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• Preparative and analytical methods based on antibody-antigen interactions (ELISA) • Functional investigations of the cells of the immune system (2nd part) The sensitivity of immunoassays Measuring the functional activity of Band T-lymphocytes Topics: 1. Polyclonal activation of T- and B-lymphocytes lectin induced activation α-IgM, α-CD3 or α-TCR induced activation 2. Investigation of the T- and B-cell response activation markers proliferative response 3H-thymidine incorporation CFSE fluorescence dilution cell cycle antibody or cytokine production (ELISA) 3. Determination of the activated T- or B-cell number ELISPOT Intracellular cytokine staining (review) Phases of the humoral immune response (review) Phases of T cell response (review) Signalling of the B-cell antigen receptor complex membrane adaptor protein (NTAL) (review) Signalling of the T-cell antigen receptor complex LAT (in the cell membrane) SLP-76(LCP2) cytoplasmic adaptor All known steps can be examined Investigations of the cellular functions of the immune system Immunodeficiencies mainly characterized by different functional immunoassays Lymphocyte activation by specific antigen is hardly detected, because of the low number of the antigen specific cells Lymphocyte function can be investigated by polyclonal T/B-lymphocyte activator materials – polyclonal activators Polyclonal activation of lymphocites by LPS, lectins, PMA/ionomycin B cell (mouse) T cell T cell TLR4 (PMA activates protein kinase C) BCR- or TCR-specific antibodies may also activate the lymphocytes (Which antibody types can effectively activate the B- and T-cells?) Polyclonal B cell activators Activator T cell dependency Ig secretion Human B cells PWM (pokeweed mitogen) no yes SpA (superantigen, staphylococcus protein A) no yes EBV (transforming effect) yes yes Anti-Ig yes In the presence of cytokines Mouse B cells LPS no yes PWM (pokeweed mitogen, lektin) yes yes PPD (purified protein derivate, mycobacterium) no yes Anti-Ig (antibodies) In the presence of cytokines no Polyclonal T cell activators Phytohaemagglutinin (PHA) lectin Phaseolus vulgaris Concanavalin A (ConA) lectin Canavalia ensiformis anti-CD3, anti-TCR antibody Pokeweed (PWM) (Phytolacca americana) – formerly used for colouring red wine (toxic: triterpene saponin) Chenopodiales Phytolaccaceae Phytohaemagglutinin (PHA) Canavalia ensiformis – Jackbean or Sword bean Concanavalin A (ConA) Phaseolus vulgaris – common bean A bab-félék nyersen, nem rendesen megfőzve a lektin tartalmuk miatt mérgezőek! (klasszikus ételmérgezési tünetek – rosszullét, hányás, hasmenés) A bab beáztatása, és az áztatóvíz leöntése jelentős mennyiségű lektintől szabadít meg, és a forralás inaktiválja (irreverzibilisen denaturálja) a lektineket. (Ezért nem célszerű a babféléket „sous-vide” –olni) Biswas S, Kayastha AM.: J Biochem Mol Biol. 2002 Sep 30;35(5):472-5. Thermal stability of Phaseolus vulgaris leucoagglutinin: a differential scanning calorimetry study Receptor crosslinking (immediate) phosphorilation steps - Western blot - Bead array (seconds-minutes) Antigen receptors (TCR, BCR), and different other receptors (e.g. cytokine receptors)ic Ca2+ increase - FACS, microscopy Gene activation - RT-PCR Cytokine synthesis - IC cytometry Cytokine secretion - ELISA, ELISPOT - DNA content - IN antigens Cell-cycle/apoptosis Lymphocyte activation Cell division - H-thymidine, CFSE, MTT and the examination possibilities The examination often requires specific 3 Ag-Ab reactions A jelenlevő vagy termelt „antigének” (pl. fehérjék, citokinek, ellenanyagok, ….) vizsgálata ELISA Enzyme Linked Immune Sorbent Assay ELISA plate well Different plastic surfaces for different materials enzyme linked immune sorbent enzyme Antibody conjugated with enzyme Antigen/antibody adsorbed to solid surface ENZYME ACTIVITY IN ELISA IS DIRECTLY PROPORTIONAL TO THE AMOUNT OF ANTIGEN PRESENT Enzyme activity is measured by the color reaction due to conversion of substrate Similar principle applies to many other antibody-based detection methods Miért enzimet használunk jelzésként? Jelölésekről/jelzésekről az előző szemináriumban esett már említés: • radioaktív izotópok RIA főleg régen használták (ELISA elődje) • fluoreszcens festék • …. egy jelzés egy jel (fluoreszcencia: fakulás (fading) ) enzim előnye: sok szubsztrát átalakítás (turnover szám) jel felerősítése ÉRZÉKENYSÉG