Enzyme-Linked Immunosorbent Assays (ELISA)

The first step in any ELISA assay is the immobilization of the antigen within the sample to the wall of the wells within a microtiter plate. These microtiter plates are usually 96-wells. This is by direct adsorption to the plates surface or by using a capture antibody. The capture antibody has to be specific to the  target antigen. After immobilization, another antibody is added called the detection antibody. This detection antibody binds to the adsorbed antigen which forms an antigen:antibody complex. This detection antibody is either directly conjugated to an enzyme, such as horseradish peroxidase (HRP), or provides an antibody-binding site for a secondary labeled antibody. There are four different types of ELISAs which will all be discussed below. ELISAs take advantage of an enzymatic label to produce a signal that can be quantified and correlated to the binding of an antibody to an antigen. The final assay signal is measured using spectophotometry.

Direct ELISA

In the direct ELISA, the detection antibody is conjugated with either alkaline phosphatase (AP) or horseradish peroxidase (HRP). These substrates produce a colorimetric output that is then measured. The advantages of a direct ELISA is that it is a short protocol which saves time and reagent, and money. There is no cross-reactivity from a secondary antibody that can cause interference. The disadvantages are that there is no signal amplification, so the primary antibody must be conjugated for it to work.

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Indirect ELISA

In the indirect ELISA, antibodies can be conjugated to biotin, which is then followed by a streptavidin-conjugated enzyme step. This is becoming more common place within the clinical laboratory. Alternatively, the detection antibody is typically a human IgG antibody that binds to the antigen within the wells. This primary antibody has multiple antibody-binding sites on it. A secondary rabbit anti-human IgG antibody conjugated with an enzymatic substrate is added. This secondary antibody binds to the first antibody and gives off a colorimetric signal which can be quantified by spectrophotometry. There are advantages over the direct ELISA, mainly that there is signal amplification by using several antibodies, allowing for high flexibility. This also creates a longer protocol, and increases the chances for cross-reactivity, which can be deemed disadvantages.

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Sandwich ELISA

The sandwich ELISA is less common, but is highly efficient in antigen detection. It quantifies antigens using multiple polyclonal or monoclonal antibodies. Monoclonal antibodies recognize a single epitope, while a polyclonal antibody recognizes multiple antigen epitopes. The antigen that is to be measured must contain at least two antigenic epitopes capable of binding to an antibody for this reason. The first step is to coat the microtiter plate wells with the capture antibody within a carbonate/bicarbonate buffer (pH 9.6). Proceed to incubate the plate overnight at 4 degrees Celsius. Wash the plate twice using PBS. Incubate the plate again for at least 2 hours at room temperature. Wash the plate again using PBS. The next step is to add diluted unknown samples to each well. Its important to run unknown samples against those of a standard curve by running standards in duplicates or triplicates. Incubate for 90 minutes at 37 degrees Celsius. then remove the sample and wash with PBS again. Next, add diluted detection antibody to each well. Its important to make sure that the detection antibody recognizes a different epitope on the target antigen than the capture antibody. The prevents interference with antibody binding. To maximize specificity and efficiency, use a tested matched pair. Once the detection antibody has been added, incubate for 2 hours at room temperature. Wash once again with PBS. After washing, add conjugated secondary antibody to each well. Incubate once again at room temperature, then proceed to wash. Once again, horseradish peroxidase and alkaline phosphatase are used as enzymes conjugated to the secondary antibody. The substrates for HRP are called HRP chromogens. Cleavage of hydrogen peroxide is coupled to an oxidation reaction which changes color. Another common substrate used is ABTS. The end product is green.

Sandwich-ELISA

The sandwich ELISA employs high specificity, even when using complex samples. Within the sandwich ELISA, both direct and indirect methods can be used. It can be challenging to find two different antibodies against the same target the recognize different epitopes.

