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UCONN / Physiology and neurobiology / PNB 2265 / What are the funcitons of blood in maintaining homeostasis?

What are the funcitons of blood in maintaining homeostasis?

What are the funcitons of blood in maintaining homeostasis?

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School: University of Connecticut
Department: Physiology and neurobiology
Course: Human Physiology and Anatomy
Professor: Kristen kimball
Term: Spring 2016
Tags: PNB and Practical
Cost: 50
Name: Practical 1 Study Guide
Description: Study guide for PNB 2265 practical 1
Uploaded: 02/24/2017
16 Pages 526 Views 0 Unlocks
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○ What events occur during the QRS wave?




○ What event occurs during the P wave?




Right atrium ● What chamber pumps oxygenated blood to the blood?



PNB Practical 1 Lab 1: Blood Part 1: Formed Elements of Blood ● Overview of components of blood and their general functions in maintaining homeostasis ○ Type of connective tissue ○ Regenerate itself on a regular basis ○ Responsible for transporting things ○ Centrifuge = blood separated ■ Plasma 55% (mostly of water andDon't forget about the age old question of How do we determine hormonal stimulus?
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also proteins ​ ) ■ Buffy coat​ (platelets and leukocytes) ■ RBCs​ 45% - erythrocytes ■ Erythro and bluffy ​= formed cells ■ Buffy coat​ = larger than Eryth. ● Most glucocytes are found in body tissues ● Never Let Monkeys Eat Bananas (most abundant to least) ​ ○ Erythrocytes (RBCs) ○ Thrombocytes (platelets) ○ Leukocytes (WBC) ■ Granulocytes ● Clearly have granules (clumps) ● Neutrophil - Most abundant, Nuclei multiple lobed (5), Post morpho ​ nuclear ● Eosinophil - Red or pink, Bilobed nuclei (2) ​ ● Basophil - ​Least, Bilonuclei, Blue violet granules ■ Agranulocytes ● Still have them but much smaller (no clumps) ● Lymphocytes - ​Large round nuclei ● Monocytes - ​C or kidney shaped ● Histology of blood disorders ○ Eosinophilia ■ All eosinophils ■ Should only make up 2-4% of leukocytes ■ Allergic or parasitic infection ○ Sickle cell anemia■ Shape of RBCs are abnormal/sickle shaped ■ Inherited disease ■ Inter cell protein does this ■ Causes them to carry less oxygen ■ Sickle cell hematocrit = blood is lower ● Shape change packs it more, compact more ○ Polycythemia ■ Packed with RBCs ■ Density of RBCs is higher ■ Increase in RBCs or decrease in blood plasma ■ Result in high BP and blood clots ■ Primary polycythemia - rare condition ■ Chronic hypoxia and Steroid hormones can contribute to primary polycythemia ■ Polycythemia hematocrit = blood is higher ● Absolute = increase in number of RBCs ● Relative = reduction in blood plasma levels, ratio is low because blood and plasma ○ Infectious Mononucleosis ■ Viral disease ■ Fever ■ Sore throat ■ Fatigue ■ Caused by epstein barr virus ● Atypical lymphocytes ● Mononucleosis ● Large with abnormally shaped nuclei ● Bright blue cytoplasm ● Spreading borders ■ Mononucleosis hematocrit = slight higher WBCs ○ Leukemia ■ Unregulated overproduction of immature leukocytes ■ Release from bone marrow and circulate blood ■ Myelogenous = affects myelogenous ■ Too many WBCs, not functioning correctly Part 2: Blood Typing ● ABO blood types ○ Presence or absence of two antigen variants ○ B antigen expressed = blood type B ○ A and B codominant = AB blood type ○ Neither A or B antigen = O blood type ○ Patient receive in compatible blood type = fatal ● Antibodies ○ Immune system produces antibodies when person is exposed to foreign substance ○ Produced by B lymphocytes in response to exposure to foreign antigens ○ Released into the blood plasma ○ Bind specifically to the inducing antigen ○ Two antigen binding sites per antibody ○ Protein on bacterial cell wall○ Bacterial protein = antigen ○ Make antibody specific to antigen ○ Specific antigen identifies a pathogen ○ Antibody = Y shaped (can bind to two different binding sites) ○ Blood Type A = B antibodies ● Hemagglutination ○ Pre formed antibodies against incompatible blood type antigens ○ Antibodies have two binding sites ■ Each antibody ca bind to two blood cell ■ Each blood cell has multiple antigens ○ Exposure to incompatible blood type results in hemagglutination ○ Hemagglutination may cause hemolysis (rupture of RBCs) and death ● Identification of Blood Type of Unknown Sample ○ Agglutination = certain antigen present ○ Rh factor (anti D) ■ Binds to antigen D (rH factor) ■ Expressed on surface of cell = agglutination ■ Rh positive or Rh negative ○ Demonstration of Blood Typing Technique ■ Two unknown ■ Separate blood typing tray ○ Rh = no clumping means - ○ A or B = clumping means A or B ● Part 3: Hemoglobinometer and Hematocrit ○ Hemoglobin ■ RBCs are filled with hemoglobin protein (Hb) ■ Transports oxygen and CO2 ■ Composed of: 2 alpha and 2 beta polypeptide chains ■ Heme: porphyrin ring with rion in the center ■ Transport oxygen dissolved into blood ■ In heme group = iron ● Iron binds O2 ○ Hemoglobinometer ■ An easy to use diagnostic tool to measure hemoglobin levels in blood ■ Hb levels are measured in grams per 100 mL or gm% ■ Typical range of values: ● 13.0 - 18.0gm% for men ● 11.0 - 16.0 gm% for women ○ Hematocrit ■ Fraction of whole blood volume that consists of red blood cells ■ Collect blood; then centrifuge ■ Measure and divide RBC volume by total blood volume ■ Expressed as a percentage ■ Normal range of values: ● 47+- 7% for men ● 42 +- 5% for women■ Hematocrit values provide useful information for clinical diagnoses ○ Hemoglobin and hematocrit = express RBC levels ○ Low = anemia ○ Normal hematocrit, low hemoglobin = anemia ● Hemoglobinometer and Hematocrit Techniques ○ Hemoglobinometer ■ Insert test card ■ 12 mL of blood ■ grams/decaliter ■ Lower = <6 g/dL ■ High = >21 g/dL ○ Hematocrit Technique ■ What portion is RBCs ■ Swirling ■ One end of capillary tube into blood sample ■ Sealed end = outside of center ■ Balance tubes ■ Height of packed RBCs layer / total height = hematocrit Lab 2: Electrocardiogram (ECG) ● Sheep Heart Dissection ○ pulmonary trunk, interventricular sulcus, apex, right and left ventricle, tricuspid valve, bicuspid valve, mitral valve, right and left atrium, auricle (opening in right atrium) - pectinate muscles, inferior and superior vena cava, pulmonary veins, aorta, chordae , papillary muscles, semilunar valve ● Three layers of blood vessels​, called tunics ○ Tunica intima​: endothelium and subendothelial layer ○ Tunica media (thickest layer in arteries): smooth muscle (vasoconstriction) ​ ○ Tunica externa ​(thickest layer in veins): areolar connective tissue ● Arteries ​- more elastic and collagen fibers ● Veins​ - collapse without blood ● Wall of arteries are thicker than veins ● Cardiac Tissue ● Normal cardiac tissue ● Most myocardial infarction ○ Heart attack ○ One of the arteries are blocked → damaged tissue ○ Many purple cells (neutrophils) ○ Loss of cross striation ○ Blood vessels ■ Arteries have thick walls and do not collapse when cross sectioned ■ Atherosclerosis​ - large deposition of lipid and other debris on the inside ■ Normal Artery ○ View, Artery, nerve bundle ■ Nerve bundle = no openings, ​ many nerve fibers■ Arteries​ = Thick tunica media ■ Veins = larger lumen and collapse whe ​ n no blood inside ● Innermost = simple squamous ● Thick tunica externa (areolar connective tissue) ● Have valves ● Human Electrocardiogram ○ Measures electrical activities of the heart from electrodes attached to the outer surface of the skin ○ Surface electrode at different parts of body → ECG or EKG ■ Sinoatrial (SA) node - pacemaker cells that pr ​ opagates action potentials through muscle fibers of atria ■ Atrioventricular (AV) node ​- electrical connection between atrial muscle and ventricular muscle, also serves as backup pacemaker ● AP pass slowly ● Then rapidly to AV bundle ● Purkinje fibers → contraction ○ Lead: ​a pair of electrodes ○ ECG:​ electrical activities recorded from a lead ○ Einthoven’s triangle ■ Right arm (-) negative → left leg (+) positive → left arm (+) positive ■ Right left = ground ○ Positive vector (positive current) ■ When positive charges moves from negative (-) to positive (+) or negative charges moves from positive (+) to negative (-) ○ Negative vector (negative current) ■ When positive charges move from positive (+) to negative (-) or negative charges moves from negative (-) to positive (+) ○ What each lead detect is the projection of the actual wave ■ Positive to negative → upward ○ Ex: Depolarization is a positive wave from