NÖVEKEDÉSE! BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY Direct method Indirect method (simplicity) (more label sensitivity) (“modularity”) Label Label Secondary antibodies Primary antibodies Antigen surface BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY Enzyme/anti-enzyme system (generally applied in microscopic immunohistochemistry) PAP – peroxidase / anti-peroxidase APAAP – alkaline phosphatase / anti- alkaline phosphatase Enzyme Enzyme-specific antibody, same (iso)type as the primary antibody Primary antibody Antigen Secondary antibody Az ELISA, immunhisztokémia, citometria során használt módszerek alap jellegzetességei Biotin-avidin rendszerrel kombinált indirekt módszerek Az avidin nagy affinitással képes a biotint kötni Egyszerű Avidin-enzim komplexek „ABC” Avidin Biotin enzimComplexek Jel felerősítése (arányok!!! mint a precipitációnál) Biotin-enzim komplex Avidin Biotinált ellenanyag Antigén (szabad) BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY Indirect systems combined with biotin-avidin signal amplification (Avidin binds biotin with very high affinity ) Basic Avidin-enzyme complexes ABC Avidin-biotin enzyme complexes Biotin-enzyme complex Avidin Biotinylated antibody Antigen Protein biotinylation is easy Modified biotins with different reactive chemical functional groups can be bound to diverse materials M.Wilchek, EA. Bayer et al. Biotin ELISA systems can be used not only for the detection of a material, but its exact concentration can also be measured by the help of a known concentration standard The different direct or indirect, biotin-avidine methods can be used in combination See the next examples! EXAMPLES FOR DIRECT, INDIRECT AND COMPETITIVE ELISAs STEPS OF COMBINED SANDWICH ELISA Goal: examination of antigens present at low concentration in complex biological and other samples (body fluids, natural waters, foods) e.g. hormons, cytokines, etc. Coating with Agspecific „capture” antibody Blocking free plastic surface with inert protein Removal of excess enzyme Removal of unbound material Removal of unbound protein Removal of unbound material Addition of antigencontaining solution Addition of biotinylated antibody specific to a different epitope on target protein Addition of avidinconjugated enzyme Addition of substrate Antigen specific antibody pair is a prerequisite TUMORDIAGNOSTICS Tumor specific (TSA) and tumor associated (TAA) antigen recognizing antibodies can be used in diagnostics The antigens can be detected by sandwich techniques Tumor Ag specific detecting/reporter antibody (suplemented) Tumor antigen (patient serum) PROSTATE CANCER: Tumor Ag specific capture antibody precoated plate (as a part of the kit) Prostate-Specific Antigen (PSA) derives its name from its first known site of origin, the prostate gland. Serum concentrations of PSA are elevated in patients with prostate cancer, benign prostatic hypertrophy (BPH) and prostatitis. In addition, PSA serum levels appear to correlate with the volume and clinical stage of prostate cancer. Human prostatic acid phosphatase (PAP) in human serum can be used similarly in quantitative measurement. Bladder cancer: NMP22 is a Nuclear Matrix Protein found in human epithelial cells. In the urine of healthy individuals, the protein is present at low levels. The majority of patients with bladder cancer release large quantities of NMP22 into their urine, that can be detected by immunoassay Thyroid Cancer : Increased Serum Thyroglobulin (sTG) can be detected by immunoassay Alpha Fetoprotein (AFP) is a 68 kDa protein, which is produced primarily during fetal life by the fetal liver and yolk sac. AFP also appears in the maternal serum, presumably by transplacental transfer . After birth, serum AFP levels decline rapidly during the first year of life and low basal levels are then apparently maintained throughout childhood and adult life. Its normal concentration in serum is below 9 ng/mL; higher concentrations are associated with hepatoma and ovarian, testicular and presacral teratocarcinomas, and other cancers. Carcinoembryonic Antigen (CEA) is a glycoprotein involved in cell adhesion. It is normally produced during fetal development. serum from individuals with colorectal and other carcinomas had higher levels of CEA than healthy individuals and can be used to monitor the response to colon cancer treatment. Her-2/neu protein is a 185 kD trans-membrane glycoprotein associated with tyrosine