[1]

Technical report

Transmittal Letter

Date: 1/11/2014

Researcher name: Amina Abdel Aziz Aly Hassan College: Applied Medical Sciences Department: Clinical Laboratory Sciences Address: Box 10219, Riyadh 11433 E-mail: aminaalyhassan@yahoo.com

Dear Sir

We are submitting to you the report, due on 1-11-2014, that you requested. The report is entitled: Determination

of Alpha-1-antitrypsin Deficiency at the Phenotypic and Genotypic level: Association with Chronic Obstructive

Pulmonary Disorder and Liver Cirrhosis Patients and some healthy groups in Saudi Arabia. The purpose of the

report is to inform you about the progress made in the project in the last year. The content of this report

concentrates on Alpha 1 antitrypsin deficiency at the phenotypical and Genotypical levels. This report also

discusses evaluation of analytical techniques used in detection of the deficiency. If you should have any questions

concerning our project and paper please feel free to contact aminaalyhassan@yahoo.com.

Sincerely, Amina Abdel Aziz Aly Hassan (PI)

King Saud University

Applied Medical Sciences

Clinical Laboratory Sciences

New Girls Campus

Riyadh, KSA

[2]

Submitted for

National Plan for Science and Technology King Saud University

Determination of Alpha-1-antitrypsin Deficiency at the Phenotypic and Genotypic level: Association with Chronic Obstructive Pulmonary Disorder and Liver Cirrhosis Patients and some healthy groups in Saudi Arabia

Project number

11-MED 1876-02

Project Investigator

Amina Abdel Aziz Aly Hassan

2014

[3]

Abstract

Alpha1Antitrypsin is a protease inhibitor enzyme whose deficiency leads to emphysema, chronic obstructive

pulmonary disease (COPD) and infantile liver cirrhosis. The deficiency is often under-recognized or misdiagnosed

by clinicians because of a lack of specific routine diagnostic tests for quantification of the enzyme levels in patient

serum. We quantitated the serum antitrypsin enzyme levels in three groups of individuals- healthy normal

individuals, Clinically diagnosed emphysema or COPD patients and smokers with a minimum of 10 pack years of

smoking history. The study was carried out in age and sex matched patients, control population and smokers in

collaboration with hospitals in Saudi Arabia. We explored antitrypsin deficiency phenotypically in all the three

sample cohorts.

The initial phase of the research was focused on comparing the different quantification techniques of serum

antitrypsin level in COPD patients, Smokers and normal individuals. We evaluated three different methods of

quantification viz, Immune nephelometry (BN Prospec), ELISA and Real time PCR. We conducted a protein profiling

of COPD and Normal controls by serum protein electrophoresis to explore if any novel biomarkers can be

identified. We compared the data of quantification of A1AT by nephelometry with quantitative Enzyme linked

Immuno sorbent assay technique (ELISA). The method with better precision was determined by this comparative

study. We investigated the genotype of the alpha1 antitrypsin deficient samples using polymerase chain reaction.

[4]

Acknowledgements

I would like to thank God Almighty for the abounding blessing that he showered on us.

This project would not have materialized if it weren’t for the unfailing support of our Vice Dean

of College of Applied Medical sciences, Dr. May Muammar, the administrative staff, the

constant encouragement of our department heads Dr. Noura Al-Jameil and Dr. May Al-Rashid. I

gratefully acknowledge the cooperation of the Pulmonologists from King Khalid university

hospital Dr. Mohammed Ibrahim, and the Pulmonologists at the Chest Hospital, Riyadh. The

services of staff members Mrs. Dalia Al Eradi, Rajah, Eman and Mrs HajeraTabassum in assisting

the project at various stages is gratefully acknowledged.

The financial support extended to the project by National Plan for Science and

Technology (NPST), King Saud University through KACST project # (11 MED 1876-02) is

gratefully acknowledged.

