Transcript Immunoassays , ELISA part two + growth hormon

Detection systems
• For all enzyme-linked immunoassays, the final stage is the addition
of the enzyme substrate. The substrate is chosen for its
quantitative yield of a colored, fluorescent or luminescent reaction
product.
• The decision as to which substrate is the best for any type of assay
depends on the sensitivity desired, the timing requirements, and
the detection device to be used.
• Types of detection systems:
– Colorimetric Assays
– Fluorescent Assays
– Luminescent Assays
Colorimetric assays
• Colorimetric assays result in a colored reaction
product that absorbs light in the visible range.
• Fluorescent immunoassays (ELFIA) are simply a
variation of colorimetric ELISA. An enzyme
converts a substrate to a reaction product that
fluoresces when excited by light of a particular
wavelength.
– In comparison to the colorimetric ELISA, fluorescent
immunoassays are only slightly more sensitive.
• Luminescent immunoassays, like fluorescent
immunoassays, are variations of the standard
ELISA. An enzyme converts a substrate to a reaction
product that emits photons of light instead of
developing a visible color.
– It is believed that luminescence is the most sensitive
detection method currently in use due to the ability of
signal multiplication and amplification
Microplate Reader
• The microplate reader also known as “Photometric microplate reader or ELISA reader” is a specialized
spectrophotometer designed to read results of the ELISA
test
• This instrument works on the principle of "Photoelectric
Colorimetry"
• Unlike the conventional spectrophotometer which facilitates
readings on a wide range of wavelengths, the microplate
reader has filters or diffraction gratings that limit the
wavelength range to that used in ELISA, generally between
400 to 750 nm (nanometres).
• Some readers operate in the UV range and carry out analyses
between 340 to 700 nm.
• The optical system uses optic fibers to supply light
to the microplate wells containing the samples.
• A detection system (sensors) detects the light
coming from the sample, amplifies the signal and
determines the sample’s absorbance.
• The location of the optical sensors of the microplate
reader varies depending on the manufacturers: these
can be located above the sample plate, or directly
underneath the plate’s wells.
• Structure of antibody-coated microplate and
treatment
– 12 strips of 8 wells connected each other are set in a
microplate frame.
– The surface of the plate is covered with a seal to
avoid dryness. Remove the seal after the plate gets to
the room temperature.
– Remove the seal just before starting assay.
– If you want to use only a part of the wells, cut the
part of the seal, and take out the strips and transfer
them to another frame, and remove the seal.
– store the rest of the wells at 2-8C.
• Washing
– The first washing after the first reaction with
standards or samples: Shake off the reaction mixture
from the plate onto a sink.
– Then add washing solution using a multi-delivery
pipette set at 250mcl to avoid carry-over caused by
flowing out the buffer to other wells. Then shake off
the buffer.
– Add new washing buffer from the washing bottle with
nozzle, and shake off.
– Repeat filling and shaking off as many times as
indicated.
• Washing solutions are usually concentrated and
need to be diluted. 20X : (1:20)= 1 part of
washing concentrate with 19 part of D.W, using
volumes equivalent to number of wells to be
washed.
• For 8 wells, you require (8*350ul = approx. 3ml)
• Take 3.8ml of DW with 200 ul of washing
concentrate. (total= 4ml).
• After final shaking off the buffer from the plate,
strike the plate on to some sheets of paper towel for
several times to remove the residual buffer
Automatic plate washers are also commercially
available
• Shaking of the well-plate for mixing
– After addition of a reagent solution, the microplate
should be shaken to mix the solutions. A short
shaking is enough because of the small volume of the
reaction mixture.
microplate mixer (microplate shaker)
• Careful addition of reaction stopping solution
– As the reaction stopper is strongly acidic, please,
be careful in treatment of the solution.
– After addition of the stopping solution the color
of the reaction mixture in the well will change
from blue to orange-yellow.
• Measurement of absorption using a plate reader
– After stopping the enzyme action, absorbance is read
using a densitometer for microplates (microplate
reader).
– If possible measure absorbance at both 450nm and
620nm, and calculate the difference between
absorbance at 450nm and 620nm.
– By doing this, we can compensate influences of some
non-specific factors of wells like small scar on the
bottom of well, and the variation in the structure of
wells, e.g., thickness of the bottom.
– The difference is expressed as Abs.450(∆620)nm.
• Preparation of standard curve and calculation of
assay values
– In manual calculation, prepare a standard curve using
bi-logarithmic section paper by plotting
Abs.450(∆620)nm on Y-axis against standard
concentration (ng/ml) on X-axis.
