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Substrates of cytochemical reactions in this immunostaining

Substrates of cytochemical reactions in this immunostaining


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Expression of extracellular protein Laminin 9 alpha-4 chain in human skeletal muscle. Indirect immunostaining with HRP immunostain marker. Ob.x40.

I have unsuccessfully searched NCBI -database, JSTOR and other major Biology databases for an answer. This suggests to me that I do not understand what is going on.

1. What are the substrates of above cytochemical reactions for the given immunostaining above?


I need to give a simpler question where I apparently know the reaction exactly, since it is possible that people cannot answer the above question.

Mitochondria in Hep-2 cell line cells. Cytochemical test for mitochondria specific enzyme NAHD dehydrogenase. Ob.x40.

2. What is the substrate of the cytochemical reaction?

My answer:

The reaction of NADH dehydrogenase is:

NADH + H+ + CoQ → NAD+ + CoQH2

Substrate: CoQ


It's hard to understand the question, but in any immunocytochemical staining such as the above, you have two different types of reactions:

  • the antibody binding to the target (in this case, some laminin)
  • the peroxidase-based colorimetric reaction with DAB. DAB (3,3'-diaminobenzidine tetrahydrochloride) is oxidized in the presence of hydrogen peroxide to form a brown precipitate, which becomes the stain.

The fact that is an indirect immunostaining implies that you have a non-conjugated primary antibody against laminin and a secondary antibody against the Fc part of immunoglobulins of the species in which the first antibody was raised. This secondary antibody will be conjugated with HRP (horseradish peroxidase, the enzyme responsible of generating the peroxides that will oxidize the DAB).

For a more complete reference of immunocytochemical stainings, you can read: http://www.ihcworld.com/_books/Dako_Handbook.pdf


20.4: Enzyme Immunoassays (EIA) and Enzyme-Linked Immunosorbent Assays (ELISA)

  • Contributed by OpenStax
  • General Biology at OpenStax CNX
  • Explain the differences and similarities between EIA, FEIA, and ELISA
  • Describe the difference and similarities between immunohistochemistry and immunocytochemistry
  • Describe the different purposes of direct and indirect ELISA

Similar to the western blot, enzyme immunoassays (EIAs) use antibodies to detect the presence of antigens. However, EIAs differ from western blots in that the assays are conducted in microtiter plates or in vivo rather than on an absorbent membrane. There are many different types of EIAs, but they all involve an antibody molecule whose constant region binds an enzyme, leaving the variable region free to bind its specific antigen. The addition of a substrate for the enzyme allows the antigen to be visualized or quantified (Figure (PageIndex<1>)).

In EIAs, the substrate for the enzyme is most often a chromogen, a colorless molecule that is converted into a colored end product. The most widely used enzymes are alkaline phosphatase and horseradish peroxidase for which appropriate substrates are readily available. In some EIAs, the substrate is a fluorogen, a nonfluorescent molecule that the enzyme converts into a fluorescent form. EIAs that utilize a fluorogen are called fluorescent enzyme immunoassays (FEIAs). Fluorescence can be detected by either a fluorescence microscope or a spectrophotometer.

Figure (PageIndex<1>): Enzyme immunoassays, such as the direct ELISA shown here, use an enzyme-antibody conjugate to deliver a detectable substrate to the site of an antigen. The substrate may be a colorless molecule that is converted into a colored end product or an inactive fluorescent molecule that fluoresces after enzyme activation. (credit: modification of work by &ldquoCavitri&rdquo/Wikimedia Commons)

The MMR vaccine is a combination vaccine that provides protection against measles, mumps, and rubella (German measles). Most people receive the MMR vaccine as children and thus have antibodies against these diseases. However, for various reasons, even vaccinated individuals may become susceptible to these diseases again later in life. For example, some children may receive only one round of the MMR vaccine instead of the recommended two. In addition, the titer of protective antibodies in an individual&rsquos body may begin to decline with age or as the result of some medical conditions.

To determine whether the titer of antibody in an individual&rsquos bloodstream is sufficient to provide protection, an MMR titer test can be performed. The test is a simple immunoassay that can be done quickly with a blood sample. The results of the test will indicate whether the individual still has immunity or needs another dose of the MMR vaccine.

Submitting to an MMR titer is often a pre-employment requirement for healthcare workers, especially those who will frequently be in contact with young children or immunocompromised patients. Were a healthcare worker to become infected with measles, mumps, or rubella, the individual could easily pass these diseases on to susceptible patients, leading to an outbreak. Depending on the results of the MMR titer, healthcare workers might need to be revaccinated prior to beginning work.

Immunostaining

One powerful use of EIA is immunostaining, in which antibody-enzyme conjugates enhance microscopy. Immunohistochemistry (IHC) is used for examining whole tissues. As seen in Figure (PageIndex<2>), a section of tissue can be stained to visualize the various cell types. In this example, a mAb against CD8 was used to stain CD8 cells in a section of tonsil tissue. It is now possible to count the number of CD8 cells, determine their relative numbers versus the other cell types present, and determine the location of these cells within this tissue. Such data would be useful for studying diseases such as AIDS, in which the normal function of CD8 cells is crucial for slowing disease progression.

Immunocytochemistry (ICC) is another valuable form of immunostaining. While similar to IHC, in ICC, extracellular matrix material is stripped away, and the cell membrane is etched with alcohol to make it permeable to antibodies. This allows antibodies to pass through the cell membrane and bind to specific targets inside the cell. Organelles, cytoskeletal components, and other intracellular structures can be visualized in this way. While some ICC techniques use EIA, the enzyme can be replaced with a fluorescent molecule, making it a fluorescent immunoassay.

