Nitrocellulose Strips Research Articles

Liposome Dehydration on Nitrocellulose and Its Application in a Biotin Immunoassay

The feasibility of utilizing dehydrated liposomes in the development of a simple immunoassay device for point-of-care diagnostics or field assays was demonstrated. The recovery of liposomes after a cycle of dehydration and rehydration was studied using biotin-tagged, dye-loaded liposomes with antibiotin antibodies immobilized in a defined zone on nitrocellulose strips. Liposomes were vacuum-dehydrated on the strip at a location below the antibiotin zone. The strip was placed in a tube containing a carrier solution and capillary action brought the solution to the dehydrated liposomes, rehydrated them, and caused them to migrate to the antibody zone where intact liposomes were captured and measured optically. High concentrations of either trehalose or sucrose external to the liposomes and both polyvinylpyrrolidone and gelatin in the membane blocking reagent were essential for preservation of the dehydrated/rehydrated liposomes on nitrocellulose. Between 70 and 80% of the liposomes were recovered on the nitrocellulose strips after a cycle of dehydration and rehydration. The dehydrated liposomes on the strips were stable for at least 1 year when stored in vacuum-sealed plastic bags at 4°C. The technique was successfully applied to the development of a rapid one-step strip immunoassay for biotin.

Evaluation of a rapid immunochromatographic test for detection of antibodies to human immunodeficiency virus.

A new immunochromatographic rapid test, Determine HIV-1/2, for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1) and HIV-2 in human whole blood, serum, and plasma was evaluated. Determine HIV-1/2 is a sandwich immunoassay and uses a nitrocellulose strip with a capture site for the patient's results and a procedural control site to confirm the validity of the assay. The results can be read visually, and a positive result is indicated by the formation of a red line within 15 min after sample application. The test showed 100% sensitivity for HIV-1 with 102 whole-blood, 152 serum, and 144 plasma samples obtained from Ramathibodi Hospital, Bangkok, Thailand. The sensitivity of the test for HIV-2 was 100% with 100 serum or plasma samples obtained from Ivory Coast. The sensitivity of the test with 4 anti-HIV-1 seroconversion panels from Boston Biomedica Inc. was equivalent to or better than those of another agglutination assay with serum or plasma and the enzyme immunoassay licensed by the U.S. Food and Drug Administration. The specificity was 100% with 367 sets of whole-blood, serum, and plasma samples from Ramathibodi Hospital. This method had an analytical sensitivity for the detection of HIV-1 equivalent to or better than that of another agglutination assay with serum or plasma. This test had an analytical sensitivity for the detection of HIV-1 better than that of another immunochromatographic test with whole blood. This evaluation demonstrated the excellent performance of this immunochromatographic test with EDTA-anticoagulated whole-blood, serum, and plasma samples. We conclude that this test is suitable for use in emerging countries and is an excellent alternative to HIV antibody testing at remote sites, as well as in traditional laboratories.

Development of a new cytomegalovirus (CMV) immunoglobulin M (IgM) immunoblot for detection of CMV-specific IgM.

We developed a new cytomegalovirus (CMV) immunoglobulin M (IgM) immunoblot to detect CMV-specific IgM in human sera. The new test contains four viral proteins (vp150, vp82, vp65, and vp28) purified from viral particles and four recombinant proteins (rp150, rp130, rp52, and rp38) purified from Escherichia coli. These antigens were individually loaded onto nitrocellulose strips, and the strips were then used to detect CMV-specific IgM by using a mu-specific conjugate. The new assay was evaluated in parallel with one or two IgM enzyme-linked immunosorbent assays (ELISAs) to test 592 serum samples from different groups of latently or acutely infected individuals. The sensitivity of the new assay with respect to the consensus of two ELISAs was 100%, the specificity was 98.6%, the positive predictive value was 96.9%, and the negative predictive value was 100%. We also evaluated the new test by testing sera from pregnant women and transplant recipients with a known clinical history. Our results suggest that the new test combines high sensitivity with high specificity, characteristics that are mutually exclusive with the other commercially available tests. Furthermore, a statistically significant correlation was observed between the number of IgM-reactive bands and the elevated risk of transmission from CMV-infected pregnant women to their offspring.

