Immunohistochemistry Detection Methods PDF

detection methods in immunohistochemistry Lect3 Assistant Professor Dr. Enas Fadhil Kadhim 20-21  ▪ Direct detection methods are known as a one-step process applying a primary antibody, which is directly labeled with reporter molecules, s...

detection methods in immunohistochemistry Lect3 Assistant Professor Dr. Enas Fadhil Kadhim 20-21  ▪ Direct detection methods are known as a one-step process applying a primary antibody, which is directly labeled with reporter molecules, such as biotin, colloidal gold, fluorochromes, or enzymes. The conjugated antibody makes a direct contact with cognate antigen in histological or cytological Direct method preparations. Direct detection methods are widely used for detecting highly expressed antigens. Furthermore, when the use of the secondary antibodies causes nonspecific and unwanted reactions, owing to the histological nature of the tissue and/or host spe- cies of the primary antibody, direct detection could be the technique of choice. ▪ One of the advantages of direct detection is that the incubation step with a secondary reagent is eliminated. Hence, this method is time saving and easy to perform ▪ ▪ In addition, due to the wide range of fluorochromes that are commercially available, direct detection is vastly used in multicolor experimental designs. ▪ It is important to note that insufficient sensitivity to detect most of the antigens found in routinely processed tissues is one of the drawbacks of using direct detection method. Furthermore, each primary antibody needs to be individually conjugated with fluorophores or enzymes, which increases considerably the cost of the whole process. Another concern with direct staining methods is the possibility of functional impairment of the antibody affinity if the process of antibody labeling is nonoptimal. This is especially case for monoclonal antibodies in which all antibody molecules in a given preparation have almost the same affinity and so are most likely to be affected all together by improper labeling. This issue is less problematic for polyclonal antibodies in which antibodies with diverse physicochemical proper- ties are produced against an antigen Direct detection methods are the method of choice in such high-sensitive protein detection systems as flow cytometry. Although this system is the simplest and the most convenient method for detection of a given marker expression, it is not routinely employed in clinical and research applications due to the limitations mentioned above.  ▪ This system employs an unlabeled primary antibody as the first layer and the secondary antibody, which is Indirect method raised against the primary antibody and is labeled with different fluorophores or enzymes ▪ In indirect methods, primary antibodies retain full avidity because they remain unlabeled. Indeed, higher number of labels per molecule of primary antibody is achieved in indirect compared to direct detection methods. The later stems from the fact that at least two labeled secondary antibodies can bind to each primary antibody molecule. These factors result in increased reaction intensity and the higher sensitivity in indirect staining methods. Accordingly, indirect methods are able to detect fewer number of antigens with less primary antibody. Moreover, indirect methods are more practical than direct methods since the same secondary antibody can be applied for detection of different sets of primary antibodies if they have been raised in the same species. Another benefit of indirect method in IF stainings is possibility to select secondary antibodies with fluorophores of different colors. ▪ if the tissue shows strong endogenous red autofluorescence, the secondary antibody labeled with green ▪ fluorophore could be a right choice. Previously mentioned advantages of indi- rect detection systems eventually led to its widespread applications in research and clinical settings. ▪ Despite advantages mentioned above, indirect immunostaining methods suffer from some shortcomings. First, additional controls and blocking steps are inevitable when using secondary antibodies. Indeed, there is possibility of nonspecific staining that happens when the secondary antibody interacts with unwanted tissue targets. If nonspecific staining is noticed, blocking reagents have to be used to treat the tissue sections that could be time-consuming and cause additional costs to IHC experiment ▪ This results in competitive blocking of the nonspecific binding sites for secondary antibody in the target tissue by the unlabeled antibodies from the same species. The addition of further layers beyond their use in the two-step indirect method for increasing