8,9) The technique involves the use of red cell membranes bound onto the surface of polystyrene microtitration strip wells, which are meant to capture the IgG globulin antibodies (if present in the patient’s serum). (9)

The patient’s serum is then added to the wells coated with screen cells; incubation was done at 37oC for 15 min where the unbound IgG antibodies are washed away. (8, 9) The indicator (anti-IgG-coated red cells) is added, centrifuged and the results of the test recorded; in interpreting the results if the patient IgG has attached on to the cell membrane, the anti-IgG coated indicator red cells will form anti-IgG-IgG complexes. (9) The indicator cells will attach to the wells as the second immobilized cell layer forming a dispersed conflux or a lawn of cells indicating positive results. Mostly, any negative test will show the indicator cells at the bottom of the well forming a distinct but smaller cell button. (9)

Indirect anti-globulin test (IAT)

This is also one of the new techniques employed and well established in the serological blood group diagnosis which is easy to handle and has no washing procedures. (10) For this diagnosis, a plastic chip similar to an ID-Micro typing card in size is required; this plastic chip contains an intrinsic microcapillary system. (10) During antibody screening, 50µl of 0.8% screening cells and 25 µl of the patient’s plasma are both pipetted into the reaction chambers of the chip, which also contains coombs reagents. (10) Incubation at 37oC for 15 min is done followed by centrifugation for 10 min. (9,10) A positive result is displayed macroscopically as hemagglutinates retained within the capillary system or as buttons of red cells deposited in the negative chambers (for negative results). (10)

Saline Polybrene test (for blood cross-matching)

This is another newest method of performing blood cross-matching tests done to check blood compatibility before blood transfusion is done. (10, 11) The saline polybrene test detects both IgM and IgG immunoglobin antibodies; this procedure is very simple, reliable, and bears clinical safety. (11) The main objective of this new technique is to ensure blood safety before transfusion to prevent after transfusion allergic reactions from occurring to the patient. (11) This method is again efficient in detecting the presence of all incomplete antibodies found in the blood. Procedures for saline polybrene test also involve mixing one drop of the donor’s blood with two drops of 5% patient serum in vitro; after centrifugation, shake the content and observe. (11) Absence of agglutination means proceeding to step two while agglutination reveals blood incompatibility.

The advanced step again involves, adding 0.7ml of low ionic solution mixed and added to the polybrene solution. (11) Centrifugation at 3400 r/min for 15 sec follows after which the supernatant is drained out leaving only about 0.1ml residual. (11) Finally, 2 drops of the suspension are added and mixed gently, results for compatible blood shows spread in the agglutination within 30seconds. (11)

Conclusion

Throughout this paper, we have seen the scientific revolution of antigen-antibody detections techniques employed currently for more accurate diagnosis in hospitals and research centers. Several cases have been raised on deaths of patients due to inaccurate diagnosis and poor interpretation of results especially in blood transfusion using old techniques, but the currently advanced techniques are more accurate, easy to perform consuming little time, and easier for results to be interpreted has largely solved this problem. Some of these techniques include the column agglutination technologies which we have discussed. (12)

References

Abou, A., Jabal. T., Shubeilat, J. & Hajjiri, F. Evaluation of 2-Column Agglutination Versus conventional tube technique for antibody screening. Eastern Mediterranean Health Journal. 2003; 9(3): 407.

Tremblay, L. Antibody Detection Methods. 2010. Web.

Linjawi, M. Simple Serological Techniques. 2010. Web.

Turgeon, M. Immunology and Serology. Laboratory Medicine textbook 2^nd Edition. 2010; 6: 111-131.

Abbas, K. & Litchman H. Cellular and Molecular Immunology. Laboratory Medicine textbook 5^thEdition. 2010; 522-534.

Rao, V. Immunology. Alpha Science International. 2005; pp 112.

Miles, I. What Is Fluorescent Antibody Technique? 2010. Web.

Lapierre, Y., Rigal, D. & Adam, J. The Gel Test: a New Way to Detect Red Cell Antigen-Antibody Reactions. 2010. Web.

Plapp, V., Sinor, T. & Rachel, M. A solid phase antibody screening. Clinical Pathology. 1984; 82: 719.

Schwind, P., Fasel, A. & Monod P. New Format for Blood Group Serology Diagnostics. 2004. Web.

Eng.hi138.com. Polybrene test in the cross salt water with the clinical significance of blood. 2011. Web.

Lamy, B., Tissot, C., Heyd, C. & Lamy, C. Red Cell Antibody Screening; Red Cell Antibody Identification and Compatibility Testing with the Column Agglutination Technology. 1994. Web.

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