[5]

Table of Contents Page No

1.0 Introduction 7

2.0 Objectives of the study 8

3.0 Background of the study 9

3.1 Alpha 1 antitrypsin deficiency

3.2 Phenotypes and deficiency

3.3 Smoking as a factor of deficiency

4.0 Materials and Methods 11

4.1 Sample collection and Processing 12

4.2 Quantitation by Immune Nephlometry

4.3 Quantitation by ELISA

4.4 Serum protein Profiling

4.5 Genotyping of SERINA 1 gene

5.0 Results

5.1 Immune nephelometric Analysis

5.2 Quantification by ELISA

5.3 Serum Protein Electrophoresis

5.4 Amplification of exons of SERPINA 1 gene

5.5 Smoking and Enzyme deficiency

6.0 Results and Discussion

7.0 Future work plans

8.0 References

9.0 Publication

[6]

List Of Tables:

1. Sample cohorts and Selection Criteria

2. Inclusion and Exclusion Criteria

3.

List of Figures:

1. Quantitaion of Alpha 1 antitrypsin by Nephelometry and Sandwich ELISA

2. Serum profiling of COPD patients

3. Serum Profiling of Smokers

4. Amplification of Exons of SERPINA 1 gene

[7]

1.Introduction

Alpha-1-antitrypsin (A1AT) is a serine protease inhibitor secreted by liver cells. This enzyme acts as an inhibitor of a

number of proteolytic enzymes, the most prominent of which is elastase. Elastase is secreted by neutrophils when

they disintegrate or are activated, and it degrades the elastin and other host extracellular matrix constituents (Lee

2001). A1AT protects the lower respiratory tract from damage due to the elastase enzyme. The normal serum level

of A1AT is 2–4 mg/L. A deficiency of this enzyme is a risk factor for development of pulmonary emphysema in

adults and liver cirrhosis in infants (Coakley et al,2001). A1AT deficiency is often unrecognized or wrongly

diagnosed in emphysema and liver cirrhosis patients, and thus they fail to have the appropriate treatment. The

only specific treatment for A1AT deficiency available at present is augmentation therapy by intermittent

intravenous administration of A1AT. The prevalence of A1AT deficiency in chronic obstructive pulmonary disease

(COPD) and liver cirrhosis patients is worth exploring in this context. Our research therefore focuses on quantifying

the serum level of A1AT in these patients and comparing it with that of normal healthy individuals to determine

the level of enzyme deficiency. A combined effort to study the epidemiology of this enzyme deficiency will be

carried out in population cohorts in collaboration with hospitals in different provinces of the Kingdom of Saudi

Arabia. This study may help to bring out the phenotypic variants of A1AT present in the Saudi population as well as

reveal a clear picture of the prevalence and the magnitude of enzyme deficiency associated with it. The current

practice of A1AT measurement is only semi quantitative. Our research explores alternate quantification techniques

which are cost effective and more sensitive.

The second phase of the research will determine the genetic makeup of the patients with A1AT deficiency. The

gene that codes for the A1AT enzyme is SERPINA1, which is located on the long arm of the 14th

chromosome. This

is a highly polymorphic gene with more than 120 variants reported to date. Liver diseases result from abnormal

proteins produced by mutant alleles of the gene coding for an altered A1AT enzyme. Based on the variant protein

function and its plasma concentration, they are designated as normal (M), deficient (Z), null and dysfunctional

alleles (ATS/ERS, 2003). The presence of the normal allele M is correlated with a normal level of A1AT in blood. The

presence of a homozygous Z allele (ZZ) is correlated with severe deficiency of A1AT, whereas the S mutation in

combination with Z contributes to an increased risk of lung disease (Carrell, 1986). Null variants are associated with

trace amounts of plasma A1AT. Dysfunctional variants of the protein may result in a change of function of the

protein from elastase inhibitor to thrombin inhibitor, called A1AT Pittsburg (Owen et al., 1983).

Genotyping of the samples will be performed by isolating DNA from cheek swabs of patients, with PCR

amplification of the gene and sequencing of the purified PCR product in a DNA analyzer. The DNA sequence will

then be analyzed by software to detect any alterations in the DNA. Genotyping will help to understand A1AT

deficiency at the molecular level. The molecular level study of a single gene disorder may also help to determine

the genetic makeup of the native Arabs of Saudi Arabia, thus revealing the most prevalent alleles present in the

population.

