Immunochemistry
Techniques
Electrophoresis, immunoelectrophoresis, immunofixation, isoelectric focusing, and immunoblotting
Serum electrophoresis
The most basic technique for the detection of serum proteins.
Serum is applied to an electrolyte containing agarose gel and a current is applied across the gel. Proteins are separated largely on the basis of their surface charge.
Separated proteins are then visualized by incubating the gel with a protein-binding dye. As the amount of dye bound is proportional to the protein present, the amount of protein present in each band can be calculated from the absorbance of the dye if the total protein concentration is known. This is the method used for paraprotein quantitation.
Faster semi-automated systems use capillary zone electrophoresis (CZE).
Immunoelectrophoresis/immunofixation
Techniques for identifying the nature of the proteins separated in an electrophoretic strip.
Individual proteins can be identified by immunoelectrophoresis, in which troughs cut parallel to the electrophoretic strip are filled with antisera, which are allowed to diffuse towards the separated proteins.
Reaction at the point of equivalence gives an arc of precipitation.
A faster technique is immunofixation, in which the antisera are laid over the electrophoretic separation and unreacted proteins washed out before staining with a protein-binding dye.
Both these techniques are used principally to identify monoclonal immunoglobulins. Serum is preferred as the mobility of fibrinogen is such that it runs in the same region as some immunoglobulins.
CZE can also be adapted to immunofixation.
Isoelectric focusing and immunoblotting
More sensitive techniques are required for CSF, as the concentrations of immunoglobulins are lower.
Electrophoretic separation is carried out by isoelectric focusing. The gel contains ampholytes, which move under the current to set up a pH gradient across the gel.
Proteins applied subsequently then move to the part of the gel where the pH ensures that they become electrically neutral. They then cease to move in the current.
The gel is blotted on to nitrocellulose filters, which are reacted with antisera against immunoglobulins (immunoblotting).
Radial immunodiffusion (RID)
A simple, although slow, method for the measurement of any protein for which an antiserum exists.
Antiserum is incorporated into an agar gel, which is poured onto a plate and allowed to set. Regular holes are then cut into the gel and
the serum containing the protein of interest is placed in the holes. The serum diffuses into the gel and forms an immunoprecipitate that can be seen as a white halo around the well.
The log[concentration] is proportional to the diameter of the ring. The ring diameter can be measured using an eyepiece graticule.
If this method is used to measure concentrations of immunoglobulins, caution is required as variations in molecular weight (e.g. if serum contains monomeric rather than polymeric IgM, as may happen in Waldenström’s macroglobulinaemia) or the presence of immune complexes may cause falsely low or falsely high values.
IgA-deficient individuals often have antibodies to ruminant proteins, including immunoglobulins, and as the antisera that are incorporated in the gel are often raised in ruminants, this may cause reverse precipitation in the gel and lead to entirely spurious results.
Ouchterlony double diffusion
A technique used for non-quantitative identification of proteins.
Wells are cut in agar, and test serum and antisera are placed in the wells and allowed to diffuse towards each other.
Lines of precipitation will form, which can be seen using a light box. When multiple samples are tested, lines of identity and partial identity can be recorded between sample wells.
The technique is sensitive and can be used for low-level IgA detection.
A variant in which the antigen and antibody are forced together by application of a current speeds the process up, but depends on the electrophoretic mobility of the antibodies and antigens. It is used for detection of antibodies to ENA (see Chapter 18)—this is called countercurrent immunoelectrophoresis.
Nephelometry and turbidimetry
These techniques are the mainstay of automated specific protein measurement.
As with other techniques, they rely on immune complex formation when antibodies and antigens react. This alters the optical properties of the solution, and the light absorbance or scatter can be measured.
The reactions can be enhanced using reaction diluents containing polyethylene glycol (PEG), which stabilizes the immune complex.
Accuracy can be enhanced using kinetic assays (rate nephelometry).
This technique is suitable for automation, but depends on high-grade monospecific antisera of high potency. Monoclonal antibodies are rarely suitable for this type of system.
Anything that causes the serum to be turbid before the reaction begins, or interferes with the optical properties of the solution, will cause difficulties. This includes lipaemic sera or haemolysed samples with excess free haemoglobin (with haemoglobin-haptoglobin complexes).
α1-acid glycoprotein
Units: g/L.
Normal range:
male, 0.6-1.2g/L
female, 0.4-1.0g/L.
Principles of test
Measured by nephelometry/turbidimetry.
Indications for testing
Also known as orosomucoid, an acute-phase protein, induced by IL-1, IL-6, and TNFα.
It used to be used extensively in the diagnosis and monitoring of inflammatory bowel disease, and it was felt that it was more specific for this category of disease. There is no strong evidence to support this assertion and CRP probably provides all the information required.
