Vet360 Vol 4 Issue 3 June 2017 Vet360 - Page 32

CLINICAL PATHOLOGY

Serum Protein

Electrophoresis

Dr Emma Hooijberg BVSc GPCert ( SAP ) DipECVCP Department of Companion Animal Clinical Studies , University of Pretoria emma . hooijberg @ up . ac . za
How does this method work ?
Serum proteins come in different shapes and sizes , and also have different charges . Albumin , for example , is the most negatively charged protein , while immunoglobulins have a slight positive charge . Serum protein electrophoresis ( SPE ) separates proteins based on their size and charge . The most common method uses agarose gel as a platform . The gel provides a 3-D matrix with large pores ( compared to the size of proteins ). A serum sample is placed into the gel , and an electrical current is applied across it . The more negatively charged proteins migrate through the gel to the cathode ( positive charge ) and the more positively charged proteins , to the anode ( negative charge ).
This results in a separation of all the proteins into bands . A stain is applied to the gel , making the bands visible . The gel is then placed into a scanner , which converts the density of each band into curves , which appear as the typical fractions of the electrophoretogram ( Figure 1 ). / Each curve / fraction represents a percentage of all protein present in serum . This percentage is calculated by a computer programme , and is multiplied by the total protein concentration to give the concentration of each fraction . The fractions , apart from albumin , have traditionally been allocated a Greek letter and a number – alpha-1 , alpha-2 , beta-1 , beta-2 and gamma . It has been determined ( through proteomic studies using other methods ), which major proteins are found in each fraction , as illustrated below in Figure 1 .
Figure 1 : SPE tracing showing the different fractions . Specific proteins migrate to certain fractions based on their charge and size
SPE can be used to give an indication of which groups of proteins are increased or decreased , but does not generally help to identify changes in specific proteins . It should be noted that SPE is not a direct measurement of the concentrations of different proteins , although it is a fairly accurate estimate . It is also important to remember that each fraction contains many different proteins of similar but not identical charge and size – this is why the fractions have broad bases .
