On approaches to ameliorate the insidious and pernicious problem of interference and inaccuracy in

immunoassays

Adel A.A.Ismail

To discuss the following

How common.

Suspecting interference.

Current follow-up confirmatory tests and their limitations.

Approaches to minimise interference and improve immunoassays accuracy and utility. Personal view

How common ?

• 0.5% to 4.0%; (0.05%)

• Could worsen in the future, with aging population, the wider use of immunotherapies and vaccination.

Suspecting inaccuracy and Interference

• Very unusual/odd result(s)

• Believable/acceptable results. (Clin Med 2007; August issue)

Tests for detecting interference; its limitations and misinterpretation

(Clin Chem 2002; 48: 2023-9)(Ann Clin Biochem 2006; 43: 249-51)

Doubling dilutions, linearity and parallelism.

Blocking antibodies (commercial) or addition of native non-immune serum.

Repeat analyses using different platforms.

Repeat analyses by another method

Bias; conventional/Routine vs immunoassays

The assumption

• Raised TSH of 22 in WF lab and 11 iu/L in LDS in a patient with normal fT4 and fT3.

• Investigated and treated with thyroxine for 18 months.

• Potential treatment for life if interfering antibodies was transient. Cost!

Limitation and interpretation

If bias is “reversed or exaggerated” ?

If results are similar or even identical ?

Blocking Antibodies (Commercial) and the

addition of non-immune serum

The assumption

Subjective vs Objective/Statistical application

The findings: ~ 20% of samples with know endogenous interfering antibodies gave results within statistical “norm”.

Addition of native non-immune serum reduced interference from 4% to 3.9% in the Norwegian study on a CEA Immunoassay.

Limitation and Interpretation

If similar results, statistically not different?

If statistically different before and after incubation ?

Doubling dilutions, linearity and parallelism

The assumption

Visual/subjective assessment !!

Objective & statistical approach: Altman and Bland plot.

Using Altman&Bland plot: ~ 40% of samples with known endogenous antibodies showed perfect linearity and parallelism.

Limitation and Interpretation

If statistically non-linear ?

If linear/parallel ?

The current state

Annually ~ 10 M immunoassay tests in UK

Mechanisms of potential interferences are numerous (CCLM, July 2007)

Cannot be predicted a priori

Screening every sample, unproductive

Monospecificity of immunoassay is

untenable

Immunoassay 1- Capture antibody

Antigen

Capture antibody

Erase the lock-key depiction and think induced-fit.

The fundamental mechanism of the immune system is the binding between Ag & Ab ; evolved over millions of years, to bind known and unknown Ags (pathogens)

Not for analytical immunoassays.

Probability of detecting interference using existing follow-up tests

Assumptions:

(1) Repeat analysis would identify ~ 50% (E)

(2) Doubling dilutions would identify ~60%

(3) Blocking antibodies would identify ~80%

Normal results = 50 X 40 X 20 / 100 X 100 X 100

The statistical probability of simultaneously normal results in the three tests would be 4% despite potential inaccuracy caused by the presence of endogenous interfering

antibodies.

Analytical approaches to ameliorate interference

and inaccuracy (A personal view)

(1)Assessed in Leeds and Wakefield the removal of immunoglobulin prior to immunoassays of AFP, FSH and PRL.

(2)Extending the utility of doubling dilutions test

(1) Removal of endogenous immunoglobulins prior to analyses

using polyethylene glycol (PEG)

(Clin Chem 2005; 51: 25-6)

Instant precipitation at room temperature.

PEG at 125 g/L final concentration quantitatively precipitated IgM, IgG and 70% of IgA.

• No precipitation occurred in the three analytes tested, namely FSH, PRL, AFP.

• Significantly, no adverse effect on any of the assay’s accuracy, precision or sensitivity.

However, 30-40% co-precipitation occurred in LH and TSH immunoassays.

Preliminary studies on 11nother widely different analytes were encouraging e.g. cortisol, testosterone, oestradiol, T4, T3, SHBG, GH, CA125.

• PEG is inexpensive, flexible, stable and mild chemical. Its mechanism of action suggests a wider utility.