SA node to AV node. What is detected on lead I is an upward deflection. ○ Major Components of ECG ■ P wave ​- atrial depolarization ■ QRS complex - ventricular depolarization (cont ​ ains atrial repolarization) ■ T wave​ - ventricular repolarization ■ Electrical events of a cardiac cycle ■ EXTRAcellular recording that reps the sum of multiple action potentials of many diff cells Calculation of the net direction of an electrical event ● Provide important physiological information and can indicate myocardial damage ● PQRS complex ● P wave ​- whether or not SA ● QRS wave axis ​- ventricular mean axis ● Height of the R wave = height of the Q wave - height of the S wave ○ 3.5-1 = 2.5 mm ○ Subtract the heights of the negative waves from the positive waves ● Amplitude of QRS complex ○ 3.5 - 2 = 2.5 mm● Normal range of net direction of the ventricular depolarization = 0 - 90 degrees Quiz 2 ● Which of the four chambers does deoxygenated blood enter first? Right atrium ● What chamber pumps oxygenated blood to the blood? Left ventricle ● Which chamber pumps deoxygenated blood to the lungs? Right ventricle ● Which chamber receives oxygenated blood from the lungs? Left atrium ● Blood flows from the left ventricle to the — to enter the body. Aorta ● Blood flowing from the left atrium to the left ventricle must pass through the Bicuspid valve ● Deoxygenated blood from the body enters the right atrium via the — and the — inferior vena cava, superior vena cava ● Waveform questions (P, QRS, T) ○ What event occurs during the P wave? Atrial depolarization ○ What events occur during the QRS wave? The atria depolarize and the ventricles depolarize ○ What events occur during the T wave? Ventricular repolarization ○ Electrical activity in the heart begins in the SA node The funny current (I​F​)​ is due to HCN channels and mediates the rhythmic electrical activity of the heart via the influx of sodium at ~60mv (when the membrane is hyperpolarized). HCN channels allow the flux of sodium and potassium ions. Calcium current​ (both T type and L type channels) allows the depolarization of pacemaker cells and Sodium and L-type Calcium ​current allows the depolarization of myocytes. ● AV valves (LUB) and semilunar valves (DUB) ● Heart and Major Arteries ○ Aortic arch​- Portion of the aorta between the ascending and descending aorta○ Pulmonary trunk branches off into the pulmonary arteries (L+R)- Brings blood from the right ventricle to the lungs (left to left lung etc) ○ Ascending aorta-​ Begins at the base of the left ventricle and brings blood to the aortic arch ○ Descending aorta –​ Two parts to it / upper part is the thoracic aorta and lower portion is the abdominal aorta ○ Common Carotid artery-​ Arteries that branch off from the aortic arch and feed the brain with blood (left and Right) ○ Internal jugular veins-​ Brings blood back from the brain to the brachiocephalic vein then to superior vena cava ○ Cephalic vein-​ Brings blood from the arm to the subclavian vein then to the brachiocephalic vein then to the superior vena cava ○ Subclavian Artery-​ (Right below the clavicle) (Left and Right) takes blood from the aortic arch (Right come off of the brachiocephalic artery) to the arms (Left and Right) ○ Subclavian Vein- Takes blood back from the arm (Axill ​ ary vein) and brings it to the brachiocephalic vein then to the superior vena cava ○ Brachiocephalic Trunk-​ First branch of the aortic arch and branches into the right common carotid artery and the right subclavian artery (One artery and two veins) ○ Axillary Vein- ​Brings blood from the armpit area (blood from brachial artery) to the subclavian vein then to the brachiocephalic vein then to the superior vena cava ○ Superior Vena Cava-​ Larger of the Vena Cava/ brings blood from the brachiocephalic veins (left and right) to the right atrium ● Upper body blood supply ○ Common Carotid artery​- Arteries that branch off from the aortic arch and feed the brain with blood (left and Right) ○ Internal jugular veins​- Brings blood back from the brain to the brachiocephalic vein then to superior vena cava ○ Cephalic vein- Brings blood from the arm to the subcla ​ vian vein then to the brachiocephalic vein then to the superior vena cava ○ Subclavian Artery-​ (Right below the clavicle) (Left and Right) takes blood from