Quantitative investigation of Immunoglobulins is the basis of the standard laboratory technique within the field of clinical immunology. Immunoglobulins can be measured quantitatively through the use of nephelometry, such measurements are vital in the instance of a suspected immunodeficiency within a patient.
For this reason the test is accurate and rapidly measures the amounts of IgM, IgG and IgA proteins within the blood of the patient and from such determine if a number of conditions or disorders are present. The role of such antibodies is within fighting infections and allergies as part of the normal immune response.
A disease (or disorder) can be identified through the measurement of such protein levels. IgM for example can appear during an initial infection and then reappear to a lesser extent upon secondary exposure. (Weir, 1978)
Nephelometry is usually performed by drawing blood from a vein on the back of the hand or if not possible the inside of an elbow. The needle draws the blood into an airtight vial or tube attached to it. Removal of the needle is followed by sterilisation and covering of the incision site. (Stanley, 2002)
Nephelometry is an automated system that measures antigen or antibody solution in very limited concentrations by the amount of light scatter. The principle is that when the light comes into contact with the solution it will not be absorbed but scatter away from the main beam and measured at angles between 0-90 from a predefined curve. The subsequent amounts are proportionate to that of the concentration of molecules. As well as dilute solutions there also needs to be a linear correlation between molecules formed and optical density. For this reason several dilutions measurements are recorded and also during the formation of molecules. This process is known as rate nephelometry.
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When considering this technique it is vital that the relative amount of antigen and antibody must be small enough so that precipitation does not occur but also large enough to allow the formulation of small immune complexes. Immunoprecipitation results are achieved through the use of monoclonal antibodies (MCAs) allowing epitopes to react with the antiserum and MCAs forming immune precipitates with their antigens.
- IgG: 560 to 1800 mg/dL
- IgM: 45 to 250 mg/dL
- IgA: 100 to 400 mg/dL
Evaluation- the automated nature of this technique means that it is both fast accurate with results available within 1-2 hours. Its wide spread use is mainly down to this factor but its simplicity and low sample size and volumes also make it a valued technique with the clinical laboratory setting. (Diamandis et al…1996).
It is however, as with most techniques not without its draw backs. The usual precautions should be taken as when taking any blood sample. Although rare excessive bleeding, fainting and infection should all be considered as risk factors when taking samples. (Drexel, 14/06/08)
The presence of dust particles and other debris can be cause for distorted readings and lead to higher values than expected. This can be addressed through centrifugation of the specimen (Diamandis et al…1996). In addition to this air bubbles can cause similar effects on results. To ensure readings are as accurate as possible, the specificity should be at optimum level, as set on the nephelometer and controls should be carried out wherever it is used. (Palmer, 1992) Although this method does determine the amount of each immunoglobulin it does not possess the ability to identify antibodies.
Another method that can be used to quantitatively investigate Immunoglobulins in serum, saliva, cerebrospinal fluid (CSF), amniotic fluid, and gastrointestinal juice is Radial Immunodiffusion (Chapel et al…1999). This technique allows for the adding of a sample to a well in a gel made up of the antibody specific for the substance being tested for. This then moves through the gel leading to the formation of a visible precipitate around the optimum concentration sample well.
The interpretation of such results however is subjective and results are delayed as the process takes several days and as such nephelometry is recommended for greater precision, automation, objectivity and speed and is suitable for large throughput tests (Keogan et al… 2006). It is also hard to quantitatively analyse the results using very small samples and a calibration curve. (Chapel et al., 2006).
2. Quantitative Other Serum Proteins-Radial Immunodiffusion
Radial Immunodiffusion acts upon the antigen- antibody complex precipitation reaction. It is used within the field of neurology and oncology. This involves passive diffusion of immunoreactants through an agar matrix. An electrical current is not required for such process to occur as is reliant upon the physiochemical relationships.
Radial Immunodiffusion works via the mixing of antiserum with agar and pouring it on a glass plate to allow it to solidify. The antiserum must be specific for the class of immunoglobulin that is being measured for the technique. The agar mixture is then punctured and the subsequent holes filled using the sera from the test samples. Diffusion of the Immunoglobulins radially causes the formation of precipitate at the point which the number of antibody and antigen are identical. As with previous techniques a calibration curve us used that has been made up from known set of solution to determine the amount of Immunoglobulins present within the sample.
