- Email: [email protected]
Measurement of antibodies to Candida albicans as a screening test for humoral immunodeficiency
Journal of lmmunological Methods, 105 (1987) 127-131
127
Elsevier JIM04563
Measurement of antibodies to Candida albicans as a screening test for humoral immunodeficiency D.I.M. Phillips and N. Matthews Department of Medical Microbiology, Universityof Wales College of Medicine, Cardiff, U.K.
(Received 10 April 1987, revised received 7 July 1987, accepted 16 July 1987)
In screening for defects in humoral immunity, as well as measuring serum immunoglobulins it is advisable to have some measure of antibody production, e.g., immunising with a test antigen and measuring the antibody response some days later. However, this delay may be inconvenient and requires a second blood sample. An alternative is to test for antibody to a widespread commensal organism to which the patient must have been repeatedly exposed. The measurement of antibody to E. coli by haemagglutination has been previously used for this purpose. We report here that an equally good and simpler alternative is to measure antibodies to the commensal Candida albicans by immunofluorescence. Using a polyvalent conjugate, all 114 blood donors tested had antibody titres > 8 to C. albicans; similar responses were noted in 20 children (aged 6 m o n t h s - 1 6 years) without recurrent infections. In contrast, anti-candida responses were low or absent as expected in patients with hypogammaglobulinaemia but also in some patients with other immunodeficiency diseases. Overall candida immunofluorescence closely paralleled E. coli haemagglutination. Key words: Candida albicans; Immunodeficiency;Immunofluorescence
Introduction Patients with quantitative defects of antibody synthesis can be diagnosed by measuring serum immunoglobulin levels. However, qualitative defects of antibody are not so easily diagnosed; inoculation with a test antigen and measurement of an antibody response days or weeks later is possible (Wedgewood, 1975). A quicker and noninvasive alternative is to test for antibodies to an antigen to which the patient has been previously exposed, either as a result of earlier active immunisation (Hong, 1976) or after natural exposure Correspondence to: N. Matthews, Department of Medical Microbiology, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, U.K.
to a commensal organism. With antigens such as diphtheria and tetanus toxins there is a wide variation in antibody levels within the population because of different immunisation histories. In contrast, natural antibodies to the commensal E. coli are more uniformly high and measurement of anti-E, coli antibodies is a well established test of humoral immunity (Webster et al., 1974). This test is not without drawbacks and prompted us to look for another suitable commensal. We selected Candida albicans as a possible candidate in that, firstly, the majority of individuals have encountered and responded to it (Cobb et al., 1978) and, secondly, simple immunofluorescence techniques exist for measuring antibodies to the organism (Lehner, 1965, 1970; Esterley, 1968; Dolan et al., 1973).
0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
128 In this study, blood donors without recurrent infections and a group of patients with suspected immunodeficiency were compared for natural antibody production to C. albicans to see whether this test is of use in screening for humoral immunodeficiency.
Materials and methods
Patients Sera were obtained from three groups. Group 1. 114 unselected blood donors (from the Blood Transfusion Service, Rhydlafar, Cardiff). Group 2. 20 children aged 6 m o n t h s - 1 6 years without recurrent infection but who had been referred for serological testing. None of these children had positive viral serology or raised ASO titres. Group 3. 12 serum samples received at this laboratory from patients with suspected immunodeficiency. These patients are an unrepresentative group but were selected to give a range of abnormalities. C. albicans A single clinical isolate was used throughout and maintained by aerobic culture on blood agar. The organism was germ tube positive and fermented glucose and maltose but not lactose and sucrose. Organisms from 5-day-old cultures were suspended in isotonic phosphate-buffered saline, p H 7.2 (PBS) and washed three times at 450 x g. The C. albicans was suspended in 0.4% formol saline at 7 x 106 o r g a n i s m s / m l and stored at 4 ° C. Preparation of slides 10 /~1 of C. albicans suspension (7 X 106/ml) was spotted onto wells of PTFE-coated 15-well multispot slides (Flow) and air-dried. The slides were fixed in acetone for 10 min at 4 ° C , allowed to air-dry and stored at - 8 0 ° C until required. Slides prepared in this way could be used for 6 months without apparent deterioration. lmmunofluorescence test Control and test sera were diluted two-fold in PBS to 1/32. 10 ~1 amounts of each dilution were spotted onto labelled wells of C. albicans-coated
slides. After incubation in a moist box at 3 7 ° C for 30 rain the slides were washed twice by immersion and agitation in 100 ml PBS for 5 rain each wash and then allowed to air-dry. Goat anti-human F I T C conjugate (Sigma) (10 /~1) of the appropriate isotype specificity (previously titrated for optimum dilution) was then spotted onto the slides and incubated at 3 7 ° C in a moist box for 30 rnin. The slides were then washed twice in PBS as before followed by a further wash for 2 min in distilled water. After air-drying and mounting in 50% PBS/glycerol ( v / v ) the slides were examined for surface immunofluorescence by incident light microscopy with narrow band blue illumination. A positive result was taken as > 50% of cells with a rim of fluorescence around the entire perimeter of the cell. The highest serum dilution giving a positive result is defined as the titre. On each slide two unknown samples ( 1 / 2 - 1 / 3 2 ) and a positive control ( 1 / 8 - 1 / 3 2 ) were included with two negative (PBS) controls.
