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.

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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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Streptococcus Identification and Work Up

Streptococcus is a genus of coccus gram positive bacteria. They typically grow in chains of pairs as cell division occurs along a single axis in this family of bacteria. Most species in the streptococcus genus are oxidase negative, catalase negative, and most are facultative anaerobes. Many streptococcal species are not intact pathogenic and are part of the commensal human microbiota of the skin, mouth, intestine and upper respiratory tract. However, certain streptococcus species are the pathogenic agent for many cases of pink eye, meningitis, bacterial pneumonia, endocarditis, erysipelas, and necrotizing fasciitis.

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Species of Streptococcus are classified based on their hemolytic properties. Alpha-hemolytic species cause oxidation of the iron within the red cells, giving a greenish color to appear on the blood agar. The bacteria produces hydrogen peroxide which oxidizes the hemoglobin to biliverdin. This is also referred to as incomplete hemolysis or partial hemolysis. Beta-hemolytic streptococci cause complete degradation of the red cells, appearing as wide clear areas surrounding the bacterial colonies. Gamma-hemolytic species cause no hemolysis.

The beta-hemolytic species are further classified by the Lancefield grouping. This is a serotype classification that describes the specific carbohydrate present on the bacterial cell wall. There are serotypes for Lancefield groups A-V. For times sake, the only ones that will be discussed are Group A, and Group B.

Alpha-Hemolytic Strep

Strep pneumoniae, often referred to as pneumococcus is the leading cause of bacterial pneumonia. It can also be the etiological agent for otitis media, sinusitis, meningitis and peritonitis. The viridian’s group of alpha-hemolytic streptococci are a large group of commensal organisms. They possess no Lancefield antigens (carbohydrates) and can or can not be hemolytic.

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Beta-Hemolytic Strep

Streptolysin which is an exotoxin is the enzyme produced by the strep species that causes complete hemolysis of the red cells in the media. There are two types of streptolysin; Streptolysin O (SLO) and Streptolysin S (SLS). Streptolysin O is an oxygen-sensitive (labile) cytotoxin which is secreted by most of the Group A Streptococcus (GAS) which interacts with cholesterol in the membrane of the red cells. Streptolysin S is an oxygen-stable cytotoxin also produced by GAS species that affects the innate immune system of the host. It works in preventing the host immune system from clearing the infection.

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Group A Strep

Group A strep, otherwise known as S. pyogenes is the causative agent for strep infections, invasive and non-invasive. The most common infection is pharyngitis, otherwise known as strep throat, impetigo, and scarlet fever. All these infections are non-invasive. Invasive GAS infections include necrotizing fasciitis, pneumonia, and bacteremia (bacteria present in the blood). Complications can arise from GAS infections. Rheumatic fever is a disease that affects the joints, kidneys, and the heart valves. It is the consequence of an untreated strep A infection. Antibodies created by the immune system cross-react with proteins in the body which causes an immune-mediated attack on the hosts own cells. Essentially an acquired auto-immune disease. GAS is also implicated in pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS). Autoimmune antibodies affect the basal ganglia causing rapid onset of psychiatric, motor, sleep and other neurological symptoms, primarily in the pediatric population.

GAS is diagnosed by either an rapid strep test or by culture.

Group B Strep

GBS, otherwise known as S. agalactiae, causes pneumonia and more importantly meningitis in neonates and the elderly. The American Congress of Obstetricians and Gynecologists now recommends that all women between 35 and 37 weeks gestation to be tested for group B strep. Those who test positive should be given prophylactic antibiotics during labor. The bacteria can cause premature rupture of the membranes during pregnancy and can colonize and cross the placenta to infect the fetus.

Laboratory diagnosis and Workup

After initial culture and preliminary identification of beta-hemolytic streptococcus is suspected there are further identification tests to make an accurate diagnosis. The first is by Lancefield antigen determination. Commercially available Lancefield antisera is used for the differentiation of beta-hemolytic species. These usually come as small kits and are directed to Lancefield groups A, B, C, F, and G. Antigen detection is demonstrated by agglutination by specific antibodies that are provided.

The PYR test is a rapid colormetric method most often used to distinguish S. pyogenes from other beta-hemolytic species. The PYR tests for the presence of the enzyme pyrrolidonyl aminopeptidase. This enzyme hydrolyzes L-pyrrolidonyl-b-naphthylamide (PYR) to B-naphthylamide, which produces a red color when a specific reagent is added. The test can be performed on paper strips that contain dried chromogenic substrates for the pyrrolidonyl aminopeptidase. S. pyogenes is PYR positive and in the case of an unknown organism displaying S. pyogenes morphology plus being PYR positive, it is acceptable to presumptively identify as S. pyogenes.

Bacitracin susceptibility is another test that can be used to differentiate S. pyogenes from other beta-hemolytic strep species. S. pyogenes has an increased susceptibility to bacitracin. A pure culture must be obtained and streaked on a sheep blood agar plate. A small disk containing 0.04 U of bacitracin is placed on the plate and incubated overnight at 35 degrees celsius in 5% CO2. A zone of inhibition surrounding the disk indicates susceptibility.