Dynamic range is only twofold, compared with >100-fold for CRP.
There is a sex difference, with levels in females being lower. Levels are also reduced in pregnancy and in patients receiving oestrogens.
It is known to bind certain drugs such as propranolol.
There are no routine indications for its regular measurement at present.
α1-antichymotrypsin
Units: g/L.
Normal adult range: 0.3-0.6g/L.
Principles of test
Measured by nephelometry/turbidimetry.
Indications for testing
This protein is one of the SERPIN family (serine protease inhibitors) whose major role is the protection of tissues from proteolysis by neutrophil and macrophage enzymes. It is particularly active against cathepsin G.
It rises rapidly during an acute-phase response (within 8 hours) and has a dynamic range of fivefold (much less than CRP). It remains elevated for longer.
It has been suggested as a useful marker for inflammatory bowel disease, but there is little evidence to support this and it is not widely used.
α1-antitrypsin (α1-AT)
Units: g/L.
Normal adult range: 1.0-1.9g/L.
Indications for testing
α1-AT is a proteolytic inhibitor with a wide range of inhibitory activities. It is a member of the SERPIN family.
Low levels are associated with cirrhosis, emphysema, and neonatal jaundice, and also with adult liver disease. Deficiency may also be associated with vasculitis (particularly of the skin) and with membranoproliferative glomerulonephritis.
There are a number of deficiency alleles, and when a low value is detected the phenotype should be determined. Other members of the family should be screened and phenotyped. This represents the only clinical utility of α1-AT measurements.
Interpretation
α1-AT comprises the major part of the α1 fraction on serum electrophoresis, and deficiency can be detected readily on an electrophoretic strip.
Occasionally split α1 bands due to allelic variants of α1-AT with different mobilities may be noted.
In conditions where there are high levels of circulating proteases, protease-α1-AT complexes may form, with loss of the α1 band. The complex moves in the α2-β region of the electrophoretic strip.
High levels are seen as part of the acute-phase response, particularly in chronic infections (bronchiectasis).
Levels are also increased in pregnancy, in patients on oestrogens, and in certain malignant diseases, including some germ-cell tumours. However, it is not useful clinically in any of these situations.
It has been suggested that measurement of α1-AT in faeces may give information on gut protein loss as it is resistant to degradation, although there appears to be a high false-negative rate. It may be a useful alternative to radio-isotopic methods for proving the presence of a protein-losing enteropathy. Normal levels are <5mg/g dry weight of faeces.
α1-antitrypsin (α1-AT) genotype (PI typing)
The gene for α1-AT is located on chromosome 14, close to the immunoglobulin heavy-chain locus (PI gene system, for protease inhibitor). A large number of variant alleles have been described.
The identification is usually undertaken by isoelectric focusing, but functional studies may also be required for some rare alleles.
In the UK, deficiency alleles occur with an incidence of about 1 in 2000. Many variants are seen in other racial groups and, as a consequence, are found only exceedingly rarely in the UK.
The important deficiency alleles are S, P, W, Z, Mmalton, and null (no expressed α1-AT). Homozygosity for one or heterozygosity for any two of these alleles leads to severe deficiency. Heterozygosity with any nondeficiency allele leads to a reduction in, but not absence of, α1-AT.
The allele Mduarte gives rise to a functional deficiency with normal antigenic concentrations.
Studies should always be undertaken on family members, and referring clinicians should always be asked for a family tree.
Antenatal diagnosis is also possible by PI typing a fetal blood sample.
In the UK, PI typing is undertaken by the Protein Reference Units (PRUs) in Birmingham and Sheffield, and their excellent handbook gives more detail on the genetic system (see Introduction, ‘Useful handbooks’ p.xxvii for details).
α2-macroglobulin
Units: g/L.
Normal ranges (quoted by PRU):
<15 years. 2.8-6.7g/L
>15 years, 1.3-2.0 -3.5-5.0g/L depending on age and sex.
Indications for testing
No value in routine measurement at present.
Interpretation
α2MG has anti-protease activity. It is an acute-phase protein and also acts as the carrier for IL-6.
In adults, levels are slightly lower in females and fall with age.
It is a major component of the α2 band on electrophoresis and contributes significantly to the elevation of α2 seen in chronic inflammatory conditions.
As it has a very high molecular weight (725kDa), this protein is preferentially retained in the nephrotic syndrome, giving a relative increase in relation to other areas of the electrophoretic strip.
Levels are also reported to be elevated in ataxia telangiectasia, diabetes mellitus, oestrogen therapy, and pregnancy.
Reduced levels have been found in pre-eclampsia and acute pancreatitis. Deficiency has been reported but is exceptionally rare.