the aortic arch (Right come off of the brachiocephalic artery) to the arms (Left and Right) ○ Subclavian Vein-​ Takes blood back from the arm (Axillary vein) and brings it to the brachiocephalic vein then to the superior vena cava ○ Brachiocephalic Trunk​- First branch of the aortic arch and branches into the right common carotid artery and the right subclavian artery (One artery and two veins) ○ Axillary Vein- ​Brings blood from the armpit area (blood from brachial artery) to the subclavian vein then to the brachiocephalic vein then to the superior vena cava ○ Superior Vena Cava​- Larger of the Vena Cava/ brings blood from the brachiocephalic veins (left and right) to the right atrium ● Aorta is the main supply of blood in upper body and all other artery structures listed below originate from it ○ Celiac artery ​– blood supply to stomach (NOT SHOWN ON DIAGRAM) ○ Superior mesenteric artery​- blood supply to duodenum ○ Renal arteries-​ blood supply to kidneys – 1/3 of blood flow from heart ○ Inferior mesenteric artery-​ blood supply to the large intestine ○ (The mesentery is a fold of membranous tissue that arises from the posterior wall of the peritoneal cavity and attaches to the intestinal tract) ● Lower body blood supply ○ Common Iliac artery-​ Brings blood from the aortic bifurcation to both the internal and external iliac artery ○ Internal Iliac artery- ​Brings blood from the common iliac artery to the but and reproductive organs ○ External Iliac artery- ​Brings blood from the common Iliac artery to the femoral artery and supplies blood to the legsLab 3: Frog Heart ● Comparative anatomy of the Frog Anatomy ○ Pacemaker cells in SA node ■ Frog heart - pacemaker cells in SV node ○ Oxygenated and deoxygenated blood ■ Two ventricles divided by a septum ■ Frog​ - one ventricle, mixing of oxygenated and deoxygenated ■ Trabeculae (sight of attachment for papillary muscles, prevention de and oxy mix) and spiral folds (maintaining separation of de and oxy ■ Deoxygenated - systemic circulation returns to ​ right through SC ● Lungs and skin (skin is another respiratory surface) ■ Oxygenated ​- left atrium through pulmonary vein ● Blood and tissue ■ Separate until ventricle ■ Ventricle pumps one artery until further separation ■ Anatomical and internal allow small mixing ● Factors Impacting Cardiac Function ○ Physical that affect heart rate and contractile forces ■ Temperature ● Q10 (temperature coefficient): ​a measure of the rate of change of a biological or chemical system as a consequence of increasing the temperature by 10 degrees C ● For most biological systems, the Q10 values is about 2 to 3 ● Raising the temp 10 degree can cause catalyze reaction ■ Ionic concentration ● Changing K ion ○ Inside cell are relatively high than outside ○ High in to out concentration gradient ○ Spontaneous potassium efflux ○ Slight to moderate increase in K ions should decrease concentration gradient reduced K efflux ○ Moderate increase in extracellular K increase action potential firing ○ Extreme increase in extracellular potassium diminish action potential firing○ Cardiac myocyte not repolarize ● Ca2+ enter cytosol through receptor of sacro reticulum ○ Cardiac muscle → Ca enters the cyto through L type Ca channels in plasma membrane ○ Activates receptors in SR ○ Influx of extra Ca through surface membrane channels important ○ Strength and speed of myocyte is proportional to Ca ■ Parasympathetic and sympathetic innervation ● Major and opposite effects on heart rate ● Sympathetic = Epi (agonist at beta receptors) ● Parasympathetic = ACh ■ Mechanical stretch ■ Electrical blockage ○ Refractory period of cardiac muscle ■ AP in myocardial cells have a very long plateau phase before repolarization ■ Ca channels in myocardial membrane ■ Sustained depolarization prolongs activation of sodium channels ■ Longer refractory period → preventing tetanus ■ Muscle stretch affects cardiac function ● Normal physiological ranger, high sensitivity to changes in RAP ● Heart has reached limit of its response, insensitive to changes in RAP ● Cardiac output increases up to max value ● Cardiac output high sensitive to changes in right atrial pressure ● Additional blood enters → blood volume stretches so heart contracts forcefully and pump more blood ○ ECG measurement ■ Keep