Other tests Antibodies to E. coli were measured as described by Webster et al. (1974). Immunoglobulins were measured by radial immunodiffusion and I g G subclasses by semi-quantitative, simple immunodiffusion using monoclonal antisera (Oxoid).
Results
If the detection of antibodies to C. albicans is to be used as a screening test for immunodeficiency then it is necessary to show that healthy individuals have antibodies to the organism. When unselected blood donors were tested by immunofluorescence using a polyvalent conjugate, all 84 individuals had detectable antibody with titres of > 8 (Fig. 1). Comparable amounts of anti-candida antibody were also seen in the sera of children from 6 months of age (Fig. 2). The predominant isotype in the anti-candida response seems to be IgG, although a minority of the 30 blood donors tested in this manner additionally had IgM antibodies (Fig. 3). These 30 donors were a separate group from those in Fig. 1 but again all 30 had titres of > 8 with the polyvalent conjugate, Because of the higher titres with
129 >32
x
XXXXXXXXXXXXX
XXXXXXXXXXXXXXX
XXXXXXXXXXXXXXX X
32
X
XXX
X
X
XXXXXXXX
16 XXXXXXXX
TITRE
XXXXXXXX
16
TITRE
XXXXXXXXX
¢,4 I
I
I
I
I
0
4
8
12
16
AGE (YR)
XXXXXXXX
Fig. l. Anti-C. albicans titres of 84 blood donors as measured with a polyvalent (anti-IgA,G,M) immunofluorescent conjugate.
the polyvalent conjugate this was preferred for subsequent routine use. Presumably the polyvalent conjugate is more sensitive because it can react with more epitopes per I g G molecule than its IgG-specific counterpart. This isotype distribution is similar to that described previously (Lehner, 1965; Esterley, 1968). These initial observations encouraged us to apply the technique to sera f r o m patients with i m m u n o d e f i c i e n c y (Table I): all of these patients had a history of recurrent infections. N o t surprisingly, those individuals with severe h y p o g a m m a globulinaemia (e.g., patients 1, 10 and 12) had low titres of a n t i b o d y to candida. However patients 3,
Fig. 2. Anti-C. albicans titres of 20 children (without recurrent infections) as a function of age. A polyvalent immunofluorescent conjugate was used.
6 and 8 with normal or near normal I g G concentrations also had reduced anti-candida responses. The two patients (7 and 9) with a seco n d a r y defect due to protein-losing enteropathy h a d normal titres of anti-candida antibody. In all serum samples except that f r o m patient 3, antib o d y responses to E. coli and candida correspond.
32
XXXXX
16
TITRE
8
XX
XXXXXX XXXXXX
XXXXXXXXX XXXXXXXXX XXXXXXXXXX
XXXXXX XXXXXX
XXXXXXXXX XXXXXXXXX XXXXXXXXX
IgG
IgM
IgA
XX
Fig. 3. Anti-C. albicans titres of 30 blood donors as measured with immunofluorescent conjugates specific for either IgA, IgG or IgM.
