SCCL MODULE 3:

Antigen Antibody Reactions

NAME VENUKUMAR SANGGAR TEY WEI SHAN CHONG WAN CHIEN CHEE XIN YING TAN JOON LI YAN YIE TING LIM JIE QI

ID NUM SCM 022525 SCM 024795 SCM 028376 SCM025309 SCM 023669 SCM 024014 SCM028115

1. Consider antigen A with 4 antigenic determinants (epitopes) and antigen B with 8 antigenic determinants. Which one will have a higher avidity to an IgM molecule? If the above two antigens are exposed to IgA, IgG and IgM, the affinity to which antibody will be highest? Why? Antigen B with 8 antigenic determinants will have a higher avidity to an IgM molecule. IgM will be highest, because it is pentamer, it has 10 paratope.

2. Lattice formation between antigen and antibody occurs in the zone of antigen excess. Is this statement true? Give your reasons. False. Zone of equivalence. It present in the middle of antibody.

3. In a lab assay to look for Rota virus in a child, stool sample was collected. A test tube with anti rota virus antibody solidified in agar was taken .It was layered on top with the serum. What test is being performed here? How long will it take to get the result? Is the antigen being tested of the soluble variety? The result of the test above was as follows:

1. Over a _________ period of time the result

b) appearance at the time of reading

Interpret the images. Why are there two bands in Fig .b? How do you interpret its significance? Double dimension ring precipitation test. It takes about 10 minutes to get the result of the test. There are two bands because there are two different antigens with two different molecular weight. The agar with antiserum has reacted with two different antigens. This means that the child has two different antigens in her body.

4. Explain the lattice hypothesis. Is it applicable to both precipitation and agglutination? How is precipitation and agglutination different from each other? Lattice hypothesis- Multivalent antigens combine with bivalent antibodies in varying proportions depending on the antigen and antibody ratio in the reacting mixture. Lattice hypothesis holds good for both precipitation and agglutination

Precipitation vs Agglutination Reactions : Treatment of infectious diseases is dependent upon their correct diagnosis. Antigen-antibody reactions are techniques using which antigens and antibodies are measured. Among these antigen-antibody reactions, serological reactions are in vitro reactions that are most popular methods for diagnosis of diseases and for identification of antigens and antibodies. Precipitation reactions and agglutination reactions are some of the common examples of these serological reactions. There are differences in these tests that will be explained in this article. Mixing of antibodies with their matching antigens on a surface such as animal cell, erythrocytes, or bacteria results in antibodies cross linking the particles forming visible clumps. This reaction is termed as agglutination. This serological reaction is very similar to precipitation reaction though both are highly specific depending upon specific antibody and antigen pair. The main difference between these two serological reactions pertains to the size of antigens. In the case of precipitation, antigens are soluble molecules while in the case of agglutination; antigens are large, insoluble molecules. Another difference between precipitation and agglutination is that agglutination reaction is more sensitive than precipitation reaction because a lot of soluble antigens and antibody molecules are required to form a visible precipitation reaction. However, it is possible to make a precipitation reaction sensitive by converting it into agglutination reaction. This can be achieved by attaching soluble antigens to large, inert carriers such as erythrocytes or latex beads. In clinical medicine, agglutination reactions have many applications. They can be used to type blood cells for transfusion, for identification of bacterial cultures and to detect the presence of a specific antibody in the serum of the patient. Agglutination is primary used to check if a patient has a bacterial infection or not.

Agglutination reaction vs Precipitation reaction Summary Agglutination reaction and precipitation reaction have great importance in immunology as they are serological reactions that help in the detection of bacterial infection in the serum of a patient. Major difference between precipitation and agglutination is the size of antigens involved. Antigens are soluble in case of precipitation while they are insoluble in agglutination Agglutination is more sensitive than precipitation. 5. Shiny, a pregnant lady underwent an ultrasound examination. Anencephaly was suspected and amniocentesis was done. The following test was done to look for a marker. The result was obtained in an hours time. Name the test; its principle &

procedure. What relevant marker was tested for?