kinase activity. Approximately 20-30% cases of breast cancer show an amplification and/or over-expression of Her-2/neu in tumor cells. Since the introduction of Herceptin as a targeted therapy for breast cancer, the clinical testing of Her-2/neu in breast carcinoma has become very important in patient care. It can be shown by immunohistochemistric or immunofluorescent methods from biopsy. Detection of antigen specific antibodies • Tracking the effectiveness of an immunisation • Findig cell microcultures which produce antigen specific antibodies (monoclonal antibody production / hybridome technics) • Detection of antigen specific antibodies in clinical samples (infections, autoimmune process – see the forthcoming examples) (simple) ELISA STEPS OF BASIC INDIRECT ELISA Goal: Detection of antigen specific antibody (This type will be performed at the practice) Adsorption of antigen (coating) Saturation of uncovered surface area with proteins Removal of excess antibody Removal of excess antigen Removal of excess protein Addition of Agspecific antibodies Addition of Secondary Ab conjugated with enzyme Addition of chromogenic substrate e.g. pure protein antigens, or large synthetic peptides (at least µg/ml concentration) TESTING VIRAL INFECTION Viral antigens cannot be detected efficiently in lot of case (latency), but you can efficiently detect the antibodies that were produced by the body in response to the viral infection. These antibodies can serve as diagnostic markers. term: SEROPOSITIVITY Human Ig specific labeled antibodies indicate the presence of the virus specific antibodies. The serum of the infected person with virus specific antibodies. The antibodies could bind the virus antigens. viral antigen precoated test plate EXAMPLES: • Epstein-Barr Virus (EBV) test kit → ELISA method for the qualitative detection of IgG antibody to Epstein-Barr Virus nuclear antigen-1 (EBNA-1) in human serum • HIV assay kit → enzyme-linked immunosorbent assay for the detection of antibody to HIV-1 in serum, plasma or dried blood spots •Toxoplasmosis (Toxoplazma gondii – parasitic protozoa) → chromatographic immunoassay for the qualitative detection of human IgM antibodies against Toxoplasma in serum or plasma AUTOIMMUNITY Autoantibodies from different autoimmune diseases can be detected similarly. In 60% of SLE patients autoantibodies can be detected against double strand DNA. Anti-ENA antibodies can be seen in many systemic AIDs. (See Ouchterlony method in the previous practice) Type I (autoimmune) diabetes can be detected by the presence of antibodies against islet-cell specific antigens. Glutamic acid decarboxylase (GAD65), specific antibodies have been found in 70-90% of prediabetic and Type 1 diabetic patients (including approximately 7-10% of adult onset diabetics with Type 1 diabetes) IA-2 (a tyrosine phosphatase-like protein) specific Ab. are found in 50-75% of Type 1 diabetic patients at and prior to disease onset, are generally more prevalent in younger patients, and are associated with rapid progression to overt disease. These autoantibodies have been detected in some ICA positive/GAD Ab negative patients, and therefore can be considered independent markers of disease. Insulin: anti-insulin ABs are found predominantly, though not exclusively, in young children (<5 years) developing Type 1 diabetes. In insulin-naive (untreated) patients, the prevalence of autoantibodies to insulin is almost 100% in very young individuals and almost absent in patients with adult onset of Type 1 diabetes. (It should be noted that insulin autoantibodies are indistinguishable from insulin antibodies that commonly develop with insulin therapy). SERUM ANTIBODY ISOTYPE DETERMINATION Sometimes the antigen specific antibodies could refer the presence of the antigen in the body (see the ”viral infection testing” part ) The isotypes of these antibodies additionally could refer the fresh/persistent (IgM dominance) or repeated/memory immune response (IgG dominance) against the parasite. The possibilities can be discriminated by the use of isotype specific secondary antibodies. α-IgM α-IgG Memory response IgM IgG Antigen Increased IgE level can be seen in atopic allergy cases, and some autoimmune process, and in the case of some parasite infection Abnormal levels of serum immunoglobulin isotypes can be seen in the case of some immunodeficiencyies (e.g. hyper IgM syndrome, multiplex myeloma (case study!)) isotype