Sarah Sasson November 2014 1

Principles of nephelometry and turbidimetry focusing on IgG subclasses

Principles of nephelometry and turbidimetry focusing on IgG subclasses

Nephelometry and turbidimetry are liquid based immunoassays based on the measurement of
scattered or absorbed light.

Light scattering is the physical phenomenon resulting from the interaction of light with particles in
solution and is dependent on:

• Particle size, shape, concentration, MW

• Wavelength and intensity of light (short wavelengths have increased scatter)
• Distance of observation
• Angle of detection
• Refractive index of media

The Antigen-Antibody Complex

• When the number of Ab binding sites is significantly greater than antigen binding sites
relatively small complexes are formed.
• When the number of Ab binding sites is only slightly greater than antigen binding sites there
is a higher chance of cross-linked aggregations forming.
• When antigen is in excess the Ab binding sites quickly become saturated again leading to
smaller non-aggregating complexes.
o This antigen excess results in the possibility of two widely different antigen
concentrations corresponding to the same light scattering value.
• The equivalence zone represents the ratio of Ab:Ag binding sites at which maximum cross-
linking occurs and the largest aggregate complexes exist.
• The kinetics of the immunoprecipitin reaction are important. After antibody and antigen are
mixed, light scattering complex formation begins, and peak rate of formation is reached around
20s after which there is a steady state reached. Methods that use the rate of increase in light
scattering are known as “rate” or “kinetic” methods
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Principles of nephelometry and turbidimetry focusing on IgG subclasses

Nephelometry
Initially fluoremeters were employed as nephelometers and scattered light was measured at 90°
Subsequently scattered light has been detected at 1-30°
General principles:
• Virtually any body fluid may be studied
• Dilute serum/fluid (approx. 1 in 50) is combined with an optimised dilution of specific antiserum in
appropriate buffer
• Small volumes eg 300-400μL (includes dead volume) are usually sufficient
• Ag-Ab reaction is allowed to proceed
• The degree of measured light-scatter is compared with that obtained with a set of standards or
calibrators
• The concentration of the analyte in the sample of interest is determined

May be “End-point” or kinetic

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Principles of nephelometry and turbidimetry focusing on IgG subclasses

Endpoint nephelometry
• Simpler
• Less sophisticated instrumentation
• Must cope with correction of background/blank scatter
Kinetic/Rate Nephelometry:
• More sensitive
• Less effected by background scatter
• May be affected by spurious Ag-Ab complexes eg M-proteins

Causes of high background light scatter in serum:

• Chylomicrons
• LDL/VLDL
• Circulating immune complexes
• Repeated freeze-thaw cycles
• (CSF and urine less effected)
• Note kinetic or rate nephelometers reduce the need to eliminate background light scatter

Antigen Excess
A major requirement of all immunoprecipitin techniques is means to detect antigen excess. The
intention of immunoassays is to read values up to the point of equivalence and not beyond it in the
“antigen-excess” zone. A commonly used method is to prepare two dilutions of sample and measure
the light scatter of both. In antigen excess situations more dilute samples will have higher light
scatter. Prescreening on serum EPG has also been employed.

Monoclonal Proteins

Can result in under and over estimation errors as the clonal protein may bind weakly or strongly and
the reaction may be slow or fast.

Turbidimetry:

• Subject to the same considerations as nephelometric systems

• Detection signal is absorbance rather than light scatter. Therefore general chemistry
analysers may be employed.
• Both End-point and Rate methods may be used
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Principles of nephelometry and turbidimetry focusing on IgG subclasses

Parameters to be considered:

• Ag: Ab ratio
• Limit of detection
• Dynamic range
• Instrument sensitivity and capacity
• Background light scattering
• Kinetic of the reaction
• Range of concentration of clinical samples
• Polymer enhancement
• Antiserum characteristics and quantity/quality

Assay development experiments:

• Antiserum dilution
• Dynamic range
• Sample dilution
• Lower limit of detection
• Standard curve (Ag-Ab precipitin curve)
• Effect of antigen excess
• Parallelism

Assay Trouble Shooting

Problem Solution(s)
Excess scatter • Dilute sample
• Dilute antibody further
Insufficient scatter • Increase sample fraction
• Increase antibody fraction
Insufficient scatter change • Adjust Ab:Ag ratio
• Use polymer enhancement
Excessive background • Filter reagents
• Ultracentrifugation of lipemic samples
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Principles of nephelometry and turbidimetry focusing on IgG subclasses