[8]

2. PROJECT OBJECTIVES:

1. Estimate the incidence of Alpha 1 antitrypsin (A1AT) enzyme deficiency in Saudi Arabian population by

quantitation of A1AT enzyme level in clinically diagnosed (COPD) patients and some healthy groups.

2. Study the correlation of AAT enzyme deficiency with respiratory disorders like chronic obstructive

pulmonary disorder (COPD), Emphysema, liver cirrhosis and individual factors such as genetic factors or

smoking habit.

3. Evaluate the use of molecular methods of quantification of enzyme deficiency like quantitative real time

PCR.

4. Phenotypic exploration of the alleles of SERPINA1 gene in normal control and patient cohorts of Saudi

Arabian population in different provinces and statistical evaluation of data collected will be carried out.

5. Genotypic Identification of any alteration in the gene or exploring the presence of novel or rare genetic

variants (if any) in Saudi Arabian population that contributes to enzyme deficiency.

6. Statistical evaluation of the enzyme deficiency in different sample cohorts

[9]

3.0 Background of the study

3.1 Alpha-1-antitrypsin

A1AT deficiency is one of the most common hereditary disorders worldwide, comparable in frequency to cystic

fibrosis (WHO, 1997; De Serres, 2002). A1AT is a 52-kD glycoprotein made up of 394 amino acid residues and three

asparagine-linked complex carbohydrate side chains (Brantly, 1988). The gene coding for this enzyme is about 12.2

kb, located on human chromosome 14q31-32.3. This gene has three noncoding exons and four (2, 3, 4, and 5)

coding exons. A1AT is produced primarily by liver cells, and lesser amounts of the protein are also produced by

mononuclear phagocytes and epithelial cells of lungs and intestine. A1AT in the serum ranges between 1.5 and 3.5

g/L under normal health conditions (Fagerhole,1967). A1AT is primarily a serine proteinase inhibitor. Kinetic

studies have revealed that neutrophil elastase is the preferential target of A1AT enzyme. Inhibition of elastase

occurs when it binds to the active site of the A1AT enzyme by forming stable equimolar complexes (Travis et al,

1983). Another elastase designated as proteinase-3 (PR-3) is also secreted by human neutrophils and is also a

serine protease. PR-3 degrades elastin in vitro and is reported to cause emphysema when given intratracheally to

hamsters (Kao et al, 1988).

3.2 A1AT phenotypes and deficiency

Normal: The plasma levels of A1AT are normally more than 20 µmol/ L and designated as M type. Deficient:

Plasma levels of A1AT less than 20 µ mol/L. Denoted as Z variant, which is the commonest deficient phenotype.

The plasma levels of A1AT among ZZ homozygotes are about 5–6 µmol/L. The Z mutation is associated with early

onset panlobular emphysema. The S variant in the homozygous condition has A1AT plasma levels at about 60% of

normal. This phenotype is common in the Mediterranean area. “M-like” or “S-like” variants are rarely reported.

Null: An A1AT variant that is associated with no detectable A1AT enzyme in the plasma. Dysfunctional: Some A1AT

variants were reported that transformed A1AT protein from an elastase inhibitor to a thrombin inhibitor (A1AT

Pittsburgh) (Owen et al, 1983). A rare variant of A1AT called PI*F variant was reported where the association of

enzyme with elastase was much reduced (Okayama et al, 1991). The most common cause of A1AT deficiency is

conformational changes of the Z A1AT, which under physiological conditions transform its reactive loop into a β

sheet polymer (Lomas et al, 1992). The local deficiency of Z A1AT is enhanced by the formation of polymers within

the lung (Lomas 2000). Scientific reports say that this polymerization inactivates and also converts the molecule to

a chemoattractant for human neutrophils (Parmar et al., 2002). Polymeric A1AT has chemotactic properties, which

causes excessive accumulation of neutrophils in the lungs of Z A1AT homozygotes. This phenomenon suggests a

new paradigm for the pathogenesis of emphysema in Z phenotype patients (Parmar et al., 2002). Abnormal A1AT

accumulation as in patients with the PiZ-type A1AT allele is thought to initiate the pathologic response causing

juvenile and adult cirrhosis. Abnormal A1AT is also implicated in hepatocellular carcinoma because of its

accumulation in the endoplasmic reticulum of hepatocytes (Lomas et al., 1992).