Figure (PageIndex<2>): Enzyme-linked antibodies against CD8 were used to stain the CD8 cells in this preparation of bone marrow using a chromogen. (credit: modification of work by Yamashita M, Fujii Y, Ozaki K, Urano Y, Iwasa M, Nakamura S, Fujii S, Abe M, Sato Y, Yoshino T)

  1. What is the difference between immunohistochemistry and immunocytochemistry?
  2. What must be true of the product of the enzymatic reaction used in immunohistochemistry?

Enzyme-linked Immunosorbent Assays (ELISAs)

The enzyme-linked immunosorbent assays (ELISAs) are widely used EIAs. In the direct ELISA, antigens are immobilized in the well of a microtiter plate. An antibody that is specific for a particular antigen and is conjugated to an enzyme is added to each well. If the antigen is present, then the antibody will bind. After washing to remove any unbound antibodies, a colorless substrate (chromogen) is added. The presence of the enzyme converts the substrate into a colored end product (Figure (PageIndex<1>)). While this technique is faster because it only requires the use of one antibody, it has the disadvantage that the signal from a direct ELISA is lower (lower sensitivity).

In a sandwich ELISA, the goal is to use antibodies to precisely quantify specific antigen present in a solution, such as antigen from a pathogen, a serum protein, or a hormone from the blood or urine to list just a few examples. The first step of a sandwich ELISA is to add the primary antibody to all the wells of a microtiter plate (Figure (PageIndex<3>)). The antibody sticks to the plastic by hydrophobic interactions. After an appropriate incubation time, any unbound antibody is washed away. Comparable washes are used between each of the subsequent steps to ensure that only specifically bound molecules remain attached to the plate. A blocking protein is then added (e.g., albumin or the milk protein casein) to bind the remaining nonspecific protein-binding sites in the well. Some of the wells will receive known amounts of antigen to allow the construction of a standard curve, and unknown antigen solutions are added to the other wells. The primary antibody captures the antigen and, following a wash, the secondary antibody is added, which is a polyclonal antibody that is conjugated to an enzyme. After a final wash, a colorless substrate (chromogen) is added, and the enzyme converts it into a colored end product. The color intensity of the sample caused by the end product is measured with a spectrophotometer. The amount of color produced (measured as absorbance) is directly proportional to the amount of enzyme, which in turn is directly proportional to the captured antigen. ELISAs are extremely sensitive, allowing antigen to be quantified in the nanogram (10 &ndash9 g) per mL range.

In an indirect ELISA, we quantify antigen-specific antibody rather than antigen. We can use indirect ELISA to detect antibodies against many types of pathogens, including Borrelia burgdorferi (Lyme disease) and HIV. There are three important differences between indirect and direct ELISAs as shown in Figure (PageIndex<4>). Rather than using antibody to capture antigen, the indirect ELISA starts with attaching known antigen (e.g., peptides from HIV) to the bottom of the microtiter plate wells. After blocking the unbound sites on the plate, patient serum is added if antibodies are present (primary antibody), they will bind the antigen. After washing away any unbound proteins, the secondary antibody with its conjugated enzyme is directed against the primary antibody (e.g., antihuman immunoglobulin). The secondary antibodyallows us to quantify how much antigen-specific antibody is present in the patient&rsquos serum by the intensity of the color produced from the conjugated enzyme-chromogen reaction.

As with several other tests for antibodies discussed in this chapter, there is always concern about cross-reactivity with antibodies directed against some other antigen, which can lead to false-positive results. Thus, we cannot definitively diagnose an HIV infection (or any other type of infection) based on a single indirect ELISA assay. We must confirm any suspected positive test, which is most often done using either an immunoblot that actually identifies the presence of specific peptides from the pathogen or a test to identify the nucleic acids associated with the pathogen, such as reverse transcriptase PCR (RT-PCR) or a nucleic acid antigen test.

Figure (PageIndex<3>): (a) In a sandwich ELISA, a primary antibody is used to first capture an antigen with the primary antibody. A secondary antibody conjugated to an enzyme that also recognizes epitopes on the antigen is added. After the addition of the chromogen, a spectrophotometer measures the absorbance of end product, which is directly proportional to the amount of captured antigen. (b) An ELISA plate shows dilutions of antibodies (left) and antigens (bottom). Higher concentrations result in a darker final color. (credit b: modification of work by U.S. Fish and Wildlife Service Pacific Region) Figure (PageIndex<4>): The indirect ELISA is used to quantify antigen-specific antibodies in patient serum for disease diagnosis. Antigen from the suspected disease agent is attached to microtiter plates. The primary antibody comes from the patient&rsquos serum, which is subsequently bound by the enzyme-conjugated secondary antibody. Measuring the production of end product allows us to detect or quantify the amount of antigen-specific antibody present in the patient&rsquos serum.

  1. What is the purpose of the secondary antibody in a direct ELISA?
  2. What do the direct and indirect ELISAs quantify?

Although contacting and testing the 1300 patients for HIV would be time consuming and expensive, administrators hoped to minimize the hospital&rsquos liability by proactively seeking out and treating potential victims of the rogue employee&rsquos crime. Early detection of HIV is important, and prompt treatment can slow the progression of the disease.

There are a variety of screening tests for HIV, but the most widely used is the indirect ELISA. As with other indirect ELISAs, the test works by attaching antigen (in this case, HIV peptides) to a well in a 96-well plate. If the patient is HIV positive, anti-HIV antibodies will bind to the antigen and be identified by the second antibody-enzyme conjugate.