specific antibody antibody from the serum antibody capture antibody COMPETITIVE ELISA Goal: detection of small antigens (haptens) in complex biological samples when the sandwich ELISA can’t be applied (no need for antibody pairs) Antigen in solution and on the solid surface compete for the binding site of labeled specific antibody. - Coating with antigen, blocking - Addition of experimental sample that contains or lacks antigen - Addition of labeled antibody binding - Washing - Addition of enzyme substrate RIA - RadioImmunoAssay (Inconvenient because of the usage of radioactive isotopes) Usage: • Some small antigen (e.g. haptens) can’t be conjugated by large labels without the destruction of the epitope • As a competitive method one type of antigen specific antibody is enough to perform Generally 125I or 131I is the label (to tyrosine side chain in the proteins) EXAMPLE: cAMP RIA: 125I labelled cAMP: measured cAMP: 125I bound labelled or “cold” cAMP Ag specific capture antibody ELISA types (summary) capture/sandwich simple e.g. detection of antigen specific antibodies Ag specific detector/reporter detector/reporter antibody antibody Ag specific antigen antibody Ag specific antigen capture antibody soluble Ag are present in the sample competitive detection of antigens no soluble Ag Ag specific antibody antigen detection of haptens or detection of an antigen with only one type of antigen specific antibodies ELISA methodical errors Aspecific adhesion of the materials The antibodies (as proteins generally) could also bind to the surface aspecifically - directly - indirectly the color is the same Aspecific adhesion of the proteins can be reduced by: •blocking of the surface •applying detergent Hook effect - It could happen in ”one step”, ”without washing” sandwich tests. Large antigen concentration could give invalid small value. Unwashed soluble antigens compete with the captured surface bound ones. It can be avoided by efficient washing steps, or with the dilution of the sample. methodical errors of the ELISA The soluble antigen can compete with the surface bound one: • In the case of very high antigen concentration • In the case of ineffective/missing washing steps (e.g. “one step” or “no-wash” procedures) Hook effect - false negative or decreased results appropriate ELISA Soluble antigen could be present in the case of very high antigen concentration or without washing the test plates. This results false negative or diminished positive readout. 0 The competitive ELISA avoids this phenomenon The competitive tests are more reliable in the clinical diagnostics ELISA PLATES - RESULTS (Chromogenic substrates can have different colors) Diagnostic quick tests: e.g. detection of human chorionic gonadotropin in serum or urine (pregnancy test) The principles of these tools are similar to the ELISA assay you have met before. hCG Rapid One-Step Immunochromatographic Assay strip front view side view absorbtion pad (cellulose) control antibody lane (detection antibody capture) hCG capture antibody lane nitrocellulose membrane (signal detection pad) glass fiber membrane with visually labeled detection antibodies sample application pad urine detection antibody capture antibodies control antibody lane hCG capture antibody lane hCG positive hCG negative control lane (C) test lane (T) detection antibodies similar to sandwich ELISA hCG Competitive system hCG positive control lane detection antibody capture antibody ( hCG lane bound hCG) hCG negative control lane test lane similar to competitive ELISA You don’t need to use additional enzymatic incubation steps, because the labels on the detection antibodies can be observed by naked eye: • colloidal gold („surface plasmon” resonance) • colored latex beads Other uses of immunechromatographic test strips: detection of toxins in food e.g.: aflatoxins (mycotoxins of Aspergillus spp.) ELISPOT Enzyme Linked Immuno-Spot the principles are similar to ELISA capable to determine the number of cells that produce antibodies, cytokines, chemokines, granzymes and other soluble effector molecules the sensitivity allows the determination 1 activated cell among 300 000 other, so it can reveal activated effector cells not only after policlonal-, but after antigen specific activation the first steps should be done in aseptic conditions ELISPOT The process - coating with antigen specific capture antibodies - blocking - administration of the cells (activation, incubation) - washing - administration of biotin conjugated antigen specific secondary antibody - avidin-enzyme conjugate upper view of a well on an ELISPOT plate with - administration of the chromogenic the generated spots substrate which forms insoluble precipitate spot (AEC 3-amino-9-ethylcarbazol) A spot showing the place of the cytokine producing cell It can be evaluated by microscopy (slow, manual process) or you can use “ELISPOT plate reader” (fast + standardizable spot number and size determination) ELISPOT • Spot number represent the number of the effector • molecule (e.g. cytokine) producing cells A spot size represent the amount of the effector molecule which is produced by one cell • The overall/cumulative spot area calculated by the software summarise this two parameter together, which represents the overall effector molecule production Cellular ELISA Identification of cell surface or intracellular antigens • Different cell surface molecules • Intracellular molecules • Signalling ratio of the phosphorylated / nonphosphorylated signalling molecules (two color ELISA - 2 different enzyme and substrate or two different fluorescent/luminescent dyes) • Adherent cell can simply grow on the surface of the plate • Non-adherent cells can be “fixed” on the surface e.g. poly-L-lysine precoated plates Western blot It can detect the presence or even different modifications (e.g. phosphorylation state) of specific proteins The cells’ activation stage can be „frozen” at different times, so the events of the activation can be monitored in parallel samples. at least 105-106 cells required WESTERN BLOT Steps: Anode(+) 1) sample preparation (cells, tissues) 2) gel electrophoresis 3) blotting 4) labeling Cathode(-) 5) detection Usage: identification of defined components from protein mixtures by antigen specific antibodies WESTERN BLOT The use of antibodies in molecular biology is widespread It is probably most often encountered in Western analysis SDS-PAGE gel resolved into single protein bands (overlap possible) Presence of a protein is determined by hybridizing the proteins, transferred or applied to a membrane, with the relevant antibody Antibody recognizes epitope in specific protein Protein Standard sample SDS-PAGE Membrane Western blot e.g. the phosphotyrosine and ZAP-70 blot of activated T-cells non-activated ConA PHA After removing (elution) of the anti P-Y antibodies, the membrane can be incubated with anti ZAP-70 antibodies also, so the phosphorylation state of the ZAP-70 can be evaluated Y P-Y blot P-Y P-Y The usage of the WB in HIV diagnostics: Western-blot: Western-blot of the HIV-1 carrier CEM cell line (T-cell line) lysate. The blot membrane with the HIV antigens is reacted with the patient’s serum. The serum of an infected individual contains anti-HIV antibodies which react with the antigens on the blot membrane and produces specific WB lane patterns. days of seroconversion WESTERN BLOT Detection methods: Colorimetric (peroxidase enzyme – densitometry / spectrophotometry) Chemiluminescent (ECL) Radioactive (isotope – X-ray) Fluorescent (staining – CCD camera) ENHANCED CHEMILUMINESCENCE (ECL) • • Immobilized proteins • • Primary Fabantibody Fc Horseradish peroxidase H2O2 Epitope on protein surface Secondary antibody Membrane In the presence of H2O2, horseradish peroxidase (HRP) oxidizes diacylhydrazides such as luminol Directly after oxidation, luminol is in an exited state, and emits a photon to return to the ground state This photon can be detected with a film or a camera Light emission can be enhanced by ~1000-fold with phenolic compounds such as 6-hydroxybenzothiazole (enhancer) H2O luminol luminol h enhancer Detection IMMUNOPRECIPITATION DO NOT MIX this term with the precipitation reaction of the soluble immuncomplexes • isolation and concentration of a particular protein from a protein mixture • detection of protein associations (e.g. members of receptor signalization) CHROMATIN IMMUNOPRECIPITATION (ChIP) Identification of molecules (mainly transcription factors) binding to a specific site of the DNA Provides information about the link between signaling pathways and gene activation IMMUNOHISTOCHEMISTRY Labeled antibodies added to fixed tissue sections detect the distribution of the chosen antigen within the tissue or within the cells of a particular tissue • Immunofluorescence •Fluorescent dye coupled to antibody FITC – fluorescein isothiocyanate (green) PE – phycoerythrin (orange) • Immunoenzyme method • enzyme-coupled antibody P – peroxidase AP – alkaline phosphatase (Substrates converted into an insoluble compound) IMMUNOHISTOCHEMISTRY Fixation Tissue sample Freezing Sectioning Section before staining IMMUNOHISTOCHEMISTRY Enzyme X Avidin Biotin Secondary antibody Primary antibody Slide Cells Tissue sample Classical histochemistry Acute bronchopneumonia (hematoxylin-eozin staining) Only few cell types could be identified Immunohistochemistry (CD68+ macrophages and lymphocytes, granuloma) Detection of actin microfilaments (TRITC) A fixed and permeabilized skin fibroblast Mitochondria F-actin Nucleus Antinuclear (ANA) autoantibodies from the serum of a SLE patient can be visualized in cell culture (Hep-2) by indirect fluorescent labeling (immunofluorescence) Intracellular cytokine detection by immunofluorescence cytokine specific antibody with fluorescent labelling - the cell membrane should be permeabilized (detergent) - the cells should be fixed previously avoiding the decomposition of the cells (e.g. aldehyde fixation) - optionally the cells could be labelled by some cell type specific antibody in the beginning (e.g. CD4) cytokines The result: You can determine which cell type has produced the cytokines! The sensitivity could reach that of the Western blot. (e.g. with chilled CCD camera mounted microscope: You need only one cell for detection) Bioassay Cytokine dependent or cytokine sensitive cell lines can serve as “bio indicators” of that given cytokine. Those cytokines can be detected by high sensitivity with these indicator cells: e.g. CTLL2 cells IL-2 present no IL-2 TNF present no TNF e.g. Wehi 164 cells Problem: cross reactivity of different cytokines The survival/proliferation of the cells can be measured by a colorimetric assay (MTT assay) MTT assay: cytokine concentration IL-2 CTLL2 TNF Wehi 164 There could be some cross reaction between cytokines. e.g. the CTLL-2 cell line can be stimulated by IL-4 also Detection of intracellular (cytoplasmic) Ca2+ concentration An inrease in cytoplasmic Ca2+ levels can be detected by fluorescent indicator dyes. /Fluo-3 or Indo-1/ for example – ic Ca2+ signal in a single cell B cell antigen presentation by B cell to T cell (click) T cell Investigation of gene activation Activation of T cells can be monitored by the detection of the transcribed mRNA of the activated genes. e.g. activation of cytokine genes method: RT-PCR, QRT-PCR cells RNA isolation RNA (reverse transcriptase) cDNA cDNA (PCR) determination of the length and quantity RT-PCR: agarose gel (densitometry) QRT-PCR: fluorescent method (TaqMan probe (FRET) or dsNA intercalating fluorochrome SYBR green) The size of the cycling cells are increased – called blast transformation Cell-cycle Possibility of the examination Stimuli (e.g. antigen) resting lymphocyte (G0) effector cell - transcription (RT-PCR) - protein synthesis memory cell (Immunoassay) changes in the RNA- and protein synthesis, in the cell membrane and in the transports cell division change in the number of the cells (MTT, CFSE, special histone modifications) DNA-synthesis DNA quantification (fluorescent DNS intercalating agents, 3H-thymidine) The cell cycle can be examined by fluorescent dye G2 G0 M that intercalates stoechiometrically into the double stranded DNA (e.g. propidium iodide, PI) DNA analysis G1 cell number s G0G1 G2 M s 0 200 400 600 800 1000 4N 2N DNA content Distribution of a normal cycling cell-population by DNA content (flow cytometry) Methods for determinating the B/T cell proliferation 3H-labeled thymidine incorporation – measures the increasing DNA content by β decomposition, and does not answer the numbers of cell division, and the dividing cell number thymidine-analog bromodeoxyuridin (BrdU) can be administered to experimental animals, or cell cultures, and the proliferating cells can be detected by labelling with BrdU specific antibody (microscopy, FACS) Carboxyfluorescein diacetate succinimidyl ester (CFSE) fluorescent stain can be used to tracking the cell divisions: Tracking the cell divisions “Cell tracer” or “Cell tracker” dyes enter the cell, and trapped there. The apolar CFSE can bind covalently to the cellular proteins. Later the stain can only be diluted by the cell divisions: distributed equally between the two daughter cells – the fluorescence intensity decreases to the half also. cell divisions: 7 6 5 4 3 2 1 0