the connections between electrodes and frog tissue moist ■ Connections between pins/hook and wires should also be moist ○ Washout experiment - ​Allow enough time for recovery to baseline between trials Quiz 3 ● In the frog heart, the pacemaker cells are located in the sinus venosus (SV) ● Myocardial action potentials have a longer refractory period due to the presence of specialized Ca2+ ​channels ● Moderately increasing the extracellular [K+] would increase ​the AP firing rate ● Dramatically increasing the extracellular [K+] would decrease ​the AP firing rate ● Increasing the extracellular [Ca2+] would increase​ the contractile force ● What treatment should increase​ heart rate? Isuprel ● What treatment should increase ​heart rate? High temperature Lab 4: Physiology of Hemoglobin - Oxygen Binding ● Deoxy = more light through ● Part 1 - Spectrophotometry ○ Sample solution is illuminated with light source (invisible light) ○ Filtered by monochromator (normal wavelength) ○ Digital display or meter → absorbance or transmittance of light (A) ○ Absorbance = calculation concentration of sample ○ Beer’s law■ I = I0 (10-ELC) ■ Based on linear relationship between the concentration of solute in a solution and the absorbance of electromagnetic radiation (light) ■ I = final intensity of light ■ I0 = initial intensity of light ■ E = molar absorptivity (extinction coefficient) ● How strongly a substance absorbs light at given wavelength ■ L = optical path length through sample ■ C = concentration of sample ■ ELC = absorbance ○ Absorbance = ELC ■ Optical density (OD) ■ Units of A = liters per mole x cm x moles per liter ● Absorbance become unitless because all cancels out ■ E = compound/wavelength ○ Absorbance maximum ■ Lambda max is the wavelength of light at which a particular substance shows maximal absorbance ■ Need to obtain highest sensitivity of absorbance ■ Some substances may have more than one peak of absorbance ■ The peak of the greatest absorbance is the lambda max ○ Deoxygenated hemoglobin has greater absorbance at 660 nm than oxygenated hemoglobin ■ Set the spectrophotometer to absorbance mode ■ Need to use absorbance not transmittance ■ Zero the spectrophotometer with the blank tube between samples ○ Spectro must be turn on for at least 10 min to warm up ○ Before placing tubes into spectro, wipe each tube with a kimwipe ○ If you cannot close door completely cover with dark cloth ● Apply formula: (A-B/A-C) x 100; know how to calculate partial pressure of oxygen ​ ● Alpha and gamma = fetal (gamma higher affinity for oxygen) ○ Fetal higher affinity ○ Fetal = left shift ● Factors affecting p50 ○ Temperature (increased= right shift, decrease = left shift) ○ more basic (left shift), more acidic (right shift) ○ DPG ● Myoglobin ○ Binds O2 to iron in heme molecule ○ Only one heme group (bind 1 O2 molecule [er myoglobin) ○ No cooperative binding Quiz 4 ● Hemoglobin contains --- heme molecules and has the ability to bind --- oxygen molecules 4,4 ● Myoglobin contains --- heme molecules and has the ability to bind --- oxygen molecules 1,1 Lab 5: Lymphatic and Immune System Part 1 - The Lymphatic System● Structures of the lymphatic system ○ Lymph vessels ■ Transport fluid called lymph ○ Lymphoid tissues ■ Ex: tonsils ■ Collections of lymph on lining of sites ○ Lymphoid organs ■ Lymph nodes, thymus, spleen, red bone marrow ● Functions of the Lymphatic System ○ Maintain fluid balance ○ Participate in immune responses to protect against foreign pathogens ○ Absorb lipids ● The Lymphatic System Maintains Fluid Balance ○ Lymphatic capillaries collect interstitial fluid (lymph) from tissue spaces, and merge into larger lymph vessels ○ Lymph vessels ultimately merge into right lymphatic duct and thoracic duct ○ Ducts return fluid to the blood circulation at the subclavian veins ○ Lymph circulates through lymph nodes scattered through the body ● Lymphatic capillaries ○ collect interstitial fluid (lymph) from tissue spaces ○ Penetrate tissue of body where blood capillaries are present ○ Merge into larger vessels (right lymphatic duct) ○ Lymph vessels merge into right lymphatic and thoracic ducts ○ Ducts return fluid to the blood circulation at subclavian veins ■ Help maintain fluid balance in body ● Lymph circulates through lymph nodes scattered through the