  1. How accurate is an indirect ELISA test for HIV, and what factors could impact the test&rsquos accuracy?
  2. Should the hospital use any other tests to confirm the results of the indirect ELISA?

Immunofiltration and Immunochromatographic Assays

For some situations, it may be necessary to detect or quantify antigens or antibodies that are present at very low concentration in solution. Immunofiltration techniques have been developed to make this possible. In immunofiltration, a large volume of fluid is passed through a porous membrane into an absorbent pad. An antigen attached to the porous membrane will capture antibody as it passes alternatively, we can also attach an antibody to the membrane to capture antigen.

The method of immunofiltration has been adapted in the development of immunochromatographic assays, commonly known as lateral flow tests or strip tests. These tests are quick and easy to perform, making them popular for point-of-care use (i.e., in the doctor&rsquos office) or in-home use. One example is the TORCH test that allows doctors to screen pregnant women or newborns for infection by an array of viruses and other pathogens (Toxoplasma, other viruses, rubella, cytomegalovirus, herpes simplex). In-home pregnancy tests are another widely used example of a lateral flow test (Figure (PageIndex<5>)). Immunofiltration tests are also popular in developing countries, because they are inexpensive and do not require constant refrigeration of the dried reagents. However, the technology is also built into some sophisticated laboratory equipment.

In lateral flow tests (Figure (PageIndex<6>)), fluids such as urine are applied to an absorbent pad on the test strip. The fluid flows by capillary action and moves through a stripe of beads with antibodies attached to their surfaces. The fluid in the sample actually hydrates the reagents, which are present in a dried state in the stripe. Antibody-coated beads made of latex or tiny gold particles will bind antigens in the test fluid. The antibody-antigen complexes then flow over a second stripe that has immobilized antibody against the antigen this stripe will retain the beads that have bound antigen. A third control stripe binds any beads. A red color (from gold particles) or blue (from latex beads) developing at the test line indicates a positive test. If the color only develops at the control line, the test is negative.

Like ELISA techniques, lateral flow tests take advantage of antibody sandwiches, providing sensitivity and specificity. While not as quantitative as ELISA, these tests have the advantage of being fast, inexpensive, and not dependent on special equipment. Thus, they can be performed anywhere by anyone. There are some concerns about putting such powerful diagnostic tests into the hands of people who may not understand the tests&rsquo limitations, such as the possibility of false-positive results. While home pregnancy tests have become widely accepted, at-home antibody-detection tests for diseases like HIV have raised some concerns in the medical community. Some have questioned whether self-administration of such tests should be allowed in the absence of medical personnel who can explain the test results and order appropriate confirmatory tests. However, with growing numbers of lateral flow tests becoming available, and the rapid development of lab-on-a-chip technology ([link]), home medical tests are likely to become even more commonplace in the future.

Figure (PageIndex<5>): A lateral flow test detecting pregnancy-related hormones in urine. The control stripe verifies the validity of the test and the test line determines the presence of pregnancy-related hormones in the urine. (credit: modification of work by Klaus Hoffmeier) Figure (PageIndex<6>): Immunochromatographic assays, or lateral flow tests, allow the testing of antigen in a dilute solution. As the fluid flows through the test strip, it rehydrates the reagents. Antibodies conjugated to small particles bind the antigen in the first stripe and then flow onto the second stripe where they are bound by a second, fixed antibody. This produces a line of color, depending on the color of the beads. The third, control stripe binds beads as well to indicate that the test is working properly. (credit: modification of work by Yeh CH, Zhao ZQ, Shen PL, Lin YC)

  1. What physical process does the lateral flow method require to function?
  2. Explain the purpose of the third strip in a lateral flow assay.

Table (PageIndex<1>) compares some of the key mechanisms and examples of some of the EIAs discussed in this section as well as immunoblots, which were discussed in Detecting Antigen-Antibody Complexes.

Table (PageIndex<1>): Immunoblots & Enzyme Immunoassays
Type of Assay Mechanism Specific Procedures Examples
Immunoblots Uses enzyme-antibody conjugates to identify specific proteins that have been transferred to an absorbent membrane Western blot: Detects the presence of a particular protein Detecting the presence of HIV peptides (or peptides from other infectious agents) in patient sera
Immunostaining Uses enzyme-antibody conjugates to stain specific molecules on or in cells Immunohistochemistry: Used to stain specific cells in a tissue Stain for presence of CD8 cells in host tissue
Enzyme-linked immunosorbent assay (ELISA) Uses enzyme-antibody conjugates to quantify target molecules Direct ELISA: Uses a single antibody to detect the presence of an antigen Detection of HIV antigen p24 up to one month after being infected
Indirect ELISA: Measures the amount of antibody produced against an antigen Detection of HIV antibodies in serum
Immunochromatographic (lateral flow) assays Techniques use the capture of flowing, color-labeled antigen-antibody complexes by fixed antibody for disease diagnosis Sandwich ELISA: Measures the amount of antigen bound by the antibody Detection of antibodies for various pathogens in patient sera (e.g., rapid strep, malaria dipstick)
Pregnancy test detecting human chorionic gonadotrophin in urine

Although the indirect ELISA for HIV is a sensitive assay, there are several complicating considerations. First, if an infected person is tested too soon after becoming infected, the test can yield false-negative results. The seroconversion window is generally about three weeks, but in some cases, it can be more than two months.

