PLASMA PROTEINS

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PLASMA PROTEINS
M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.
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INTRODUCTION

• Total blood volume is 4.5-5 litres.
• If blood containing anticoagulants (e.g.heparin ,
  potassium oxalate) is centrifuged , the plasma
  separates out as a supernatant while the cells
  remain at the bottom.
• About 55-60 of blood is plasma
• The packed cell volume or hematocrit is about
  40-45

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Introduction

• Plasma is the clear straw coloured fluid
  portion of the blood minus its cellular
  elements.
• It constitutes about 55 of the blood volume.
• Serum is plasma minus clotting factors
  (fibrinogen prothrombin ).
• The defribrinated plasma is called serum

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COMPOSITION OF PLASMA

• PLASMA contains the following composition

• WATER
• Is the main constituent of Plasma 91

SOLIDS 9 of the plasma (1 inorganic molecules
8 organic molecules)
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OTHER ORGANIC MOLECULES

• Carbohydrates Glucose ( 100-120 mg)
• Fats neutral fats, phospholipids (150-300mg)
• Cholesterol (150-240 mg)

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• Non protein nitrogenous substances
  ammonia, amino acids, creatine, creatinine
  (0.6-1.2 mg) xanthine, hypoxanthine, urea
  (20-40 mg) uric acid (2-4 mg).
• Hormones enzymes antibodies.

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• Inorganic molecules are sodium, potassium,
  calcium, magnesium, chloride, iodide, iron,
  phosphates copper.
• Gases presents in the plasma are O2 ,Co2, N2 .

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• plasma proteins
• - forms 7 of the solids in plasma
• - their normal valves 7.4 gm
  
• ranges from (6.4 8.3 gm)
• INCLUDES
• ALBUMIN
• GLOBULINS
• FIBRINOGEN

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NORMAL VALUES

• Total protein content of normal plasma is
• 6 - 8 g/100ml
• The plasma proteins consist of
• 1)albumin (3.5-5 g/dl)
• 2)globulins (2.5-3.5 g/dl)
• 3)fibrinogen (200-400 mg/dl)

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• The albumin globulin ratio is usually between
  1.2 1 to 1.5 1
• Almost all plasma proteins , except
  immunoglobulins are synthesized in liver

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SEPARATION OF PLASMA PROTEINSUSING SALTS

• In clinical laboratory, separation is usually
  done by salts.
• Thus , fibrinogen is precipitated by 10 and
  globulins by 22 concentration of sodium sulphate
• Ammonium sulphate will precipitate
• albumin by full-saturation
• globulin by half-saturation

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ESTIMATION OF PLASMA PROTEINS

• In clinical laboratory , total proteins of
  patients are estimated by Biuret method.
• Albumin is quantitated by Bromo cresol green
  (BCG) method , in which the dye is preferentially
  bound with albumin , and the colour intensity is
  measured colourimetrically.

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• OTHER METHODS
• Lowrys method
• Kjeldahls method
• Dye-binding method
• UV-absorption method

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ELECTROPHORESIS

• The most common method of analyzing plasma
  proteins is by electrophoresis.
• The term electrophoresis refers to the movement
  of chargeD particles through an electrolyte when
  subjected to an electric field

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• In clinical laboratory , cellulose acetate is
  widely used as a supporting medium.
• Its use permits resolution , after staining , of
  plasma proteins into five bands , designated
  albumin , a1 , a2 , ß and ? fractions,
  respectively

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• The stained strip of cellulose acetate is called
  electrophoretogram.
• The amounts of these five bands can be
  conveniently quantified by use of Densitometric
  scanning machines.
• Characteristic changes in the amounts of one or
  more of these five bands are found in many
  diseases.

