Bioassay is Assessment of a biological substance. Bioassay or biological standardization is a type of scientific experiment typically conducted to measure the effects of a substance on a living organism and is essential in the development of new drugs and in monitoring environmental pollutants. Both are procedures by which the potency or the nature of a substance is estimated by studying its effects on living matter. Bioassay is a procedure for the determination of the concentration of a particular constitution of a mixture.

Bioassays are procedures that can determine the concentration of purity or biological activity of a substance such as vitamin, hormone, and plant growth factor. While measuring the effect on an organism, tissue cells, enzymes or the receptor is preparing to be compared to a standard preparation. Bioassays can be classified in two types:

Qualitative Quantitative

Qualitative bioassay is used for assessing the physical effects of a substance that may not be quantified, such as abnormal development or deformity. Example of a qualitative bioassay includes Arnold Adolph Berthold's famous experiment on castrated chickens. This analysis found that by removing the testes of a chicken, it would not develop into a rooster because the endocrine signals necessary for this process were not available.

Quantitative bioassays involve estimation of concentration/potency of a substance by measurement of the biological response it produces. These bioassays are typically analyzed using the methods of biostatistics.

Principles of bioassay

Bioassay involves the comparison of the main pharmacological response of the unknown preparation

with that of the standard.

The reference standard and test sample should have same pharmacological effect and mode of action, so that their DRC curve run parallel and their potency ratio can be calculated.

The test solution and standard should be compared for their established pharmacological effect using a specified pharmacological technique.

The method selected should be reliable, sensitive, and reproducible and should minimize errors due to biological variation and methodology. (Animals should of same species, sex and weight and number of animals should be large enough to permit statistical analysis.

Need of Bioassay

1. They not only help to determine the concentration but also the potency of the sample. Potency denotes activity of the compound i.e. if a compound shows better activity at minute concentration, greater is the potency, and if its activity is low at lower concentrations, lesser is the potency.

2. It is used to standardize drugs, vaccines, toxins/ poisons, disinfectants, antiseptics etc. as these are all used over biological system in some form.

3. These also help determine the specificity of a compound to be used ex: Penicillin's are effective against Gram+ve but not on Gram-ve. Testing of infected patients sputum helps determine which anti-biotic be given for quick recovery.

4. Certain complex compounds like Vitamin B-12 which can't be analyzed by simple assay techniques can be effectively estimated by Bioassays.

5. Sometimes the chemical composition of samples is different but has same biological activity. 6. For samples where no other methods of assays are available.

Procedure

i. Prepare the physiological salt solution

ii. Arrange the instrument and adjust the water bath. iii. Balance the lever

iv. Tissue selection

v. Surgical process and collection of required tissue. vi. Tissue attachment to the water bath

vii. Relaxation time given to the tissue

viii. Prepare the standard drug( serial dilution) ix. Select the lowest possible measurable concentration by trial and error method. x. Prepare DRC for the standard drug.

xi. Prepare DRC for the test drug.( serial dilution) xii. Select a assay method (3 point or 4 point assay)

xiii. Calculation

1. Prepare the physiological salt solution

reagent Frog-

Ringer Krebs Tyrode Ringer-

Locke

De

Jalon Mc

Ewen

NaCl 65 g 69 g 80 g 91.5 g 90 g 76 g

KCl 1.4 g 3.5 g 2.0 g 4.2 g 4.2 g 4.2 g

MgCl. 6HO --- 1.1 g 1.0 g --- --- ---

NaH2PO4.

HO

0.1 g 1.4 g 0.5 g --- --- 1.4 g

NaHCO 2 g 21 g 10 g 1.5 g 5 g 21 g

CaCl 1.2 g 2.8 g 2 g 2.4 g 0.6 g 2.4 g

Glucose 20 g. 20 g. 10 g. 10 g. 5 g. 20 g

Aerating

Gas air O +

5%CO O or air Pure O O +

5% CO

O + 5% CO

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2. Arrange the instrument and adjust the water bath.