In addition to false negatives, false positives can also occur, usually due to previous infections with other viruses that induce cross-reacting antibodies. The false-positive rate depends on the particular brand of test used, but 0.5% is not unusual. 1 Because of the possibility of a false positive, all positive tests are followed up with a confirmatory test. This confirmatory test is often an immunoblot (western blot) in which HIV peptides from the patient&rsquos blood are identified using an HIV-specific mAb-enzyme conjugate. A positive western blot would confirm an HIV infection and a negative blot would confirm the absence of HIV despite the positive ELISA.

Unfortunately, western blots for HIV antigens often yield indeterminant results, in which case, they neither confirm nor invalidate the results of the indirect ELISA. In fact, the rate of indeterminants can be 10&ndash49% (which is why, combined with their cost, western blots are not used for screening). Similar to the indirect ELISA, an indeterminant western blot can occur because of cross-reactivity or previous viral infections, vaccinations, or autoimmune diseases.

  1. Of the 1300 patients being tested, how many false-positive ELISA tests would be expected?
  2. Of the false positives, how many indeterminant western blots could be expected?
  3. How would the hospital address any cases in which a patient&rsquos western blot was indeterminant?

Computerized analysis of cytochemical reactions for dehydrogenases and oxygraphic studies as methods to evaluate the function of the mitochondrial sheath in rat spermatozoa

Cytochemical reactions for mitochondrial NADH-dependent dehydrogenases (diaphorase/NADH which is related to flavoprotein), NAD-dependent dehydrogenases (isocitrate, malate) and succinate dehydrogenase were carried out in rat spermatozoa. In addition to a morphological evaluation, the intensity of the reactions was assessed using a computer image analysing system (Quantimet 600 S). The intensity of the reactions was examined in sperm midpieces by measuring integrated optical density (IOD) and mean optical density (MOD). The activity of mitochondrial respiratory chain complexes was also analysed using the polarographic method.

In the population of spermatozoa studied, all whole spermatozoa midpieces were completely filled with formazans, the product of the cytochemical reaction. These morphological findings corresponded to the values obtained for IOD and MOD for the given enzymes. In the oxygraphic studies, the spermatozoa demonstrated consumption of oxygen in the presence of substrates for I, II and IV complexes and their mitochondria revealed normal integrity and sensitivity to the substrates and inhibitors. However, the oxygraphic studies revealed differences between the sperm and somatic cells. These differences concerned the stimulation of pyruvate oxidation by malate, the lack of an effect of malonic acid on phenazine methosulphate (an acceptor of electrons) oxidation and the lack of an effect of cytochrome c on ascorbate oxidation.

The cytochemical method, together with densitometric measurements, enables: (1) the reaction intensity to be determined objectively (2) subtle and dramatic differences in reaction intensity to be revealed between spermatozoa that do not differ under morphological evaluation of the intensity (3) possible defects within the mitochondrial sheath to be located and assessed in a large number of spermatozoa. This method can be used as a screening method alongside the routine morphological examination of spermatozoa. On the other hand, the oxygraphic method in the inner membrane of mitochondria can reveal functional changes which are related to the action of respiratory chain complexes and display characteristic features of mitochondria energy metabolism. The methods used are complementary and allow the complex evaluation of mitochondria in spermatozoa. Both methods can be used in experimental and clinical studies.


Endocrinology

3.7.9.1.2 Kinetic parameters and specificity

JHE kinetics and substrate specificity have long been a central focus of JHE studies, owing to the obvious agricultural potential of this information. Elucidation of JHE kinetic constants also permits predictions of the rate limits of JH metabolism, as well as provide valuable insight into to how peripheral JH levels are regulated. A number of reports from the 1980s indicate that among lepidopterans, the apparent Michaelis constant (Km) for the naturally occurring JHs ranged from 10 −8 to 10 −6 M ( Roe and Venkatesh, 1990 ). JH titers in the species tested are at least 10–100 times lower than the estimated Km concentration, indicating that the enzyme is very sensitive to changes in JH concentration, but suggesting that its catalytic potential is wasted. Since the Km values seem unduly high with regard to substrate concentration, investigators have examined the individual rate components of the enzymatic reaction to better explain the observed data ( Sparks and Rose, 1983 Hammock, 1985 Abdel-Aal and Hammock, 1986 ).

In the simplest of terms, the velocity of JHE or any enzymatic reaction can be viewed as the sum of various rate components and the concentration of the reactants, and can thus be defined by the familiar equation:

where E represents enzyme, S represents substrate, ES represent the Michaelis complex, and P the product. Under physiological conditions, where the concentration of JH is several orders of magnitude lower than the Km, the rate at which JH acid is formed is a function of JH interaction with JHE (k1 and k−1) and the rate of product formation (k2). Several important lessons can be drawn from the kinetic data for JHE. First, JHE has a very high affinity for JH. Second, it has a relatively low turnover number or kcat, where kcat is the maximum number of substrate molecules (JH) converted to product (JH acid) per active site per unit time. These factors make the enzyme a very effective scavenger that can “find” JH at physiological concentrations and convert it to JH acid ( Abdel-Aal and Hammock, 1986 ).

Substrate specificity of JHE represents another characteristic that seems counterintuitive. The Km and Vmax that the JHEs display towards the JH homologs is surprisingly low when compared to other substrates, such as α-naphthyl acetate. For example, recombinant M. sexta JHE displays a Km (410 μM) and Vmax (21 μmol min −1 mg protein −1 ) for α-naphthyl acetate that is considerably higher than for its natural substrate, JH III (Km = 0.052 μM, Vmax = 1.4 μmol min −1 mg protein −1 ) ( Hinton and Hammock, 2003 ). As noted by Fersht (1985) , when specificity is used to discriminate between two competing compounds, it should be determined by the kcat/Km ratios, and not Km alone. The kcat/Km ratio for JH III and α-naphthyl acetate are 27 and 0.04, respectively, underscoring the high degree of specificity of JHE for JH III ( Hinton and Hammock, 2003 ). While these kinetic parameters suggest that the enzyme has a high degree of specificity, it appears that JHEs from several species also hydrolyze methyl and ethyl esters of JH I and III at very similar rates ( Grieneisen et al., 1997 ). Moreover, even n-propyl and n-butyl esters of these homologs can serve effectively as substrates, albeit at a lower rate of hydrolysis. As might be expected, the unnatural (2Z,6E)-JH I ethyl ester isomer is not metabolized by JHEs from M. sexta or H. virescens.

