Agglutination Test Methods - PDF

# Agglutination Test * Reaction between Ag. Ab (Agglutinogen) and Agglutinin: **Agglutinate "result" = Clump** * **Conditions for Agglutination:** * **Insoluble Ag:** * **Cellular:** (RBCs + bacteria) * **Particulate** Ag: * Organic particles...

# Agglutination Test * Reaction between Ag. Ab (Agglutinogen) and Agglutinin: **Agglutinate "result" = Clump** * **Conditions for Agglutination:** * **Insoluble Ag:** * **Cellular:** (RBCs + bacteria) * **Particulate** Ag: * Organic particles * Inorganic: * Synthetic material: * Polyethylene * Latex * Charcoal * Bentonite * **Divalent or multivalent:** * **IgM:** Optimum Agglutinin for larger molecules as binds to many sites simultaneously. * **Types of Agglutination Tests:** * **Direct:** If Ag is insoluble * Blood grouping * Pullorum test * **Indirect:** Ag is soluble * **Carrier** molecule is needed for agglutination. * Examples: * Inert particle * Bacteria * RBCs * Heme * Latex * **Qualitative** * Slide or plate agglutination test: blood grouping * Milk ring test (tube test): * Pullorum test * Rose Bengal plate test (bovine brucellosis) * RBPT * **Quantitative** * **Tube** : * Bovine brucellosis * Widal test (human Salmonellosis) * Micro-titre plate (bovine brucellosis) * Microagglutination test for bovine brucellosis # Blood Grouping (ABO System) * **Slide agglutination test for determination of blood groups** * **RBC** antigens in human blood. * 4 types: (A), (B), (AB), (no-antigen, O) * **Slide agglutination:** Test uses purified anti-A and anti-B antibodies dropped on the surface of a blood sample. The reaction confirms the antigen present in the blood sample. * **Importance of Blood Grouping in Transfusion Rejection** * Not compatible blood transfusion → agglutiation shock → death * Not compatible antibodies → **anti-B** antibodies in the serum of a blood type (A)→ clumping of RBCs → death * Blood to be infused needs to be **free from any antigen**. * **Universal donor **: Blood group (O) because RBCs do not contain A or B antigen. * **Universal recipient:** Blood group (AB) because serum is free from anti-A and anti-B * **Forensic medicine**: Disputed paternity, or blood stains. * There are many cases where there are more than 10 antigens in chicken RBCs, causing issues with blood transfusion. # Uses of Slide Agglutination Test * **Pullorum test:** * **Antigen** : Stained Salmonella (Blue color) * **Serum sample** * **Result:** Clumps → Diagnosis of Fowl typhoid Pullorum Disease * **Rose Bengal test:** * **Antigen:** Stained brucella (Rose color) * **Serum** * **Result:** Clumps * **Intermittent Mediterranean Fever** * Diagnosis of human + bovine brucellosis * **Identification of mo from mixed culture:** * **Isolated unknown colony of mo** * **Known Ab** * **Result:** Unknown microorganisms * **MRT (Milk ring Test)** * **Anitgen:** Stained brucella (Blue color) * **Lipophylic Abs:** Blue color of cream layer * **Result:** Blue color of cream layer + blue color of underlaying (aquous) phase * **Milk Sample:** Mastitis Test * **Fat globules in cream layer** * **Antigen** * **Result:** * Blue in aqueous phase → Mastitis. * Homogenous bluish color of milk → Equivocal. * **Milk ring test is not used in:** * **Pasteurized milk** * **Skimmed milk** * **Collostrum** (First milk) * **Vaccination against brucella** # **Drying Off Period** * It is best performed during the **drying off period** which is ideally **2 months before birth** to avoid shedding of antibodies in milk. * **Milk ring test is not effective in sheep or goat because of the low level of fat in milk.** * It is better to perform **Rose Bengal test** on serum. # Tube Agglutination Test * **Quantitative:** * Best test for Bovine brucellosis * Series of dilutions of the serum are done and a constant concentration of Ag is added. * The **reciprocal of the lowest concentration** that shows agglutination defines the result. * This is known as **titer**. * **Qualitative:** * **Tube test**. * **Human salmonellosis** * **S-typhi:** Typhoid - enteric fever. * **S-Paratyph:** A.B * **Widal's test** # **Typhoid Fever** * **Transmission:** Fecal oral route: contaminated food or water by stool. * **Incubation period in intestine:** * **X ion transport channels** (important for nutrient absorption) * **Replication** (of bacteria) * **Disturbance of enterocyte function** * **Diarrhoea** * **Gastroenteritis** * **After 15 days, the bacteria spreads to blood:** * **Fever** * **Bacteremia** * **Rosen red spots** (on skin) * **Localization in gall