Accuracy and specificity is the most potent threat to validly of this technique. The fundamental problem is the lack of sensitivity and is not a rapid technique with results taking over 48 hours owing to reaction times. (Chapel, 2002) Whilst it does provide use in the determination of serum proteins quantitatively there are an array of factors that can lead to unreliable results. Temperature of the gel and external environment, molecular size, gel viscosity, reactant concentration and buffer pH highlights a few of the factors that will affect the rate of diffusion but is not exhaustive. (Nakamura et al, 1979)
3. Quantitative other Serum proteins
Collection of serum
- Collect blood in a glass container and allow it to clot at room temperature for an hour.
- Once the clot has formed loosen the walls of the container to aid retraction.
- Transfer to 4 degrees and leave overnight if necessary
- Collect the expressed serum and centrifuge at 150g for 5 minutes to sediment the erythrocytes, and then at 350g for 15 minutes.
- Transfer the straw coloured serum to suitable containers and heat at 56 degrees for 30 minutes to destroy the heat labile components of complement. (Hay et al., 2002)
The stages of diagnosis, determination of immunity and the susceptibility of an individual to many microbial infections, are based upon immunological tests in serum. When blood clots the fluid that remains is known as serum and as such it is rich in Immunoglobulins. Serum however is not easily accessible so other sample sources can be used. The presence of specific Immunoglobulins in urine, saliva and cerebrospinal fluid mean that such bodily fluids, inclusive of others such as semen, can be used instead. Anatomically the most readily available and less intrusive however, as with other bodily fluids, contain low concentrations of IgM and IgG. Semen is abundant with these Immunoglobulins and as such may be perceived as being the most accurate and reliable in any such investigations (PCT, 1987)
In Serum- Immunoelectrophresis
Serum Protein Electrophoresis is a qualitative investigation carried out to test for the presence of monoclonal bands (paraproteins). (Chapel et al., 2002) During electrophoresis, discrete monoclonal bands may appear (M bands). Further investigation is needed in order to determine the immunoglobulin heavy and/ or light chains through immunofixation. This is important when trying to distinguish what sort of Immunoglobulins are present. Determination is achieved through Immunoprecipitation in a gel with anti- sera that is specific for heavy and light chains of the immunoglobulin.
Immunoelectrophresis works by separating sera in agarose gel by electrophoresis. Troughs that are parallel to the unfixed electrophoretic strips have specific anti- sera added to them leading to the formulation of precipitin arcs that are clearly visible owing to the process of diffusion.
Immunofixation however tends to be more commonly used and as such will be more focused upon within the portfolio. This technique is commonly used in the diagnosis of conditions such as osteoporosis.
In the abnormal absence of a heavy chain and an abnormal reaction occurring with the ant- sera that are specific for light chains discrete (M) bands are present. It is also a highlight to the possibility of an IgD or IgE paraproteins although is far less common.
If an abnormal reaction occurs with only the heavy chain anti sera it is indicative of a rare heavy chain disorder. It is possibly to quantify individual M bands with the use of a densitometer. This acts by measuring the intensity of the stain taken up by each individual band and as such is the only method at present to be of use in the measurement of paraproteins concentration (Chapel et al…1999).
Practical Schedule – taken from Clinical Immunology. (Chapel et al., 2002)
Immunoelectrophoresis- Apply serum samples to an electrophoresis gel at the cathode end alongside a normal serum sample as a control. Apply an electric current for 45 minutes and remove the gel. Use a stain to visualize the bands.
Immunofixation-specific antisera to IgG, IgA, IgM and kappa and lambda light chains are then applied to the electrophoresed samples by soaking strips of cellulose acetate in the individual antisera and laying them on the electrophoresis gel. This is then incubated for 2 hours and all the un-fixed proteins are washed off leaving the precipate. Individual monoclonal bands can be quantitatively measured by a densitometer.
The dark areas indicate monoclonal bands. The picture above shows a positive result for the lambda chain. The presence of monoclonal bands can indicate multiple myelomas or osteoporosis.