Since hJHBPs have a significant effect on substrate availability ( Peter et al., 1979 Peter, 1990 King and Tobe, 1993 ), studies on JHE specificity must be performed with relatively pure preparations of the enzyme. In those few studies using highly purified or recombinant T. ni JHE, it was determined that the JHE hydrolyzed JH I more rapidly than JH II or III and that it displayed a faster catalytic rate for the naturally occurring enantiomer, (10R,11S)-JH II, than for the (10S,11R)-JH II form ( Hanzlik and Hammock, 1987 ). However, a comparison of the kcat/Km ratios for the enantiomers indicated that hydrolysis of the two forms was equivalent ( Hanzlik and Hammock, 1987 ). The same holds true for the gypsy moth, Lymantria dispar, in which the JHE shows no enantiomeric selectivity and appears unable to discriminate between homologs ( Valaitis, 1991 ). One of the most unexpected groups of JHE substrates is found in the naphthyl and p-nitrophenyl series. While it was originally concluded that hemolymph JHE from some species, such as M. sexta, could not use α-naphthyl acetate as a substrate ( Coudron, 1981 ), the JHEs of other species can recognize members of the naphthyl series ( Rudnicka and Kochman, 1984 Hanzlik and Hammock, 1987 ). More recently it was demonstrated that recombinant JHE from M. sexta, H. virescens, and T. molitor can hydrolyze naphthyl compounds with chain derivatives eight carbons long, thus dispelling the long-held belief that JHE from M. sexta is unable to use naphthyl derivatives as substrates ( Kamita et al., 2003 ).

Another counterintuitive observation is that while the major role of JHE is the conversion of JH to JH acid, both native and recombinant JHEs from several species can, under the appropriate conditions, transesterify JH to form the higher ester homologs, i.e., JH ethyl, JH n-propyl, and JH n-butyl esters ( Grieneisen et al., 1997 ). Although JHE-mediated JH transesterification may be a curiosity limited to the test tube, Debernard et al. (1995) demonstrated that when JH III, dissolved in ethanol (10 μl), was injected into L. migratoria , it was both converted to JH III acid and transesterified to the JH III ethyl ester. With regard to this particular study, it should be noted that care must be taken to avoid artifacts when alcohols are used as carrier solvents for the hormone in JHE assays. Nevertheless, while JHE in a biological milieu clearly serves as an esterase , these new findings imply that the enzyme may have other physiological roles (see Anspaugh et al. (1995) for other possible roles for JHE).


V. An appraisal of indigogenic reactions for esterase localization

Results are described of comparative studies of indoxyl acetates, used as indigogenic substrates, in cytochemical staining processes for esterases in formalin-fixed tissues. Correlations have been observed between the staining patterns produced, molecular structures of the substrates and derived dyes, solubilities and substantivities of the dyes, and rates of oxidation of the enzymically produced indoxyls. This has enabled an objective choice to be made of the most precise staining systems, and has led to the systematic development of 5-bromo-4-chloroindoxyl acetate, an improved substrate which enables esterases to be located with a precision of about 0⋅5 μ. Kinetics of indoxyl oxidation in tissues indicate that the staining processes have considerable quantitative potentialities.


Substrates and chromogens for IHC

The choice of chromogen used is directed by the enzyme employed in the experiment, as well as a number of other factors. Here, we review guidelines for selecting the appropriate enzyme substrate for chromogenic detection.

Mounting media

Advantages (+) and disadvantages (-)

Horseradish peroxidase (HRP)

+ Intense color, contrasts well with blue in double staining.

Horseradish peroxidase (HRP)

Horseradish peroxidase (HRP)

Horseradish peroxidase (HRP)

Horseradish peroxidase (HRP)

+ Alternate color useful in multicolor chromogenic IHC

+ Less intense, good for double staining.

- Fast Blue BB prone to fading.

+ Less intense, good for double staining.

- Fast Red TR prone to fading.

Naphthol AS-MX phosphate + new fuchsin

+ Alternate color useful in multicolor chromogenic IHC

+ No endogenous enzyme activity, so does not suffer from endogenous peroxidases causing false positive staining.