Detection and differentiation of HIV-1 group O sera from HIV-1 group M and HIV-2 using recombinant antigens and peptides

Recombinant antigens and peptides were used to develop an HIV slot immunoblot assay to confirm and differentiate infection by HIV-1 group M, HIV-1 group O or HIV-2. Recombinant antigens from the gag, pol or env regions of HIV-1 and HIV-2, in addition to synthetic peptides from the immunodominant region (IDR) of transmembrane proteins gp41 (HIV-1) or gp36 (HIV-2), were blotted on nitrocellulose strips and used as a substitute for competitive Western blots. Evaluation of a large number of samples ( N=440) from various regions of the world, using the immunoblot, showed effective differentiation of HIV-1 group M, HIV-1 group O and HIV-2. The immunoblot identified correctly all (24/24) HIV-1 group O samples that were confirmed subsequently by PCR and sequence analysis. The immunoblot is a useful tool for identifying HIV-1 group O seropositive samples and has the potential to identify other serological HIV variants that may represent detection problems for HIV screening assays using HIV-1 group M subtype B reagents.

Wheat protein proteolysis in the monoculture of rumen protozoon Entodinium caudatum

The study of the wheat protein (albumin and globulin fraction) proteolysis was performed in the culture medium of the rumen protozoon Entodinium caudatum under anaerobic conditions in vitro. The rate of the wheat protein degradation was measured by the immunological sandwich technique dot-blot. The centrifugation of the protozoal culture media was performed at 500 g for 20 min. The resulting concentration of the protozoal cells was 20,000–24,000 per ml in the experimental culture medium. The samples were taken from the culture medium at 0, 2, 4, 6 and 24 h during incubation. After centrifugation at 500 g for 20 min, the supernatants were used for the determination of: (1) the total protein concentration, (2) the nonspecific proteolytic activity (azoalbumin was used as a substrate) and (3) the wheat protein concentration in the culture medium by the immunological sandwich technique dot-blot. Subsequently, the results were used for the calculation of the degradation rate of the wheat protein. The rabbit polyclonal antiserum against the wheat protein was used as the first antibody in this method. The quantitation of this test was based on the spectrophotometric determination of the violet stain quantity on the nitrocellulose strip surfaces. The average rate of the wheat protein degradation in the culture medium E. caudatum was 0.014 mg ml −1 h −1, whereas the average rate of the azoalbumin degradation was 0.003 mg ml −1 h −1 in supernatant. Azoalbumin was more resistant to degradation in the supernatant of the culture medium in comparison with the wheat protein in the culture medium. The assay sensitivity (=the limit of detection) of the simple immunological assay dot-blot was 104 ng ml −1 (i.e., 0.52 ng wheat protein applied to nitrocellulose foil) at 88% recovery. The accuracy and reproducibility characterized as intra-assay and inter-assay CV% (coefficient of variation) were 9.75% and 14%, respectively. The screening of the samples by dot-blot procedure proved to be an efficient way for the quantitation of the proteolytic activity in the rumen protozoa monoculture cultivated in vitro.

The presence of masked antiribosomal P autoantibodies in healthy children

To determine if antiribosomal P (anti-P) autoantibodies are present in healthy children. Sera from healthy children were screened for anti-P by conventional enzyme-linked immunosorbent assay and immunoblot techniques. Sera were also treated with immobilized ribosomal P antigens on nitrocellulose strips; affinity-purified fractions were tested for anti-P by high-sensitivity immunoblot. The relative binding affinities were compared for affinity-purified anti-P antibodies from healthy children and adults, and patients with systemic lupus erythematosus. IgG fractions of anti-P-depleted sera from healthy children were assessed for inhibition of autologous anti-P activity. Conventional serologic screening showed no IgG nor IgM anti-P in 88 untreated sera. IgG anti-P were unmasked in all 79 sera treated by the membrane batch affinity technique. IgM anti-P were identified in 27 of the treated sera; the percentage of positive sera decreased with increasing age (chi(2) for linear trend P = 0.00081). Affinity-purified anti-P from children had relative binding affinities similar to those of anti-P from other groups. Sera from healthy children contained inhibitory IgG antibodies to anti-P. These results show that anti-P autoantibodies are present in all healthy children. The majority of these autoantibodies are masked by IgG antibodies, suggesting concordant development of a regulatory network.