PI*ZZ deficiency is inherited as an autosomal codominant gene. If both parents are Z allele carriers, the risk of a

homozygous offspring is 1 in 4 for each birth. If one parent is homozygous (PI*ZZ) and the other heterozygous,

then all children are either carriers or affected (PI*ZZ). The A1AT level will be monitored in patients with early-

onset pulmonary emphysema and also in patients with or without a history of cigarette smoking. The World Health

Organization (WHO) recommends quantitative A1AT determination for all subjects with COPD or asthma

characterized by incompletely reversible airflow (WHO, 1997). Individuals with evidence of liver cirrhosis of

unknown etiology (contributing phenotypes include PI*ZZ, PI*MZ and PI*Mmalton), those with Wegener’s

granulomatosis, antiproteinase-3 vasculitis (PI*ZZ and PI*MZ being contributing phenotypes), and adults with

bronchiectasis without evident etiology should be tested for serum A1AT levels (Eriksson et al, 1986).

[10]

Studies report a large variation in frequency of the Z gene in different regions of Europe (Hutchison, 1998). The

highest PI Z gene frequency is observed on the northwestern seaboard of the European continent, and the

mutation is assumed to have originated in southern Scandinavia (Hutchison, 1998). Z gene frequencies are highest

in individuals of northern and western European descent in the USA (Dykes, 1984). The S type gene frequency is

highest in the Iberian Peninsula, and the mutation is likely to have originated in that region (Hutchison, 1998).

The role of genetic factors in the etiology of COPD in nonsmokers has not been properly studied so far. However,

there is evidence of genetic factors influencing the development of COPD in response to cigarette smoking

(Silverman,2006). A1AT deficiency is a well-established risk factor for COPD in nonsmokers. Case studies have

revealed that nonsmoking protease inhibitor (PI Z) subjects are at increased risk for developing COPD, although to

a lesser degree compared with PI Z smokers (Janus, 1985,Tobin et al., 1983, Larsson, 1978). According to gene-

mapping studies, the PiZ allele probably originated in northern Europe (Cox et al., 1985; Seixas et al., 2001).

Reports of the American Thoracic Society (2010) point out that most cases of COPD present in the young

population, in women, and in people of developing nations cannot be attributed to smoking. This suggests the

presence of additional causative agents responsible for the development of COPD. It is important to determine the

role of the above mentioned nontraditional risk factors to develop good therapeutic strategies. Cutis laxa, a rare

genetic disorder, also has been clearly demonstrated to cause emphysema in childhood and adolescence in some

people, even if they are nonsmokers. Cutis laxa is a disorder of the elastic fibers, which in some cases is caused by

mutations in the elastin gene (Rodriguez-Revenga et al., 2004; Van-Maldergem et al., 1988; Turner-Stocks et al.,

1983). Genetic factors such as A1AT deficiency, tuberculosis, diet, occupational exposures, environmental

pollution, biomass smoke and traffic cannot be ruled out as contributing risk factors for COPD (ATS, 2010).

[11]

4. Materials and Methods

Study Outline

Quantification of Alpha1 antitrypsin

deficiency by Nephlometry

Quantification of Alpha1 antitrypsin

deficiency by ELISA

Sample collection & Processing

SDS

PAGE

Sequencing of exons of confirmed deficient

Phenotypes

Phenotyping by Iso electric focusing (IEF)

Amplification of SERPINA 1 gene exons (2-5)

Exploring new Biomarker if any by protein

profiling

Study of genetic patterns of enzyme

deficiency in Arabic population

Study of Prevalence of enzyme deficiency in

different sample cohorts

2 D PAGE

[12]

4.1 Sample Collection and Patient cohorts:

We conducted a case control study in collaboration with University hospitals and pulmonary specialized

hospitals in Riyadh, Saudi Arabia. Ethical approval was obtained from the Institutional review board of the

respective hospitals and informed patient consent was taken. We collected blood samples and medical

history of people belonging to 3 groups. Group1 comprised of patients who were clinically diagnosed as

having emphysema, chronic bronchitis (subsets of COPD) or liver cirrhosis. Group 2 consisted of age

matched, normal control population belonging to both sexes. Group 3 comprised of smokers/ ex-smokers

to study the effect of smoking on the enzyme deficiency.

