Immunology-Immunoglobin And Tcr Diversity, Genetics

Front 1

Q: In general, how does an organism respond to unpredictable antigens and how does it make Abs with higher affinity?

Back 1

-Physiologically, before antigens are introduced, B cells differentiate into individually different, uniquely responsive cells, each one able to respond to a unique epitope. The antigen receptors of B cells, surface Ig. These Ig receptors match the cell's initial affinity for antigens.
-Rearrangements (cutting and moving) of chromosomal DNA allow the different Ig receptors and Igs to be encoded with different antigen specificities.
-Initially, antigen-specific antibodies are absent from serum. B lymphocytes specific for particular antigens do not secrete antibody unless the B cell is specifically stimulated and supported to grow.
-After exposure to antigen, 13 cells with surface Ig's that bind to the antigen proliferate (divide) to increase in cell numbers and then secrete antibodies. The cell division requires growth factors from T cells. The antibodies they first secrete have the Fab (and therefore the antigen specificity) of the initial membrane-associated Ig receptors. The first antibodies secreted are of the same class as the Ig membranes receptor, membrane IgM.
-Later, more DNA rearrangements in these antigen-stimulated cells permiting class switches (e.g., IgM to IgG)
-After re-encounter with the same antigen, additional changes within the DNA of individual cells account for the increase in antibody affinity. This reencounter is termed 'boosting with antigen'. The antigen-specific B cells, that divided upon first encounter with antigen, mutate their antibody genes so that higher affinity antibodies can be made. The amino acid sequences of the high affinity antibodies were absent from the immunized person's initial set of B cells. This mutation occurs without cutting up or rearranging the DNA

Front 2

Q: What are the main problems posed to the generator of diversity (GOD)?

Back 2

-The body can make different antibodies to more than 10^9 different antigens, each with 5 different classes of immuglobulins. The body also makes many millions of T cells, each one recognizing a different antigen. Normally, each protein in the body is encoded by a single, separate gene. However, for antibodies and TCRs, this one gene/one protein is impossible because there is not enough DNA per cell to encode that many different proteins.
-The second problem posed is how Individual B lymphocytes change or "switch" their production from IgM antibodies to IgG or other classes while the immunoglobulin retained the same antigen specificity. (Keeping the antigen specificity would mean keeping the variable region that binds antigen, and altering the other part of the IgH chain). Thus, the rest of the heavy Ig protein chain is "switched" to another class.

Front 3

Q: Describe the germline theory.

Back 3

-in its most extreme form, originally proposed that every variable domain in every single antibody was encoded in a different segment of germline DNA. Germline DNA is DNA with the sequence found in the original embryo. All variable regions therefore would be inherited as germline material or "mini-genes". Later, DNA encoding mini-genes would have to be joined to DNA encoding constant regions of a light chain or the constant regions of a heavy chain subclass. The theory was that the '''mini-genes'' would be cut and joined together in individual cells produced in the mature animal. This theory did not fit with the one gene-one protein theory of molecular biology. It was heresy to consider cutting up, chromosomal DNA and rejoining it. It turns out that only in immunology are chromosomes cut and rejoined. DNA is cut and rejoined to make Ig and to make TCRs.

Front 4

Q: Describe the somatic mutation theory.

Back 4

-in its most extreme form, proposed that there was only one piece of DNA for each light chain and each heavy chain isotype. "Hot" spots of DNA mutation would produce the hypervariable regions in lymphocytes (while the DNA in other tissues remained unchanged). "Not so hot" spots would be framework regions and "cold" spots would be the constant regions. 'Somatic' mutations would occur only in lymphoid tissue. Each individual would acquire different mutations. This theory was also unacceptable to molecular biologists. At that time, there were no known "hot" spots of DNA where mutation occurred at a fast rate. Somatic mutation occurs In the variable region after B cells encounter antigen. The T cell receptors for antigen lack "hot spots" and stay as encoded by their mini-genes.

Front 5

Q: Which theory is right (the somatic mutation or the germline theory)?

Back 5

-Both theories are right. To make one antibody, variable regions are formed from different germline Ig "minigenes". To make one TCR, the variable regions are formed from different TCR "mini-genes". Somatic mutation improves the antigen binding strength of Ig when B cells re-encounter antigens

Front 6

Q: What problem did the two theories forget to look at?

Back 6

-Neither theory made much attempt to address the problem of the Ig switch.
-And neither theory included the random insertion of nucleotides at the sites of DNA re-joining, which introduce more diversity. These random amino acid insertions are different from the mini-genes and from mutations

Front 7

Q: Where does rearrangement occur in the body?