the sensitivity of detection can be problematic as addition of every new species of antibody considerably increases the risk of nonspecific interactions and background staining  ▪ Early conjugation protocols were not efficient and did not label all antibodies leaving a fraction of antibodies unlabeled. These unlabeled antibodies were able to compete with labeled antibodies for binding to the cognate antigen and reduced the efficiency of detection. To overcome this problem, new approaches were invented that eliminated the need for chemical Bridge methods conjugation of antibodies. In these approaches, antigen specificity of antibodies is employed to couple antibodies to the enzymes. Taking advantage of antigen specificity of antibodies, antiperoxidase or antialkaline phosphatase antibodies are easily coupled with peroxidase or alkaline phosphatase after incubation with these enzymes without need for any chemi- cal modifications of the antibody. ▪ These preformed soluble enzyme-antienzyme immune complexes are then used as the third layer reporter antibody for detection of the antigen-bound primary antibody in tissue section. Taking advantage of the bivalent properties of IgG binding, a second-step antibody with binding specificity to primary antibody and tertiary antienzyme antibody complexed with the enzyme bridges two layers. The bridge antibody is usually used in excess, so that one of its two identical binding sites interacts with enzyme-coupled tertiary anti- body, while the other site interacts with primary antibody.  Biotin- ▪ Labeled avidin/streptavidin-biotin (LAB/LSAB) avidin/streptavidin -based methods ▪ Labeled avidin/streptavidin-biotin (LAB/LSAB) are among very sensitive IHC ▪ detection methods, which take advantage of high- affinity binding of avidin/strep- tavidin to a water- soluble vitamin, ▪ The principle of labeled avidin-biotin (LAB) technique is based on sequential interaction of biotin-labeled antibody with tissue antigen and enzyme-labeled avidin with biotinylated primary antibody. In the bridged avidin-biotin (BRAB) technique, however, avidin bridges biotin-labeled primary antibody and biotin-labeled enzyme. BRAB is particularly suitable in cases where intracellular penetration and/or sensitivity of the staining reaction are the major concerns. An indirect approach of BRAB technique (IBRAB) can also be applied for identification of antigens in formalin- fixed paraffin-embedded tissues in which avidin and biotin-labeled peroxidase are added sequentially to the system after primary antibody and biotin-labeled secondary antibody. ▪ LAB/LSAB methods offer several advantages for IHC applications. Biological activities of macromolecules (e.g., enzymatic catalysis or antibody binding) are not affected when they are conjugated with biotin.  ▪ The sensitivity of an IHC staining is a function of detection method for signal amplification. ▪ The choice of a detection system is mainly determined by laboratories based on the nature of the specimen, expression level of the antigen, cost, desired sensitivity, Choice of and possible automation. Choosing an appropriate detection system detection system enables maximum sensitivity and optimum visibility of the immune reaction with the and concluding fewest steps and in the shortest time. As a general rule, remarks the more complex an IHC method, the more sensitive it is. One- or two-step IHC procedures are usually less sensitive than more complex, multistep procedures. In addition, the detection system must be accurate, reproducible, and results in a high signal-to-noise ratio ▪ When choosing a desirable detection system, several factors are needed to be taken into consideration: ▪ (1) the expertise/experience of the technician; ▪ 2) type of the antigen to be identified; for example, some antigens are widely expressed and do not need a sensitive method to be visualized; ▪ (3) number of tests and the amount of antibody that is available; ▪ (4) the affinity of the antibody: each antibody has its own affinity that requires a specific detection system, antibodies with less affinity usually need more sensitive detection systems; ▪ (5) species idiosyncrasies (does the tissue contain endogenous biotin), ▪ (6) budget; ▪ (7) localization of the antigen of interest (some detection systems do not have high cell penetration capacity due to the large size and regardless of having high sensitivity for detection of surface antigens, do not yield a high sensitivity for intracellular or nuclear antigens), (8) the need for or type of antigen retrieval; typically, a non-biotin- labeled detec- tion system is recommended if HIER is used to avoid background from endogenous avidin-biotin activity (EABA).