Competitive ELISA

The competitive ELISA is exactly what its name suggests; it is a competitive binding process which is produced by the sample antigen, and an add-in known concentration of antigen. A primary unlabeled antibody is incubated with the unknown sample antigen. This creates antigen:antibody complexes, which are then conjugated to a microtiter plate which is pre-coated with the same antigen. Any free antibody binds to the same antigen on the well. Unbound antibody is removed by washing the microtiter plate. The more antigen within the unknown sample means that less antibody will be able to bind to the antigens within the wells, hence the assay gets its name. Its a competition. A secondary conjugated antibody that is specific for the primary antibody bound to the antigen on the pre-coated on the wells is added. When a substrate is added, the reaction elicits a chromogenic or fluorescent signal. The higher the sample antigen concentration, the weaker the eventual signal.

competitive

References

https://www.bio-rad-antibodies.com/elisa-procedure.html

https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-elisa.html

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Iron Deficiency and Microcytic Anemias

Iron is an essential element for oxygen transport within hemoglobin. Oddly enough it is the element that is missed the most in regards to adequate intake and proper nutrition. Over 1.62 billion people in the world are effected by anemia, which is most commonly caused by iron deficiency. Iron deficiency can be caused by chronic blood loss, and is most common in women and teenagers from loss of blood due to menses. Iron loss leads to increased fatigue and depression, pallor, and dry and splitting hair. It can also lead to confusion cognitive effects. Hemoglobin is made of four polypeptide chains, two of which are alpha, and two are beta that come together to form a tetramer heme group with iron located in the middle. Ferrous iron within each heme molecule reversibly binds to one oxygen molecule. With iron deficiency, there becomes a hemoglobin deficiency. A decreased hemoglobin lowers oxygen-carrying capacity leading to anemia. Anemia by definition is a reduced oxygen-carrying ability. Tissue hypoxia can wreak havoc on almost every cell of the body, and can shift the oxygen dissociation curve in an unfavorable direction. The structure of hemoglobin and its function and key elements can be reviewed here.

To understand iron deficiency its important to recognize important aspects of iron metabolism and transportation in cells. Review the Iron Absorption and Metabolism article here for that information. There are also laboratory values that give a good picture of the iron status within the body that one should pay attention to. Transferrin; which is measured as the total iron binding capacity (TIBC) indicates how much or how little iron is being transported throughout the body. Serum iron is an important indicator of the tissue iron supply, and finally serum ferritin gives a picture of iron storage status within the bone marrow and cells.

Iron Deficiency Anemia

There are three stages within iron deficiency. Each comes with their own classic picture of laboratory results and worsen from stage to stage. In the first stage, there is storage iron depletion. This is mild and the patient may not even feel a difference physically. The patients hemoglobin is normal, normal serum iron, and TIBC. There is however decreased ferritin which indicates that there is decreased storage of iron. The second stage of iron deficiency is characterized by transport iron depletion. The hemoglobin may or may not be abnormal, but there is increased TIBC, and decreased serum iron. An increased TIBC, means that there are more substrate (iron) binding spots within the transferrin molecule. This implies that less iron is binding, which when coupled with a decreased serum iron makes sense. The patient may experience mild anemia which comes with increased fatigue and pallor. A peripheral blood smear will most often start to exhibit anisocytosis and poikilocytosis. These reference indicators represent abnormal sized red cells and abnormal shaped red blood cells respectively. A good indicator is an increased RDW, an increased RDW indicates some degree of anisocytosis. This is accurate because the red blood cell is realizing the loss of this oxygen-carrying capacity so its trying to release red blood cells as fast it can from the bone marrow to compensate for the loss, and as a result these red blood cells will appear smaller in diameter and hypochromic. Hypochromasia indicates that there is less hemoglobin within the cell and there is more of a central pallor. The thought is that even though there is less hemoglobin within each cell, if the bone marrow can produce more of these red blood cells than normal then that equals out. This leads to a microcytic anemia, micro meaning small. Stage three of iron deficiency is often referred to as functional iron deficiency. Within this stage there is an unmistakable decrease in hemoglobin, serum iron, and ferritin. There is also a large increase in TIBC.

The overall effect of iron deficiency anemia on the body and on the bone marrow is ineffective erythropoiesis. The red cell production within the bone marrow is compromised. As a result, the bone marrow becomes hypercellular with red cell precursors reducing the M:E (Myeloid:Erythroid) ratio.

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This picture depicts how a peripheral blood smear would illustrate iron deficiency anemia. The red cells are smaller and there is more of a central pallor to them, indicating a loss of hemoglobin. This is also called hypochromia.

normalbloodsmear

This picture depicts a normal peripheral blood smear. The red blood cells are larger in size and they have more color to them.

