Bioassay techniques

An Over view on Bioassay, structure & principles, types & methods of bioassay. Also mention of other assay's like biotechnology, microbio assay, immunoassay etc.

1. BIOASSAY TECHNIQUES Submitted to Dr. Sujarani S. Asst. Prof. Dept. of VPT, Pookode. Submitted by Dr. Sindhu K. MVSc scholar, Dept. of VPT, Pookode.

2. Biological assessment. Estimation or determination of concentration or potency of a physical, chemical or biological substance (agent) by  means of measuring and comparing the magnitude of the response of the test with that of standard over a suitable biological  system under standard set of conditions. The estimation of the concentration or potency of a substance by measurement of the biological response that it produces. The structure of bioassay: STIMULUS-applied to subject. RESPONSE-of the subject to the stimulus.

3. The purpose of bioassay. To ascertain the potency of a drug and hence it serves as the quantitative part of any screening procedure (Research). To standardize drugs, vaccines, toxins or poisons, disinfectants, antiseptics  etc., so that each contains the uniform specified pharmacological activity. ( standardization required as these are all used over biological system in some or other form.) Helps to determine the  specificity of a compound  to be used e.g. Penicillin's are effective against Gram + ve . but not on Gram – ve . From the clinical point of view, bioassay may help in the diagnosis of various conditions. e.g. gonadotrophins for pregnancy. Sometimes the  chemical composition of samples are different  but have  same biological activity. Certain  complex compounds like Vitamin B-12   which can't be analysed by simple assay techniques can be  e ffectively estimated   by Bioassays. For samples  where no other methods of assays are available .

4. Principle of Bioassay. To compare the test substance with the International Standard preparation of the same and to find out how much test substance is required to produce the same biological effect, as produced by the standard .

5. The standards are internationally accepted samples of drugs maintained and recommended by the Expert Committee of the Biological Standardization of W.H.O. They represent the fixed units of activity (definite weight of preparation) for drugs.

6. In India standard drugs are maintained in Government institutions like Central Drug Research Institute , Lucknow Central Drug Laboratory , Calcutta .

7. Classification of bioassay. Invitro. Invivo. Exvivo .

8. In vitro techniques :  These techniques  employ a cell culture of recommended biological system to study the effect of compound under standard condition  not similar to that of living environment. Here the  cell culture survives by utilization of the nutrition  in the media. Ex: use of  stem cells ,  cell culture , microbes ( bacteria )  etc.

9. In vivo techniques : These techniques employ a living animal recommended for the purpose of assay. The  techniques aims to study the biological effect or response of the compound under screening in a living system directly . Ex: By use of rodents, rabbits etc .

10. Ex vivo techniques : These techniques  employ a tissue or cells of recommended living system to study the effect of compound under test in suitable conditions within the stipulated time of organ survival outside the body. Ex: Use of any isolated organ from animals in a glass ware to study the effect of compound within the period of its survival outside the living body with provision of only oxygen, glucose and isotonic salts to maintain cell &  cell organelles integrity .

11. Types of bioassay. Qualitative bioassay  is used for assessing the physical effects of a substance that may not be quantified, such as abnormal development or deformity . Eg : 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 .

12. Bioassay Methods. 1.   Graded Response Assay: :  In these assays, as the dose increases there is an equivalent rise in response. The potency is estimated by comparing the Test sample responses with the standard response curve. Conc. of unknown= Threshold dose of standard/threshold dose of test x Conc. of standard. E.g. Acetyl-choline producing contraction in the muscle of frog Rectus abdominis .

13. 2.   End Point or Quantal Assay: As the name indicates, the threshold dose of the sample required to elicit a complete or a particular pharmacological effect is determined and compared with standard. E.g., Digitalis producing cardiac arrest. 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: Matching point or bracketing method Interpolation assay Three point (2+1) assay Four- point (2+2) assay

14. Matching point or bracketing method: 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 response of the sample is achieved. This technique is used when test sample is too small Inaccurate & margin of error difficult to estimate Eg : histamine on guinea pig ileum, Posterior pituitary on rat uterus .

15. 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 .

16. 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. It can further divided as 3 point, 4 point and 6 point bioassay.