Principle and Procedure: Principle : In counter current-line electrophoresis (CCL-electrophoresis) antigen and antibody migrate towards each other and form a line precipitate. The precipitation pattern is greatly influenced by the relative ration between antigen and antibody ranging from balanced precipitation with a thin, sharp precipitate to a gradual diffuse broadening and complete absence of the precipitate in antigen or antibody excess. This dependence on balanced precipitation makes CCL-electrophoresis suited for analysis of single precipitates in antigen antibody systems of high complexity since major part of unwanted precipitates can be eliminated by selection of the optimal antigen-antibody ratio.

Procedure : A trough of 0.5 cm width and a number of squares (0.5 x 1.0 cm) are cut out in a 1% (w/v) agarose gel of 1.5mm thickness. The length of the upper trough correspond to the total length of the squares. The distance between the upper and the lower trough can be varied. An antibody-containing gel is applied in the upper trough. Thereafter, the gel is removed from every second square and the 75 microlitre antigen-containing gel is added to each square. When this gel has solidified, the remaining gel squares are removed and the antigencontaining gel is added also to these. Finally, antigen and antibody are allowed to migrate towards each other by electrophoresis for 18 hours at 2.5V/cm. Relevant marker was tested for quantification of antibodies. The test is primarily qualitative, although from the thickness of the band you can get some measure of quantity.

6. Immunodiffusion tests are done in single dimension and double dimension. Explain the differences between the two. A single diffusion test is a precipitin tests in which antigen solutions placed into a well and antibody incorporated into agar. Precipitation rings are formed in the well depending on the antigen concentration as the antigen diffuses with the antibody with time. Double diffusion in two dimensions incorporates antigen and antibody solutions placed in separate wells in a sheet of plain agar, permitting radial diffusion of both reactants; this method is widely used to determine antigenic relationships; the bands

of precipitate that form where the reactants meet in optimal concentration are of three patterns, referred to as reaction of identity, reaction of partial identity and reaction of nonidentity. 7. The test given below was performed after Kim who was transfused blood developed allergic reaction. Upon development of reaction, the transfusion was stopped. What test is depicted here? Explain its principle.

Since he still required blood to be transfused, care was taken to perform the test depicted below. It was done using Kims serum and the donors RBCs. How is this test different from the above test? Name this test and its underlying principle.

The test is the direct Coombs test. The direct Coombs' test is used to detect IgG or C3 antibodies that are stuck to the surface of red blood cells. The antiserum against human immunoglobulin is used to agglutinate the red blood cells. The other test is different because the test is an indirect Coombs test. The indirect Coombs' test looks for free-flowing antibodies against certain red blood cells. In clinical practice this is referred to as the "antibody screen" and is part of the type and screen procedure. It is performed by mixing a patients serum in to normal red blood cells and antiserum to human immunoglobulin is added. Agglutination occurs if antibodies are present in patients serum. Approximately 5% of patients have a positive result due to IgG antibodies, IgM antibodies, or both. Most clinically significant alloantibodies are IgG antibodies that react best at 37C and are formed as a result of previous exposure via transfusion or pregnancy.

8. Herpes virus infection was confirmed in Joshua using a serological test where a cell line was used. Virus was cultured in the cell line and Joshuas serum was added. The virus stopped multiplying. Name the test and its underlying principle.

Virus Neutralization test.

Neutralization of a virus is defined as the loss of infectivity through reaction of the virus with specific antibody. Virus and products containing a neutralizing antibody are mixed under appropriate conditions and then inoculated into cell culture. The virus stop multiplying because the antibodies will react to the virus and neutralize the reaction while the cell remain unchanged. If it does not contain antibodies, cell will bind to toxin and cause cell damage.