With the introduction and advancement of nucleic acid detection and serological methods in the laboratory it is now getting easier and faster to detect strep species without relying on culturing and further confirmation testing. It should be important to note that this does not replace the use of a culture or any of the further testing mentioned above. Serological testing relies on antibodies to anti-streptolysin O and anti-DNase B. The antibody levels against streptolysin O rise within one week of infection and peak around 3-6 weeks. DNase B is a nuclease among many that S. pyogenes uses to escape neutrophil extracellular nets. DNase B is specific for S. pyogenes. The antibody levels to DNase B rise post two weeks infection and reach maximum titers at around 6-8 weeks.

The Optochin test is used to differentiate alpha-hemolytic streptococci. This is either S. pneumoniae or strep viridians. S. pneumoniae species are sensitive to the chemical ethylhydrocupreine hydrochloride, otherwise known as optochin. Optochin disks, sometimes called P disks can be obtained from a commercial vendor. A pure culture is obtain and incubated to allow growth overnight. When growth has occurred, a subculture is plated and a P disk is placed on the agar and incubated overnight at 35 degrees celsius with 5% CO2. The zone of inhibition is then measured and a zone greater than 14 mm indicates sensitivity and allows for the presumptive diagnosis of pneumococci.

The bile solubility test is usually used as confirmatory test for S. pneumoniae that distinguishes it from other alpha-hemolytic species. Sodium deoxycholate (Bile) will lyse the cell wall of pneumococcal species.

-Caleb

PCR, A Surge Forward

What is it?

PCR or polymerase chain reaction is a technique used in molecular biology and diagnostics to amplify a single or few copies of a sequence of DNA or RNA into thousands to millions. Developed in 1983 by Kary Mullis, who also went on to win the Nobel Prize in 1993.

It changed the way for a lot of different domains. PCR is an indispensable technique used in clinical and research laboratories with a broad span of applications. PCR is used for DNA cloning, gene cloning and manipulation, gene mutagenesis and functional analysis of genes for diagnostic or monitoring purposes.

PCR is dependent on thermal cycling; that is exposing the reactants to cycles of repeated heating and cooling which allows different temperature dependent reactions to take hold.

So, how does it work?

The basic PCR set-up requires several specific components and reagents. There needs to be a DNA template that contains the DNA target sequence that is targeted for amplification. DNA polymerase is needed, more specifically taq polymerase as it is heat-resistant. Taq polymerase is an enzyme that is isolated from the thermophilic bacterium Thermus aquaticus. It can survive the high-temperature DNA denaturation phase. Primers that are complementary to the 3′ ends of each sense and anti-sense strands of the DNA target. Primers are specific and complementary to the target sequence and are often selected beforehand. More than likely the primers are artificially synthesized from a commercial biochemical supplier. Deoxynucleoside triphosphates of dNTPs are the building blocks from which taq polymerase synthesizes a new strand.

There are three steps to PCR; The first step, denaturation is the first step in the natural cycle of events and consists of heating the reaction to 94-98 degrees Celsius for 20-30 seconds. This causes DNA denaturation of the dsDNA template by breaking the hydrogen bonds between the complementary base-pairs. The result is two, ssDNA molecules. Annealing is the second step in PCR and the temperature is lowered to 50-65 degrees Celsius (122-149 F) for 20-40 seconds. This allows annealing of the primers to each of the ssDNA molecules. The temperature in the annealing step is critical because you must select a temperature low enough to allow hybridization of the primer to the strands, but high enough for the hybridization to be specific to the target sequence selected to amplify. The primer should only bind to the complementary part of the strand and no where else. If the temperature is too low the primer may bind improperly and cause issues or may not bind at all. Extension and elongation is the final step in PCR where the DNA taq polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding free dNTPs from the reaction mixture in a 5′ to 3′ direction. The reaction is raised to about 72 degrees Celsius. The 5′-phosphate group is condensed to the 3′-hydroxyl group at the end of the nascent or elongating new DNA strand.

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At the elongation step in each cycle, the number of DNA copies is doubled. Denaturation, annealing, and elongation constitute a single cycle.

PCR can fail for various reasons. PCR is very sensitive to contamination causing DNA amplification of erroneous DNA products. Primer-design techniques are important to improving PCR product yield and in avoiding the production of wrong DNA products. There are multiple primer rules that should be followed; Primers should be between 22-26 bases in length with optimization at 24. Specificity and Tm (Melting temperature) should be between 58-66 degrees Celsius. Both primers should have a similar Tm (+/- 2 degrees). Keep the G-C base pair content between 40-60% , optimization at 50% if possible. Avoid repetitive sequences (AAAA, TATATA) as they can cause mis-priming. Because annealing of the primer is most critical at its 3′ end, a primer that has a high G-C content at its 3′ end is more likely to cause mis-priming.

Some notable analogs of PCR are RT-PCR (Reverse transcriptase PCR) and qPCR (quantitative PCR). RT-PCR is used for amplifying DNA from RNA. Reverse transcriptase enzyme transcribes RNA template into cDNA, which is then amplified. This is widely used for gene expression profiling or to identify the sequence of an RNA transcript. This determines the expression profile of a gene if known. It can be used to map the exons and introns in the gene. qPCR is used to measure the quantity of the target sequence. It quantitatively measures starting amounts of DNA, cDNA, or RNA and measures the amount of copies in the sample. qPCR is highly specific and precise. Usually qPCR methods use fluorescent dyes, most commonly Sybr green or Taqman which measures the amount of amplified product in real time.

PCR and its PCR derivatives have evolved and changed the way clinical labs and diagnostics are performed. There is a wide range of use in medicine.

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