In this example, the M band is identified as IgG of kappa type. Concentration of the M band is determined using a densitometric trace, as demonstrated in the second image.
Evaluation- The presence of air bubbles will distort the formation of protein bands and as such the gel must be degassed. The method detailed above is much quicker and far more sensitive than the singular use of immunoelectrophresis. Its cheapness and low hazard level mean it is a desirable technique in the detection of Immunoglobulins within serum. (Zola et al. 1999)
Qualitative Immunoglobulins in Urine- Electrophoresis and Immunofixation
Normal physiology of the kidney dictates that protein is usually excreted within the urine in minimal amounts. Higher levels can lead to the suspicion of multiple myeloma that can lead to irrapairable damage to the kidneys as nephritic cells are non replaceable with chronic lymphocytic leukaemia and hypogammaglobulninaemia being suspects. Kidney disorders such as IgA nephropathy may also be a causation of such symptoms.
All humans produced excessive amounts of free polyclonal light chains in accompaniment to normal immunoglobulin synthesis with these being secreted into the urine and are detectable in low amounts in all samples (Thompson, 1981). If the normal range of this is exceeded however it is indicative of renal damage. This method is often used in order to detect these small free monoclonal light chains that are also called “Bence- Jones Proteins” owing to the fact that normal parameters of testing fail to pick them up. (Chapel, 2005). Bence- Jones Proteins are distinguishable by the fact they possess unusual thermal properties, for example they precipitate out of the urine solution at 56 degrees and redissolve upon further heating. (Thompson, 1978)
Determine concentration by ultrafiltration, absorption of water, or by freeze-drying. There are several commercially available kits for determining the concentration of urine. This involves concentrating the urine, then using electrophoresis to determine the presence of monoclonal bands. Then using immunofixation to establish what the monoclonal band is made of. (Chapel et al., 2006)
Serum protein samples from patients with light chain multiple myeloma and one normal result on the far left.The M protein is seen as a dark dense band localised on the strip, this picture shows the different bands that can be detected.
- Decreased with malnutrition and malabsorption, pregnancy, kidney disease (especially nephrotic syndrome), liver disease, inflammatory conditions, and protein-losing syndromes
- Increased with dehydration
- Alpha1 globulin
- Decreased in congenital emphysaema (a1-antitrypsin deficiency, a rare genetic disease) or severe liver disease
- Increased in acute or chronic inflammatory diseases
- Alpha2 globulin
- Decreased with hyperthyroidism or severe liver disease, haemolysis (red blood cell breakage)
- Increased with kidney disease (nephrotic syndrome), acute or chronic inflammatory disease
- Beta globulin
- Decreased with malnutrition, cirrhosis
- Increased with hypercholesterolaemia, iron deficiency anaemia, some cases of multiple myeloma or MGUS
- Gamma globulin
- Decreased variety of genetic immune disorders, and in secondary immune deficiency
- Increased Polyclonal: chronic inflammatory disease, rheumatoid arthritis, systemic lupus erythematosus, cirrhosis, chronic liver disease, acute and chronic infection, recent immunization. Monoclonal: Waldenstrom’s macroglobulinaemia, multiple myeloma, monoclonal gammopathies of undetermined significance. (MGUS)
Table from lab tests UK online.
Evaluation-this method allows the determination of the different proteins in the urine and can be vital in allowing the doctor to work out a diagnosis of the condition. It is relatively simple and reliable however the results can only be read by a skilled worker and owing to its various steps is not as rapid as desired. Results show that different diagnoses are reached depending on which Immunoglobulins are increased in the urine, as indicated in the table above.