When should it be used ? SPE is indicated in the following scenarios : 1,2
1 . Determination of albumin in some exotic species : the assay ( bromocresol green reaction ) used to vet360
Issue 03 | JUNE 2017 | 32
CLINICAL PATHOLOGY Serum Protein Electrophoresis Dr Emma Hooijberg BVSc GPCert (SAP) DipECVCP Department of Com- panion Animal Clinical Studies, University of Pretoria emma.hooijberg@ up.ac.za How does this method work? Serum proteins come in different shapes and sizes, and also have different charges. Albumin, for example, is the most negatively charged protein, while immunoglobulins have a slight positive charge. Serum protein electrophoresis (SPE) separates proteins based on their size and charge. The most common method uses aga- rose gel as a platform. The gel provides a 3-D matrix with large pores (compared to the size of proteins). A serum sample is placed into the gel, and an electrical current is applied across it. The more negatively charged proteins mi- grate through the gel to the cathode (positive charge) and the more positively charged pro- teins, to the anode (negative charge). This r •ΝΥ±Ρ́₯Έ„Ν•Α…Ι…Ρ₯½Έ½˜…±°Ρ‘”ΑΙ½Ρ•₯ΉΜ)₯ΉΡΌ‰…Ή‘ΜΈΝΡ…₯Έ₯́…ΑΑ±₯•ΡΌΡ‘”•°°΅…­₯Ήœ)Ρ‘”‰…Ή‘́Ω₯Ν₯‰±”ΈQ‘”•°₯́ё•ΈΑ±…•₯ΉΡΌ)„Ν…ΉΉ•Θ°έ‘₯ ½ΉΩ•ΙΡ́ё”‘•ΉΝ₯Ρ䁽˜•… )‰…Ή₯ΉΡΌΥΙΩ•Μ°έ‘₯ …ΑΑ•…ȁ…́ё”ΡεΑ₯…°)™Ι…Ρ₯½ΉΜ½˜Ρ‘”•±•ΡΙ½Α‘½Ι•Ρ½Ι…΄€‘₯ΥΙ”€Δ€Έ(½… ΥΙΩ”Ό™Ι…Ρ₯½ΈΙ•ΑΙ•Ν•ΉΡ́„Α•Ι•ΉΡ…”)½˜…±°ΑΙ½Ρ•₯ΈΑΙ•Ν•ΉΠ₯ΈΝ•ΙΥ΄ΈQ‘₯́Α•Ι•ΉΡ…”)₯́…±Υ±…Ρ•‰δ„½΅ΑΥѕȁΑΙ½Ι…΅΅”°…Ή)₯́΅Υ±Ρ₯Α±₯•‰δΡ‘”Ρ½Ρ…°ΑΙ½Ρ•₯Έ½Ή•ΉΡΙ…Ρ₯½Έ)ΡΌ₯Ω”Ρ‘”½Ή•ΉΡΙ…Ρ₯½Έ½˜•… ™Ι…Ρ₯½ΈΈQ‘”)™Ι…Ρ₯½ΉΜ°…Α…ΙЁ™Ι½΄…±‰Υ΅₯Έ°‘…Ω”ΡΙ…‘₯Ρ₯½Ή…±±δ)‰••Έ…±±½…Ρ•„Ι••¬±•Ρѕȁ…Ή„ΉΥ΅‰•ΘƒŠL)…±Α‘„΄Δ°…±Α‘„΄Θ°‰•Ρ„΄Δ°‰•Ρ„΄Θ…Ή…΅΅„Έ%Π)‘…́‰••Έ‘•Ρ•Ι΅₯Ή•€‘ёɽ՝ ΑΙ½Ρ•½΅₯ŒΝΡՐ΄)₯•ΜΥΝ₯Ήœ½Ρ‘•Θ΅•Ρ‘½‘Μ€°έ‘₯ ΅…©½ΘΑΙ½Ρ•₯ΉΜ)…Ι”™½ΥΉ₯Έ•… ™Ι…Ρ₯½Έ°…́₯±±ΥΝΡΙ…Ρ•‰•±½ά)₯Έ₯ΥΙ”€ΔΈ)Ω•ΠΜΨΐ)%ΝΝΥ”€ΐ́π)U9€ΘΐΔ܁π€ΜΘ)₯ΥΙ”€ΔθMAΡΙ…₯ΉœΝ‘½έ₯ΉœΡ‘”‘₯™™•Ι•ΉΠ™Ι…Ρ₯½ΉΜΈMΑ•₯™₯ŒΑΙΌ΄)Ρ•₯ΉΜ΅₯Ι…Ρ”ΡΌ•ΙΡ…₯Έ™Ι…Ρ₯½ΉΜ‰…Ν•½ΈΡ‘•₯ȁ‘…ɝ”…ΉΝ₯ι”)MA…Έ‰”ΥΝ•ΡΌ₯Ω”…Έ₯Ή‘₯…Ρ₯½Έ½˜έ‘₯ Ι½ΥΑΜ)½˜ΑΙ½Ρ•₯ΉΜ…Ι”₯ΉΙ•…Ν•½Θ‘•Ι•…Ν•°‰ΥЁ‘½•ΜΉ½Π)•Ή•Ι…±±δ‘•±ΐΡΌ₯‘•ΉΡ₯™δ‘…Ή•Μ₯ΈΝΑ•₯™₯ŒΑΙ½Ρ•₯ΉΜΈ)%Ё͑½Υ±‰”Ή½Ρ•Ρ‘…ЁMA₯́Ή½Π„‘₯Ι•Π΅•…ΝΥΙ”΄)΅•ΉΠ½˜Ρ‘”½Ή•ΉΡΙ…Ρ₯½ΉΜ½˜‘₯™™•Ι•ΉΠΑΙ½Ρ•₯ΉΜ°…°΄)Ρ‘½Υ ₯Ё₯́„™…₯ɱ䁅ΥΙ…Ρ”•ΝΡ₯΅…Ρ”Έ%Ё₯́…±ΝΌ₯΅Α½Θ΄)Ρ…ΉΠΡΌΙ•΅•΅‰•ΘΡ‘…Ё•… ™Ι…Ρ₯½Έ½ΉΡ…₯ΉΜ΅…Ήδ)‘₯™™•Ι•ΉΠΑΙ½Ρ•₯ΉΜ½˜Ν₯΅₯±…ȁ‰ΥЁΉ½Π₯‘•ΉΡ₯…°‘…ɝ”)…ΉΝ₯锃ŠLΡ‘₯́₯́ݑδΡ‘”™Ι…Ρ₯½ΉΜ‘…Ω”‰Ι½…‰…Ν•ΜΈ)]‘•ΈΝ‘½Υ±₯Ё‰”ΥΝ•ό)MA₯́₯Ή‘₯…Ρ•₯ΈΡ‘”™½±±½έ₯ΉœΝ•Ή…Ι₯½Μθ€Δ°Θ(ΔΈ$•Ρ•Ι΅₯Ή…Ρ₯½Έ½˜…±‰Υ΅₯Έ₯ΈΝ½΅”•α½Ρ₯ŒΝΑ•₯•Μθ)Ρ‘”…Νͅ䀑‰Ι½΅½Ι•Ν½°Ι••ΈΙ•…Ρ₯½Έ€ΥΝ•Ρ