Anemia of Chronic Disease

Anemia of chronic disease is another form of microcytic anemia similar to iron deficiency anemia. It usually arises from a chronic infection or from chronic inflammation, but its also associated with some malignancies. A buildup in inflammatory cytokines alters iron metabolism. IL-6, which is an inflammatory cytokine inhibits erythrocyte production. It also increases hepcidin production. Hepcidin blocks iron release from the macrophages and the hepatocytes by down-regulating ferroportin. Without ferroportin there is no transportation of iron throughout the body and no production of hemoglobin or red blood cells. Laboratory findings will usually demonstrate low serum iron, low TIBC, low transferrin, and an increased to normal ferritin. The reticulocyte count is also normal, and sometimes increased. Reticulocytes are released from the bone marrow in times of red cell shortages to compensate.

This is just a brief overview of iron deficiency anemia and other microcytic anemias. This is just the beginning, follow and look forward to more in-depth reviews of each microcytic anemia. Key differences to look for is the TIBC value. In iron deficiency anemia the TIBC is increased and in anemia of chronic disease the TIBC is decreased. Ferritin is increased in anemia of chronic disease because the stored iron can’t be released from cells and the bone marrow due to the increased hepcidin production. Also the degree of anemia is mild compared to the more severe iron deficiency anemia.

 

 

Laboratory Equation Guide

SI Units: The International System of Units is a system devised around the convenience of the number ten. It is the worlds most widely used system of measurement in science.

Definitionsandabbreviations

Temperature

SI Standard units are Kelvin (K)

Celsius = K-273

Kelvin = Celsius+273

Temperature Conversions

Celsius = 5/9 (F – 32)

Fahrenheit = (9/5 x C) + 32

Solution

A solution is a homogenous mixture of two or more substances. A solute is a substance that is small part of a solution. A solvent is a substance that constitutes a large portion of a solution. Solubility refers to the solutes ability to dissolve in the solvent.

Dilutions 

A dilution is when the solute or substance of interest is combined with an appropriate volume of solvent to achieve a desired concentration. The dilution factor is the resulting total number of unit volumes in which the solute was dissolved.

For example: Dissolving one part solute into 3 parts solvent. Total dilution is 1:4. The dilution factor is 4 because there are four total parts of unit volumes.

(C1)(V1)=(C2)(V2) is used when making fixed volumes of specific concentrations.

Percent solutions refer to parts per hundred. They can either be percent by volume (% (v/v)) or percent by mass (% (m/m)).

Percent by Volume (% (v/v)) = (Volume of solute/Volume of solution) x 100

Percent by Mass (% (m/m)) = (Mass of solute/Mass of solution) x 100

Molarity is a unit of concentration equal to the number of moles of solute in one liter of solution.

Molarity (M) = Moles of solute/Liters of solution

Density is the ratio between the mass and volume of a material.

Density = Mass/Volume

Clinical Validity

Specificity: The frequency of a negative test when no disease is present.

Specificity = (True negatives/True negatives+False positives) x 100

Sensitivity: The frequency of a positive test when disease is present.

Sensitivity = (True positives/True positives+False negatives) x 100

Chemistry

Calculated plasma osmolality = 2[Na+] + Glucose/18 + BUN/2.8

Osmolar Gap: Difference between the measured and calculated osmolality.

Friedman Formula:

LDL = Total cholesterol – HDL – VLDL

Triglycerides = (Total cholesterol – HDL – LDL) x 5

Precaution: The Friedman formula is only reliable for triglyceride levels less than 400 mg/dL.

Bilirubin

Total Bilirubin = Conjugated bilirubin + Unconjugated bilirubin

Conjugated Bilirubin = Total bilirubin – Unconjugated bilirubin

Unconjugated bilirubin = Total bilirubin – Conjugated bilirubin

Creatinine Clearance

Cockcroft-Gault Equation

Creatinine Clearance = Male (1.0), Female (0.85) x (140-age) x (Serum Creatinine) x (Weight/72).

Hematology

Hematocrit: The percentage of blood that is represented by packed red cells.

Hematocrit (%) = Hemoglobin x 3

MCV: Mean cell volume refers to the average size of the red cell population within the sample.

