Philip

Planning an Experiment—Biology Trimester Investigation Plan due on Friday, April 27th

EACH STUDENT IS RESPONSIBLE FOR WRITING UP A CLEAR PLAN FOR YOUR INVESTIGATION, EVEN IF YOU ARE WORKING IN A GROUP.

Question(s) for investigation: Are antibodies absolutely specific or will they cross react with similar antigens?

Independent variable and how it will be measured: Specificity of the antibodies Epitopes on the antigen

Dependent variable(s) and how it (they) will be measured: Cross reaction of antigens Absence or presence of an immune precipitate

Variables held constant: Specificity of the antibodies will be held constant

List of Materials and Sketch of the Setup:
 * 1) Goat anti human IgG
 * 2) Rabbit IgG (similar)
 * 3) Human IgG (identical)
 * 4) Borine serum albumen (no cross reaction)

Who else will work on this investigation? Who will be responsible for collecting materials to get started and when will all materials be ready to begin?

Phil will get materials from chemical store, and will be able to get it after May break. Both Phil and Alicia will work on the investigation.

Procedure/Method: Prepare a 2% agar in phosphate buffer by dissolving 2grams of agar in 98 grams of phosphate buffer and heating until it dissolves. Add some NaN3 (0.1%) as a preservative. Pour into a Petri dish and let it set overnight.

Punch 4 holes as shown in the diagram using a glass pipette. Pipette 5 microliters of G and H IgG into one hole and 5 microliters of Rabbit IgG into another hole and 5 mL of human IgG into the other. Let the reaction stand overnight and look for immunoprecipitate lines between the holes.

Data:
 * Make a blank data table which you plan to fill out during the investigation
 * What other data do you expect to include in a report of your results?
 * sample ||
 * is immune precipitate line present or absent due to cross reactions with Goat anti human antibodies ||
 * human IgG ||
 * albumen ||
 * Rabbit IgG ||
 * albumen ||
 * Rabbit IgG ||
 * albumen ||
 * Rabbit IgG ||
 * albumen ||
 * Rabbit IgG ||
 * albumen ||
 * Rabbit IgG ||
 * Rabbit IgG ||
 * Rabbit IgG ||
 * Rabbit IgG ||
 * Rabbit IgG ||
 * Rabbit IgG ||
 * Rabbit IgG ||
 * Rabbit IgG ||

Expectations: · What do you think might happen and why?

We expect that the human and rabbit IgG will create a immunoprecipitate line because there are some common epitopes, therefore there would be some cross reactions with the goat, antihuman antibodies. The albumen, however, will not have a precipitate line because there are no common epitopes and the antibodies will not react with the albumen.

· What other results do you imagine might happen and what would they mean?

We might expect that one precipitate line would be more intense than the other because there are more common epitopes, therefore the antigen antibody lattice would be more intense.

Communication: Appendix
 * How will you plan to communicate your work clearly to others?
 * What do you expect to accomplish in EACH of the next three class periods in which you can work on this investigation?

Albumin- a simple water-soluble protein found in many animal tissues and liquids [Origin: 1590–1600; < LL, equiv. to //alb//(//us//) white, with s. in //-ū-// + //-men// n. suffix                ]

Antibodies- proteins in the blood that are produced by the body in response to specific antigens such as bacteria [Translation of German :, //antagonistic// (from Latin , //anti-//) + , //body//.] [Origin: 1895–1900; [|anti-] + [|body]  ]

Antigens- Any substances that when introduced to the body stimulates the production of an antibody. Antigens include toxins, bacteria, foreign blood cells, and the cells of transplanted organs

Agar-A gelatinous material obtained from marine algae, especially seaweed, used as a medium for growing bacterial cultures in the laboratory and as a thickener and stabilizer in food products. [Origin: 1885–90; < Malay //agaragar// seaweed from which a gelatin is rendered, or the gelatin itself ]

Diffusion- act of being spread out or dispersed [Origin: c.1374, from L. //diffusionem,// from stem of //diffundere// "scatter, pour out," from //dif-// "apart, in every direction" + //fundere// "pour"]

immunodiffusion- any of various analytical techniques that involve antigen and antibody solutions diffusing toward each other in a gel until antibody binds specifically to antigen to form a precipitate. [Origin: 1955–60; [|immuno-] + [|diffusion] ]

epitopes-A localized region on the surface of an antigen that is capable of eliciting an immune response and of combining with a specific antibody to counter that response. [**epi-** + Greek, //place, spot//.]

antiserum- Human or animal serum containing antibodies that are specific for one or more antigens. [Origin: 1900–05; [|anti-] + [|serum] ]

cross reaction- A specific reaction between an antiserum and an antigen complex other than the complex that caused the production of the specific antibodies of the antiserum [Origin: 1945–50 ]

polyclonal antibodies- a mixture of antibodies of different specificities, as in the serum of a person immunized to various antigens [Origin: 1960–65; [|poly-] + clonal ]

specificity- the selective attachment or influence of one substance on another, as an antibiotic and its target organism or an antibody and its specific antigen. [Origin: 1875–80; [|specific] + [|-ity] ]

reaction-the specific cellular response to foreign matter, as in testing for allergies. [origin: 1611, from [|re-] "again, anew" + [|action] (q.v.). Modeled on Fr. //réaction,// older It. //reattione,// from M.L. //reactionem// (nom. //reactio//), from L.L. //react-,// pp. stem of //reagere// "react," from //re-// "back" + //agere// "to do, act" (see [|act]).]

antigen antibody lattice- formed when antigens react with their antibodies and combine to form a huge long chain which makes up the immonoprecipitate line [Middle English, from Old French , from , //lath//, of Germanic origin .]

immunoprecipitate line- a visible line that is formed when a type of antigen reacts with its antibodies [Origin: immuno- + Latin praecipitāre, praecipitāt]

precipitate- A solid or solid phase separated from a solution. [Origin: Latin praecipitāre, praecipitāt-] immunodiffusion- A technique for studying reactions between antigens and antibodies by observing precipitates formed by the combination of specific antigens and antibodies that have diffused in a gel in which they have been separately placed. interaction is manifested by a precipitin line for each system. [Origin__: immuno__- + diffusion] constant- An experimental or theoretical condition, factor, or quantity that does not vary or that is regarded as invariant in specified circumstances. [Origin: Middle English, from Old French, from Latin cōnstāns, cōnstant-, present participle of //cōnstāre//, to stand firm : com-, intensive pref.; see [|com–] + stāre, to stand.]

Appendix II

Cross Reactivity: In the experiment, cross reactivity is one of the main ideas. The purpose of the experiment was to see if antibody antigen reactions are completely specific. We found that they weren’t. This proves that there was cross reactivity between the human and rabbit antigens, which reacted with the goat anti human antibody. The cross reactivity was shown by the immuno- precipitate line that formed between the three wells as shown in the figure below.

Immuno-diffusion: Immuno diffusion is the concept where the contents of the well (the human antigen, rabbit antigen, and goat antihuman antibody) diffuse out. They diffuse until they meet the contents of another well, then if there is any possible reaction between the two then they will react forming the precipitate line, if not then there is no line present.