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ABNORMAL PATTERNS IN CLINICAL DISEASES

• Various abnormalities can be identified in the
  electrophoretic pattern
• 1) CHRONIC INFECTIONS
• The gammaglobulins are increased

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• 2)MULTIPLE MYELOMA
• In para-proteinemias , a sharp spike is noted and
  is termed as M-band.
• This is due to monoclonal origin of
  immunoglobulins

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• PRIMARY IMMUNE DEFICIENCY
• The gamma globulin fraction is reduced
• NEPHROTIC SYNDROME
• All proteins except very big molecules are lost
  through urine , and a-2-fraction will be very
  prominent

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• CIRRHOSIS OF LIVER
• Albumin synthesis by liver is decreased , with a
  complementary excess synthesis by globulins by
  reticuloendothelial system

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• CHRONIC LYMPHATIC LEUKEMIA
• Gamma globulin fraction is reduced
• ALPHA-1-ANTITRYPSIN DEFICIENCY
• The alpha-1 band is thin or even missing

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ALBUMIN

• Albumin (69 kDa) is the major protein in human
  plasma(3.4-4.7 g/dl)
• It makes up approximately 60 of the total plasma
  protein.
• About 40 of albumin is present in the plasma,
  and the other 60 is present in the extracellular
  space.

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• The liver produces about 12g of albumin per day ,
  representing about 25 of total hepatic protein
  synthesis
• Albumin can come out of vascular compartment. So
  albumin is present in CSF and interstitial fluid.

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FUNCTIONS OF ALBUMIN

• 1)COLLOID OSMOTIC PRESSURE OF PLASMA
• The total osmolality of serum is 278-305
  mosmol/kg.
• This is exerted mainly by salts, which can pass
  easily from intravascular to extravascular space.
• Therefore, the osmotic pressure exerted by
  electrolytes inside and outside the vascular
  compartments will cancel each other.

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• But proteins cannot easily escape out of blood
  vessels, and therefore , proteins exert the
  effective osmotic pressure.
• It is about 25mm Hg, and 80 of it is contributed
  by albumin.
• The maintenance of blood volume is dependent on
  this effective osmotic pressure

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Regulation of colloidal pressure
Gaw Clinical Biochemistry Churchill
Livingstone (1999), p. 44.
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• TRANSPORT FUNCTION
• Albumin is the carrier of various hydrophobic
  substances in the blood such as
• i)bilirubin non-esterified fatty acids
• ii)drugs (sulpha,aspirin,salicylate,)
• iii)hormones(steroid hormones,thyroxine)
• iv)metals (calcium,copper,heavy metals)

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• 3)BUFFERING ACTION
• Albumin has maximum buffering capacity amongst
  all proteins
• It has a total of 16 histidine residues which
  contribute to this buffering action.

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• 4)NUTRITIONAL FUNCTION
• All tissue cells can take up albumin by
  pinocytosis.
• It is then broken down to amino acid level.
• So albumin may be considered as the transport
  form of essential amino acids from liver to
  extrahepatic cells.

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CLINICAL APPLICATIONS

• 1)BLOOD-BRAIN BARRIER
• Albumin-fatty acid complex cannot cross
  blood-brain barrier and hence fatty acids cannot
  be taken up by brain.

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• 2)PROTEIN-BOUND CALCIUM
• Calcium level in blood is lowered in
  hypo-albuminemia
• Thus , even though total calcium level in blood
  is lowered, ionised calcium level may be normal,
  so tetany may not occur.

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• 3) THERAPEUTIC USE
• Human albumin is therapeutically useful to treat
  burns,hemorrhage and shock.
• 4)EDEMA
• Hypo-albuminemia will result in tissue edema
• Eg a)malnutrition
• b)nephrotic syndrome
• c)cirrhosis of liver
• d)chronic congestive cardiac failure.

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HYPO-ALBUMINEMIA

• CIRRHOSIS OF LIVER
• Synthesis is decreased.
• MALNUTRITION
• Availability of amino acids is reduce and so
  albumin synthesis is affected.
• NEPHROTIC SYNDROME
• Permeability of kidney glomerular membrane is
  defective , so that albumin is excreted in large
  quantities.

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• PROTEIN LOSING ENTEROPATHY
• Large quantities of albumin is lost from
  intestinal tract.