Student Organ bath o Outer bath:-

First designed by rudolph magnus Perplex glass

Store water outside the inner bath to maintain the temperatureo Inner bath:-

Glass To observe the tissue during experiment 5-50ml (usually 10ml)

Arrange the instrument and adjust the water bath.

First designed by rudolph magnus glass

Store water outside the inner bath to maintain the temperature

To observe the tissue during experiment 50ml (usually 10ml)

Store water outside the inner bath to maintain the temperature

Kymograph: Sherrington- starling kymograph

To obtain a graphical amplified measurable response of a muscle or tissue Two important parts: motor box and drum Speed lever: 1 revolution/ 96 min. Paper: glossy side outside least resistance

Rough side inside stick to the drum. Fixing solution: shellac and colophony saturated in alcohol

3. Surgical process and collection of required tissue Animal sacrificed by cervical dislocation. Tissue identified and isolated. Carefully dissect and separate unwanted tissue. Tissue kept in a physiological salt solution.

Avoid excessive handling of tissue.

4. Tissue attachment to the water bath

Attach the ends of the tissue: - One end to tissue holder other end to lever

Method of attachment of tissue: Attach the thread at the end by a needle. Care should be taken for intestine tissue not to block the lumen.

5. Prepare DRC for the standard and test drug

Two STD doses s1& s2 from linear part of DRC

Types of Bioassays

Basically there are two types of bioassays as per the technique

1. Graded Response Assay 2. End Point or Quantal Assay

Graded Response Assay: In these assays, as the dose increases there are The potency is estimated by comparing the Test sample graded dose response relationship, relates the size of the response to the drug in a single biologic unit as the dose administered increased the pharmacological response also increases and eventually reachcalled the ceiling effect there will be on further increase in response even with an increase in dose.

Prepare DRC for the standard and test drug

part of DRC [Let the corresponding response be S1, S2] Also s2/s1 = t2/t1 = 3/2

Basically there are two types of bioassays as per the technique used in determination of the Sample under test.

In these assays, as the dose increases there are an equivalent rise in response.

The potency is estimated by comparing the Test sample responses with the standard response curve. In the graded dose response relationship, relates the size of the response to the drug in a single biologic unit as the dose administered increased the pharmacological response also increases and eventually reachcalled the ceiling effect there will be on further increase in response even with an increase in dose.

Also s2/s1 = t2/t1 = 3/2

used in determination of the Sample under test.

an equivalent rise in response.

responses with the standard response curve. In the graded dose response relationship, relates the size of the response to the drug in a single biologic unit as the dose administered increased the pharmacological response also increases and eventually reaches a steady level called the ceiling effect there will be on further increase in response even with an increase in dose.

The graded dose response curve is obtained by plotting a graph with dose on the Xaxis. It is usually sigmoid in shape however the log dose response curve is almost a straight line and particularly useful in bio assay.

Conc. of unknown= Threshold dose of standard/threshold dose of test x Conc. of standard.

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End Point or Quantal Assay

It is the simplest type of the bioassay. particular pharmacological effect is determined and compared with standard.

E.g. Digitalis producing cardiac arrest. Here the sample effect is identified by the response it produces on the biological system. Digitalis produces cardiac stim

(+)d TC (Tubocurarine) producing neck relaxation in rabbit, Here as the sample is injected to the neck muscle of the Rabbit, the neck starts to droop. On further doses there is complete hanging of the ne

The graded dose response curve is obtained by plotting a graph with dose on the X-axis and response on the Yd in shape however the log dose response curve is almost a straight line and particularly

Conc. of unknown= Threshold dose of standard/threshold dose of test x Conc. of standard.

$%&'&&&()

It is the simplest type of the bioassay. The threshold dose of the sample required elicitingparticular pharmacological effect is determined and compared with standard.

Digitalis producing cardiac arrest. Here the sample effect is identified by the response it produces on the biological system. Digitalis produces cardiac stimulation on further doses it produces cardiac arrest.