Acute-phase proteins (CRP, ESR, SAA)
Principles of test
Indications for testing
Acute and chronic infections, vasculitis, connective tissue disease, arthritis, autoinflammatory diseases.
Interpretation
CRP is a member of the pentraxin family of proteins.
Clinicians are usually confused by ESR and CRP—they do not give the same information and should be used together.
ESR is largely dependent on elevation of fibrinogen, a long-lived serum acute-phase protein, and of serum macroglobulins (α2MG, IgM). It is also affected by red cell morphology.
CRP rises within hours of onset of inflammation/infection and falls quickly once treatment is instituted. Half-life is 6-8 hours. Therefore it is useful for rapid diagnosis and for monitoring response (Table 17.1)
The ESR rises slowly, as it is dependent in part on fibrinogen, a longlived protein, and falls equally slowly. The half-life of fibrinogen is approximately a week.
CRP is driven by IL-6, as well as IL-1 and TNFα, and may be elevated in myeloma. Measurements of CRP reflect serum IL-6 levels and there is no additional value to measuring IL-6 directly.
Levels in very young children may be much lower for a given stimulus.
A very small number of patients do not make inflammatory responses which exceed the normal range, but seem to run on a lower ‘normal’ range (10-fold less). Ultrasensitive assays for low-level CRP are available.
Some patients with giant cell arteritis never show an ‘elevated’ CRP.
SAA, which is the circulating precursor of the secondary (AA) type of amyloid and whose physiological function is currently unknown, is an acute-phase protein with a very wide dynamic range (1000-fold).
Monitoring of SAA is said to be valuable in disease known to predispose to the development of AA amyloid, such as chronic infections and inflammation, particularly autoinflammatory syndromes. The CRP test is more widely available and probably gives the same information.
Serum amyloid P (SAP) is related to CRP structurally and is a member of the pentraxin family. Despite this similarity, its function is not fully understood and it does not function as a major acute-phase protein. Routine measurement is not justified at present, although labelled SAP has been used as a tracer for amyloid deposits.
Table 17.1 Levels of CRP and their common associations | ||||||||||
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Amyloid proteins
Amyloid refers to the deposition of altered proteins in tissues in an insoluble form.
The precursor protein varies according to the cause, and can often be measured specifically (Table 17.2).
Amyloid is usually confirmed by special stains on histological examination of biopsies.
Measurement of serum immunoglobulins and electrophoresis, serum free light chains, β2-microglobulin, and CRP is essential if amyloid is suspected.
Table 17.2 Clinical syndromes and associated amyloid proteins and protein precursors | |||||||||||||||||||||||||||||||||||||||
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Avian precipitins
Units: reported semi-quantitatively.
Normal range: healthy adults exposed regularly to birds may demonstrate precipitins.
Principles of test
Usually carried out by double diffusion. Fluorescence assays and ELISA are also used, and Pharmacia have an ImmunoCAP® available.
Indications for testing
Used in the investigation of bird-fancier’s lung (extrinsic allergic alveolitis).
Interpretation
IgG-precipitating antibodies to avian antigens are found in cases of bird-fancier’s lung. These react particularly with avian serum and faecal proteins.
Presence of precipitins is a marker of exposure and does not automatically mean that disease will be present.
Presence of multiple precipitin lines tends to be a feature of disease.
Any bird species is capable of inducing precipitins, but the most common causes of problems are pigeons (in pigeon breeders), psittacine cage birds, and domestic poultry (as an occupational disease).
Bacterial and viral antibodies (specific antibodies; functional antibodies)
Units are variable: U/L, IU/mL, µg/mL.
Ranges are variable: check with reporting laboratory.
Pneumococcal antibodies, >20U/L (asplenics >35U/L).
Tetanus antibodies: >0.1IU/mL (minimum protective level)
Haemophilus influenzae type B, >1.0µg/mL (full protection, asplenics >1.5µg/mL).
Viral antibodies: usually qualitatative, semi-quantitative, or titre.
Principles of test
Measurement of antibody production against defined pathogens or antigens purified from pathogens plays an important role in the investigation of suspected immunodeficiency.
Most assays are carried out by enzyme-linked immunoassay, but some viral antibodies are still measured by haemagglutination or complement fixation.
Pre- and post-immunization samples should be run together for direct comparison as the coefficients of variation for the assays tend to be high (15-25%!).
An EQA scheme exists for tetanus, pneumococcal, and Hib antibodies, and internationally agreed standards are available for tetanus and Hib. A UK standard exists for pneumococcal antibodies.
Post-immunization samples should be taken at 3-4 weeks to see the optimum response.
Assays have tended to focus on agents for which there are safe and effective vaccines.
Live vaccines should never be given to any patient in whom immunodeficiency is suspected.