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• ALBUMINURIA
• Presence of albumin in urine is called
  albuminuria.
• It is always pathological.
• Seen in
• a)Nephrotic syndrome(large quantities)
• b)Acute nephritis
• c)Inflammatory conditions of urinary
  tract.
• Detection of albumin in urine is done by heat and
  acetic acid test.

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• MICRO-ALBUMINURIA
• In micro-albuminuria or minimal albuminuria or
  plauci-albuminuria , small quantity of albumin
  (30-300 mg/dl) is seen in urine
• It is estimated by RIA
• Increased levels of microalbuminuria is an
  indication of early involvement of renal tissue
  in diabetic patients

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• Albumin-globulin ratio
• In hypo-albuminemia, there will be a compensatory
  increase in globulins which are synthesized by
  the reticulo-endothelial system(plasma cells).
• Albumin-globulin ratio (A/G ratio) is thus
  altered or even reversed.

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• Hypoproteinemia
• Since albumin is the major protein present in the
  blood, any condition causing lowering of albumin
  will lead to reduce total proteins in blood

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• HYPERALBUMINEMIA
• Increased levels of plasma albumin are present
  only in acute dehydration and have no clinical
  significance
• ANALBUMINAEMIA
• Analbuminemia is a rare hereditary abnormality in
  which plasma albumin concentration is usually
  less than 1.0gm/L

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GLOBULINS

• Globulins are bigger in size than albumin .
  Globulins constitute several fractions. These
  are
• a1- globulin
• a2- globulin
• ß- globulin
• ?- globulin

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a1- GLOBULINS

• Retinol binding protein(RBP)
• a1 fetoprotein(AFP)
• a1 protease inhibitor (API)
• a1 - acid glycoprotein (AAG)
• High density lipopprotein (HDL)
• Prothrombin

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• RETINOL BINDING PROTEIN (RBP)
• Retinol (vitamin A) is transported in plasma
  bound to RBP.
• Most retinol RBP in the plasma is reversibely
  complexed with transthyretin (thyroxine binding
  protein)
• a1- FETOPROTEIN (AFP)
• This is present in the tissues and plasma of the
  fetus
• It may play an immunoregulatory role during
  pregnancy.

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• a1- PROTEASE INHIBITOR (API) / a1-
  ANTITRYPSIN (AAT)
• API is one of the plasma proteins, that inhibits
  activity of proteases particularly elastase,
  which degrades elastin, a protein that gives
  elasticity to the lungs
• a1-ACID GLYCOPROTEIN (AAG)
• AAG also known as orosomucoid, contains a high
  percentage of carbohydrate with a large number of
  sialic acid residues
• It is synthesized by liver parenchymal cells.

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• PROTHROMBIN
• It is synthesized by liver with the help of
  vitamin K and involved in blood clotting

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a2- GLOBULINS

• Ceruloplasmin(ferro-oxidase)
• Transcortin / corticosteroid binding globulin
• Haptoglobin
• Thyroxine binding globulin(TBG)
• a2 - macroglobulin (AMG)

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• CERULOPLASMIN (FERRO-OXIDASE)
• This is a copper containing protein.
• It has oxidase activity
• Ceruloplasmin is the major transport protein for
  copper, an essential trace element.
• It is also essential for the regulation of
  oxiation-reduction , transport and utilization of
  iron
• Plasma ceruloplasmin level is reduced in Wilsons
  disease in patients with malnutrition and in the
  nephrotic syndrome.

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• TRANSCORTIN /CORTICOSTEROID BINDING GLOBULIN
• This binds cortisol
• It is synthesized in liver and synthesis is
  increased by oestrogen
• HAPTOGLOBIN
• It plays an important role in the conservation of
  iron by preventing its loss in the urine
• Haptoglobin binds free Hb to form a complex which
  is too large to be filtered by the kidney and
  thus prevents the loss of iron in the urine.

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• THYROXINE-BINDING GLOBULIN (TBG)
• TBG is synthesized in liver
• TBG has a electrophoretic mobility between a1
  a2 globulins
• It transports thyroxine hormone(T3 T4)
• a2 - MACROGLOBULIN(AMG)
• This is major a2 - globulin , which is a natural
  inhibitor of endopeptidases such as trypsin,
  chymotrypsin, plasmin, thrombin .etc.