Back 7

-DNA is not cut into pieces, rearranged or rejoined. The Ig rearrangements take place only in B lymphocytes. T cell receptor rearrangement occur only in T cells.

Front 8

Q: What are some of the characteristics of virgin B lymphocytes?

Back 8

-Each individual virgin B lymphocyte is committed to make antibody of only one antigen specificity. One B cell is initially committed to make light chains (containing only one VK or VA) and heavy chains (containing only one VH) Immunocompetent B lymphocytes have surface immunoglobulins and are ready to respond to antigens (and later make antibodies). 'Virgin' B cells are immunocompetent, have not encountered antigen, and have surface IgM and IgD that act as, receptors for antigen.

Front 9

Q: Define germline DNA.

Back 9

-DNA as inherited in the embryo.

Front 10

Q: What is somatic DNA?

Back 10

-In contrast, somatic DNA is DNA found in differentiated cells which are not geminal (sperm- or egg-forming) cells. In T and B lymphocytes, the DNA that encodes TCRs and immunoglobulin genes is rearranged with substantial deletions of DNA and differs from germline.

Front 11

Q: How are the genes selected for the chains of the TCRs and immunoglobulins?

Back 11

-For both of the chains of TCRs and Immunoglobulin, DNA segments or mini-genes are selected from the germline DNA and rearranged to form new somatic genes that will encode the TCR and light and heavy chain Ig proteins.
-The DNA coding for heavy chains undergoes a second DNA rearrangement later to switch from one Ig class to another Ig class.

Front 12

Q: What is used to make the DNA code for the light chains?

Back 12

-For light chains, 3 noncontiguous segments of DNA from the germline are used to form Ig light chains. These DNA segments are 2 mini-genes that make up the variable region that are combined with a constant region for the light chain.

Front 13

Q: What are the different types of diversity in Ig light chains?

Back 13

-Combinatorial diversity comes from selection of a mini-gene from the Vkappa group, selection of a mini-gene from the Jkappa group with attachment to the Ckappa constant DNA.
-junctional diversity arises from repair at the sites where the pieces of DNA are joined.

Front 14

Q: What are the different types of segments of light chain DNA?

Back 14

-The 3 segments of light chain DNA in the germline are called V for variable, J for joining, and C for the constant DNA segment. The V and J DNA segments encode the variable region of the light chain. [*NOTE this segment of DNA has no relation to the "J chain" protein that is found in IgM and IgA, the multimeric classes of Ig.

Front 15

Q: What are the different type of Vkappa segments for light chains?

Back 15

-For human kappa light chains, there are many different Vk segments of DNA. Each has a separate piece of DNA. One Vk must rearrange in the germline DNA to join one Jk before kappa light chain mRNA can be transcribed. The rearranged DNA in this B lymphocyte is now "somatically" rearranged.

Front 16

Q: How many different J pieces are there for the kappa light chains?

Back 16

-For human kappa light chains, there are 5 different J pieces of DNA. One J segment is selected and joined to a Vk. The DNA between the selected V and J is deleted.

Front 17

Q: At transcription, what happens to the Vk and Jk?

Back 17

-At transcription, the RNA polymerase copies the joined VK-JK and keeps copying through the end of the Ck

Front 18

Q: What happens to the transcribed mRNA?

Back 18

-the transcribed mRNA is processed to remove small pieces called introns which do not code for protein, and mature mRNA is sent to the cytoplasm. the extra J mRNA exon segments are spliced out to give a processed mRNA that encodes a complete kappa light chain.

Front 19

Q: What are some characteristics concerning the light chain?

Back 19

-For human kappa light chains, there is only one kappa constant segment of DNA.
-The V and J pieces of DNA encode the variable domain of the light chain.
-Each kappa or lambda light chain gene has its own set of V and J DNA segments.
-The DNA of only one light chain allele becomes fully rearranged within a single B cell.

Front 20

Q: how many different kappas can be formed?

Back 20

-Thus, many different human kappa light chains (-100 Vs) x (5 Js) x (1 C) = ~500 kappas can be formed, providing a "mini-gene" explanation of diversity. Furthermore, DNA repair at the V-J splice contributes additional junctional diversity. This DNA repair can be nucleotide deletions or N-nucleotide additions to the cut ends before they are joined. Any base is added by an enzyme called terminal deoxyribonucleotidyl transferase (TdT) (which you are to remember). Also contributing to diversity is imprecise DNA rearrangement, where the cuts in the DNA are not placed identically in each cell but the reading frame (3 bases per amino acid codon) is kept. The reading frame of the 5 Js can be shifted, so that new amino acid sequences appear in the J regions.