Qualitative Immunoglobulins in Cerebrospinal Fluid- immunoperoxidase and isoelectric focusing
This test allows for the differentiation between IgG and albumin concentrations. This relationship is important to differentiated as IgG is synthesised by lymphocytes within the brain where as albumin is not and is known as the CSF IgG Index that is indicative of this fact as demonstrates how much IgG within the CSF has been synthesised. (Chapel et al…2006). Unlike the before mentioned serum where single discrete (M) bands where formed the locally synthesised IgG is often oligoclonal and subsequently cannot be detected by means of electrophoresis of CSF as isn’t concentrated. (Roitt et al.. 2002)
The only available method for the detection of oligoclonal bands are isoelectric focusing and immunofixation with enzyme labelled antiserum. Investigation and diagnosis of demyelinating disorders such as Multiple Sclerosis is carried out using such tests. (Richard et al… 2002)
Isoelectric focusing and immunofixation with enzyme labelled antiserums. This involves separating the proteins within a pH gradient and transferring them to nitrocellulose membranes that have previously been immunofixed with IgG antiserum to show the specific bands. This can be compared with controls to determine the new bands. (Richard et al., 2002)
Results– A positive result is where the oligoclonal IgG bands are not found in serums, but, in Cerebrospinal Fluid. These are shown as dense dark bands on the results below. 5-10% of CSF protein tends to be IgG. If a patient has disseminated sclerosis or sub-acute sclerosing panencephalitis then the proportion of IgG in CSF is over 12%.
This is a relatively modernised method and is approved for use within a clinical setting. The older isoelectric focusing is no longer recommended as it possesses a higher degree of specific (95%) and sensitivity. In addition it is favourable as only requires low concentrations of serum samples and results are available within 2 hours and mostly work on an automated level. (Richard et al.. 2002)
Qualitative Immunoglobulins in Saliva-
Introduction– Complement components are large molecular weight proteins. Activation of these usually results in proteolytic cleavage of the molecule into fragments. (Thompson, 1978) Western blotting is used in combination with gel electrophoresis and ELISA and RIAs are used when a whole saliva sample is collected or when there are saliva fractions [Fabian et al., 2007].
Gel filtration is carried out on Sephadex G-200. Serum samples of 1.5ml were applied to and 2.5cm diameter, 40 cm length column containing the Sephadex. This is equilibriated with a buffer containing 0.14M NaCl, 0.006M NaH2PO4 and 0.035M Na2HPO at a pH value of 7.3. Fractions of 2.5ml each are collected at a flow rate of 30ml per hour and the protein content of this effluent is measured as UV transmission at 280mµ in an absorbiometer.
Results– the results are determined by using these filtrated samples and single radial diffusion, a calibration curve is needed to determine amounts. This is created by using standard solutions. (Rose et al., 1997)
Evaluation– Occur in large amounts in serum can be measured accurately precipitin reaction in gel. Detecting them as antigens however means it cannot be identified as to whether they are active or not.
Collecting specimens for complement assays can be difficult as you are to avoid inducing the complement pathway. Care should be taken to avoid false results caused by this when trying to determine the activation that was caused in vivo.
Single radial diffusion can be used to determine quantitatively. This test is rapid reliable and easy to carry out and determine results of. (Rose et al., 1997)
Complement-C3- Crossed immunoelectrophoresis
The complement system comprises of proteins (which may be membrane bound or present in plasma) that play an important role is host defences [Stanley, 2002]. The system is involved in destroying certain bacteria and viruses, and is also involved in initiating inflammatory response. Complement is also important for opsonisation of foreign materials, facilitation of phagocytosis by leukocytes, and direct cytotoxic reactions [Gaspari & Tyring, 2008]. A determinant of the amount of C3 is crossed immunoelectrophresis and has the advantage of differentiating between inactive and active forms of C3. Deficiencies in C3 can lead to systemic infections including sepsis meningitis, pneumococcal and influenza infections.
- Prepare a 2% agarose solution in the barbitone buffer containing EDTA (ethylene diamine tetra-acetic acid)
- Pour 3 ml of agarose solution onto the microscope slide and let set.
- Cut a 1mm well in the slide removing the agarose and filling with the serum sample for the C3 quantification.
- Apply a potential difference of approx. 150v for 2 hours.
- Cut a 5 mm wide longitudinal strip containing the sample.
- Prepare 12ml of an anti-C3 solution in 2% agarose solution at 56 degrees.
- Place the agarose strip at one end of the square glass plate and cover the whole slide with the agarose containing the anti-C3.
- Place the plate in the electrophoresis tank making sure it is the right way and electrophorese overnight.
- Wash and stain the precipitin arcs.