130 TABLE I Patient No.
1
2 3 4 5 6 7 8 9 10 11 12
Age (years)
0.5
1 1 2 2 2 4 6 14 14 19 35
Normal values (adult)
Anticandida titre
AntiE. coli titre
Immunoblogulins (g/l) IgG
Other Information
IgA
IgM
2
4
1.4
< 0.1
<2 <2 <2 4 <2 8 <2 32 <2 2 <2
4 32 8 16 4 256 <2 128 <2 2 4
<0.1 8.2 2.5 2.9 5.8 2.8 4.8 3.4 2.1 2.8 0.2
<0.1 0.3 0.1 0.1 0.1 0.2 <0.1 0.4 < 0.1 0.6 0.1
0.3 0.7 3.4 0.2 0.8 1.4 1.1 0.2 0.8 <0.1 0.1 < 0.1
>8
> 32
> 5.0
> 0.5
> 0.5
Discussion
This study has shown that the serological response to C. albicans is of value in screening for h u m o r a l i m m u n o d e f i c i e n c y . All 114 b l o o d d o n o r s tested reacted with titres of > 8 o n imm u n o f l u o r e s c e n c e whereas patients with hypog a m m a g l o b u l i n a e m i a a n d some individuals with suspected a n t i b o d y d y s f u n c t i o n had m u c h reduced titres. Overall c a n d i d a i m m u n o f l u o r e s c e n c e closely paralleled E. coli h a e m a g g l u t i n a t i o n , a n established m e t h o d of screening for a n t i b o d y dysfunction (Webster et al., 1974). W i t h the original E. coli m e t h o d the disa d v a n t a g e s are the need to extract antigen, the r e q u i r e m e n t for a n t i g e n from six serotypes a n d the i n c o n v e n i e n c e of c o a t i n g a fresh b a t c h of erythrocytes every few days. Also, patients of the A B b l o o d group m a y give false negative responses. W i t h the c a n d i d a m e t h o d , a single strain is used, a n t i g e n extraction a n d coating of i n d i c a t o r particles is n o t required a n d it is possible to prepare a single large b a t c h of antigen-coated slides a n d store these for u p to 6 m o n t h s in the deep-freeze u n t i l required. T h e disadvantages of the c a n d i d a technique are that firstly, there is a subjective e l e m e n t i n assessing e n d p o i n t s a n d secondly, a n expensive fluorescence microscope is needed. However, m a n y laboratories already possess such
No other defects
Protein-losing enteropathy? Protein-losing enteropathy? Wiskott-Aldrich Myotonic dystrophy?
microscopes a n d the staff are familiar with the i n t e r p r e t a t i o n of i m m u n o f l u o r e s c e n c e . A g g l u t i n a tion or E L I S A w o u l d be possible alternatives to fluorescence for m e a s u r i n g antibodies to C. albicans. A g g l u t i n a t i o n is simple b u t again there is subjectivity in reading e n d p o i n t s a n d other disadvantages are that a u t o a g g l u t i n a t i o n can be a p r o b l e m a n d that c a n d i d a p r e p a r a t i o n s for agg l u t i n a t i o n are stable for only a few weeks. Our p r e l i m i n a r y experiments with a n E L I S A assay emp l o y i n g whole c a n d i d a organisms have shown that this c o m p a r e d f a v o u r a b l y with i m m u n o f l u o r e s cence. E L I S A also has the a d v a n t a g e of being m o r e objective. Whichever detection m e t h o d is e m p l o y e d we believe that the m e a s u r e m e n t of a n t i b o d i e s to C. albicans is of real value in screening for h u m o r a l i m m u n o d e f i c i e n c y .
References
Cobb, J. and Parrat, D. (1978) Determination of antibody levels to Candida albicans in healthy and hospitalised adults using a radioimmunoassay. J. Clin. Pathol. 31, 1161. Dolan, C.T. and Stried, R.P. (1973) Serological diagnosis of yeast infections. Am. J. Clin. Pathol. 59, 49. Esterley, N.B. (1968) Serum antibody titres to Candida albicans, Am. J. Clin. Pathol. 50, 291. Hong, R. (1976) Immunodeficiency. In: N. Rose and H. Friedman (Eds.), Manual of Clinical Immunology. American Society for Microbiology, Washington, DC, p. 620.
131 Lehner, T. (1965) Immunofluorescence study of Candida albicans antibodies in human saliva. Arch. Oral. Biol. 10, 975. Lehner, T. (1970) Serum fluorescent antibody and immunoglobulin estimation in candidosis. J. Med. Microbiol. 3, 475. Webster, A.D.B., Efter, T. and Asherson, G.L. (1974)
Escherichia coli antibody: A screening test for immunode-
ficiency. Br. Med. J. 3, 16. Wedgewood, R.J., Ochs, H.D. and Davis, S.D. (1975) The recognition and classification of immunodeficiency disease with bacteriophage oX 174. Birth Defects Orig. Artic. Ser. 11, 331.