MtDNA provides the first known marker distinguishing proto-Indians from the other

a marker for neuronal loss or mitochondrial dysfunction. PMID: 9778562

Succinate Dehydrogenase (SDH)

mitochondrial marker enzyme. Cytochemical localization of SDH was between the outer and the inner mitochondrial membranes. PMID: 2138522

transiently expressed mitochondrial protein. PMID: 10336016

Magnesium Dependent Adenosine Triphosphatase (Mg-ATPase)

mitochondrial marker enzyme. Mg-ATPase was on the inner surface or matrix side of the inner membrane. PMID: 2138522

an enzyme marker for the outer membrane of rat liver mitochondria. PMID: 5460462, PMID: 4291912

a subunit of yeast mitochondrial ATP synthase. PMID: 9247150

a highly conserved, 30- to 32-kDa mitochondrial membrane protein, occurs in a number of unexpected isoforms, ranging from 64 to greater than 185 kDa in the mammalian sperm mitochondria, which are the ubiquitinated substrates. PMID: 12646488

an antiproliferative protein, is localized to mitochondria. PMID: 7843414

Electrophoretical visualization of SOD patterns provided evidence for possible migration of cytosolic Cu/Zn SOD to mitochondria. The characteristic Cu/Zn SOD profile in mitochondria of all tested strains suggested its ubiquity within the fermentative and respiratory yeasts. PMID: 14734161

UCP-3 protein (mitochondrial uncoupling protein-3)

is present in mitochondria isolated from rat thymus and mitochondria isolated from reticulocytes, monocytes and lymphocytes of rat spleen. PMID: 15262223

UCP-3 is predominantly overexpressed in skeletal muscle. PMID: 12630934

VDAC (voltage-dependent anion channel)

Immunogold labeling and EM analysis of the cerebellar molecular layer showed specific VDAC immunostaining of the mitochondrial outer membrane, highly enhanced in contact sites between mitochondria or between mitochondria and associated ER. PMID: 15238267

Other Mitochondria Markers

A cytochemical marker for epidermal differentiation, Langerhans cells, skin resident macrophages and mitochondria. PMID: 7138784

Diaminobenzidine cytochemistry in unfixed human epidermis: a marker for epidermal differentiation and for mitochondria. PMID: 6177799

Mitochondria DNA markers and genetic demographic processes in neolithic Europe. PMID: 9749344

Two- and three-point extranuclear crosses have been carried out via heterokaryons involving the three extranuclear mitochondrial markers of Aspergillus nidulans: (oliA1), (cs67) and (camA112). All three markers appear to be located on a single functional mitochondrial genome. PMID: 765725

Cobalt as a mitochondrial density marker in a study of cytoplasmic exchange during mating of Schizophyllum commune. PMID: 4127537

On the use of glutamate dehydrogenase as a mitochondrial marker enzyme for the determination of the intracellular distribution of rat liver pyruvate carboxylase. PMID: 11945602


Enzyme-linked Immunosorbent Assays (ELISAs)

The enzyme-linked immunosorbent assays (ELISAs) are widely used EIAs. In the direct ELISA, antigens are immobilized in the well of a microtiter plate. An antibody that is specific for a particular antigen and is conjugated to an enzyme is added to each well. If the antigen is present, then the antibody will bind. After washing to remove any unbound antibodies, a colorless substrate (chromogen) is added. The presence of the enzyme converts the substrate into a colored end product (Figure 1). While this technique is faster because it only requires the use of one antibody, it has the disadvantage that the signal from a direct ELISA is lower (lower sensitivity).

In a sandwich ELISA, the goal is to use antibodies to precisely quantify specific antigen present in a solution, such as antigen from a pathogen, a serum protein, or a hormone from the blood or urine to list just a few examples. The first step of a sandwich ELISA is to add the primary antibody to all the wells of a microtiter plate (Figure 3). The antibody sticks to the plastic by hydrophobic interactions. After an appropriate incubation time, any unbound antibody is washed away. Comparable washes are used between each of the subsequent steps to ensure that only specifically bound molecules remain attached to the plate. A blocking protein is then added (e.g., albumin or the milk protein casein) to bind the remaining nonspecific protein-binding sites in the well. Some of the wells will receive known amounts of antigen to allow the construction of a standard curve, and unknown antigen solutions are added to the other wells. The primary antibody captures the antigen and, following a wash, the secondary antibody is added, which is a polyclonal antibody that is conjugated to an enzyme. After a final wash, a colorless substrate (chromogen) is added, and the enzyme converts it into a colored end product. The color intensity of the sample caused by the end product is measured with a spectrophotometer. The amount of color produced (measured as absorbance) is directly proportional to the amount of enzyme, which in turn is directly proportional to the captured antigen. ELISAs are extremely sensitive, allowing antigen to be quantified in the nanogram (10 –9 g) per mL range.

Figure 3. Click for a larger image. (a) In a sandwich ELISA, a primary antibody is used to first capture an antigen with the primary antibody. A secondary antibody conjugated to an enzyme that also recognizes epitopes on the antigen is added. After the addition of the chromogen, a spectrophotometer measures the absorbance of end product, which is directly proportional to the amount of captured antigen. (b) An ELISA plate shows dilutions of antibodies (left) and antigens (bottom). Higher concentrations result in a darker final color. (credit b: modification of work by U.S. Fish and Wildlife Service Pacific Region)

In an indirect ELISA, we quantify antigen-specific antibody rather than antigen. We can use indirect ELISA to detect antibodies against many types of pathogens, including Borrelia burgdorferi (Lyme disease) and HIV. There are three important differences between indirect and direct ELISAs as shown in Figure 4. Rather than using antibody to capture antigen, the indirect ELISA starts with attaching known antigen (e.g., peptides from HIV) to the bottom of the microtiter plate wells. After blocking the unbound sites on the plate, patient serum is added if antibodies are present (primary antibody), they will bind the antigen. After washing away any unbound proteins, the secondary antibody with its conjugated enzyme is directed against the primary antibody (e.g., antihuman immunoglobulin). The secondary antibody allows us to quantify how much antigen-specific antibody is present in the patient’s serum by the intensity of the color produced from the conjugated enzyme-chromogen reaction.