– Because the coefficients of variation (CV) in
absorbance do not change so much throughout all the
assay range, it is proper to choose logarithmic
expression in both concentration and absorbance.
This expression allows easy reading of lower
concentration area.
• Read the concentrations of the substance to
measure in samples from their
Abs.450(∆620)nm, and multiply the assay
value by sample dilution rate if samples have
been diluted.
Important points in performing ELISA and
improvement of assay
performance
1. Sampling and treatments of samples
– Serum or plasma?
o serum samples is most recommended
o In getting plasma:
•
•
heparin is most often used as an anticoagulant
Use of fluoride must be avoided because
fluoride ion is a potent inhibitor of peroxidase.
o An important phenomenon with frozen
plasma is that an insoluble substance (fibrin)
will be formed when thawed.
In this case, the sample must be vortexed and
centrifuged, then the insoluble cluster flowing in the
plasma should be taken out by a thin wire needle
sharply bent at an end. If such fibrin remains in the
sample, it may clog the tip of a pipette and influences
assay variability.
• Storage temperature and freezing-thawing
– Sample storage temperature is better to be lower
than -35C.
– Ultra-low temperature such as -80C is
recommended for a long-term storage.
– A long storage in a home freezer is not
recommended because the sample is frozen very
slowly and the solute may separate from solvent,
and is concentrated at to the bottom of the
storage tube
– Repeated freezing and thawing is also harmful to
the protein, and may cause inactivation.
• When samples are taken out from the freezer and
thawed, never forget to vortex these samples because
the solution after thawing is not homogeneous, and
the bottom area contains more solute.
• Stability of assay samples
• In assay, the problem of sample stability, i.e. how
long the substance to be measured can keep its
immunoreactivity, in serum or plasma, is very
important. Blood samples also contain enzymes to
destroy peptides or proteins, and stability against
those enzymes differs from substance to substance
• Most Kits have their own stability ranges;
most of them have the following ranges:
– Stability at room temperature: 2 hours
– Stability at refrigerator temperature: 24 hours
– Stability against freezing-thawing: 3 times
– Stability for long period: at –20℃ or-80℃
• Influence of humidity and air stream
As ELISA is carried out in small volume of reaction
mixture in shallow wells, the ratio of surface area to
the liquid volume is large. This means that
evaporation of water from the surface during
incubation cannot be ignored. During all the
incubation process, the well-plate should be covered
using the attached plate cover.
• Pipettes
• Selection of pipettes
– Pipettes for samples and standard solutions are
especially important because their precision
directly influence the assay precision. When you
add samples or standards at a volume of 5 or 10
µl, for example, use a pipette with the maximal
setting volume of 5 or 10µl, and never use with
the maximum setting volume of 100µl or more.
In other words, use volume-matched pipette
Trouble shooting in ELISA
1st , Coloration
Troubles
Possible Causes and Measures
Poor or no
coloration after the
last step
1) The standard or samples might not be added.
2) 2) Reagents necessary for coloration shown below might
not be added.
①Biotin-labeled antibody
②HRP-conjugated avidin
③TMB or OPD
3) Contamination of enzyme inhibitor(s).
4) Influence of the temperature under which the kits had
been stored. Freezing and thawing might cause denaturation
of HRP-avidin conjugate.
5) Excessive hard washing of the well plate.
6) Addition of TMB solution soon after taking out from a
refrigerator might cause poor coloration owing to low
temperature.
All the reagent solutions for ELISA should be used after fully
warmed up to roomtemperature (20-25°C)
Satisfactory absorbance was not
obtained by the plate reader; though
the coloration is apparent to the
eye.
The plate-reader might not be
adjusted to the correct wavelength
(450nm for TMB). ⇒ Check the
reader and adjust the wavelength. In
the case of a filter-type plate-reader,
check the wavelength of the filter,
and replace for right one when
wrong.
All the wells showed high coloration. TMB solution might have been
oxidized by some agents..
2nd standard curve
Flat standard curve.
All the wells for the standard curve
showed very low coloration, though
sample wells gave good coloration.
Standard solutions are not added.
In the dilution of original standard
solution, other solution
than the right original standard
solution might be used, or
simply the original standard solution
was forgotten
Absorbance of the standard
decreased when standard
concentration increased.
Order of the standard solutions might
be reversed.
The standard curve obtained
was not smooth
There might be some mistake in the
serial dilution of the original standard
solution. Pay attention to volumes of
standard and buffer solutions and
enough mixing
The standard curve obtained
moved toward right than that
shown in the instruction
paper.
1) The standard preparation might
have been denatured.
2) The wrong initial dilution of the
original standard
solution.