The presence of masked antiribosomal P autoantibodies in healthy children

Objective To determine if antiribosomal P (anti-P) autoantibodies are present in healthy children. Methods Sera from healthy children were screened for anti-P by conventional enzyme-linked immunosorbent assay and immunoblot techniques. Sera were also treated with immobilized ribosomal P antigens on nitrocellulose strips; affinity-purified fractions were tested for anti-P by high-sensitivity immunoblot. The relative binding affinities were compared for affinity-purified anti-P antibodies from healthy children and adults, and patients with systemic lupus erythematosus. IgG fractions of anti-P-depleted sera from healthy children were assessed for inhibition of autologous anti-P activity. Results Conventional serologic screening showed no IgG nor IgM anti-P in 88 untreated sera. IgG anti-P were unmasked in all 79 sera treated by the membrane batch affinity technique. IgM anti-P were identified in 27 of the treated sera; the percentage of positive sera decreased with increasing age (χ2 for linear trend P = 0.00081). Affinity-purified anti-P from children had relative binding affinities similar to those of anti-P from other groups. Sera from healthy children contained inhibitory IgG antibodies to anti-P. Conclusion These results show that anti-P autoantibodies are present in all healthy children. The majority of these autoantibodies are masked by IgG antibodies, suggesting concordant development of a regulatory network.

Activated macrophage/microglial cells can promote the regeneration of sensory axons into the injured spinal cord.

A prominent role for phagocytic cells in the regenerative response to CNS or PNS injury has been suggested by numerous studies. In the present work we tested whether increasing the presence of phagocytic cells at a spinal cord injury site could enhance the regeneration of sensory axons from cut dorsal roots. Nitrocellulose membranes treated with TGF-beta or coated with microglial cells were cotransplanted with fetal spinal cord tissue into an injured adult rat spinal cord. Cut dorsal roots were apposed to both sides of the nitrocellulose. Four weeks later, animals were sacrificed and spinal cord tissue sections were processed for immunocytochemical detection of calcitonin gene-related peptide (CGRP-ir) to identify regenerated sensory axons. Adjacent sections were processed with the antibody ED-1 or the lectin GSA-B4 for detection of macrophage/microglial cells in association with the regrowing axons. Qualitative and quantitative data indicate a correlation between the pattern and extent of axonal regeneration and the presence of phagocytic cells along the nitrocellulose implant. Axonal regeneration could be experimentally limited by implanting a nitrocellulose strip treated with macrophage inhibitory factor. These results indicate that increasing the presence of activated macrophage/microglial cells at a spinal cord injury site can provide an environment beneficial to the promotion of regeneration of sensory axons, possibly by the release of cytokines and interaction with other nonneuronal cells in the immediate vicinity.

A simple dot immunoassay for detecting antibodies to the lipopolysaccharide of Verocytotoxin-producing Escherichia coli in patients with haemolytic uraemic syndrome

A simple method for detecting patients' antibodies to the lipopolysaccharide (LPS) from Verocytotoxin-producing Escherichia coli (VTEC) is described. LPS was prepared from a panel of VTEC serotypes by the digestion of whole bacteria with proteinase-K, preparations were `dotted' onto strips of nitrocellulose and reacted with sera from patients with haemolytic uraemic syndrome. The use of whole-cell LPS in the form of strips of stable VTEC antigens, provides a rapid procedure for diagnosing infection with VTEC belonging to any serogroup.

Rapid method for visual identification of specific DNA sequences based on DNA-tagged liposomes

We describe a rapid method for visually determining specific DNA sequences at femtomole concentrations. Liposomes, encapsulating a red dye and labeled with oligonucleotide, were used in a capillary migration-sandwich hybridization assay. Capture probe was immobilized on nitrocellulose strips, and liposomes, migrating along each strip, formed a visually discernible band in the presence of target DNA. One femtomole of synthetic target sequence could be detected in < 10 min. Sufficiently stringent hybridization conditions can be used to allow the discrimination of a 10% mismatch sequence from perfectly complementary DNA. A 366-base PCR product was detected at 200 fmol.