Antibodies normally run in immunology laboratories include pneumococcal polysaccharides, which may be further differentiated as IgG1 and IgG2, Haemophilus influenzae type B (Hib), and tetanus.
Assays to individual serotypes of Pneumococcus are available in reference laboratories. This is a time-consuming and expensive test and should only be used by experienced immunologists investigating suspected immune failure.
Diphtheria antibodies are not run by many laboratories as the assay’s EQA performance has been so poor.
Antibodies to Pseudomonas and Burkholderia are used in cystic fibrosis.
Antibodies to Salmonella Vi antigens are being studied as a potential for evaluating immune response to polysaccharide antigens (alternive to pneumococcal antibodies).
Meningococcal C polysaccharide antibodies are run by a few specialized laboratories, but correspondence with known clinical status has been poor and there is no EQA.
ASOT may be helpful. However, antibodies to staphylolysin are not useful, as they may be negative in normal individuals.
Viral antibodies may be valuable—to natural exposure and immunization antigens such as polio, measles, mumps, rubella, chickenpox, EBV, and hepatitis B (if immunized).
Isohaemagglutinins are naturally occurring IgM antibodies (in patients of a suitable blood group) and should be measured as part of a functional antibody screen.
Indications for testing
These assays should be used in the work-up of patients with suspected immunodeficiency, or in monitoring change in such patients.
Responsiveness to immunization is a helpful marker of immunological recovery following bone marrow transplant.
Annual monitoring of levels may be valuable in asplenic patients, as such patients lose immunity more rapidly than a eusplenic population.
Interpretation
Interpretation is entirely dependent on the context!
Assays for pneumococcal polysaccharides measure a composite of responses to the 23 strains in the Pneumovax II® vaccine. This can be misleading as not all strains represented in the vaccine are equipotent
as immunostimulators. This means that a ‘normal’ response may actually mean a good response to the immunogenic strains, masking failure of response to the less immunogenic strains.
Evaluation of the response to conjugated pneumococcal vaccines and the measurement of serotype-specific responses may be helpful.
Therefore evaluation of such patients should be carried out by an immunologist with an interest in immunodeficiency. More weight should be placed on changes in response to immunization than to actual values—hence the need to run pre- and post-immunization samples together.
A ‘normal’ response to immunization has never been standardized. Publications frequently use different criteria, rendering comparison impossible. The following is a useful working definition:
a fourfold rise in titre that rises to well within the normal range.
All antibody responses must be interpreted in the light of the clinical history and previous infections/immunizations.
Some patients lose specific antibody rapidly, within 3-6 months of immunizations (i.e. poor long-term immunological memory). If this is suspected, repeat testing after 6 months.
Measured responses if conjugated pneumococcal vaccines (Prevenar®), which contain fewer serotypes, are used may be lower if the laboratory uses assays based on Pneumovax II® as the test antigen mix.
Bence-Jones proteins
β2-microglobulin (β2MG)
Units: mg/L.
Normal range: 1-3mg/L.
Principles of test
Test measures free β2-microglobulin, which normally forms the light chain of HLA class I molecules but is shed when there is increased lymphocyte turnover and therefore is present in serum in soluble form. It is usually rapidly cleared by the kidneys.
It has a molecular weight of 11-12kDa.
Measurement is usually by automated analyser, nephelometry, or turbidimetry.
RID is still used, but it is slow.
Indications for testing
The main indication is as part of the routine monitoring of patients with myeloma, lymphoma, and HIV. However, its non-specific nature and the influence of renal function on results mean that it is less widely used.
Serum free light chains are replacing β2MG in the monitoring of myeloma.
Elevated levels can be seen in dialysis patients.
Interpretation
Levels are elevated in the following.
HIV infection (surrogate marker of progression).
In HIV disease, β2MG is said to be a useful surrogate predictor of disease progression, and gives similar information to the absolute CD4+ T-cell count. However, the dynamic range in HIV is small compared with that in lymphoproliferative diseases. The CD4+ T-cell count and viral load are preferred in the UK.
Other viral infections.
Myeloma (marker of tumour mass).
In myeloma, serial measurement of β2MG is a useful adjunct in terms of monitoring tumour burden and cell turnover. Interpretation of levels is complicated by the need to consider renal function, particularly as free light chains are nephrotoxic and may damage tubules, thus inhibiting reabsorption, and there may also be glomerular damage preventing filtration. Therefore the effects on β2MG serum levels in myeloma are complex.
Levels below 4mg/L are associated with a good prognosis, while levels above 20mg/L are associated with a poor prognosis, although this may well represent the effects of renal damage.
Treatment with α-interferon elevates serum levels, which needs to be considered in monitoring levels.
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