(+)d TC (Tubocurarine) producing neck relaxation in rabbit, Here as the sample is injected to the neck muscle of the Rabbit, the neck starts to droop. On further doses there is complete hanging of the ne

axis and response on the Y-d in shape however the log dose response curve is almost a straight line and particularly

Conc. of unknown= Threshold dose of standard/threshold dose of test x Conc. of standard.

&()

eliciting a complete or a

Digitalis producing cardiac arrest. Here the sample effect is identified by the response it produces on the ulation on further doses it produces cardiac arrest.

(+)d TC (Tubocurarine) producing neck relaxation in rabbit, Here as the sample is injected to the neck muscle of the Rabbit, the neck starts to droop. On further doses there is complete hanging of the neck and rabbit has no

ability to lift the neck. Even the Determination of LD50 (LD=Lethal dose) or ED50 (ED= effective dose) is done by this method.

Based on the method used during the grade point assay procedure for determination of Type of activity and Potency of the Sample, four methods of assays are classified as:

1. Matching point or bracketing method2. Interpolation assay

3. Three point (2+1) assay 4. Four- point (2+2) assay

1. Matching point or bracketing methodvarying dose of sample until an exact matching between the standard dose responses and the particular dose response of the sample is achieved.

This technique is used

i. when test sample is too small

ii. Inaccurate & margin of error difficult to estimateiii. Eg: histamine on guinea pig ileum, Posterior pituitary on rat uterus

Even the Determination of LD50 (LD=Lethal dose) or ED50 (ED= effective dose) is

Based on the method used during the grade point assay procedure for determination of Type of activity and otency of the Sample, four methods of assays are classified as:

Matching point or bracketing method

Matching point or bracketing method: Here a constant dose of the standard is bracketedvarying dose of sample until an exact matching between the standard dose responses and the particular dose

est sample is too small

Inaccurate & margin of error difficult to estimate histamine on guinea pig ileum, Posterior pituitary on rat uterus.

Even the Determination of LD50 (LD=Lethal dose) or ED50 (ED= effective dose) is

Based on the method used during the grade point assay procedure for determination of Type of activity and

: Here a constant dose of the standard is bracketed by

varying dose of sample until an exact matching between the standard dose responses and the particular dose

2. Interpolation assay: Bioassays are conducted by determining the amount of preparation of unknown potency required to produce a definite effect on suitable test animals/organs/Tissue under standard conditions. This effect is compared with that of a standard. Thus the amount of the test substance required to produce the same biological effect as a given quantity the unit of a standard preparation is compared and the potency of the unknown is expressed as a % of that of the standard by employing a simple formula.

3. Multi point Bioassay: This method incorporates the principle of interpolation and bracketing. 2+1 indicates- Two response of Standard and one response of Test respectively. This procedure of 2+1 or 2+2 is

repeated 3 times or 4 times based on the method with crossing over of all the samples. & *+(,%

Three point assay [2+1 dose assay] is Fast & convenient: Log dose response [LDR] curve plotted with varying conc of std drug solutions and given test

solution

Select two std doses s1& s2 [ in 2:3 dose ratio] from linear part of LDR [ Let the corresponding response be S1, S2]

Choose a test dose t with a response T between S1 & S2 Record 4 sets data as follows

s1 s2 t

t s1 s2

s2 t s1

s1 s2 t

Log Potency ratio [M] = [(T S1) / (S2-S1)] X log (dose ratio)

4 point assay [2 +2 dose assay] [E.g. Ach bioassay] Log dose response [LDR] curve plotted with varying conc of std Ach solutions and given test

solution

Select two std doses s1& s2 from linear part of DRC [ Let the corresponding response be S1, S2]

Choose two test doses t1 & t2 with response T1 &T2 between S1 & S2 ; Also s2/s1 = t2/t1 = 2/3

Record 4 data sets s1 s2 t1 t2

s2 t1 t2 s1

t1 t2 s1 s2

t2 s1 s2 t1

The enzyme-linked immuno-sorbent assayand chemicals to detect immune responses in the body. The ELISA test involves an enzyme (a protein that catalyzes a biochemical reaction). It also involves an antibody or antigen (immunologic molecules).popular format of a "wet-lab" type analytic (EIA) to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample.