3) The vortex mixing in the dilution
of original standard
solution might not be enough, and
the mixture might not be
homogeneous.
4) The reaction period might not be
enough by any mistake.
3rd samples
Assay values were not
obtained because
samples gave lower
absorbance than that
of blank though the
standard curve
seemed to be normal.
1) In case NaF-coated tubes are used, or NaN3 is added
to assay sample as a preservative, they may influence
the enzyme action of HRP though wells are washed
before the addition of HRP-conjugate. The best way is
not to use NaF and NaN3.
2) Inactivation of the substance to be measured e.g
during storage
3) presence of any interfering substances in blood
samples=> serial dillution
4) Hemolysis.
A slight hemolysis will not influence on assay value,
however, too much hemolysis (hemoglobin
concentration more than 40mg/ml) may interfere
significantly with the assay reaction.
5) pH of serum/plasma samples.
Blood samples like serum or plasma easily lose carbon
dioxide soon after preparation, and pH will become
alkaline (> pH 8) which may interfere with antigenantibody binding.
4th, assay precision
Big variation between two
wells in duplicated assay was
observed.
1) Scratching the bottom of the well
by aspirator tip during aspiration
of washing buffer.
2) Scratching the bottom of the well
by pipette tip during addition of
standards, samples, or reagents.
3) Insufficient removal of washing
buffer from the wells might dilute
reagent solution added in the
following step of the procedure.
4) Assay variation due to pipetting
Advantages of ELISA 
 High sensitivity
 High specificity
 relatively cheap and require small amounts of reagents
 rapid and can give both qualitative and quantitative results
 Detection is easy: results can be detected visually or using
special readers
 Results are reproducible
 Automated, high throughput and manual methods are
available,
 Very versatile
 No problems with radiation or disposal of waste
 Can use monoclonal or polyclonal antibodies.
• Human Growth Hormone (hGH) is a polypeptide chain,
composed of 191 amino acids and with a molecular mass
of 21,500 Da. It is released by the anterior pituitary of
both men and women. The secretion is stimulated 3 –4
hours after a meal, about 1 hour after the beginning of
sleep, and after physical exercise.
• Hyposecretion of hGH becomes apparent in infants a few
months after birth and may result in dwarfism. In the
opposite case, hypersecretion of hGH results in
gigantism and may be due to hypophysic tumors.
• In adults, when epiphyses are closed,
hypersecretion of hGH provokes an increase
in volume of soft tissues (hands, feet, lips), a
proliferation of bones (acromegalysyndrome),
and a limited tolerance of glucose Plays an
important role in growth control.
• Its major role in stimulating body growth is to stimulate the
liver and other tissues to secrete IGF-1.
• It stimulates both the differentiation and proliferation of
myoblasts.
• It also stimulates amino acid uptake and protein
synthesis in muscle and other tissues.
• Growth hormone secretion is regulated by three feedback
loops, including both long, short and ultrashort loops.
1. GHRH inhibits its own secretion from the hypothalamus
via an ultrashort-loop feedback.
2. Somatomedins, which are by-products of the growth
hormone action on target tissues, inhibit secretion of
growth hormone by the anterior pituitary.
3. Both growth hormone and somatomedins stimulate the
secretion of somatostatin by the hypothalamus.
• Growth hormone is secreted in a pulsatile pattern, with
bursts of secretion occurring approximately every 2 hours.
• The largest secretory burst occurs within 1 hour of falling
asleep (during sleep stages III and IV). The bursting pattern,
in terms of both frequency and magnitude, is affected by
several agents that alter the overall level of growth hormone
secretion.
• The normal values often increase to as high as 50 ng/ml
after depletion of the body stores of proteins or carbohydrates
during prolonged starvation.
• In brief, it has been found that growth hormone causes the
liver (and, to a much less extent, other tissues) to form
several small proteins called somatomedins that have the
potent effect of increasing all aspects of bone growth.
• Many of the somatomedin effects on growth are similar to
the effects of insulin on growth. Therefore, the
somatomedins are also called insulin-like growth
factors (IGFs). At least four somatomedins have been
isolated, but by far the most important of these is
somatomedin C (also called IGF-1).
• The molecular weight of somatomedin C is about 7500,
and its concentration in the plasma closely follows the
rate of growth hormone secretion.
• Some aspects of the somatomedin hypothesis are still
questionable. One possibility is that growth hormone can
cause the formation of enough somatomedin C in
the local tissue to cause local growth.
• It is also possible that growth hormone itself is directly
responsible for increased growth in some tissues and
that the somatomedin mechanism is an alternative means
of increasing growth but not always a necessary one
Short Duration of Action of Growth Hormone but
Prolonged Action of Somatomedin C.