17. Three point assay [2+1 dose assay] 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)

18. 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

19. Other bioassay’s. Immunological assay (ELISA). Micro-bioassay . Radioimmunoassay. Biotechnology.

20. ELISA (immunological assay) ELISA is a popular format of a "wet-lab" type analytic biochemistry assay that uses a solid-phase enzyme immunoassay (EIA) 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. The substances detected by ELISA tests include hormones, bacterial antigens and antibodies .

21. Types of ELISA. Direct ELISA: 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: 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: 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: 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 .

22. Applications of ELISA ELISA Test Applications in Antibody Concentration Determination ELISA Test Applications in Monoclonal Antibody Screening ELISA Test Applications in Virus test (HIV, West Nile Virus, NDV) ELISA Test Applications in Home Pregnancy Test ELISA Test Applications in Food industry (detecting potential food allergens such as milk, peanuts, walnuts, almonds and eggs) ELISA Test can be used to diagnostic diseases

23. Micro-bioassay (antibiotics) The potency (activity) of an antibiotic product is expressed as the ratio of the dose that inhibits the growth of a suitable susceptible microorganism to the dose of an International Biological Standard, an International Biological Reference Preparation, or an International Chemical Reference Substance of that antibiotic that produces similar inhibition . To carry out the assay a comparison is made between the inhibition of the growth of microorganisms produced by known concentrations of the reference material and that produced by measured dilutions of the test substance . This response can be measured by the diffusion method or in a liquid medium by the turbidimetric method .

24. 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. It is the estimation of the concentration of the substance in a unit quantity of preparation using radiolabelled antigens. It requires special precautions and licensing, since radioactive substances are used. A number of drugs are estimated now days by radioimmunoassay methods because these methods are highly specific and highly sensitive. Eg : the radioimmunoassay of insulin is based on the ability of human insulin ( unlabelled ) to displace beef’s insulin (which may be labelled) from the binding sites (i.e. antibodies).

25. 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].

26. Method of RIA. 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: Drug Antibody Labeled Drug

27. 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.

28. Biotechnology. “Any technological application that uses biological systems, living organisms or derivatives thereof, to make or modify products or processes for specific use" (UN Convention on Biological Diversity, Art. 2) The   American Chemical Society  defines biotechnology as the application of biological organisms, systems, or processes by various industries to learning about the science of life and the improvement of the value of materials and organisms such as pharmaceuticals, crops, and livestock. The   European Federation of Biotechnology defines Biotechnology is the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services. Biotechnology also writes on the pure biological sciences ( animal cell culture ,  biochemistry ,  cell biology ,  embryology ,  genetics ,  microbiology , and molecular biology ).

29. B ranches of biotechnology. Bioinformatics Blue biotechnology   Green biotechnology   Red biotechnology   White biotechnology

30. Bioinformatics Is an interdisciplinary field which addresses biological problems using computational techniques, and makes the rapid organization as well as analysis of biological data possible. It plays a key role in various areas, such as functional genomics, structural genomics, and proteomics, and forms a key component in the biotechnology and pharmaceutical sector.

31. Blue biotechnology  is a term that has been used to describe the marine and aquatic applications of biotechnology .

32. Green biotechnology is biotechnology applied to agricultural processes. Eg : 1. The selection and domestication of plants via  micropropagation . 2. The designing of transgenic plants to grow under specific environments in the presence (or absence) of chemicals

33. Red biotechnology  is applied to medical processes. Eg : 1.The designing of organisms to produce  antibiotics, 2.The engineering of genetic cures through genetic manipulation.

34. White biotechnology A lso known as industrial biotechnology, is biotechnology applied to  industrial processes. Eg : 1. The designing of an organism to produce a useful chemical. 2. The using of enzymes as industrial catalysts to either produce valuable chemicals or destroy hazardous/polluting chemicals. White biotechnology tends to consume less in resources than traditional processes used to produce industrial goods.

35. Applications H ealth care (medical ), C rop production and agriculture, N on food (industrial) uses of crops and other products ( e.g.  biodegradable plastics ,  vegetable oil ,  biofuels ), E nvironmental uses.

36. To Sum - up Bioassay & its principles, structure. Types & methods of bioassay. Immunological assay (ELISA). Micro-bioassay. Radioimmunoassay. Biotechnology.

37. Thank-you