Select two std doses s1& s2 from linear part of DRC [ Let the corresponding response be S1,

Choose two test doses t1 & t2 with response T1 &T2 between S1 & S2 ; Also s2/s1 = t2/t1 = 2/3

ELISA

assay (ELISA). An ELISA test uses components of the immune system and chemicals to detect immune responses in the body. The ELISA test involves an enzyme (a protein that catalyzes a biochemical reaction). It also involves an antibody or antigen (immunologic molecules).

lab" type analytic biochemistry assay that uses a solid-phase to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample.

Select two std doses s1& s2 from linear part of DRC [ Let the corresponding response be S1,

Choose two test doses t1 & t2 with response T1 &T2 between S1 & S2 ;

. An ELISA test uses components of the immune system

and chemicals to detect immune responses in the body. The ELISA test involves an enzyme (a protein that catalyzes a biochemical reaction). It also involves an antibody or antigen (immunologic molecules). ELISA is a

phase enzyme immunoassay

to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample.

The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality-control check in various industries.

Use of an ELISA test

ELISA tests are widely utilized to detect substances that have antigenic properties, primarily proteins (as opposed to small molecules and ions such as glucose and potassium). The substances detected by ELISA tests include hormones, bacterial antigens and antibodies

Principle of ELISA test

There are variations of the ELISA test, but the most basic type consists of an antibody attached to a solid surface. This antibody has affinity for the substance of interest, E.g. human chorionic gonadotropin (HCG), the commonly measured protein which indicates pregnancy. A mixture of purified HCG linked to an enzyme and the test sample (blood, urine, etc) are added to the test system. If no HCG is present in the test sample, then only HCG with linked enzyme will bind. The more HCG which is present in the test sample, the less enzyme linked HCG will bind. The substance the enzyme acts on is then added, and the amount of product measured in some way, such as a change in color of the solution.

As a wet lab analytic biochemistry assay, ELISA involves detection of an "analyte" (i.e. the specific substance whose presence is being quantitatively or qualitatively analyzed) in a liquid sample by a method that continues to use liquid reagents during the "analysis" (i.e. controlled sequence of biochemical reactions that will generate a signal which can be easily quantified and interpreted as a measure of the amount of analyte in the sample) that stays liquid and remains inside a reaction chamber or well needed to keep the reactants contained; It is opposite to "dry lab" that can use dry strips - and even if the sample is liquid (e.g. a measured small drop), the final detection step in "dry" analysis involves reading of a dried strip by methods such as reflectometry and does not need a reaction containment chamber to prevent spillover or mixing between samples.

Types of ELISA

Four kinds of ELISA here are here illustrated as you may concern:

Direct ELISA

Direct ELISAs involve attachment of the antigen to the solid phase, followed by an enzyme-labeled antibody. This type of assay generally makes measurement of crude samples difficult, since contaminating proteins compete for plastic binding sites.

Indirect ELISA

Indirect ELISAs also involve attachment of the antigen to a solid phase, but in this case, the primary antibody is not labeled. An enzyme-conjugated secondary antibody, directed at the first antibody, is then added. This format is used most often to detect specific antibodies in sera.

Competitive ELISA

The third type of ELISA is the Competition Assay, which involves the simultaneous addition of 'competing' antibodies or proteins. The decrease in signal of samples where the second antibody or protein is added gives a highly specific result.

Sandwich ELISA

The last type of assay is the sandwich ELISA. Sandwich ELISAs involve attachment of a capture antibody to a solid phase support. Samples containing known or unknown antigen are then added in a matrix or buffer that will minimize attachment to the solid phase. An enzyme-labeled antibody is then added for detection.

The ELISA method is a benchmark for quantization of pathological antigens and there are indeed many variations to this method. ELISAs are adaptable to high-throughput screening because results are rapid, consistent and relatively easy to analyze. The best results have been obtained with the sandwich format, utilizing highly purified, pre matched capture and detector antibodies. The resulting signal provides data which is very sensitive and highly specific.