Sample Inclusion and exclusion criteria:

Sample type Inclusion & Exclusion Criteria Age group Sample size

1.COPD Patient Cohort Inclusion Criteria

Smoking history of ≥ 10 pack-years

Diagnosis of COPD Stages 2, 3 and 4 by

GOLD criteria (post-bronchodilator

FEV1/FVC < 0.70 and FEV1 < 80%

predicted)

Exclusion Criteria

Any respiratory disorder other than

asthma or COPD such as diffuse

bronchiectasis, cystic fibrosis, or interstitial

lung disease.

Lung surgery with removal of a lobe or

more

Suspected or confirmed Lung cancer

Surgical or bronchoscopic lung volume

reduction

Pregnancy

ongoing radiation therapy or

chemotherapy

Patients with recent history of surgery,

heart attack or any transplant

30-50 years 62

[13]

1. Smokers Inclusion Criteria:

History of 10 pack years or more of cigarette

/ hukka smoking

Exclusion criteria:

Any other disease condition as in COPD

exclusion criteria

30-80 years 72

2. Normal,

unaffected

group(male &

female)

No respiratory illness, never smokers.

Normal level of Alpha 1 antitrypsin in serum.

30-80 years 30

6 samples in

each age

group 30-80

Table:1 Sample cohorts and selection criteria

4.2. Sample Processing:

Two sets of blood samples were collected from the study cohorts – whole blood and blood with anticoagulant,

EDTA. The whole blood sample was collected in yellow or red vacutainer tubes (Becton Dickinson, USA) and was

used for serum preparation. The tube was left at room temperature for 30 minutes, allowed to form clot and was

centrifuged at 2,000 rpm for 15 minutes. The yellow serum that separated at the top was separated into

microtubes and stored at -20C. The whole blood sample with EDTA was used for extraction of genomic DNA.

Genomic DNA was isolated using Qiagen kit for tissue and DNA isolation.

4.3 Quantitation of Alpha 1 antitrypsin by Immnue Nephlometry:

The serum samples were collected from COPD patients, Smokers and healthy normal individuals and analysed for

alpha 1 antitrypsin enzyme using Immune nephlometric analysis. The analysis was performed in BN Prospec

Immune nephlometer (Siemens Health care, USA) as per the instructions on the manual. Alpha 1 antitrypsin was

measured after appropriate dilution of the serum sample in diluent buffer. Diluted sample was mixed with

antibody specific for alpha 1 antitrypsin. Antigen antibody complexes were formed after the reaction. A light beam

having a wavelength of 840nm was passed through a solution containing antigen antibody complex and the light

scattered by the antigen antibody complex was measured. The amount of light scattered was proportional to the

amount of antigen antibody complex in the solution.

4.4 Quantitation of Alpha 1 antitrypsin by ELISA:

Serum samples were analysed for alpha1 antitrypsin levels using sandwitch Enzyme linked immuno sorbent assay

(ELISA). Human alpha1-AT (Alpha 1-Antitrypsin) ELISA Kit (MY BIOSOURCE, CA, USA) was used for this experiment.

The kit contained microplates pre coated with antibody specific to alpha 1 antitrypsin. The kit had standard protein

sample which were added to first column of the microplate in varying dilutions. The diluted patient samples are

then added to the remaining wells and allowed to bind to the bound antibody in the well. A biotynylated detection

antibody specific for alpha1 antitrypsin and Avidin horseradish peroxidase (HRP) conjugate were added and the

plate was incubated for one hour at 37C. The microplate was then washed to remove the excess unbound

components. Substrate solution was added. The enzyme substrate reaction was then stopped by adding Sulphuric

acid solution and the colour turns yellow. The optical density (OD) of the solution was measured

[14]

spectrophotometrically in ELISA plate reader (IRE 96, SFRI reader, France) at 450nm. A standard curve was plotted

using the OD value and concentration of the standard sample. Concentration of the protein in the patient samples

were calculated from the standard curve using the OD values observed.