This method works by using the electric field to separate the complement components.
Evaluation- as with many of the before mentioned techniques it requires a skilled technician in order to carry out such a test and can edge on the side of time consuming owing to its numerous steps and incubation periods set out in the methodology. (Hay et al.. 2002)
Complement- nephritic factor
Introduction– nephritic factor is an autoantibody to activated C3, it breaks down C3 in the alternate pathway by cleaving it into two fragments that are inactive forms (C3d and C3c) of the normal version of C3b. It binds and stabilises the alternative pathway C3 convertase (that is present in all sera) in the presence and absence of serum proteins. The alternative pathway C3 convertase blocks inhibitors from acting on and destroying C3 convertase.. The autoantibody (the C3 nephritic factor) reacts in the complement system not by blocking the enzyme active site but instead, block the site where inhibitors limit the action of and destroy the enzyme. Tests to determine the C3 nephritic factor are performed in patients that possess a C3 concentration that is below normal and is unexplained, with normal C4 levels [SAS Centre, 2009]. This is because the presence of the C3 nephritic factor in a patient means that C3 is continuously broken down and depleted. Low levels C3 can be associated with kidney disorders or recurrent infections. (Chapel et al., 2006)
Practical Schedule-The practical schedule is similar to the before mentioned. It used samples with the suspected nephritic factor and other normal serum samples. They are incubated together and if the nephritic factor is present, it breaks down the C3 in the normal sample. (Chapel et al., 2006)
Results-As expected from the similarity in methodology the results are similar to the detection of C3 in the picture above using crossed immunoelectrophoesis. If only inactive forms are present owing to inactivation from nephritic factor than the result is deemed positive. A negative result is when there is no nephritic meaning that none of the C3 has been inactivated.
Evaluation-This method is useful in the detection of nephritic factor only and it is not a very direct test as it is carried out to determine the amount of C3.
Complement-functional assay CH50
Introduction– complement functional assay are the basis for the diagnosis of complement deficiency disorders. They are divided into subcategories dependent upon their relation to another disease.
Primary complement deficiencies are genetic based and secondary refer to those that are acquired. Functional assays play a pivotal role within the assessment of the classical, alternative and terminal pathway of complement activation.
The most common haemolytic assay used within the laboratory setting is the CH50 assay as it is both the simplest and easiest to carry out. The functional integrity of the classical complement pathway, C1, C2, C3, C4 is measured using CH50 along with total haemolytic complement.
This is achieved by measuring the required quantity of serum in order to cause haemolysis of half the quantity that had been stabilised and sensitised red blood cells (Chapel et al..2006). Classical components become activated to lyses sheep erythrocytes that are coated in rabbit anti- sheep E antibodies (Rose.. 1997)
Practical Schedule– Add to microtiter wells the sera to be tested along with a buffer in different concentrations. Then add the sheep erythrocytes. Cover and incubate at 37dgrees for 1 hour. Then centrifuge and carry out ELISA to detect results. (Rose et al., 2002)
The method is generally sensitive and reliable providing the specimen is tested quickly and all reagents are kept on ice. (Chapel et al., 2006) The problem arises in availability as they are not widely available and as such functional assays for complement are limited to laboratories that have the equipment (Gaspan and Tyring 2008). As with many immunological techniques the fundamental threat to validity is improper sample collection, this can occur easily in the onsite environment where it can be left to stand for considerable periods of time at room temperature. (Rose, 1997)
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5. Microbial Antigens – ELISA
By coupling the antigen to an insoluble adsorbent it is possible to detect human antibodies to specific antigens using this technique. Elevated levels of antibody titre remains a reliable indication to the presence and measurement of an active infection within the diagnostic process. ELISAs provide highly sensitive and precise methods for the estimation of biological parameters, with the added advantage that they can handle large numbers of samples that may then be analysed rapidly and are useful in detection of a range of viruses and bacterial infections inclusive of TB and pneumonia and viral antigens. (Chapel et al..2006)
Many types of immunoassays can be used to detect and quantitative both antigens and antibodies, but there are differences in the avidity requirements for the antibodies, the signal strengths of the labels, and the amount of background for each of these types of assays. Antibody capture assays are the most appropriate for measuring the titre of the antisera you have generated.