Multiple and portable ELISA

A new technique uses a solid phase made up of an immunosorbent polystyrene rod with eight to 12 protruding gives. The entire device is immersed in a test tube containing the collected sample and the following steps (washing, incubation in conjugate and incubation in chromo gens) are carried out by dipping the ogives in micro wells of standard micro plates filled with reagent.

Applications of ELISA

1. ELISA Test Applications in Antibody Concentration Determination 2. ELISA Test Applications in Monoclonal Antibody Screening 3. ELISA Test Applications in Virus test (HIV, West Nile Virus, NDV) 4. ELISA Test Applications in Home Pregnancy Test 5. ELISA Test Applications in Food industry (detecting potential food allergens such as milk, peanuts,

walnuts, almonds and eggs) 6. - .

Radioimmunoassay

RIA is a very sensitive in vitro assay technique used to measure concentrations of antigens (E.g. hormone levels in the blood) by use of antibodies. As such, it can be seen as the inverse of a radio binding assay, which quantifies an antibody by use of corresponding antigens. Although the RIA technique is extremely sensitive and extremely specific, requiring specialized equipment, it remains among the least expensive methods to perform such measurements. It requires special precautions and licensing, since radioactive substances are used. The unique ability of RIA to measure small molecules can nowadays be achieved in many cases by non-radioactive methods such as ELISA, where the antigen-antibody reaction is measured using colorimetric, such as absorbance, fluorescence intensity or polarization.

Principle of radioimmunoassay

It uses an immune reaction [antigen-antibody reaction] to estimate a ligand. Ag+Ag*+Ab [Ag -Ab+ Ag*Ab + Ag + Ab*]

- Unbound Ag* and Ag washed out

- Radio activity of bound residue measured.

- Ligand concentration is inversely related to the radio activity.

- [Ag: ligand to be measured; Ag*: radiolabelled ligand].

Method

Requirements: 1. Preparation and characterization of an antigen 2. Radio labeling of the antigen 3. Preparation of the specific antibody 4. Development of assay system.

Components of RIA Assay Kit 1. Drug

2. Antibody

3. Labeled Drug

To perform a radioimmunoassay, a known quantity of an antigen is made radioactive, frequently by labeling it with gamma-radioactive isotopes of iodine, such as 125-I, attached to tyrosine. This radio labeled antigen is then mixed with a known amount of antibody for that antigen, and as a result, the two specifically bind to one another. Then, a sample of serum from a patient containing an unknown quantity of that same antigen is added. This causes the unlabeled (or "cold") antigen from the serum to compete with the radio labeled antigen ("hot") for antibody binding sites. As the concentration of "cold" antigen is increased, more of it binds to the antibody, displacing the radio labeled variant, and reducing the ratio of antibody-bound radio labeled antigen to free radio

labeled antigen. The bound antigens are then separated from the unbound ones, and the radioactivity of the free antigen remaining in the supernatant is measured using a gamma counter. Using known standards, a standard curve can then be generated which allows the amount of antigen in the patient's serum to be derived. The radioactivity for each of a series of known concentrations of standards is used to derive terms for en equation, often sigmoid curve fit. Once the terms for the equation are derived, the radioactivity is fed into the equation to obtain concentrations of the unknowns. This function is often integrated into the radioactivity counter, along with functions indicating coefficient of variation and properties of the binding of tracer to antibody. A typical RIA will have somewhere around 50% of tracer bound to antibody in a sample with no analyte. Samples are often run in duplicate to ensure a correct reading.

Applications of RIA

Endocrinology Insulin, HCG, Vasopressin

Detects Endocrine Disorders Physiology of Endocrine Function

Pharmacology

Morphine

Detect Drug Abuse or Drug Poisoning Study Drug Kinetics

Epidemiology Hepatitis B

Clinical Immunology Antibodies for Inhalant Allergens Allergy Diagnosis

Oncology

Carcino embryonic Antigen Early Cancer Detection and Diagnosis