MCV = (Hematocrit (%) x 10)/RBC (x10^12/L)

MCH: Mean cell hemoglobin refers to the average weight of hemoglobin within the red cell population.

MCH = (Hgb x 10)/RBC (x10^12/L)

MCHC: Mean cell hemoglobin concentration refers to the average concentration of hemoglobin within the red cells constituting the sample.

MCHC: (Hgb x 100)/Hematocrit (%)

Corrected WBC: Nucleated RBCs are counted as white blood cells regardless of which method is utilized. For this reason when a differential is performed and there is presence of NRBCs a corrected WBC must be calculated. The number of NRBCs per 100 leukocytes is recorded during the differential leukocyte count when performing a blood smear examination. This number is then used;

Corrected WBC = (WBC x 100)/(NRBC + 100)

 

 

 

 

Liver Enzymes

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The liver is the largest internal organ. It is incredibly functionally complex. It carries out more than 500 different functions that include detoxification, protein synthesis and chemical synthesis to aid in digestion. Some of the livers main functions will be talked about. Bile produced by the liver aids in digestion in the small intestine. It breaks down and absorbs fats, cholesterol, and occasionally some vitamins. Bile is produced by the liver and is stored in the gallbladder. The liver also absorbs and metabolizes bilirubin. Bilirubin is caused by the breakdown of hemoglobin in red cells. This can either be extravascular or intravascular hemolysis and can either be normal breakdown as red cells only live for 120 days before being recycled or it can be not normal. The iron that is released by the bilirubin is stored in the liver or the bone marrow that is used to make new red cells. Bilirubin is metabolized to its direct conjugated form where it is excreted by the urine and feces. The liver produces clotting factors. Vitamin K is necessary for some clotting factors to be produced and in order for vitamin K to be absorbed from the diet the liver needs to produce bile. Without the bile clotting factors would not be produced. The liver aids in filtering the blood. It filters and removes different waste material produced from the body as well as exogenous compounds like alcohol and other drugs. The liver also has a immunological function in that it contains Kupffer cells. These Kupffer cells are the macrophages of the liver and are part of the mononuclear phagocyte system that destroy any foreign antigen that enters the liver. The liver produces albumin which is arguably the most important protein in the body. Albumin is used as a transport protein and maintains colloid pressure in the blood vessels. Angiotensinogen is produced by the liver that is used in the angiotensinogen-Renin system in the kidneys that raises blood pressure by vasoconstriction.

Regeneration is a unique feature of the liver. Its important to the body is unmatched and it has evolved the ability of regeneration. Regeneration is the ability for the organ to regrow rapidly as long as it is kept healthy. During this process of regeneration the function of the liver is not compromised. If needed there are a number of compounds that can aid in the regeneration process like hepatocyte growth factor, insulin, epidermal growth factor, IL-6, and even norepinephrine.

Analysis of bilirubin is based on the reaction of bilirubin with a diazotized sulfanilic acid. Three fractions of bilirubin is measured. Conjugated, unconjugated, and delta bilirubin. Delta bilirubin is bilirubin bound to albumin. A fasting sample is preferred and hemolyzed samples should be avoided. Bilirubin is sensitive to light so care should be taken to shield the sample from the light.

Liver enzymes are relatively nonspecific indicators that can indicate tissue destruction in several organs. Alkaline phosphatase is a group of isoenzymes that are found on the membranes of cells in almost every tissue. Alkaline phosphatase can become elevated in many different conditions including bone diseases, puberty, and even in late pregnancy. One important feature of alkaline phosphatase is that the isoenzyme found in bone is the most heat labile. Differentiation in the laboratory includes heating the sample and then remeasuring the alkaline phosphatase and measuring the difference.