Front 21

Q: What are the components of one human kappa light chain?

Back 21

-YOU ARE EXPECTED TO KNOW ONE Vkappa, ONE Jkappa, AND the Ckappa DNA SEGMENT ARE USED TO MAKE ONE HUMAN KAPPA LIGHT CHAIN.

Front 22

Q: Describe the V-J splice site of kappa DNA.

Back 22

-The V-J splice cite of kappa DNA encodes amino acid 'position 96 at the 3rd hypervariable region of the kappa light chain. Since the first two hypervariable regions are within the V-kappa DNA mini-genes, it is still a mystery how these hypervariable regions happened. Since all three hypervariable regions form the three complementarity detennining regions where antigens bind, only part of GOD has really been explained for the light chain.

Front 23

Q: What are the different components of the kappa and lamba light chains?

Back 23

-note that Vkappa and Jkappa, Vlambda and Jlambda mini-genes form kappa or lambda light chains

Front 24

Q: Describe heavy chain DNA.

Back 24

-rearrangement of Germline heavy chain DNA is similar, but uses V-D-J-C with D as a fourth DNA mini-gene segment to form the somatic DNA that encodes heavy chains

Front 25

Q: What is the order of chromosome rearrangement in heavy chain DNA?

Back 25

-In a human pre-B lymphocyte, the heavy chain DNA on chromosome 14 rearranges first, then the cell tries for kappa on chromosome 2, and if that doesn't work, goes for lambda on chromosome 22. The cell stops trying as soon as it gets one functional rearrangement for a heavy chain and one for a light chain.

Front 26

Q: What are the components of heavy chains?

Back 26

-4 segments of DNA are used to make heavy chains, V for variable, D for diversity, J for joining, and C for constant. [Note: Once again, the J region of heavy chain DNA has no relationship to the "J" protein of IgM and IgA.]

Front 27

Q: How many different forms of V-heavy regions of DNA are there?

Back 27

-For human heavy chains, there are MANY (--200) V-heavy regions of DNA each with a piece encoding the leader piece of protein.

Front 28

Q: What are the first heavy chains that a B lymphocyte makes?

Back 28

-The first heavy chains that a B lymphocyte makes are membrane delta and mu chains. Extra 3' mRNA. encodes a membrane spanning piece of the heavy chain so that monomeric surface IgM units and IgD can be the initial B cell receptors for antigen.

Front 29

Q: What is the order of things that happen during formation of heavy-DNA?

Back 29

-The first thing that happens is that one D piece of DNA is joined to one J-heavy piece of DNA, with removal of the intervening segments of the D and J heavy DNA. (The 5' flanking pieces of extra D and the 3' flanking extra J DNAs will remain.)
-Next, a V-heavy piece of DNA is joined to the D-J piece, with removal of all other segments of D. THRS THE END OF THE FIRST DNA REARRANGEMENTS OF HEAVY CHAINS. At this point, the B cell can make IgM and IgD.
-Now the V-D-J DNA can transcribed with all the extra J segments straight through to the end of the terminus of mu.
-After transcription, introns and the extra J mRNA segments are spliced out to give processed mu heavy chain message.

Front 30

Q: How many of the different segments for the heavy chains are there?

Back 30

-For human heavy chains, there are hundreds of V H genes, several (>20) D segments, ~6 J segments and one C-mu segment. There are also the extra, junctionally introduced somatic changes by nucleotide excision and by N nucleotide addition (by the enzyme TdT). Terminal deoxynucleotide transferase (TdT) is found only in developing lymphocytes. The D segments can also be used in several different open reading frames, contributing even more diversity.

Front 31

Q: Where does transcription continue past?

Back 31

-Transcription can alternately continue right through the end of the Cdelta membrane DNA. The membrane pieces are not illustrated in the figure. This one big piece of mRNA can be processed to give four different mRNAs that will be translated into four proteins: secreted mu, membrane mu, secreted delta or membrane delta.
-The result of this process is an immunocompetent B lymphocyte that has membrane-bound and secreted IgM and IgD immunoglobulins, all with the same variable domains.

Front 32

Q: What is the combinatorial diversity of heavy chain?

Back 32

-The combinatorial diversity of this rearrangement is great (several hundred V-heavy) x (20 D) x (6 J) x (1 C) = thousands of possibilities. Additional junctional diversity is added by repairs at the splice sites.