bladder:** * **Salmonella** can be isolated from bile. * **Shedding in the small and the large intestine. Stool Carrier**. * **Widal test:** * Test that detects agglutination of Salmonella Ag. * **Direct agglutination test for diagnosis of typhoid fever** # Widal Test * **Qualitative** * **Slide Widal test:** * **Somatic Ag (O Ag/ Flagella Ag (H Ag)** * **Standard Ab** * **Controls:** * **Ctrl+** (Serum + control) * **Ctrl-** (Serum + saline) * **Results:** * **O Ag** * **H Ag** * **A** * **B** * **Quantitative:** * **Tube Widal test** * **Double fold serial dilution** # Validation of Results * **Control+** * **Validation of result** * **Evaluation of reagent quality** * **Hemolysis** * **Improper reagent** * **Ctrl-** * **Contamination with other sample** * **Improper reagent** # False Results * **Widal test:** * **Cross reactivity:** Test is positive for other microorganisms than Salmonella. * **Vaccinated person:** Vaccinated person in an endemic area. * **Vaccines against malaria:** Shared epitopes with Salmonella typhi. * **Acute malaria infection** * **Autoimmune disease - Chronic Liver Disease** * **Increase in globulins and Abs:** non specific binding * **Previous infection with Salmonella Typhi** * **True infection** * **Vaccination** * **Titre:** * The difference between the 2 titers should be high, usually 2 fold. * **General:** * **Early (Recent) infection** (prozone) * **Long T3 of Antibiotics** * **Immune compromised patients** (Cancer, Diabetes, Chemotherapy) * **Long TIII of Cortisole** * **Recovery (Post zone)** * **Latent infection (Carrier state) ** * **Shedding in Stool ** * **Expired reagent- Technical error** # Hemagglutination * **Agglutination of RBCs** * **Biological:** * OAI (Ornithosis) * OND (Ornithosis) * **Immunological:** * Due to **Ag Ab reaction** * **Direct Hemagglutination** * **Indirect or passive Hemagglutination** * Soluble Ag * Carrier molecule # Disadvantage of Using RBC as Carrier Molecules (Carrier Particle HA Test) * **Short durability** of RBCs * **Fragile cell membrane** * **Hemolysis** (destruction of RBCs) * **Formalization of RBCs** (chemical treatment) ## Serum Complement * It is one of the **humoral immunity** components. * It can **lyse** (destroy) RBCs before the Ab molecule binds to Ag. * **Overcomed by** heat treatment of serum at **56°C for 30 minutes**. * **IgM** gets deactivated at **65°C** ## Heterophilic Antibodies * **Specificity:** * **Broad specificity** (Polyclonal) - it doesn't have affinity to one type of epitope * **Naturally present** in serum due to infection by: * **HIV** * **Epstein Barr virus** * **Infectious mononucleosis** * **Cancer myeloma** * **Sensitization** of the immune system to sheep wool. * it may **cross-react** with other antigens. * **Non-specific binding** * **Anti- animal Ab** is produced in the immune system by handling animal with other animals. * **Hyperimmune Serum** can be used to treat cancer. * **Hyperimmune serum** will coat the cancer cells, and NK cells will be attracted by the increased affinity. * **Autoimmune Disorders** * **Rheumatoid arthritis** # Heterophilic Antibodies * **Bind with Fc region** → **False result** * **Species Specific Abs** ## Cross Reaction * Ags of a species can react with Abs of another animal or another species. * Sheep Abs can react with Goat, Cattle, and Buffalo Ags. * **Has ability to block** all binding sites of heterophilic Abs (paralobes) * Use **heterophilic blocking reagents** to prevent cross-reaction. * HBRs in serum bind with **Fab region of assay Ab** to prevent it from binding with Ag. * **Serial dilution** is also used for blocking of heterophilic antibodies. # Monospot Test (IM Test) * **Epstein Barr virus** * **Kissing disease "** * **Infectious mononucleosis** * It transmits through **saliva (direct contact)** * It is also called **mononuclear cells in liver, spleen, and lymph node**. * **Infection of B cells:** * **Malfunction:** * Defective Abs (heterophilic Abs) * **Hemolytic anemia** * **Detected by:** * **Monospot test** * **Lateral flow immune assay.** ## Monospot Test (IM Test) * **Pregnancy** * **Hepatitis B-C** * **Assay:** * **Sandwich ELISA** * **Control:** * Serum sample and RBCs with chromatic dye * **Test:** Serum * **Chromatic dye** * **Sheep RBCs** * **Result:** * **Release of chromatic dye** * **Line in control** * **Line in control + test** ## False Results * **False positive:** * **For children under 5 years old:** Immune system is still developing and it is not uncommon to have heterophilic Abs. * **False negative:** #