As with several other tests for antibodies discussed in this chapter, there is always concern about cross-reactivity with antibodies directed against some other antigen, which can lead to false-positive results. Thus, we cannot definitively diagnose an HIV infection (or any other type of infection) based on a single indirect ELISA assay. We must confirm any suspected positive test, which is most often done using either an immunoblot that actually identifies the presence of specific peptides from the pathogen or a test to identify the nucleic acids associated with the pathogen, such as reverse transcriptase PCR (RT-PCR) or a nucleic acid antigen test.

Figure 4. Click for a larger image. The indirect ELISA is used to quantify antigen-specific antibodies in patient serum for disease diagnosis. Antigen from the suspected disease agent is attached to microtiter plates. The primary antibody comes from the patient’s serum, which is subsequently bound by the enzyme-conjugated secondary antibody. Measuring the production of end product allows us to detect or quantify the amount of antigen-specific antibody present in the patient’s serum.

Think about It

  • What is the purpose of the secondary antibody in a direct ELISA?
  • What do the direct and indirect ELISAs quantify?

Clinical Focus: HIV Part 2

This example continues the story that started in Polyclonal and Monoclonal Antibody Production.

Although contacting and testing the 1300 patients for HIV would be time consuming and expensive, administrators hoped to minimize the hospital’s liability by proactively seeking out and treating potential victims of the rogue employee’s crime. Early detection of HIV is important, and prompt treatment can slow the progression of the disease.

There are a variety of screening tests for HIV, but the most widely used is the indirect ELISA. As with other indirect ELISAs, the test works by attaching antigen (in this case, HIV peptides) to a well in a 96-well plate. If the patient is HIV positive, anti-HIV antibodies will bind to the antigen and be identified by the second antibody-enzyme conjugate.

  • How accurate is an indirect ELISA test for HIV, and what factors could impact the test’s accuracy?
  • Should the hospital use any other tests to confirm the results of the indirect ELISA?

We’ll return to this example later on this page.


Order Details

To use as substrate/chromogen in conjunction with peroxidase based immunostaining systems.
Note: The working chromogen solution is stable for 6 hours. Any solution not used after this period should be discarded.
1. Take 5 ml of distilled or de-ionized water in a test tube.
2. Add two drops of concentrated buffer and mix.
3. Add two drops of concentrated AEC Chromogen and mix.
4. Add two drops of 3% H2O2 substrate solution and mix.

Procedure:
1. Once tissue sections have been incubated with peroxidase, wash them with buffer thoroughly.
2. Wipe the class to remove excess buffer and add enough drops of the working AEC solution to cover the tissue sections.
3. Incubate for 10-20 mintues at room temperature. For the best results, look under the microscope for the signal development. Once desired signal to noise ratio is achieved, stop the reaction by washing slides in wash buffer.


Materials and methods

DNA constructs and transfections

pEGFP-HA-KAP1wt (wild type) and pEGFP-HA-KAP1S824A were a gift from Y Shiloh (Tel Aviv University, Israel). pEGFP-HA-KAP1S473A and pEGFP-HA-KAP1S473D were made by site-directed mutagenesis of pEGFP-HA-KAP1wt using the primers: KAP1-S473A-F, 5'-GAAACGGTCCCGCGCAGGTGAGGGCGAG-3' KAP1-S473A-R, 5'-CTCGCCCTCACCTGCGCGGGACCGTTTC-3' KAP1-S473D-F, 5'-GGTGTGAAACGGTCCCGCGACGGTGAGGGCGAGGTGAGC-3' KAP1-S473D-R, 5'-GCTCACCTCGCCCTCACCGTCGCGGGACCGTTTCACACC-3'.

Plasmid DNA was transfected with FuGENE 6 reagent (Roche Diagnostics Ltd., Burgess Hill, UK)) following the manufacturer's instructions.

Expression and purification of recombinant proteins

pFastBac-TEV-SBP-Chk1wt was prepared by amplifying Chk1 from pCIneo-FLAG-Chk1 (provided by J Bartek, Institute of Cancer Biology, Copenhagen, Denmark) and cloning it into pFastBac1-TEV-SBP (gift from P Marco-Casanova, Gurdon Institute, Cambridge, UK) via EcoRI and XbaI restriction sites. Bacmids were prepared in DH10Bac™ Escherichia coli cells (Invitrogen, Carlsbad, CA, USA)) following the manufacturer's protocol. Primers for site-directed mutagenesis of Chk1 Leu84 were: Chk1L84G-F, 5'-GCAATATCCAATATTTATTTGGGGAGTACTGTAGTGGAGGAGAGC-3' Chk1L84G-R, 5'-GCTCTCCTCCACTACAGTACTCCCCAAATAAATATTGGATATTGC-3' Chk1L84A-F, 5'-GCAATATCCAATATTTATTTGCGGAGTACTGTAGTGGAGGAGAGC-3' and Chk1L84A-R, 5'-GCTCTCCTCCACTACAGTACTCCGCAAATAAATATTGGATATTGC-3'. SBP-tagged wild type and mutated Chk1 proteins were expressed in Sf9 insect cells and purified to homogeneity as described for SBP-tag purification [53]. pGEX20T-Cdc25A was a gift from J Bartek (Institute of Cancer Biology, Copenhagen, Denmark). GST-Cdc25A was expressed in BL21 E. coli cells and purified with glutathione sepharose beads following the manufacturer's instructions.

Protein kinase assays

All in vitro kinase assays were done in Chk1 kinase buffer (50 mM HEPES, pH 7.4 13.5 mM MgCl2 and 1 mM dithiothreitol) in the presence of 1 mM Na3VO4 and 1 mM ATP or ATP analogue. Reactions were incubated for 30 minutes at 30°C and stopped by addition of 10 mM EDTA, pH 8. For western blotting, proteins were mixed with Laemmli buffer and separated on 9% SDS-polyacrylamide gels.