Serum protein Electrophoresis:

Serum samples stored at -20C was thawed and diluted 1: 1 with 2x sample buffer (Novagen, USA),and heated at

80C in waterbath for 3 minutes and loaded into the Precasted ready gel (Mini- ProteanTGX stain free, BIORAD Inc,

CA, USA). Unstained molecular weight marker (Invitrogen, USA) was loaded to the first well of the precast gel.

Denatured samples of COPD patients were loaded and Sodium dodecyl sulphate Polyacrylamide gel

electrophoresis was done in Mini Protean gel assembly (BIORAD Inc, CA, USA) using 1x Tris Glycine running buffer

at a voltage of 170 for 1 hour. The gel was taken out of the casting frame and gel image was captured using Gel

Doc EZ image lab software ( Biorad, CA,USA).

PCR amplification and restriction enzyme digestion:

Genomic DNA is isolated from whole blood according to the standard method (Maniatis et al., 1989) and was preserved in sterile distilled water at 4°C until the PCR assay. For PI genotyping, PCR-mediated mutagenesis is performed as described in the protocols (Andresen et al., 1992). Briefly, Target DNA (0.2 g) from the PI genome was amplified by PCR using two sets of mutated primers, Genbank accession no: K-02212 (MWG Biotech,Germany), (PCR kit of Fermentas, U. S), in a DNA thermal cycler (ABI Gene Amp 700, U.S). The sequence of primers used for PiS and PiZ typing are shown in Table 1. This table shows that mutated bases in the primer sequences as underlined. The fragment was amplified in a reaction volume of 25 µl containing 10 pm of primers, 200 mM dNTPs of 1U of Taq polymerase. Each reaction mixture was subjected to 35 cycles of 1 min at 95°C, 1 min at 50°C and 2 min at 72°C, followed by initial denaturation for 5 min at 95°C, and final extension for 9 min at 72°C. PCR products (148 bp for the S-type and 97 bp for the Z-type) were checked by electrophoresis on a 3% agarose gel. In order to test for the S and Z allele mutations, PCR products were digested by XmnI and TaqI as previously described (Andresen et al., 1992; Ferrarotti et al, 2004). Upon digestion, the cleaved products were submitted to electrophoresis on 20% polyacrylamide gel and visualized after staining by ethidium bromide. Depending on the

band pattern generated, six possible different genotypes; PiMM, MS, MZ, SS, ZZ, and SZ, can be distinguished. Genotyping of Deficient samples:

Genotyping by PCR-DNA sequencing was performed according to the procedure reported by Costa et al.,2000. The

amplification of the four coding exons 2, 3, 4 and 5 of the SERPINA1 gene was done in a thermalcycler (ABI , Gene

amp 9700, U.S). Amplified product is purified and sequencing PCR is performed with big dye sequencing kit (ABI,

Califf, U.S). Each exon is then sequenced in a Genetic Analyser (ABI,310) and the genotypes of SERPINA1 gene are

analysed using the DNA analyser (Applied Bio systems, Calif, U.S) using terminator Big dye sequencing. The

sequence of the genotypic variants from the analysis are fed into a software (Codon code Aligner, U.S) and the

genetic variations are noted.

[15]

0

0.5

1

1.5

2

2.5

3

3.5

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61

Red---Nephlometry (g/L)

Blue------sandwitch ELISA (g/L)

Fig1: Alpha 1 antitrypsin quantitation by Nephlometry and ELISA technique compared

4.6 Polymerase chain reaction:

Polymerase chain reaction was carried out to amplify all the 4 coding exons of SERPINA 1 gene viz., exon 2,3,4 and

5 in a single multiplex reaction. The reaction was performed using Illustra ready to go PCR beads (GE health care,

USA). Each microtube was used for a 25 microlitre reaction by adding DNAse free water 1 microlitre each of the

forward and reverse primers of the gene and 1 microlitre of the DNA sample. The conditions of the reaction are as

follows

Initial denaturation of 95 C for 5minutes followed by 35 cycles of 95 C for 1.0 min, 55C for 1min,72C for 2.0min

denaturation, annealing and extension and a final extension of 72 C for 10 minutes was carried out in Thermal

cycler ABI 9700 (Life technologies, USA).