9. Given below is an image. Identify the test based on the principle shown.

Immunofluorescence Primary (direct) Primary, or direct, immunofluorescence uses a single antibody that is chemically linked to a fluorophore. The antibody recognizes the target molecule and binds to it. This technique has several advantages over the secondary (or indirect) because of the direct conjugation of the antibody to the fluorophore. This reduces the number of steps in the staining procedure making the process faster and can reduce background signal by avoiding some issues with antibody cross-reactivity or non-specificity. However, since the number of fluorescent molecules that can be bound to the primary antibody is limited, direct immunofluorescence is less sensitive than indirect immunofluorescence. Secondary (indirect) Secondary, or indirect, immunofluorescence uses two antibodies; the unlabeled first (primary) antibody specifically binds the target molecule, and the secondary antibody, which carries the fluorophore, recognises the primary antibody and binds to it. Multiple secondary antibodies can bind a single primary antibody. This provides signal amplification by increasing the number of fluorophore molecules per antigen. This is more complex and time consuming than the primary (or direct) protocol above, but it allows more flexibility because a variety of different secondary antibodies and detection techniques can be used for a given primary antibody. Activity 10

In testing for Brucella infection, the lab reported the result as negative due to prozone phenomenon. What is meant by prozone phenomenon? How do you overcome it?

Prozone phenomenon, prozone effect or the Hook effect is a common false negative result that happens in immunoassays (biochemical tests that involves antibody), such as serological tests for Brucellosis (Brucella infection). This is due to the very high concentrations of particular antibodies, or the antibodies are said to be in excess. Hence, prozone is the zone of antibody excess. In this zone, there is too much antibodies for efficient lattice formation, lead to lesser amount of reactions or proper precipitations to take place. Consequently, prozone phenomenon often leads to the occurrence of false negative results. In order to overcome this situation, serial dilutions of the serum can be done. The concentration of antibodies will be decreased following serial dilutions, therefore proper precipitation reactions able to occur effectively with more appropriate ratio of antibodies to antigens.

Activity 11 This ELISA test given below is done for four patients (each row represents one patient ROW1, ROW2, ROW3,ROW4). It was done in serial dilutions. Discuss its test principle. Which patient has a negative result among ROW1, ROW2, ROW3 and ROW4? Which patient has the highest load of infection? Give reasons for your answer.

Enzyme-linked immunosorbent assay (ELISA) is an immunoassay that involves the reactions of antigen and antibody in vitro (isolated from usual biological surrounding for more detailed and convenient analysis), especially for HIV screening. It is used for the quantitation of either antigens or antibodies in patients specimens, which usually performed in microwell plates.

The underlying principles and mechanisms of ELISA are: 1. An enzyme (such as Horse Radish Peroxidase) is covalently linked to a known antibody (mainly antibody to humanIgG). 2. The complex is allowed to incubate and react with dilutions of the patients serum that contain antibodies (IgG) and the specimen that contain antigens on the microwells. 3. After that, a substrate (such as tetramethylbenzidine) that specifically cleaved by the enzymes will be added to the mixture to react with the enzymes. 4. Then, colored products will be formed as reaction progresses, which will then be measured by a spectrophotometer. 5. The amount of antibodies bound to the antigens is proportional to the enzyme activity, which can be judged via observing the intensity of the colored products. For the situation as above, result shown in ROW4 represents a negative result. This is because none of the dilutions shows a colored result, except the controlled set. On the other hand, patient that produces result shown in ROW3 has the highest load of infection. This is due to the fact that consecutive positive color reactions have appeared following several serial dilutions of the patients serum. The result indicates relatively high amount of antibodies are present in his serum, even in serums with further dilutions, which concordant with the greater load of infections in the body.