Front 33

Q: What adds more diversity to the heavy chain?

Back 33

-Even more diversity is added by the random selection of one possible Land one possible H chain per cell to combine into a single immunoglobulin molecule.

Front 34

Q: What are we expected to remember concerning heavy chain composition?

Back 34

-a) The first heavy chain DNA rearrangement involves 4 pieces of DNA -- one VH, one DH, one JH and the CH and that there are many different Vs, some Ds and some Js which can be joined together randomly and added to a C segment to create diversity and b) RNA splicing leads to the messages for the delta chains.

Front 35

Q: What is needed for the mu and delta Ig heavy chain to change to other heavy chains?

Back 35

-A second round of DNA rearrangement is needed to change the mu and delta Ig heavy chains to the other heavy chain classes. This second DNA rearrangement is called Jg class switching. The heavy chain class is switched and the DNA encoding the variable domain (V-D-J) is unchanged and maintainedfor the next class of Ig. The immunoglobulin class changes but the antigen specificity of the individual cell's antibody does not change.

Front 36

Q: Describe Ig class switching.

Back 36

-On the chromosome with the heavy Ig chain, IgH chain constant genes are available to be switched and attached to the previously rearranged variable region V-D-J DNA.
-The VH-DH-JH rearranged DNA that encodes the variable domain of the antibody heavy chain is now "switched" from the Cmu to other C regions of DNA encodes each immunoglobulin isotype
-The result is that a single B lymphocyte changes from making IgD and IgM antibody of a particular antigen specificity to antibodies of the IgG, IgA or IgE classes with the same antigen specificity.

Front 37

Q: Describe somatic mutations.

Back 37

-Scattered somatic mutations occur within the DNA of the rearranged V segment as B cells respond to renewed antigen stimulation and undergo multiple rounds of cell divisiono
-The somatic mutations happen after antigen boosting. The molecular basis for these mutations is still uncertain.

Front 38

Q: How are somatic mutations found?

Back 38

-These mutations are found by comparing the Vheavy DNA sequences of mature immunoglobulins of the antibodies developed after each boost with the germline DNA for the same particular V heavy DNA segment. The somatic mutations provide a repertoire of mutant B cells which can be selected by the booster antigen. The antibodies secreted by these mutant B cells contributes to the increased IgG affinity for the antigen used for boosting.

Front 39

Q: Where do somatic mutations appear?

Back 39

-Somatic mutations appear in a single V region after an immune response is boosted-by new exposure to antigen. The mutations are found in the V regions of both the light and heavy chains. Note how the affinity increases (Kd indicated on the right side of figure, gets smaller) after the 3rd boost.

Front 40

Q: How are T cell receptors formed?

Back 40

-The receptors for T cells are also formed by combinatorial rearrangements with junctional repair

Front 41

Q: Do T cells have different receptors?

Back 41

-T lymphocytes are antigen specific (just as B cells are). T cells each have different clonotypic receptors, each T cell having only one T cell receptor (TCR) for one distinct small peptide antigen. The predominant T cell receptors, on 90-99% of T lymphocytes, are composed of TCR alpha and beta chains, which are rearranged at the DNA level similarly to the Ig light and heavy chains

Front 42

Q: What are the components of TCRs?

Back 42

-The TCR alpha chain has a molecular weight of ~49 kD and is encoded by Valpha-Jalpha-Calpha regions of DNA. The TCR beta chain has a m.wt. of 40-43 kD and is encoded by Vbeta-Dbeta-Jbeta-Cbeta regions of DNA. The alpha chain has 3 regions of DNA that form half of an antigen-binding receptor, similar to the Ig L chains

Front 43

Q: Describe the second type of T cell receptor.

Back 43

-Some T cells use a different kind of TCR. These T cell antigen receptors are composed of dimers of TCR delta & gamma chains. Between 1-10% of the T lymphocytes of an individual will have delta-gamma TCR receptors. One T cell will have either alpha and beta chains or delta and gamma chains, not all four chains. The gamma and delta chains are also rearranged at the DNA level. The cells that have gamma-delta receptors may bind a limited subset of antigenic peptides, which includes the heat shock proteins of intracellular bacteria.

Front 44

Q: How many different loci are there that makes up the different chains?

Back 44

-Thus there are only 7 loci in humans where chromosomal germline DNA is cut up and rearranged: the loci for kappa and lambda Ig light chains, heavy Ig chains, and TCR alpha, beta, gamma and delta chains.