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ß- GLOBULIN

• Haemopexin
• Transferrin
• ß2 -microglobulin(BMG)
• C-reactive protein(CRP)
• Low density lipoprotein

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• HAEMOPEXIN
• Like haptoglobulin, haemopexin also plays an
  important role in the conservation of iron by
  preventing its loss in urine
• TRANSFERRIN
• Is synthesized in liver
• It transports iron(2 molecules of Fe3 per
  molecule of transferrin) through blood to the
  sites where iron is required

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• C-REACTIVE PROTEIN(CRP)
• CRP is involved in the bodys response to
  inflammations .mainly bacterial..
• It is useful in differentiating bacterial from
  viral infections because the level of CRP is
  increased in bacterial infections only.

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• MICROGLOBULIN
• This protein forms part of the human leucocyte
  antigen(HLA) system
• Plasma levels are increased whenever, there is
  malignant lymphoid or myeloid proliferation and
  renal failure

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ACUTE PHASE RESPONSE

• The acute phase response is a non-specific
  response to the stimulus of tissue following
  trauma, infection ,inflammation, burn, etc
• Following trauma etc , the body responds by
  initiating a series of mechanisms that lead to
  rapid decrease in the concentration of many
  proteins,eg
• Albumin
• Prealbumin
• Transferrin
• These are termed negative acute phase reactants
  

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• An increase in the concentration of several
  specific proteins occur some hours after the
  injury. These proteins are called the positive
  acute phase proteins

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IMMUNOGLOBULINS(Ig)

• Definition
• The Igs constitute a heterogenous family of serum
  proteins, which either function as antibodies or
  are chemically related to antibodies
• The immunoglobulins are ?- globulins , called
  antibodies. All antibodies are immunoglobulin but
  all immunoglobulins may not be antibodies

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• They constitute about 20 of all the plasma
  proteins
• Igs are produced by plasma cells to some extent
  by lymphocytes

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STRUCTURE OF IMMUNOGLOBULIN

• Immunoglobulins are glycoproteins made up of
  light(L) and heavy(H) polypeptide chains.
• All Igs have the same basic structure.
• The basic Ig is a Y shaped molecule and
  consist of 4 polypeptide chains
• 2 H chains
• 2 L chains
• The 4 chains are linked by disulfide bonds

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• An individual antibody molecule always consists
  of identical H chains identical L chains
• L chain may be either of 2 types, kappa(?) or
  lambda(?) but not both
• The heavy chains may be of 5 types and are
  designated by greek letter
• Alpha(a)
• Gamma(?)
• Delta(d)
• Mu(µ)
• Epsilon(e)

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Five Classes of Immunoglobulin
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• Igs are named as per their heavy chain type as
  IgA , IgG , IgD , IgM IgE
• The L and H chains are subdivided into variable
  and constant regions
• L chain consists of one variable(VL) and one
  constant (CL) domain or region
• Most H-chains consist of one variable(VH) and 3
  constant(CH-1,CH-2 CH-3) domains
• IgG IgA have 3 CH domains whereas IgM IgE
  have 4

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• Each Ig molecule has hinge region between CH-1
  CH-2, which allows better fit with the antigen
  surface.
• The variable regions of both L H chains have 3
  extremely variable amino acid sequences at the
  amino terminal end called hypervariable region
• Enzyme(papain) digestion splits the Ig molecule
  into 2 fragments named as Fab (Fragment for
  antigen binding) and Fc (crystallizable fragment)
  

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FUNCTIONS OF Ig

• The primary function of antibodies is to protect
  against infectious agents or their products.
• Igs provide resistance because they can
• Neutralize toxins viruses
• Opsonize microbes so they are more easily
  phagocytosed
• Activate complement prevent the attachment of
  microbes to mucosal surfaces

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• In addition to these functions, antibodies can
  act as an enzyme to catalyze the synthesis of
  ozone (O3) that has microbicidal activity.