127
Elsevier JIM04563
Measurement of antibodies to Candida albicans as a screening test for humoral immunodeficiency D.I.M. Phillips and N. Matthews Department of Medical Microbiology, Universityof Wales College of Medicine, Cardiff, U.K.
(Received 10 April 1987, revised received 7 July 1987, accepted 16 July 1987)
In screening for defects in humoral immunity, as well as measuring serum immunoglobulins it is advisable to have some measure of antibody production, e.g., immunising with a test antigen and measuring the antibody response some days later. However, this delay may be inconvenient and requires a second blood sample. An alternative is to test for antibody to a widespread commensal organism to which the patient must have been repeatedly exposed. The measurement of antibody to E. coli by haemagglutination has been previously used for this purpose. We report here that an equally good and simpler alternative is to measure antibodies to the commensal Candida albicans by immunofluorescence. Using a polyvalent conjugate, all 114 blood donors tested had antibody titres > 8 to C. albicans; similar responses were noted in 20 children (aged 6 m o n t h s - 1 6 years) without recurrent infections. In contrast, anti-candida responses were low or absent as expected in patients with hypogammaglobulinaemia but also in some patients with other immunodeficiency diseases. Overall candida immunofluorescence closely paralleled E. coli haemagglutination. Key words: Candida albicans; Immunodeficiency;Immunofluorescence
Introduction Patients with quantitative defects of antibody synthesis can be diagnosed by measuring serum immunoglobulin levels. However, qualitative defects of antibody are not so easily diagnosed; inoculation with a test antigen and measurement of an antibody response days or weeks later is possible (Wedgewood, 1975). A quicker and noninvasive alternative is to test for antibodies to an antigen to which the patient has been previously exposed, either as a result of earlier active immunisation (Hong, 1976) or after natural exposure Correspondence to: N. Matthews, Department of Medical Microbiology, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, U.K.
to a commensal organism. With antigens such as diphtheria and tetanus toxins there is a wide variation in antibody levels within the population because of different immunisation histories. In contrast, natural antibodies to the commensal E. coli are more uniformly high and measurement of anti-E, coli antibodies is a well established test of humoral immunity (Webster et al., 1974). This test is not without drawbacks and prompted us to look for another suitable commensal. We selected Candida albicans as a possible candidate in that, firstly, the majority of individuals have encountered and responded to it (Cobb et al., 1978) and, secondly, simple immunofluorescence techniques exist for measuring antibodies to the organism (Lehner, 1965, 1970; Esterley, 1968; Dolan et al., 1973).
0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
128 In this study, blood donors without recurrent infections and a group of patients with suspected immunodeficiency were compared for natural antibody production to C. albicans to see whether this test is of use in screening for humoral immunodeficiency.
Materials and methods
Patients Sera were obtained from three groups. Group 1. 114 unselected blood donors (from the Blood Transfusion Service, Rhydlafar, Cardiff). Group 2. 20 children aged 6 m o n t h s - 1 6 years without recurrent infection but who had been referred for serological testing. None of these children had positive viral serology or raised ASO titres. Group 3. 12 serum samples received at this laboratory from patients with suspected immunodeficiency. These patients are an unrepresentative group but were selected to give a range of abnormalities. C. albicans A single clinical isolate was used throughout and maintained by aerobic culture on blood agar. The organism was germ tube positive and fermented glucose and maltose but not lactose and sucrose. Organisms from 5-day-old cultures were suspended in isotonic phosphate-buffered saline, p H 7.2 (PBS) and washed three times at 450 x g. The C. albicans was suspended in 0.4% formol saline at 7 x 106 o r g a n i s m s / m l and stored at 4 ° C. Preparation of slides 10 /~1 of C. albicans suspension (7 X 106/ml) was spotted onto wells of PTFE-coated 15-well multispot slides (Flow) and air-dried. The slides were fixed in acetone for 10 min at 4 ° C , allowed to air-dry and stored at - 8 0 ° C until required. Slides prepared in this way could be used for 6 months without apparent deterioration. lmmunofluorescence test Control and test sera were diluted two-fold in PBS to 1/32. 10 ~1 amounts of each dilution were spotted onto labelled wells of C. albicans-coated
slides. After incubation in a moist box at 3 7 ° C for 30 rain the slides were washed twice by immersion and agitation in 100 ml PBS for 5 rain each wash and then allowed to air-dry. Goat anti-human F I T C conjugate (Sigma) (10 /~1) of the appropriate isotype specificity (previously titrated for optimum dilution) was then spotted onto the slides and incubated at 3 7 ° C in a moist box for 30 rnin. The slides were then washed twice in PBS as before followed by a further wash for 2 min in distilled water. After air-drying and mounting in 50% PBS/glycerol ( v / v ) the slides were examined for surface immunofluorescence by incident light microscopy with narrow band blue illumination. A positive result was taken as > 50% of cells with a rim of fluorescence around the entire perimeter of the cell. The highest serum dilution giving a positive result is defined as the titre. On each slide two unknown samples ( 1 / 2 - 1 / 3 2 ) and a positive control ( 1 / 8 - 1 / 3 2 ) were included with two negative (PBS) controls.