Front 45

Q: Describe monoclonal antibodies.

Back 45

-A monoclonal antibody is pure with only one amino acid sequence, generated from one single B cell clone. It is produced from hybrid cell or hybridoma made from a tumor cell and a normal B cell. The hybrid cells secrete only one antibody. One wants to use this antibody to detect a particular antigen.

Front 46

Q: What is a hybridoma?

Back 46

The hybridoma cells are made from normal mature B cells that originally made the antibody and myeloma tumor cells, hence the term" hybridoma". The nuclei of the two cell types fuse to form one larger nucleus with the DNA from both cell types. The myeloma tumor is the fusion partner of choice because it is a tumor of B lymphocytes, which secrete Ig.

Front 47

Q: What poperities does the hybrid cell show?

Back 47

-The hybrid cell will have properties of both fusion partners and have one huge nucleus containing the chromosomes of both parents. Because one of the parents was a tumor, the chromosomal polyploidy will be tolerated. The resulting hybridoma will have the immortal growth characteristics of a tumor.

Front 48

Q: Where are monoclonal antibodies made from?

Back 48

-made from the B cells of immunized animals. The initial hybribomas have to be screened with antigen to find those few that are making antibody to the antigen. (Many hybridomas will be to previous antigens). Often the desired clone is one screened from a million different hybridomas.

Front 49

Q: Do all the Abs from one hybridoma have the same protein sequence? Are they identical?

Back 49

-YES, They will all bind to the same antigens. Keep in mind that even though monoclonal, these antibodies can have cross-reacting specificity if provided with several structurally similar epitopes.

Front 50

Q: What organism made the polyclocal Abs?

Back 50

-The donor animal made polyclonal antibodies. The cultured and cloned hybridoma selected for its antigen-specific antibody makes a monoclonal antibody.

Front 51

Q: Why are humanized monoclonal Abs made?

Back 51

-made so that one has the specificity of a mouse monoclonal in a human antibody that will be treated as self by humans. These antibodies are made by recombinant DNA technology

Front 52

Q: What method is used to characterize cells by their different cell surface antigens?

Back 52

-flow cytometry used with monoclonal Abs

Front 53

Q: What is the purpose of flow cytommetry?

Back 53

-One purpose of flow cytometry is to determine the number (or %) of blood lymphocytes that bear cell surface marker proteins that indicate their cellular function. For example, helper T lymphocytes have a plasma membrane protein called CD4 that other lymphocytes lack. Antibodies to CD4 will identify the cells. Fluorescence is a signal that can be used to track where the antibody is bound. Fluorescent chemicals are attached covalently to the antibodies. The more antibody bound, the more fluorescent a single cell. Each single cell can be measured for how bright its fluorescence is. After the cells are measured one at a time and data are stored, the frequencies and relative protein expression of the positive cells can be determined.

Front 54

Q: What does flow cytometry use?

Back 54

-Flow cytometry uses a laser for excitation of the fluors, which gives a single wavelength for excitation. The absence of other wavelengths for excitation prevents molecules other than the fluors from excitation and limits background noise at the fluorescent emission wavelength. Thus weak positive signals can be detected because there is very little background noise. Five hundred molecules of fluorescein can routinely be detected by flow cytometry.

Front 55

Q: Describe the absorption of the fluorescent molecules.

Back 55

-Fluorescent molecules absorb light at one wavelength and emit fluorescence at another. For example, the molecule fluorescein absorbs light at 488 nm and emits it at 514 nm (green). A different molecule called phycoerythrin also absorbs light at 488 run but emits it at 578 nm (red). Texas red is another fluor than can be used to tag proteins, absorbs some at 488 nm and emits at ~630 nm

Front 56

Q: Describe the interaction between small fluorescent molecules and Abs.

Back 56

-they are coupled covalently so that where the antibodies bind, there can be fluorescence. The more fluorescence produced by a cell, the more antibody that was bound to it. Frequently, the antibodies are mouse monoclonal antibodies to human antigens (e.g., mouse anti-human CD4).

Front 57

Q: What is a flow cytometer?