Proteins measureable by Nephelometry/Turbidimetry

α1- Acid glycoprotein

α1-Antichymotrypsin
α2-Antiplasmin
α1-Antitrypsin
α2-Macroglobulin
α1-Microglobulin
Albumin
Antithrombin III
Apolipoprotein A1
Apolipoprotein B
B2 Microglobulin
C1 inhibitor
C3
C4
Ceruloplasmin
CRP
Fibrinogen
Fibronectin
Gc globulin
Haptoglobin
IgA
IgG
IgE
IgM
Serum free light chains
Prealbumin
Retinol binding protein
RF
Transferrin
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Principles of nephelometry and turbidimetry focusing on IgG subclasses

Quality Control and Assurance:

Commercial platforms usually include a

• calibrator
o Involves approx. 6 dilutions
o run approx. once a month
o Recalibration with new Lot #, machine servicing etc
• controls
o low and high
o In house controls may also be run

CAPIR preparation for proteins in human serum contains: prealbumin, albumin, α1 Acid
glycoprotein, α1 Antitrypsin, α2 Macroglobulin, CRP, caeruloplasmin, Haptoglobin, transferrin, C3,
C4, IgG, IgA, IgM.

Diagnostic Laboratories should be participating in QAP programs

Test Advantages Disadvantages

Nephelometry Higher sensitivity Need dedicated analyser (cost
Higher precision implications)
Reliable Slower turn-around-time
Calibrator problems
Turbidimetry Can use routine analysers Inferior sensitivity
High throughput
Fast turn-around-time
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Principles of nephelometry and turbidimetry focusing on IgG subclasses

Measurement of IgG subclasses by Nephelometry/Turbidimetry

In 1981 Beck and Kaiser reported the use of rate nephelometry to measure IgG subclasses.

Prior to this other methods were used including:

• Radial immunodiffusion (RID)

o costly (due to the need of large quantities of antiserum)
o time-consuming
o Inherent error of around 10%.
• Radioimmunoassay
o Uses less antisera
o Still costly

There was a recognised need for a reliable, inexpensive and fast method for determining IgG
subclasses

(Beck and Kaiser Clin Chem 1981)

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Principles of nephelometry and turbidimetry focusing on IgG subclasses

Since this early work it has been established that there are 4 subclasses of IgG (1-4) named in order
of frequency in human serum and occurrence as paraproteins.

Differences between total IgG and sum of the IgG sunclasses in clinical samples.

McLean-Tooke et al Pathology 2013: Retrospective analysis of 571 consecutive serum samples.

IgGsum was a mean 3.7% higher than total IgG:

(McLean-Tooke et al Pathology 2013)

In general Total IgG correlated well with IgGsum. There were 4 main outliers

• When samples were repeated 2 outliers returned very different results (i.e. were no longer
outliers) while the other two remained anomalies:

(McLean-Tooke et al Pathology 2013)

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Principles of nephelometry and turbidimetry focusing on IgG subclasses

Of 568 samples 60 (10.6%) had a difference of IgG from IgGsum >15%

• 51(9.0%) had an IgGsum >15% above IgG

• 9(1.6%) had an IgGsum >15% below IgG

(McLean-Tooke et al Pathology 2013)

Authors suggest samples showing >15% difference should be repeated at dilution and if
discrepancies still occur samples should undergo further analysis e.g. Alternate platforms, EPG/IFX
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Principles of nephelometry and turbidimetry focusing on IgG subclasses

Pediatric Reference intervals for IgG subclasses:

• IgG and subclass reference ranges are much lower in the first year of life and may display
geographical variation.

(Lepage et al Clin Biochem 2010)

IgG subclasses in the elderly:

It was previously thought that IgG and IgG subclasses plateaued around 15-18 yrs and remained
fairly constant in adult life.

More recent data suggests adult reference ranged derived for 18-60 yrs age group may not be
applicable to elderly patients and populations.

Lock and Unsworth Ann Clin Biochem 2003: studied 1146 patients >60yrs and compared values to
those from 925 patients 18-60yrs:

• Serum IgG and IgM were reduced in the elderly

o Changes in IgG subclasses were not consistently shown
• Serum IgA was maintained
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Principles of nephelometry and turbidimetry focusing on IgG subclasses

(Lock and Unsworth Ann Clin Biochem 2003)

References:

Beck and Kaiser 1981 Clin Chem 27(2) 310-313.

Lepage et al Clinical Biochemistry 2010 43 694-696

Lock and Unsworth Ann Clin Biochem 2003 40 143-148

McLean-Tooke et al Pathology 2013 45 (7) 675-677

Rose et al Manual of Clinical Laboratory Immunology 5th ed