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Ig CLASSES

• IgG (HEAVY CHAIN ? )
• Is a monomeric molecule with 2 antigen binding
  sites
• There are 4 subclasses, IgG1 to IGg4 based on
  antigenic differences in the H-chains and on the
  number and location of disulfide bonds
• It is produced mainly in the secondary response
  and constitutes an important defence against
  bacteria viruses

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• IgG is the major class of immunoglobulin found in
  the serum which accounts for 70 of the total
• IgG is the only antibody that crosses the
  placenta therefore is the class of maternal
  antibody that protects the fetus
• Functions
• Neutralizes bacterial toxins and viruses
• Opsonises bacteria, making them easier to
  phagocytize
• Activates complements which enhances bacterial
  killing

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IgA (HEAVY CHAIN a)

• IgA is the 2nd most abundant class constituting
  about 20 of serum immunoglobulins
• IgA occurs in 2 forms
• Secretory IgA
• Serum IgA
• Secretory IgA is a dimeric molecule formed by 2
  monomer units, joined together at their carboxy
  terminals by a protein termed J-chains

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• Additionally secretory IgA has a secretory
  component attached to dimer
• Secretory IgA is found in external secretions
  such as colostrum,saliva,tears and respiratory ,
  intestinal genital tract secretions
• Serum IgA exists as monomeric form( found in
  internal secretions such as synovial,amniotic,pleu
  ral CSF )

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• Functions
• Secretory IgA prevents attachment of bacteria and
  viruses to mucous membranes and helps protect
  mucous surface from antigenic attack
• Prevents access of foreign substances to
  circulation

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IgM (HEAVY CHAIN µ)

• It is a pentamer consisting of 5 identical Ig
  molecules, joined together by disulfide bridges.
• IgM accounts for some 10 of normal Ig
• IgM is the main Ig produced early in the primary
  response
• As it is pentamer, it has 10 antigen binding
  sites is the most efficient Ig in
  agglutination, complement activation other
  antibody reactions is important in defence
  against bacteria viruses

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• The natural blood group antibodies, anti-A
  anti-B are IgM
• IgM present on the surface of B lymphocytes is
  monomer, where it functions as an antigen binding
  receptor for antigen recognition
• IgM can be produced by fetus in certain
  infections.
• Functions
• Activate complement, promotes phagocytosis
  causes lysis of antigenic cells(bacteria)

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• Waldenstorms macroglobulinaemia
• It is a malignant disease of lymphoid elements,
  characterized by high serum concentrarion of IgM

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IgD (HEAVY CHAIN d)

• It is a monomer and resembles IgG structurally
• IgD has no known antibody function but may
  function as an antigen receptor
• Like, IgM, it is present on the surface of many B
  lymphocytes
• The circulating concentration of IgD in blood is
  very low
• IgD is labile

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IgE (HEAVY CHAIN e)

• IgE is a monomeric molecule similar to IgG. It is
  sometimes called reagin
• Although IgE is present in trace amounts, in
  normal persons with allergic activity have
  greatly increased amounts
• Functions
• Antiallergic antiparasitic

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• IgE is responsible for anaphylactic(immediate)
  type of hypersensitivity allergy. Its main
  activity is mediated by mast cells or basophils
• Defends against worm infections by causing
  release of enzymes from eosinophils
• Main host defence against parasites like
  helminthus, provides protection in the disease
  schistomiasis

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MULTIPLE MYELOMA(MYELOMATOSIS)

• A malignant proliferation of plasma cells
• Results in an abnormally high concentration of
  serum immunoglobulins, usually IgG or IgA

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BENCE JONES PROTEINS

• In multiple myeloma, more light chains are
  produced than heavy chains and enter the
  bloodstream
• Because they are of relatively low m.wt, they
  pass through glomerular membrane and appear in
  the urine, these protein chains of low m.wt are
  known as Bence Jones Proteins

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• Bence Jones proteins have the remarkable
  characteristic of precipitating on heating urine
  from 450 600 C and redissolve when the heating
  is continued above 800 C
• Multiple myeloma with Bence Jones proteins in the
  urine is called light chain disease

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