Gamma-glutamyltransferase (GGT) is a membrane enzyme that is important in glutathione metabolism. It transfers glutamate to the amino acid peptide chain. GGT is among one of the first enzymes to become elevated in acute liver diseases such as hepatitis. Its important to note that GGT is normal in patients with bone diseases making it one of the most clinically specific of all the liver enzymes. Lactate dehydrogenase is a cytosolic enzyme that interconverts pyruvate and lactate. There are five isoenzymes. Isoenzymes 4 and 5 are increased in viral or toxic hepatitis, biliary obstructions and cirrhosis. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) convert aspartate and alanine to oxaloacetate and pyruvate respectively. ALT is very specific for liver pathology. AST is found in liver tissues, but also present in heart and muscle tissue. The AST/ALT ratio is an important factor to look at. A ratio less than 1.0 indicates viral hepatitis. This is an increase in ALT greater than the increase in AST. An AST/ALT ratio greater than 1.0 usually means that the ALT is elevated to a lesser degree than the AST and this can be found in cirrhosis, bile duct obstruction, or metastatic cancer of the liver.

-Caleb

Acid-Base Balance

An acid is any compound that can donate H+ when dissolved in water. A base is any compound that can donate OH- ions. A buffer system is a combination of a weak acid or base and its salt or conjugate that resists changes in pH. The human body has incredible mechanisms to maintain an acid-base balance. Changes in pH put the body in different physiological states that can cause an array of problems. Acidosis is when the pH falls below the reference range of 7.34. Alkalosis is when the pH increases above the reference range of 7.44.

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The most important buffer system in the body is the bicarbonate (HCO3)/carbonic acid (H2CO3) system. Carbonic acid works to allow the human body to rid of toxic CO2 via respiration to maintain a normal pH of 7.4. There normally is a 20:1 ratio of bicarbonate to carbonic acid.

The red cells pick up CO2 from tissues and throughout its travel through the blood vessel its converted to carbonic acid. That carbonic acid is then broken down into bicarbonate and hydrogen. The excess hydrogen ions are buffered by hemoglobin. Bicarbonate leaves the red cell and goes into circulation. Bicarbonate enters the plasma through an exchange mechanism with chloride to maintain a state of electroneutrality in the cell. When the red cells reach the lung the hemoglobin will release the excess hydrogen ions by the binding of oxygen to hemoglobin. The excess hydrogen ions bind to bicarbonate to form carbonic acid. Carbonic acid then dissociates into H20 and CO2 which is expelled.

As mentioned above, an individual can be in a state of acidosis or alkalosis. This can be caused by ventilation and is called respiratory acidosis or respiratory alkalosis or it can either be caused by HCO3-. This is called metabolic acidosis or alkalosis.

Respiratory acidosis is an increase in PCO2. Conversely respiratory alkalosis is a decrease in PCO2. Metabolic acidosis is a loss of HCO3- or an addition of H+. Metabolic alkalosis is a loss of H+ or an increase of HCO3-. The body will naturally compensate for the pH changes. Some of the compensatory mechanisms are increasing respiration in metabolic acidosis. Hyperventilation increases the amount of CO2 that is expelled and raising the pH. In respiratory acidosis the kidney will increase its reabsorption of HCO3-.

Metabolic acidosis can be caused by multiple different disease states. Excessive loss of HCO3- by diarrhea can cause metabolic acidosis. Diabetic ketoacidosis can cause it. Other causes are ingestion of acids or renal tubular failure where there is no renal reabsorption of HCO3-.

Metabolic alkalosis is caused by excess or an overdose of HCO3-. Excessive vomiting causes a loss of hydrochloric acid with the stomach contents. Vomiting also results in hypokalemia and hyponatremia which are both positively charged ions (acids) leading to an increase in the pH. Excessive diuretic use can sometimes initially cause an increase in chloride, but most commonly results in hyponatremia and causing a contractile alkalosis.

Respiratory acidosis is most commonly caused by CO2 retention usually due to ventilation failure. Decreased cardiac output and hypotension also cause acidosis. Less blood is pumped to the heart so less CO2 is getting transported to the lungs to be expelled. Chronic lung conditions such as COPD result in an inability to ventilate properly and to expel CO2. Certain drugs cause depression of the respiratory center in the brain and can cause respiratory acidosis. Some of these drugs are barbiturates, opiates and ethanol (alcohol).

Respiratory alkalosis is primarily caused by hyperventilation (increased alveolar ventilation). This results in a decreased arterial PCO2. Any condition which decreases pulmonary compliance causes a sensation of dyspnea. Dyspnea is not a single sensation and there are at least three distinct sensations including air hunger, work/effort, and chest tightness. These sensations cause a state of hypoxia which is caused by the hyperventilation.

-Caleb