12)Muah, a 21-year old girl did a urine test to rule out pregnancy using a kit purchased at the local pharmacy. She followed the instructions and observed 2 lines ion the kit after adding 2 drops of urine. What is the principle of the test? Which substance in urine is she testing for? What is incorporated in the visible strip to produce the two lines upon adding the urine? Urine HCG (Urine Pregnancy Test) is performed by collecting a sample of urine, which is usually applied to a test kit using a disposable plastic dropper. A colour change will indicate a positive pregnancy test, and there is almost always a control area or symbol which changes colour as a double-check that the test is actually working.. Substance tested: human chorionic gonadotropin hormone Substance incorporated: mouse monoclonal antibody(1st line), polyclonal antibody and dye(2nd line), anti mouse antibody and dye(3rd line)

13. Mr. M was diagnosed with HIV infection 5 years ago. He is clinically healthy; on HAART therapy and regular follow up. Every year his blood is tested to check if his CD4 T cell count is within the required limits. Why is the CD4 cell count important? What test principle is depicted it? Explain its principle. The importance:

CD4 cells or T-cells are the generals of the human immune system. These are the cells that send signals to activate your bodys immune response when they detect intruders, like viruses or bacteria. Because of the important role these cells play in how your body fights off infections, its important to keep their numbers up in the normal ranges. This helps to prevent HIV-related complications and opportunistic infections. Principle: The use of potent combination therapy to suppress HIV replication below the limits of detection (optimally < 50 copies/cc) decreases or may prevent the production of mutant, drug resistant strains in the large proportion of patients treated. A. The goal of therapy is to suppress HIV to undetectable levels, ideally < 50 copies/cc.

B. If undetectable viral load can not be achieved, the goal of therapy should be to minimize the HIV viral load. Clinical and immunologic improvement is uniformly seen at or below a HIV RT-PCR viral load of 5000 copies/cc and may also be seen at somewhat higher viral load levels. C. HIV suppression below detectable ranges does not equate with eradication. Cessation of therapy almost always results in rebound viremia followed by loss of CD4 lymphocytes.

14) Sara is suspected to have Salmonella typhi infection. A Widal tube agglutination test was performed which required multiple serial dilutions of her serum. What disease is she suffering from? Explain the principle of the test. An infection caused by bacteria from the Salmonella genus which causes typhoid fever, with symptoms such as gastroenteritis. Infection is caused by consuming contaminated food or drinks. Donor sera were screened by slide agglutination with Salmonella enterica serotype Typhi O and H antigens (Difco). The positive donor sera and all patients' sera were serially diluted in tubes with 08.5% NaCl from 1/50 to 1/6,400, and antigens (H and O) were added. The tubes were incubated at 37C for 2 h and then at room temperature overnight and examined for agglutination by an agglutinoscope Principle: Patients suffering from enteric fever would possess antibodies in their sera which can react and agglutinate serial doubling dilutions of killed, coloured Salmonella antigens in a tube agglutination test.

Question 15: In a case of cholera, a Vibrio cholera inhibition test is used to identify the strain of cholera bacteria. Explain the principle of the test.

Vibrio Cholera is taken and observed under microscope. This type of bacteria is highly motile. Human serum which contain antibody is injected onto the slide of Vibrio Cholera. Then, antigen which found in Vibrio Cholera bind with human antibodies. Thus, agglutination occur and immobilization of Vibrio Cholera. This test is known as immobilization. Question 16: ELISA can be performed by direct or indirect methods. List the difference between the two. Direct ELISA Indirect ELISA -faster -slower because involve one more steps -hard and specific as we need to find the -less specific exact antibodies that bind with that antigen -expensive -cheaper -test for antigen(hormone, drugs and -test for antibodies(serum) viruses) -antigen attached to the bottom of the -antigen attached to the bottom of the tank and enzyme-linked antibodies bind tank. Then, antibodies (IgG) bind to directly to antigen antigen. After that, enzyme linked anti-Ig G bind to IgG

For direct ELISA, antigen is attached to the bottom and enzyme-linked antibody will directly bound to the antigen. For indirect ELISA, antigen is attached to the bottom and antibody of the patient(IgG) is attached to antigen. Then, another enzyme-linked antibody to human IgG attach to the patient IgG. For direct ELISA, the complex forming consisting immobilized antigen but for indirect ELISA, the complex containing immobilized antibody. Direct ELISA is more simple while indirect ELISA is more complicated and involve more procedure. Direct ELISA is used to test antigens but indirect ELISA is used to test antibodies.