Other tests Antibodies to E. coli were measured as described by Webster et al. (1974). Immunoglobulins were measured by radial immunodiffusion and I g G subclasses by semi-quantitative, simple immunodiffusion using monoclonal antisera (Oxoid).
Results
If the detection of antibodies to C. albicans is to be used as a screening test for immunodeficiency then it is necessary to show that healthy individuals have antibodies to the organism. When unselected blood donors were tested by immunofluorescence using a polyvalent conjugate, all 84 individuals had detectable antibody with titres of > 8 (Fig. 1). Comparable amounts of anti-candida antibody were also seen in the sera of children from 6 months of age (Fig. 2). The predominant isotype in the anti-candida response seems to be IgG, although a minority of the 30 blood donors tested in this manner additionally had IgM antibodies (Fig. 3). These 30 donors were a separate group from those in Fig. 1 but again all 30 had titres of > 8 with the polyvalent conjugate, Because of the higher titres with
129 >32
x
XXXXXXXXXXXXX
XXXXXXXXXXXXXXX
XXXXXXXXXXXXXXX X
32
X
XXX
X
X
XXXXXXXX
16 XXXXXXXX
TITRE
XXXXXXXX
16
TITRE
XXXXXXXXX
¢,4 I
I
I
I
I
0
4
8
12
16
AGE (YR)
XXXXXXXX
Fig. l. Anti-C. albicans titres of 84 blood donors as measured with a polyvalent (anti-IgA,G,M) immunofluorescent conjugate.
the polyvalent conjugate this was preferred for subsequent routine use. Presumably the polyvalent conjugate is more sensitive because it can react with more epitopes per I g G molecule than its IgG-specific counterpart. This isotype distribution is similar to that described previously (Lehner, 1965; Esterley, 1968). These initial observations encouraged us to apply the technique to sera f r o m patients with i m m u n o d e f i c i e n c y (Table I): all of these patients had a history of recurrent infections. N o t surprisingly, those individuals with severe h y p o g a m m a globulinaemia (e.g., patients 1, 10 and 12) had low titres of a n t i b o d y to candida. However patients 3,
Fig. 2. Anti-C. albicans titres of 20 children (without recurrent infections) as a function of age. A polyvalent immunofluorescent conjugate was used.
6 and 8 with normal or near normal I g G concentrations also had reduced anti-candida responses. The two patients (7 and 9) with a seco n d a r y defect due to protein-losing enteropathy h a d normal titres of anti-candida antibody. In all serum samples except that f r o m patient 3, antib o d y responses to E. coli and candida correspond.
32
XXXXX
16
TITRE
8
XX
XXXXXX XXXXXX
XXXXXXXXX XXXXXXXXX XXXXXXXXXX
XXXXXX XXXXXX
XXXXXXXXX XXXXXXXXX XXXXXXXXX
IgG
IgM
IgA
XX
Fig. 3. Anti-C. albicans titres of 30 blood donors as measured with immunofluorescent conjugates specific for either IgA, IgG or IgM.