Table2: Sequence of primers used for amplification of s, z alleles.

[16]

5.0 Results and Discussion

Estimation of Alpha1 antitrypsin:

Estimation of alpha 1 antitrypsin enzyme was done using serum samples of patients who were clinically diagnosed

with Chronic Obstructive pulmonary Disease (COPD), smokers who had a smoking history of at least 10 pack years

and normal healthy individuals. Patient cohorts and the prevalence of deficiency of protease inhibitor in the serum

samples were separately plotted graphically as shown in fig:2.

The quantification of alpha1 antitrypsin enzyme was done using two methods Immnue Nephlometry (BN Prospec,

Siemens, Erlangen, Germany) and Sandwitch Enzyme linked Immuno sorbent assay (IRE96 Reader,SFRI,France).

The results of both methods were compared. Immune nephlometric readings were higher than that of ELISA. Both

techniques being immune complex reactions ELISA seemed to be more sensitive.

Serum Protein Electrophoresis:

Protein profile of Serum samples were analysed and interpreted. There were five prominent protein bands-

Albumin, alpha1 zone, alpha 2, beta1, beta 2 and gamma globulin region. Alpha 1 region included the protease

inhibitor called alpha1 antitrypsin which showed up on the gel as a 52 kDa protein band. We analyzed serum

samples of COPD patients by SDS Page and found that the enzyme deficient samples had a heterozygous genotype

as compared to the normal. This was seen clearly as two separate protein bands in the gel (Fig: 1).

Fig2: Alpha 1 antitrypsin defiency in COPD patients. Protein marker,

Lane1-9: COPD patient samples which shows 2 bands in alpha 1 region

[17]

Fig3: Serum protein profile of Smokers

N-Normal Control Serum profile, Lane 1-3 Smoker serum profile showing changes in protein bands of alpha 1,

alpha2, beta1 and beta 2 regions.

Polymerase chain reaction for SZ alleles:

Fig4: Amplicons of exons of SERPINA 1 gene. Lane1-

4:exons4,exon2(595bp),exon3(383bp),exon5(332bp),Lane5:100bpDNA ladder

[18]

Discussions

The current method of A1AT quantification in Saudi Arabia is by immune nephelometry; but this may overestimate

A1AT levels because of interference with lipids or hemoglobin (ATS, 2003).

Readings of alpha 1 antitrypsin were higher by Immune nephlometry than the values obtained by using ELISA. Both

techniques being immune complex reactions ELISA seemed to be more sensitive. The detection limit of ELISA is

lower compared to that of nephlometry. There fore any deficiency will be detected more accurately determined

using ELISA than by nephlometry. The sandwitch ELISA that we used seemed to the detect alpha1 antirypsin

protein specifically and the optical density obtained was the indication of the actual amount of protein in the

serum. In nephlometry the light scattered by the antigen antibody complex was measured. However additional

components in the serum may also contribute to the values estimated by this technique. Therefore sandwich ELISA

seems to be more sensitive and cheaper than Nephlometry. Cost effectiveness can make ELISA a preferred

analytical technic even in ordinary clinical settings accessible for the common man.

Serum protein profile was analyzed as per the literature on serum proteins of O’ Connell et al 2005.Serum protein

profiling of the COPD patients showed two protein bands in the alpha 1 zone indicating heterozygosity (Fig2). This

preliminary screening technique can be used as a guide for further analysis and genotyping of the samples. In the

second category of samples, Smokers it was found that there was a marked reduction in the protein concentration

of alpha 1 antitrypsin band (Fig3).

Polymerase chain reaction was used for amplification of all four coding exons 2,3,4 and 5. These products were

subjected to clean up reaction (Qiagen PCR clean up). This product was then frozen a -70 C for sequencing.

Deficient samples were analyzed for the presence of the S and z alleles and further exon sequencing is planned for

these samples in the next phase of investigation.

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chronic obstructive pulmonary disease. 2010, Am J Respir Crit Care Med, Vol. 182. PP. 693-718.

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