body ● Lymph Nodes ○ Inside are lymphatic modules ○ B lymphocytes, T lymphocytes, macrophages ○ Detect pathogen → immune response ○ Lymph into nodes via afferent lymphatic vessels ○ Lymph exits through 1-3 efferent lymphatic vessels ○ Secondary lymphoid organs ● Lymphoid organs ○ Lymphatic organs ○ Primary ■ Sites where cells of immune system are generated and mature ■ Ex: red bone marrows (all form elements of blood) ■ Ex: thymus ○ Secondary lymphoid organs ■ Mature cells of the immune system aggregate and initiate a specific immune response ○ Thymus: primary lymphoid organ ■ Site of maturation of T lymphocytes ■ Lobule - lobe of the thymus ■ Histology of the thymus ● Distinct thymus lobules● Hassall’s corpuscle - thymic corpuscle ○ Spleen: A Secondary Lymphoid Organ ■ Filters blood and sites of initiation of immune responses ■ Red pulp - serve as reservoir of stored WBCs and platelets (fetus and adults with severe anemia: new RBCs) ■ White pulp - circular or oval clusters of lymphatic cells (T, B, macro) ● Lymphatic nodules ● Mount immune response against foreign pathogens filtered by spleen ● Central artery - WBcs exit blood circulation to populate white pump ● PALs - T lymphocytes, germinal center that contains B lymph and macrophages ■ Sites where mature functional cells of immune system aggregate during response Part 2: ELISA (Enzyme Linked Immunosorbent Assay) ● Immune system protects against pathogens ○ Bacteria, viruses, fungi, parasites (protozoa, worms) ● Pathogen - an infectious microbe that causes disease ● Antigen - molecule that elicits the production of antibodies as part of the immune response ○ “Antibody generator” ○ Exotoxin - toxin molecules are antigens ○ HIV binds to host cells - protein expressed by HIV virus also an antigen ● WBCs aka leukocytes are main players in immune response ○ Large round nucleus and thin cytoplasm = lymphocytes ● Functions of Lymphocytes in the immune system ○ T lymphocytes (T cells) - macrophage (phagocytosis) ■ Helped (CD4) T cells secrete cytokines that regulate the function of cells of the immune system ■ Cytotoxic (Cd8, killer) T cells directly kill cells infected with viruses and tumor cells ○ B Lymphocytes (B cells) - antibodies ■ Production of antibodies ● Antibodies are produced by B cells in response to infection with a pathogen ○ Infection with a pathogen ○ Antigens on the pathogen are detected by B cells in the lymphoid organs ○ B cells differentiate into plasma cells and secrete antibodies that bind only to that specific antigen on the invading pathogen ○ The specific antibodies travel by the blood to the site of infection by the pathogen ■ Antibodies neutralize or target destruction ● Antibody structure ○ Fab = antigen binding region, variable region (unique that only binds to one antigen) ○ Fc = constant region ● Enzyme Linked Immunosorbent Assay (ELISA)○ Utilized the basic principles of antibody mediated immunity to detect antibodies or antigens in a sample ○ Important diagnostic tool to detect infection with specific pathogens ■ Extremely sensitive - infected with pathogen, ELISA will detect, low incidence of false negative ■ Extremely specific - unlikely of false positive ○ Use wells with series of reagents ○ Indirect ELISA ■ Step 1 ● Purified antigen proteins are added to the wells of the microtiter plate ● These antigens bind to the bottom of the plasmic wells ○ Antigen bind to bottom via hydrophobic reactions ● Between each step, the wells are washed with a mild detergent solution to remove excess proteins that are not bound to the plastic ■ Step 2 ● Primary antibody that is specific for the antigen of interest is added to the wells ● In clinical tests patient serum is added to the wells ○ If the patient has been exposed to an antigen, and has antibodies to the specific antigen in their blood serum, these antibodies will bind specifically to the antigen coated on the wells ○ If the person has not been exposed, there will be no antigen specific antibodies in their serum, and no antigen binding ● Negative sample = not this step ■ Step 3 ● Secondary antibody is added to the wells ○ Secondary antibody recognizes/binds the Fc region of human antibodies ○ Secondary antibody is linked to an enzyme ● Secondary antibody is in another specific ■ Step 4 ● Chromogenic substrate