Electrophoresis Separation of Glycosaminoglycans on Nitrocellulose Membranes

A rapid and simple electrophoretic separation method on nitrocellulose membranes by a single run of glycosaminoglycans, mainly slow-moving and fast-moving heparins, dermatan sulfate, and chondroitin sulfate, is reported. Different dyes, azure A, toluidine blue, and Alcian blue, were used to stain the nitrocellulose strips, and azure A showed the highest sensitivity. The mobility for slow-moving heparin was 0.00 cm/A/min, for fast-moving species about 2.87 × 10−3cm/A/min, for dermatan sulfate 4.00 × 10−3cm/A/min, and for chondroitin sulfate about 5.75 × 10−3cm/A/min. Calibration curves were determined by the increasing amounts (from 0.1 to 2.5 μg) of single glycosaminoglycan species. The curves demonstrated a linear relationship between the amount of glycosaminoglycans (slow-moving and fast-moving heparin, dermatan sulfate, and chondroitin sulfate) and the optical density at 600 nm calculated by densitometric scanning. The limit of detection for fast-moving heparin, dermatan sulfate and chondroitin sulfate was about 0.2–0.3 μg, while that for slow-moving heparin was 0.1 μg. Hyaluronic acid, keratan sulfate, and highly sulfated chondroitin sulfate from shark cartilage were also separated electrophoretically on nitrocellulose. No difference in mobility was detected for chondroitin sulfates with different structure and physicochemical properties. Hyaluronic acid has about the same mobility as that of dermatan sulfate, while keratan sulfate has a mobility intermediate between those of dermatan sulfate and chondroitin sulfate (about 4.75 × 10−3cm/A/min). Glycosaminoglycans extracted and purified from bovine aorta and bovine lung were submitted to electrophoresis on nitrocellulose and the amount of each polysaccharide species was calculated by densitometric scanning. By this analysis bovine aorta glycosaminoglycans consist of about 11% fast-moving heparin (heparan sulfate), 23% dermatan sulfate, and 65% chondroitin sulfate. Bovine lung polysaccharides are composed of about 16% slow-moving and 46% fast-moving species (62% heparin), 15% dermatan sulfate, and 22% chondroitin sulfate.

Evaluation of immunochromatographic assay systems for rapid detection of hepatitis B surface antigen and antibody, Dainascreen HBsAg and Dainascreen Ausab.

We evaluated two immunochromatographic assays (ICAs), Dainascreen HBsAg for detecting human hepatitis hepatitis B surface antigen (HBsAg) and Dainascreen Ausab for detecting human hepatitis B surface antibody (anti-HBs) in human serum. The ICA systems are composed of a comb-shaped device that contains nitrocellulose strips on which complexes of HBsAg and anti-HBs can be visualized. The results can be read within 15 min of incubation. The limit of detection for HBsAg was 3.1 ng/ml and that for anti-HBs was 42 mIU/ml. Results of HBsAg detection agreed completely with those of conventional enzyme immunoassays (EIAs) and showed a 100% sensitivity (158 of 158 samples) and a 100% specificity (304 of 304 samples). The Dainascreen Ausab detected 184 of the 199 EIA-positive samples (sensitivity, 92.5%) and yielded 6 positive results among the 281 EIA-negative samples (specificity, 97.9%). The ICA systems are rapid and sensitive methods for detecting HBsAg and anti-HBs. They are low-cost systems that need no complex instrumentation for analysis and can be recommended for routine use in clinical microbiology laboratories.

Ein Vergleich von vier Immunoblot-Systemen zur Differenzierung von Antikörpern gegen nukleäre und zytoplasmatische Antigene

Since recently, immunoblots for detection and differentiation of antibodies against nuclear antigens (ENA), other nuclear antigens and cytoplasmic antigens, blotted on nitrocellulose strips (Western blot technique) became commercially available. The aim of our analysis was to compare the specifity and sensitivity of four different immunblot systems (blots A, B, L, V). We examined 20 sera of patients with various clinically confirmed connective tissue diseases. The sera contained a total of 32 different antigens. The antibodies in the sera were defined by double immunodiffusion (Ouchterlony) test, immunofluorescence (HEp2 cells) and by comparison with WHO reference sera.

Novel Optical Measurement Approach for the Quantitation of Liposome Immunomigration Assays

Abstract In a recent series of papers1–3 the use of dye-containing liposomes as a component of immunochemical field assay methods for the detection and measurement of environmental contaminants has been demonstrated. These assays produce a colored measurement zone on a plastic-backed nitrocellulose strip, with the amount of color in the zone being proportional to the amount of analyte present in the test sample. The color in this zone has been measured using scanning densitometry of the strips. While giving a quantitative measurement, such a system is not field-portable and cannot differentiate between multiple colors in the measurement zone. The human eye is also limited in the case of mixed colors. In this report, the use of a ColortronTM 32-band digital color sensor for the quantitation measurement is demonstrated. This instrument is designed for inexpensive color analysis in the desktop publishing industry, but the fact that it can be used as a portable scanning reflectometer means that it has great p...