•
• Growth hormone attaches only weakly to the plasma
proteins in the blood. Therefore, it is released from the
blood into the tissues rapidly, having a half-time in the
blood of less than 20 minutes.
• By contrast, somatomedin C attaches strongly to a carrier
protein in the blood that, like somatomedin C, is produced in
response to growth hormone. As a result, somatomedin C is
released only slowly from the blood to the tissues, with a halftime of about 20 hours. This greatly prolongs the growthpromoting effects of the bursts of growth hormone secretion
shown in Figure
• The serum level of insulin-like growth factor-1 (IGF-1)
and of its major circulating binding protein IGFBP-3 are
types of these of biomarkers. They are used to diagnose
and assess the degree of the human growth hormone
(hGH)-deficient state, and to measure responsiveness
and predict the outcome of hGH therapy of a patient.
They are also used in the differential diagnosis of growth
disorders in order to elucidate the etiology of the disease.
In addition, IGF-1 and IGFBP-3, i.e. their ratio, can
serve as safety biomarkers of hGH therapy.
• IGF-1 measurement before and after growth hormone
therapy.
• The Human Growth Hormone (hGH) ELISA is a solid
phase sandwich ELISA method. The samples and antihGH-HRP conjugate are added to the wells coated with
hGH MAb. hGH in the serum binds to anti-hGH MAb
on the well and the anti -HGH second antibody then
binds to hGH. Unbound protein and HRP conjugate are
washed off by wash buffer. Upon the addition of the
substrate, the intensity of color is proportional to the
concentration of hGH in the samples. A standard curve
is prepared relating color intensity to the concentration
of the hGH.
• The diagnosis of growth hormone defiance cant be
made in a single random growth hormone level
because growth hormone is secreted in pulses.
• Some pediatric endocrinologists diagnosis growth
hormone defeciency based on an extremely low level
of insuline like growth hormone which varies much
less in the course of the day than growth hormone.
• IGF1 levels are dependent on the amount of growth
hormones in the blood but can also be low in normal,
young children, so the test must be interpreted
carefully.
• A more accurate but still imperfect way to diagnosis
growth hormone deficiency is a growth hormone
stimulation test. In this test, your child has a blood
drawn for about 2 to 3 hours after being given
medications to increase growth hormone release.
• If the child does not produce enough growth
hormone after this stimulation, then the child is
diagnosed with growth hormone deficiency.
However, growth hormone stimulation tests can
over diagnose growth hormone deficiency. Growth
hormone stimulation tests vary and are complicated,
so they are usually performed under the guidance of
a pediatric endocrinologist.
• Usually, other tests to check the pituitary or to
evaluate the brain MRI are performed when
treatment is considered.
Clinical significance
• In children, ascertaining linear bone growth along
the epiphyseal plate. Abnormally elevated levels lead
to gigantism while complete absence slows the rate
of growth one third to one half of normal.
• In adults, the epiphyseal growth plates had fuse so
hGH excess gradually produces acromegaly, a coarse
thickening of the bones of the skull, hands and feet.
Growth hormone test
• The specimens shall be blood, serum in type and the
usual precautions in the collection of venipuncture
samples should be observed.
• For accurate comparison to established normal
values a fasting morning serum sample should be
obtained.
• The blood should be collected in a plain redtop
venipuncture tube without anticoagulant or additive.
Allow the blood to clot. Centrifuge the specimen to
separate the serum from the cells.
Sample storage
• Samples may be refrigerated at 2-8 C for maximum
period of five days.
• If the specimen can not assayed within this time, the
samples may be stored at temperatures of -20C for
up to 30 days.
• Avoid use of contamination devices. Avoid repetitive
freezing and thawing. When assayed duplicate, 0.100
ml of the specimen is required.
Principle
• In this method, GH calibrator, patient specimen or
control is first added to streptavidin coated well.
• Biotinylated monoclonal and enzyme labeled
antibodies(directed against district and different
epitopes of GH) are added then the reactants mixed.
• Reaction between the various GH antibodies and
native Gh forms a sandwich complex that binds with
streptavidine coated to well.
• After completion of the required incubation period,
the enzyme-growth hormone antibody bound
conjugate by aspiration or decantation.
• The activity of the enzyme present on the surface of
the well is quantized by reaction with a suitable
substrate to produce color.
• Hyperglycemia inhibits growth hormone secretion.
Age is an important factor in growth hormone
concentrations. At birth, GH is high and generally
declines with the exception of a burst during the
growth phase of adolestronce.
• Women typically have a 50% higher level than their
age-mached males.