PEG’S LIMITATIONS

• Pre-analytical step requiring re-engineering of immunoassay analysers

• Can not be used when the analyte itself is an immunoglobulin e.g. TPO, RF…etc

• May interfere with some signal e.g. flouresence.

(2) Extending the utility of doubling dilutions test

(Clin Chem & Lab Med 2007; 45: July)

As mentioned before, non-linearity/non parallelismoccurs only in 60% of samples.

Could interference be detected in the other 40% despitelinearity and parallelism ?

IF yes, then many more samples with interfering antibodiescould be identified even when linear and parallel.

Immunoassay kinetics expressed as a Scatchard Plot

Scatchard plot is a linearized non-linear regression analysis

Parameters needed at different concentration (mole/L):

Amount of reagent antibody [Ab]

Amount of antigen [Ag] at each concentration.

Amount of free antigen “c “ at each concentration.

Amount of bound antigen [Ag-Ab] at each concentration

To depict Scatchard plot

Calculate the ratio “r “ from [Ag-Ab] / [Ab]

Plot r / c against r to produce the Scatchard plot

The slope deduces Ka

The intercept deduces the number of binding sites (n) per Ab molecule

Scatchard plot:

In immunoassays without interference,

the Scatchard plot is straight line.

When endogenous interfering Ab is present, the line becomes concave, broken into two or more slopes.

This causes average affinity (K0) to change (deduced from r /c value of the curve at r= 0.5)

“n” value also changes if interfering antibody has different valency. Monomeric IgG (bi-valent) is nearly always used as reagent. Interfering antibody however may be IgG, IgA or IgM class.

Monitoring in real-time Ag-Ab binding kinetics in immunoassays

• Binding kinetics in the absence of interference is known because it is necessary for developing/ formulating immunoassays. This information is not provided to the user nor utilised in every day analyses.

• If used, a distorted data on binding kinetics would point to the potential presence of another interacting antibody(s) in the assay cocktail, warranting further validation of such immunoassay result.

Well developed affinity analyses mathematical models are available.

Embedded in modern immunoassay analyzer could help identifying potential interference when the endogenous antibody is sufficiently different from reagent antibody even when doubling dilutions is linear and parallel.

Limitations:

Endogenous interfering antibodies from the same class and with similar affinity may no be detected.

• Free hormones e.g. fT4 ; fT3

Take-home message

• Interference is as old as the technique itself. Why?

• The very primary role of the clinical lab is

to provide analytically timely, precise and accurate results.

• Clinicians perceive all results in the same light.

• Automation of immunoassays has ensured timeliness and precision, but inaccuracy remained a major problem.

• Inaccuracy could be ameliorated analytically. This would enhance the efficacy of clinical validation and utility of immunoassays.

Ethylene and Polyethylene Glycol

• Ethylene CH2=CH2

• Ethylene glycol HO-CH2-CH2-OH

• HO-CH2-CH2-OH HO-CH2-CH2- OH

• PEG HO-(CH2-CH2-O-CH2-CH2-O)n -H

• “n” is The number of subunits in the polymer .• If n = 80, PEG will have MW of 3500 i.e. PEG 3500

PEG mechanisms of action

• Changes in the microenvironment causes protein to precipitate.

• Steric exclusion i.e. proteins are excluded from the regions of the solvent occupied by PEG, thus increasing proteins’ concentration until their solubility is exceeded.

• pH of the buffer and the pI (isoelectric point) of the protein.

• PEG is soluble in water and methanol/ethanol.

• Ions such as calcium and strontium could modify its action on protein solubility.

• PEG is a member of very similar polymers e.g. polyethylene oxide (PEO) and polypropylene glycol (PPG).

Factors affecting binding Kinetics of Ag to Ab in immunoassays

Concentration and diffusion rate determinebumping into each others.

Association and dissociation rates (K1 & K -1 ) ;

their ratio is the affinity rate constant (Ka ).

Proximity and closer interaction between epitopeand paratope depends on conformationalcomplexity and spatial complementarities.

Non-covalent bonds

• Electron cloud shape• Hydrogen bonds• Van der Waals forces• Hydrophobic interactions• Hydrophilic interactions (major water role)• Electrostatic

• More bonds = Stronger binding or Ka

• Ka is expressed as 10* (i.e. ten to the power of the digit).