Improved liposome immunomigration strip assay for alachlor determination

The feasibility of a simple, single-use immunomigration strip assay for alachlor was previously demonstrated. In the device, capillary action caused alachlor and alachlor-tagged, dye-containing liposomes to migrate through an anti-alachlor antibody zone, on a plastic-backed nitrocellulose strip, where competitive binding occurred. Unbound liposomes continued migration to a liposome capture zone, where they were quantified by densitometry. The amount of liposome-entrapped dye measured in this zone was directly proportional to the alachlor concentration in the sample. This report describes modifications to various components of the system, leading to improvement in the sensitivity of the assay to the point where the maximum contaminant level of alachlor, 2 ppb, can be easily detected. Measurements of liposome size and antibody cross-reactivity are also presented. The new methodology involves acid treatment of the antibody and the use of preincubation of the analyte-tagged liposomes, free analyte and anti-alachlor before initiating migration. This results in strong interactions between the anti-alachlor, liposome and strip such that liposomes that have bound to antibody do not migrate. This inhibition of migration is reversed by free analyte, which competes with the liposome for the available antibody binding sites. As in the previous assay, unbound liposomes migrate to a capture zone where they can be quantified, and the color intensity of this zone is directly proportional to the amount of analyte present. This technique produces an assay capable of detecting alachlor at levels down to 1 ppb.

Production of a Human Anti-CD4 Monoclonal Antibody with Antiidiotype to Anti-HIV Type 1 Glycoprotein 120

Human anti-CD4 IgG antibodies from 3 HIV-1-infected patients were affinity purified and shown to inhibit HIV-1 binding and infection of HBP-T cells. Lymphocytes from patient A, whose anti-CD4 inhibited HIV-1 binding by 68% and infection by 72%, were cultured and transformed with EBV. A human monoclonal antiidiotype antibody against anti-HIV-1 gp120 (2B) was produced, which inhibited infection of HBP-T cells by 68% at 1 microgram/ml. Mice were immunized with 2B to determine whether this anti-CD4 could be an internal image antiidiotype against anti-HIV-1 gp 120 (Ab1). Two mice produced antisera reactive with rgp120 on ELISA, whereas immunization with normal IgG produced minimal reactivity compared to unreactive normal mouse sera. However, immunoblot competition studies in which affinity-purified anti-HIV-1 gp120 (Ab1) bound to the gp120 band on nitrocellulose strips in the presence of 2B demonstrated enhancement of signal (i.e., binding of Ab2 to Ab1), rather than inhibition of Ab1 binding. Thus 2B is not an internal image of the paratope of anti-HIV-1 gp120 but yet it is capable of inducing an antibody against rgp120. This indicates that the anti-CD4 (Ab2) does bind to the binding site of Ab1, but not as a complete internal image. These data indicate the production of a human monoclonal antiidiotype antibody that inhibits binding of HIV-1 to CD4 and induces the production of antibody against HIV-1 gp120, without being an internal image antiidiotype (Ab2 beta).

A Double Labeling Procedure for Lipoproteins: Independent Visualization of Dual Ligand-Receptor Interaction with Colloidal Gold- and 125I-Labeled Ligands

The analysis of multiple ligand binding to a single receptor molecule poses a methodological challenge. The chicken oocyte 95-kDa receptor for the uptake of the two major yolk lipoprotein precursors very low-density lipoprotein (VLDL) and vitellogenin (VTG) is such a multipotent transport receptor. Here we describe methods for rapid independent and simultaneous analysis of VLDL and VTG binding to this receptor, termed VLDL/VTG receptor. First, further development of a one-step labeling protocol for chicken lipoproteins with colloidal gold (Au) to visualize independently the binding of VLDL and VTG to the chicken VLDL/VTG receptor is reported. The advantage of this protocol is that the preparation of the Au-lipoprotein conjugates is rapid, and utilization of Au-lipoprotein complexes in ligand blots does not require their further purification, while signal enhancement by silver staining is still applicable. Second, the simultaneous use of 125I- and Au-labeled ligands in a one-step ligand blotting procedure facilitates the direct demonstration of the competitive interaction of VLDL and VTG with the receptor. Following sequential processing of the nitrocellulose strips, binding of differently labeled ligands can be studied independently. In summary, we describe a procedure for differential labeling of lipoproteins and its application toward the analysis of receptors that bind more than one ligand.