Back 57

-A flow cytometer is an instrument that measures the fluorescent signal from single cells that are separated in a liquid stream (the plumbing, figure 7). Lasers are used as the source of light for flow cytometry because they generate only one wavelength and are very bright. (The stronger the excitation, the more light that can be absorbed and then emitted.) The cells are run single file through the machine so that each cell is separated from the others by distance. The distance between the cells become differences in time as they pass sequentially before the fluorescence light detector. Each cell passed first through the laser light (excitation) and then immediately through light detector, where its fluorescence is measured (the optics). Two or more colors of fluorescence can be detected on each cell. Each cell trips a signal as it goes through so that its signals are recorded as a separate unit of sample, at about 300 cells per second. If the instrument has a plate with inducible electric charges, the individual cells in individual droplets of liquid can be sorted. The instrument is sometimes called a fluorescence-activated cell sorter (FACS) but this term should really be reserved for when the cells are sorted into groups of cells with different fluorescent signals.

Front 58

Q: How is the information from the flow cytometer measured?

Back 58

-use dot plots and histograms. Flow cytometry information is often presented as a "dot plot" in which each cell is a dot. Where the cell is placed on the plot depends on its brightness in two colors, or its measurement by any 2 parameters. In the figure below, CD3 cells would be green if fluorescein mouse monoclonal anti-CD3 (fluor I) had been used to label the cells and then the unbound antibody washed away. The more antibody bound to the cell, the brighter the cell. In figure 9, cells were stained simultaneously with anti-CD3, a marker used to detect T cells and with anti-CD25 to detect cells with the alpha chain of the receptor for interleukin 2. A different fluor was used with each antibody, green fluorescein (FITC) with anti-CD3 and red PE for anti-CD25. In class, mark up the illustration. The cells on the left (9A) are freshly taken from blood, the ones on the right (9B) were stimulated in vitro with antigen. The antigen-specific T lymphocytes will respond and make high affinity receptors for the growth factor interleukin 2 that are called CD25

Front 59

Q: Describe data from flow cytometry put on a histogram.

Back 59

-The data from flow cytometry can be presented a different way, as histograms of a single parameter (e.g., brightness of a fluor) that was measured, to allow more ready assessment of the number of cells and the amount of antibody bound to them. The X axis indicates the extent of brightness of the fluorescence of single cells on a log scale. The further right on the plot, the more antibody bound per cell. The Y axis indicates the number of cells at each degree of brightness. The values representing the number of cells for each of >200 levels of fluroescent brightness are plotted and the connect to form a line. The solid line indicates the fluorescence when the lymphocytes were reacted with phycoerythrin-coupled anti-CD25 antibody. The dashed line indicates the fluorescence when they were reacted with an irrelevant phycoerythrin-conjugated antibody of the same isotype (called the isotype control, which indicates nonspecific binding). There will always be some sticking of the irrelevant antibody or autofluorescence of the cells, to give a background signal indicated the dashed line. You will draw the isotype control signal in class with a dashed line.

Front 60

Q: What is the medical and other relevancy of immunogenetics and monoclonal Abs?

Back 60

-The essentials of light and heavy chain immunoglobulin and TCR DNA rearrangements are likely to be board questions. They also explain how one lymphocyte makes antibody of only one specificity.
-Events responsible for antibody affinity maturation occur in vivo where B cells somatically mutate and then are selected by the boosting antigen. B cells do not somatically mature well in vitro. Therefore, the best antibodies are still made by directly immunizing people or animals. Expression libraries of heavy and light chain cDNAs (to produce antibodies without using live animals or people) will be from Igs that did not undergo affinity maturation for particular antigens.
-Monoclonal antibodies are an incredible advance for medical diagnostics in vitro and in vivo. Given the enormous number range and exquisite specificities of antibodies, monoclonal antibodies can be used to distinguish between different strains of virus, different bacteria, etc. Since the hybridomas are immortal and unchanging they do not need to be recharacterized like antisera raised to one antigen in different animals. Furthermore, monoclonal antibodies can be use in vivo to detect just one antigen, like a tumor-associated antigen or a viral antigen to see where the tumor or virus is sequestered in the body
-Monoclonal antibodies are an incredible advance to science of all kinds, from plant biology to archeology. The 1975 Nature paper (Kohler, G. and C. Milstein. "Continuous cultures of fused cells secreting antibody of predefined specificity" Nature 256: 495, 1975) ultimately led to Nobel prizes for the authors. It was rejected as a full paper because it was of 'insufficient general interest' to be a full article and shortened to a letter in Nature. The last sentence, however, was prophetic: "Such cultures could be valuable for medical and industrial use."
-For therapy in humans, one would like to have humanized monoclonals. An example of a humanized monoclonal antibody in use is Rituximab, an anti-human CD20 monoclonal that reacts with B cells and is used to treat B cell leukemias and lymphomas.
-Flow cytometry has greatly advanced our knowledge of immunology and revolutionized characterization of leukemias.