ELISAs by definition exploit the use of an enzyme attached to one of the reagent utilized in the test. Subsequent addition of the relevant enzyme substrates/ chromogens cause a colour change: the results can be read both by eye and quantified using specially designed spectrophotometers. The fact that proteins (including antibodies) and carbohydrates can be passively attached to plastics has been exploited in most applications of ELISA. Since one of the components is attached to a solid phase by passive absorption, subsequent reagents can be added, and after a period of incubation, unreacted material can be simply washed away. Such assays are termed Heterogeneous ELISAs. The plastic surface is known as the solid phase and plastic in the form of 96-well microtiter plates has proved highly practical for the following reasons.
- A large number (96) of sample wells are available in a highly practical from;
- Multichannel pipets (4,8,12 channels) designed for use with such plates are available, making reagent handling rapid and simple;
- Test volumes are small (e.g. 50uL, 100uL);
- Comparative readings of coloured products can be made by eye or by specially designed multichannel spectrophotometers (96 wells are read in 2-5s)
The above facts afford the potential to rapidly handle numerous plates, and hence numerous samples may be examined, e.g., 20 plates/person= 1920 sample points/ person.
Attachment of reagents also allows great versatility for ELISA since the various components of assays may be used in different combinations and in different phases to investigate their potential. It is difficult to generalise about the potential performance of the various ELISA systems. There is a wide range of configurations available and probably no two scientific groups attempting to perform the same task by ELISA will use identical configurations.
- Dissolve antigen in carbonate-bicarbonate buffer.
- Add 200 µl to each well of a micro-ELISA plate cover and incubate overnight at 4 degrees.
- Wash so that unbound antigen is removed and fill with casein to block remaining binding sites.
- Incubate at room temperature for 1 hour.
- Add 200 µl of test serum and incubate for 2 hours at room temperature in a humid chamber.
- Wash the plate three times
- Prepare the peroxidase-antibody conjugate, mix 100microlitres of casein with 1 ml of serum, 100µl Tween 20 with 50 µl peroxidase-antibody and stir gently.
- Add 200µl to each well and incubate at room temperature for an hour.
- Wash three times.
- Prepare the substrate solution and add200 µl substrate to each well. Leave in the dark and allow colour to develop.
- Stop the reaction by adding 50 µl of sodium fluoride to each of the wells.
- An ELISA reader can then be used to quantify the colour reaction.
General Method from Hay et al., 2002.
A positive result would be characterised by the reaction that causes colour showing the presence of antibodies to the specific type of bacteria highlighted by a dark band.
On the whole this method remains largely specific and rapid. The major cause of problem is the scientist(s) involved. The main problem is the lack of close- contact training in the fundamentals of ELISA, so that the scientist has the experience to identify and then solve the problems in the use of reagents. The results yielded cannot have the biological implications assessed without general knowledge of several field of science, e.g., epidemiology, immunochemistry, biochemistry and immunology. This however should not be considered too problematic as the ELISA should be a tool for the investigation of specific problems rather than an end in itself. Whilst it in comparison to immunoflourence in the detection of TB and flow cytomentry it may lack sensitivity, it does remain the cheapest and easiest to carry out. (Rose et al..1997)
It is becoming increasingly evident that the presence of tissue auto antibodies is not in itself pathognomic of disease. Improvements in the technique in the last few years have led to increased sensitivity and detection of weak antibodies in sera which would hitherto have been reported negative. As a large series of patients are tested in an increasing number of laboratories previously held views on the specific clinical association of particular antibodies are being revised and reference to early literature may therefore be misleading.
Since antigens and antibodies are defined by their mutual interactions, they can be used to quantify each other. At a practical level in a diagnostic laboratory, the functional tests are labour intensive and therefore expensive, and a compromise is usually sought by using immunochemical assays which measure composite of medium to high affinity antibodies and their abundance.
The antibody has become the scientist’s “flexible friend”! For example, antibodies raised against hormones, serum proteins, cell constituents, cytokines, or even immunoglobulin’s themselves, allow these parameters to be measured in immunoassays. Immunoassays form the backbone of tests used in the study