Investigation of liposome-based immunomigration sensors for the detection of polychlorinated biphenyls.

The use of immunospecific liposome migration offers many advantages for the extralaboratory detection of environmental contaminants. Devices utilizing this technique are fast, easy to use, and robust and respond to the presence of analyte at low-parts-per-billion concentrations. Investigations have been carried out that determine optimal interactions for key components of these assays, and techniques are presented for the development of generalized liposome immunoassays. Two complementary prototype liposome-based immunomigration techniques have been developed for the detection of polychlorinated biphenyls (PCBs). The liposome immunocompetition assay format measures the competitive reaction between analyte-tagged liposomes and the sample analyte for immobilized antibodies and can detect 0.4 nmol of PCB in less than 8 min. A more sensitive format, the liposome immunoaggregation (LIA) assay detects the inhibition of immunospecific liposome aggregation in solution and can detect 2.6 pmol of PCB in less than 23 min. Laser diffraction particle sizing has been used to study LIA-induced increases in liposome size over time and to determine optimal conditions for the application of this technique. Both formats utilize capillary action to transport liposome-containing solutions along strips of nitrocellulose. Measurement of color intensity is then carried out visually or with a desktop scanner.

Dynein inner arm heavy chain identification in cAMP‐activated flagella using class‐specific polyclonal antibodies

While studying cAMP-dependent dynein alpha-heavy chain phosphorylation, we found previously [Stephens and Prior, 1992: J. Cell Sci. 103:999-1012] that high salt extraction of sperm flagella from the mussel Mytilus edulis or the clam Spisula solidissima removed most visible dynein arms, accompanied by an amount of Mg+2-ATPase that correlated with the mass of dynein alpha- and beta-heavy chains removed. However, although almost devoid of ATPase activity, such extracted axonemes retained one third of the heavy chain mass as two sets of electrophoretically-distinct, vanadate-cleavable, non-phosphorylated proteins. To explore the nature of these dynein-like proteins, antibodies to the alpha- and beta-heavy chains were blot affinity-purified from a rabbit antiserum raised against gradient-purified Spisula 18-20S flagellar outer arm dynein. Although able to recognize common epitopes of the opposite chain type, neither the alpha- nor the beta-heavy chain antibody recognized the tightly-bound proteins in either species, proving that they are immunologically distinct. While the beta-antibody recognized its heavy chain homolog in gill cilia, the alpha-antibody did not, demonstrating immunological distinction between flagellar and ciliary dynein alpha-heavy chains. Immunization of a mouse with nitrocellulose strips containing one of the two tightly-bound Spisula flagellar proteins produced an antiserum that cross-reacted with each tightly-bound protein in both species and also recognized alpha- and beta-heavy chains. The anti-molluscan serum cross-reacted strongly with sea urchin sperm flagellar dynein B-, C-, and D-bands, considered to be inner arm components, but not with sea urchin outer arm alpha- or beta-heavy chains. These data indicate that the electrophoretically and immunologically distinct, tightly-bound proteins of molluscan flagella are inner arm dynein heavy chains.

8] Identification of receptors for bacterial lectins by blotting techniques

Publisher Summary This chapter discusses the identification of receptors for bacterial lectins by blotting techniques. Because of the phenomenon of phase variation in the production of adhesins, however, the bacteria must be grown under conditions that are optimal for phenotypic expression of the adhesin. Also, for application to nitrocellulose strips, the bacteria must be suspended in a buffer that is optimal for adhesin activity. The latter can be determined by testing the ability of the bacteria to adhere to suitable target cells. Autoradiography is the most sensitive means to detect the receptors on the blots. Exposures can be repeated until optimal signals are obtained. However, the resolution of enzyme-linked assays appears to be superior to that of autoradiography. Whenever possible, the specificity of the binding should be ascertained. In the case of the bacterial surface lectins, this is readily done by establishing whether binding of the probe to the blotted glycoproteins is inhibited by the mono- or oligosaccharides for which the bacteria are specific. Another way is to test the effect of treatment of blots in situ with carbohydrate modifying reagents, such as glycosidases or periodate.