130 TABLE I Patient No.
1
2 3 4 5 6 7 8 9 10 11 12
Age (years)
0.5
1 1 2 2 2 4 6 14 14 19 35
Normal values (adult)
Anticandida titre
AntiE. coli titre
Immunoblogulins (g/l) IgG
Other Information
IgA
IgM
2
4
1.4
< 0.1
<2 <2 <2 4 <2 8 <2 32 <2 2 <2
4 32 8 16 4 256 <2 128 <2 2 4
<0.1 8.2 2.5 2.9 5.8 2.8 4.8 3.4 2.1 2.8 0.2
<0.1 0.3 0.1 0.1 0.1 0.2 <0.1 0.4 < 0.1 0.6 0.1
0.3 0.7 3.4 0.2 0.8 1.4 1.1 0.2 0.8 <0.1 0.1 < 0.1
>8
> 32
> 5.0
> 0.5
> 0.5
Discussion
This study has shown that the serological response to C. albicans is of value in screening for h u m o r a l i m m u n o d e f i c i e n c y . All 114 b l o o d d o n o r s tested reacted with titres of > 8 o n imm u n o f l u o r e s c e n c e whereas patients with hypog a m m a g l o b u l i n a e m i a a n d some individuals with suspected a n t i b o d y d y s f u n c t i o n had m u c h reduced titres. Overall c a n d i d a i m m u n o f l u o r e s c e n c e closely paralleled E. coli h a e m a g g l u t i n a t i o n , a n established m e t h o d of screening for a n t i b o d y dysfunction (Webster et al., 1974). W i t h the original E. coli m e t h o d the disa d v a n t a g e s are the need to extract antigen, the r e q u i r e m e n t for a n t i g e n from six serotypes a n d the i n c o n v e n i e n c e of c o a t i n g a fresh b a t c h of erythrocytes every few days. Also, patients of the A B b l o o d group m a y give false negative responses. W i t h the c a n d i d a m e t h o d , a single strain is used, a n t i g e n extraction a n d coating of i n d i c a t o r particles is n o t required a n d it is possible to prepare a single large b a t c h of antigen-coated slides a n d store these for u p to 6 m o n t h s in the deep-freeze u n t i l required. T h e disadvantages of the c a n d i d a technique are that firstly, there is a subjective e l e m e n t i n assessing e n d p o i n t s a n d secondly, a n expensive fluorescence microscope is needed. However, m a n y laboratories already possess such
No other defects
Protein-losing enteropathy? Protein-losing enteropathy? Wiskott-Aldrich Myotonic dystrophy?
microscopes a n d the staff are familiar with the i n t e r p r e t a t i o n of i m m u n o f l u o r e s c e n c e . A g g l u t i n a tion or E L I S A w o u l d be possible alternatives to fluorescence for m e a s u r i n g antibodies to C. albicans. A g g l u t i n a t i o n is simple b u t again there is subjectivity in reading e n d p o i n t s a n d other disadvantages are that a u t o a g g l u t i n a t i o n can be a p r o b l e m a n d that c a n d i d a p r e p a r a t i o n s for agg l u t i n a t i o n are stable for only a few weeks. Our p r e l i m i n a r y experiments with a n E L I S A assay emp l o y i n g whole c a n d i d a organisms have shown that this c o m p a r e d f a v o u r a b l y with i m m u n o f l u o r e s cence. E L I S A also has the a d v a n t a g e of being m o r e objective. Whichever detection m e t h o d is e m p l o y e d we believe that the m e a s u r e m e n t of a n t i b o d i e s to C. albicans is of real value in screening for h u m o r a l i m m u n o d e f i c i e n c y .
References
Cobb, J. and Parrat, D. (1978) Determination of antibody levels to Candida albicans in healthy and hospitalised adults using a radioimmunoassay. J. Clin. Pathol. 31, 1161. Dolan, C.T. and Stried, R.P. (1973) Serological diagnosis of yeast infections. Am. J. Clin. Pathol. 59, 49. Esterley, N.B. (1968) Serum antibody titres to Candida albicans, Am. J. Clin. Pathol. 50, 291. Hong, R. (1976) Immunodeficiency. In: N. Rose and H. Friedman (Eds.), Manual of Clinical Immunology. American Society for Microbiology, Washington, DC, p. 620.
131 Lehner, T. (1965) Immunofluorescence study of Candida albicans antibodies in human saliva. Arch. Oral. Biol. 10, 975. Lehner, T. (1970) Serum fluorescent antibody and immunoglobulin estimation in candidosis. J. Med. Microbiol. 3, 475. Webster, A.D.B., Efter, T. and Asherson, G.L. (1974)
Escherichia coli antibody: A screening test for immunode-
ficiency. Br. Med. J. 3, 16. Wedgewood, R.J., Ochs, H.D. and Davis, S.D. (1975) The recognition and classification of immunodeficiency disease with bacteriophage oX 174. Birth Defects Orig. Artic. Ser. 11, 331.