Western blotting

Proteins were separated by SDS-PAGE. Antibodies used were: Chk1 (1:100 mouse G4 Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Chk1 phospho-Ser317 (1:1,000 rabbit Cell Signaling Technology, Danvers, MA, USA), Chk1 phospho-Ser345 (1:5,000 rabbit Cell Signaling), Chk2 phospho-Thr-68 (1:1,000 rabbit Cell Signaling), Cdc25A (1:100 mouse Santa Cruz), Cdc25A phospho-Ser123 was provided by E Appella (National Cancer Institute, Bethesda, US), GFP (1:1,000 mouse Roche), histone H3 phospho-Ser10 (1:5,000 mouse Abcam, Cambridge, UK), KAP1 (1:500 rabbit Santa Cruz), KAP1 phospho-Ser824 (1:1,000 rabbit Bethyl Laboratories, Inc., Montgomery, TX, USA), KAP1 phospho-Ser473 (1:1,000 rabbit BioLegend), tubulin (1:5,000 mouse Sigma Aldrich Company, Ltd., Dorset, UK), thiophosphate-ester-specific antibody (1:5,000 Epitomics, Inc., Burlingame, CA, USA) according to the manufacturers' instructions.

Large-scale kinase assay, purification of phospho-peptides and mass spectrometry

Large-scale Chk1 kinase assay and subsequent peptide enrichment was as previously described [54]. Briefly, 1 mg of HeLa nuclear extract (Cil Biotech, Mons, Belgium) was incubated with 10 μg of SBP-Chk1L84G in the presence of 1 mM Na3VO4 and 1 mM N6B-ATPγS in 1× Chk1 kinase buffer for 30 minutes at 30°C. Reactions were stopped by addition of EDTA. Trypsin digestion was done in denaturing buffer following a standard protocol. Phosphopeptides were enriched using a previously described method [16]. Briefly, 100 μl of iodoacetyl-agarose beads (SulfoLink gel, Thermo Fisher Scientific Inc., Rockford, IL, USA) in 100 μl of 50% acetonitrile were added to trypsin-digested peptides. The beads were extensively washed with 2 ml each of water, 5 M NaCl, 50% acetonitrile, and 5% formic acid in water, sequentially. Phosphopeptides were eluted using 200 μl of a 1 mg/ml solution of Oxone, and purified on C18 StageTips [55]. Phosphopeptides were analyzed on a linear ion trap/Orbitrap mass spectrometer (LTQ-Orbitrap XL), as described previously [56]. Raw MS data were processed using MaxQuant [57]. Data were searched using the Mascot search engine (Matrix Science Ltd., London, UK), and peptides were identified using MaxQuant at a false discovery rate of 1% for peptides and proteins. Cysteine carbamidomethylation was searched as a fixed modification, whereas amino-terminal protein acetylation, phosphorylation of Ser, Thr, and Tyr, and oxidation of Met were searched as variable modifications. Raw MS data are available at the PeptideAtlas repository [58].

Cell culture and reagents

U2OS cells were used throughout and grown in DMEM supplemented with 10% fetal bovine serum, penicillin, streptomycin, and glutamine. Stable clones expressing GFP-KAP1 were selected adding G-418 (0.5 mg/ml) to the medium. Aphidicolin, caffeine, etoposide, hydroxyurea and camptothecin were from Sigma-Aldrich phleomycin was from Melford Laboratories Ltd., Ipswich, UK. IR was applied with a Faxitron X-ray cabinet. UV irradiation was done on cells covered in 1× PBS at a rate of 0.7 J/m 2 per second. AZD7762 was provided by AstraZeneca and used at 50 nM. KU55933 [36] was used at 20 μM. Caffeine was used at 4 mM. All incubations with inhibitors started 1 h before any other treatment was applied. N-6-Benzyladenosine-5'-O-triphosphate (N6B-ATP) and N-6-benzyladenosine-5'-O-(3-thiotriphosphate) (N6B-ATPγS) were from BIOLOG Life Science Institute Forschungslabor und Biochemica-Vertrieb GmbH, Bremen, Germany.

SiRNAs and transfections

siChk1 and siChk2 were with siGENOME SMARTpool siRNA (Thermo Fisher Scientific Dharmacon Products, Lafayette, CO, USA) siLuc (5'-cguacgcggaauacuucgatt-3') and siKAP1 [31] were from Eurofins MWG Operon, Ebersberg, Germany. Transfections were done with Lipofectamine RNAiMAX (Invitrogen). Cells were treated 12 h (siChk1 and siChk2) or 48 h (siKAP1) afterwards.

Immunofluorescence

Cells were grown on poly-L-lysine-coated coverslips, fixed with 2% paraformaldehyde for 10 minutes and permeabilized with 1× PBS containing 0.2% (v/v) Triton X-100 for 5 minutes. Primary antibody staining was for 1 h in 5% fetal bovine serum in 1× PBS with KAP1 phospho-Ser473 (1:100 rabbit BioLegend) and γH2AX (1:1,000 mouse Millipore, Billerica, MA, USA). Secondary antibody staining was with goat anti-mouse Alexa Fluor 488 or goat anti-rabbit Alexa Fluor 594 (1:1,000 Invitrogen, Carlsbad, CA, USA) for 30 minutes. Coverslips were washed three times with 1× PBS and mounted on slides with Vectashield solution (Vector Laboratories Ltd., Peterborough, UK) containing 4',6-diamidino-2-phenylindole (DAPI) to stain DNA. All incubations were done at room temperature.



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