required. Most nephelometers measure light scattering at a 90° angle. However, in order to measure the forward scatter intensity caused by light scattering from large

 

Certain reactions, however, may not follow the BeerLambert law within the pathophysiological concentration

for an analyte and, hence, do not provide a straightline graph. For such analytes, the unknown cannot

be determined using a single standard. A graph using

different concentration of standards vs absorbance has to

be plotted on a graph paper. The plotted curve is known as

the standard curve (Fig. 23.1B).

The concentration of the unknown can be interpolated

from this standard curve.

Measuring Principles in Biochemistry

Criteria for Wavelength Selection

It has been established that when the wavelength of

light used is complementary to the color of the chemical

complex to be measured, peak absorbance is obtained.

Thus, selection of the wavelength depends on the color of

the complex to be measured.

Complementary Filters for Measuring Color Complexes

Color of the complex Wavelength/Color

Yellow 405/Violet:

Red 505/Green

Blue-violet 546/Green

Green 630/Red

FIG. 23.1A: Illustration of a straight-line graph obtained by plotting

absorbance vs concentration of analyte for reactions which obey

Beer-Lambert law

FIG. 23.1B: Illustration of a standard curve obtained by plotting

absorbance vs concentration of analyte for reactions which do not

obey Beer-Lambert law

Diagnostic Immunology 703

For example, for the estimation of hemoglobin using

cyanmethemoglobin method, a red colored complex,

which is formed during the reaction, is measured using a

green filter.

Reading Methods

The measurements of biochemical reactions using

enzymes, substrates or specific chemicals are read by

methods mentioned below.

1. Equilibrium Methods

Also known as end point methods. Here, the absorbance

of end product is measured when the reaction between

the reagent and sample has virtually come to equilibrium

(end) and the substrate has been converted into a stable

end product. The reaction ceases when equilibrium is

reached. The concentration of the test specimen can be

calculated by using the equation 2 as described earlier.

2. Kinetic Methods

Also known as rate methods where the rate of change

of absorbance (∆A) produced in a fixed time interval is

measured.

The kinetic measurements are of two types:

¾ Fixed time analysis

Where the ∆A produced by the reaction between the

reagent and the substrate is measured by stopping the

reaction at a fixed time interval.

¾ Continuous monitoring

Where the ∆A produced is monitored continuously as the

reaction proceeds.

Results of the unknown are derived using a factor (K)

in the kinetic methods, which is usually provided by the

manufacturer or can be calculated as:

 Vtotal× 1000

K = _________________

 Vsample × t × ∈× d

Vtolal= total volume of the reaction mixture, Vsample= Volume

of sample, t = time, ∈ = molar extinction coefficient of the

chromogen, d = length of the light path

Standardization of Time Interval for Rate Reactions

Determination of the reaction rate involves the measurement of the amount of change in absorbance (∆A)

produced in a defined time interval. Depending on the

reaction, kinetics between a specific reagent and the

substrate, the time interval for reading the ∆A can be

selected to measure the reaction rate. The different types

of reaction curves, which can be obtained as the reaction

progresses, typically follow the following patterns

(Fig. 23.2).

Curve A

If a graph similar to ‘curve A’ is obtained then any time

interval can be selected for reading reactions, as the rate of

change is constant during the entire reaction run.

Curve B

Correct results can be obtained only if the rate is measured

along segment II. Incorrect results are obtained if the ∆A is

measured during the lag phase (I) or during the phase III.

Curve C

Deviates from linearity over its entire course and ∆A fall off

with time. At no time does it give rate of constant changes.

Such reaction curves are not suitable for measurements

and the reagent systems have to be optimized to obtain

correct reaction curves.

Measurement of Immune Complexes by

Spectrophotometry

Unlike in classical biochemistry, where the reactants

are clear and endpoints are expressed as absorbances,

the behavior of light differs for solutions containing

suspensions or particulates. Such particles (insoluble

immune complexes) are formed as the reaction between

antigens and antibodies takes place.

When light of suitable wavelength is allowed to pass

through a reaction solution containing antigens (analytes)

and the initial absorbance is measured, the absorbance is

minimum at this point (Fig. 23.3).

Subsequently, the reagent containing corresponding

antibody solution is then added to the antigen in the

cuvette and allowed to react. An agglutination reaction

begins when a single molecule of antibody binds to at

least two corresponding binding sites on different antigen

particles. As the reaction proceeds, the agglutinating

particles aggregate and form immune complexes. Immune

complexes increase in size, become larger, resulting in

an increase in turbidity and the scattering of the incident

light. Thus, a decreasing part of the incident light (Io) is

transmitted as the reaction proceeds. Spectrophotometers

read this decrease in the intensity of the transmitted light as

absorbance (Fig. 23.4).

This measurement of reduction in the intensity of the

transmitted light at 180° is defined as turbidimetry. The

turbidity is proportional to the analyte concentration,

which in turn is proportional to the amount of

agglutination. Based on this proportional relationship,

the amount of analyte in the sample causing the turbidity

can be easily determined. It should be noted that the

nature of immunochemical reaction is exactly the same

704 Concise Book of Medical Laboratory Technology: Methods and Interpretations in turbidimetry and nephelometry. However, it is the

detection principle applied for measurement, which

differentiates turbidimetry from nephelometry (Fig. 23.5):

Nephelometry measures light scattered or reflected

towards the detector, which is away from direct path of

the transmitted light. Routine spectrophotometers cannot

be used for nephelometry and hence, nephelometers are

required. Most nephelometers measure light scattering

at a 90° angle. However, in order to measure the forward

scatter intensity caused by light scattering from large

particles, some nephelometers are designed to measure

scattered light at an angle other than 90°.

Selection of Wavelength for Measuring Immune

Complexes

The optimum wavelength for optical measurement of

immune complexes increases with the size of immune

complex to be measured. In general, if the size of the immune

complex formed is less than 1/10th the size of the wavelength of incident light, then the light scattering is relatively

symmetrical (Fig. 23.6). This uniform scattering of light is

known as Rayleigh scattering.