is added to the wells ● In positive wells, the enzyme will convert the substrate to the a colored end product ● In negative wells, no color will develop ● Lab ○ Antigen BSA ■ Wash wells to remove excess antigen ■ Wash and block with gelatin ■ Testing unknown samples for presence or absence of ● Antibody rabbit anti BSA● Antibody goal anti rabbit Fc-HRP ● Substrate ABTS ● Positive result = green ○ Indirect ELISA ■ Detects antibodies made in response to a pathogen ● HIV infection ● Lyme disease ● Systemic lupus erythematosus ■ Used for low level pathogen or hiding ○ Direct ELISA ■ Detects antigens directly ■ E. coli 0157 antigen test kit for food samples, animals feeds, and human stool samples ■ HCG pregnancy test ○ We are using direct ELISA because we don’t have blood samples so would not have antibodies so test for antigens Indirect ELISA (enzyme linked immunosorbent assay) to Measure Specific Serum Antibodies VS Direct ELISA (enzyme immunoassay aka EIA) to detect antigens ● Indirect (look for antibodies) ○ Wells of plates have assay ○ Filled with solution of present serum ○ Antigen specific antibodies bind to well ○ Wash ○ Animal antibody against human ○ Bound to antibodies ○ Substrate = color ○ Reaction is less intense if diluted ○ HIV test: ​antibodies because pathogen, don’t use direct because viruses can go dormant, might have antibodies produced yet ● Direct (take antibodies and look for antigen) ○ Detect antigens ○ Enzyme can be done in multi well microtiter ○ Plate is coated by antibodies ○ Well filled with sample ○ Antigen present in sample = bind to antibodies ○ Specific molecules bind to antibody coat ○ Wells washed and antigen stuck ○ Second antibody add conjugated covalently ○ Bind to specific ○ Washed ○ Solution of chlorogenic enzyme substrate added ○ Color = positive (contain antigen of interest) ○ No color = negative ○ Pregnancy test: ​use direct because no antibodies in urine (only blood) ● ELISA Procedure ○ Indirect, detect antigen specific antibodies ○ BSA○ 4 unknown samples and positive and negative control ○ Wash between each incubation step ○ Empty solution into sink ○ Gelatin blocks sites on plastic not bound to antigen ○ Incubate at room temp ○ Positive = primary anti BSA antibodies ○ Negative = not ○ Which is positive or negative? ○ Add secondary antibodies to each well ○ Final reagent = ABTS ○ Positive = dark green Part 3 - Hemolytic Plaque Assay ● Hemolytic Plaque Assay ○ Used to detect antibody producing plasma cells ○ Mice are immunized with sheep RBCs ○ Stimulated B cells in the mouse spleen to differentiate into plasma cells and secrete antibodies against SRBC ○ Mix together ■ Mouse spleen cells ■ SRBCs ■ Complement ○ Incubate in slide chambers ● Complement System ○ 30 proteins synthesized primarily in liver ○ Circulate in blood plasma as inactive form ○ Make diverse contributions to innate and adaptive proteins ○ Classical ■ Depend update presence of antibodies ○ Innate immunity, disposal system, adaptive immunity ○ Classical Complement Pathway ■ Invading bacterium that expresses foreign antigens ■ C1 attach to binding sites in antibody ■ Complement fixation ● C3b activates other complement protein ● C3b initiates MAC (membrane attack complex) ○ Form pore in phospholipid bilayer ○ Electrolytes leak out of cell and water into ○ Cell ruptures ○ Hemolytic plaque assay■ ■ Antibodies binding to antigen → complement attaches to antibodies → MAC → lysis of SRBC ■ Each plaque = 1 antibody producing cell ■ RBCs are rupturing around antibody producing cells ● Plaque forming ■ # of plaques = # of antibody producing cells ■ SRBC (5 days before) in mice → spleen remove → harvest spleen cells → SRBC+complement and other RBC+ complement (chicken rabbit etc) ● Won’t know which contains which ● Use hemolytic plaque assay to determine ● Incubate on heating tray → look for plaque in sheep RBCs ○ Demonstration of Hemolytic Plaque Assay ■ Two unknown blood samples ■ Which contains sheep which contain guinea pig ■ Pipette in each tube and mix well ■ Obtain slide with two micro chambers A and B ■ 50 mL in A and 50 mL in B ■ Seal chamber in hot wax ■ Place chambers on warmer for an hour ■ Examine slide chambers in microscope (gaps or clearings) ■ Plaques = sheep RBCs

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